gene specific dna methylation  (New England Biolabs)


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    New England Biolabs gene specific dna methylation
    EpiMark 5 hmC and 5 mC Analysis Kit
    EpiMark 5 hmC and 5 mC Analysis Kit 20 rxns
    https://www.bioz.com/result/gene specific dna methylation/product/New England Biolabs
    Average 92 stars, based on 111 article reviews
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    gene specific dna methylation - by Bioz Stars, 2020-05
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    Images

    1) Product Images from "RG108 increases NANOG and OCT4 in bone marrow-derived mesenchymal cells through global changes in DNA modifications and epigenetic activation"

    Article Title: RG108 increases NANOG and OCT4 in bone marrow-derived mesenchymal cells through global changes in DNA modifications and epigenetic activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207873

    Changes in DNA modifications at gene-specific regulatory elements in response to RG108 treatment. Methylation and hydroxymethylation levels were analyzed by DNA glycosylation followed by restriction enzyme analysis and qPCR of promoter sequences, after 3 days of 50 μM RG108 treatment and compared to DMEM and DMSO controls. The relative levels were determined using the cycle threshold (Ct) method and the methylation results are presented as HpaII levels— MspI levels/control levels and the hydroxymethylation results are presented as MspI levels/control levels (% of control). Two biological replicates were performed for each group and each of the biological replicates was done in technical triplicates. (A) All DNMT genes are mostly unmethylated (20–30% methylation level) and undergo only small (2–3%) methylation after RG108. (B) The hydroxymethylation levels at DNMTs’ promoters are low and do not change after RG108 treatment. (C) TET genes have similar 20–30% methylation levels; TET1 and TET2 undergo small but significant demethylation after RG108. (D) Hydroxymethylation levels at TET genes are low and do not change with RG108. (E) RG108 treatment resulted in 40% and 40% loss of methylation and hydroxymethylation, respectively at NANOG regulatory element. (F) For OCT4, the methylation loss was 48% and hydroxymethylation was 32%. For all graphics, *, ** or *** above the bars represent significant inter-group differences when compared to DMEM. Other significant differences are represented by * symbol above the linkers. *, ** and *** indicate, respectively, p ≤ 0.01, p ≤ 0.001 p ≤ 0.0001 by ANOVA One Way followed by the Tukey test.
    Figure Legend Snippet: Changes in DNA modifications at gene-specific regulatory elements in response to RG108 treatment. Methylation and hydroxymethylation levels were analyzed by DNA glycosylation followed by restriction enzyme analysis and qPCR of promoter sequences, after 3 days of 50 μM RG108 treatment and compared to DMEM and DMSO controls. The relative levels were determined using the cycle threshold (Ct) method and the methylation results are presented as HpaII levels— MspI levels/control levels and the hydroxymethylation results are presented as MspI levels/control levels (% of control). Two biological replicates were performed for each group and each of the biological replicates was done in technical triplicates. (A) All DNMT genes are mostly unmethylated (20–30% methylation level) and undergo only small (2–3%) methylation after RG108. (B) The hydroxymethylation levels at DNMTs’ promoters are low and do not change after RG108 treatment. (C) TET genes have similar 20–30% methylation levels; TET1 and TET2 undergo small but significant demethylation after RG108. (D) Hydroxymethylation levels at TET genes are low and do not change with RG108. (E) RG108 treatment resulted in 40% and 40% loss of methylation and hydroxymethylation, respectively at NANOG regulatory element. (F) For OCT4, the methylation loss was 48% and hydroxymethylation was 32%. For all graphics, *, ** or *** above the bars represent significant inter-group differences when compared to DMEM. Other significant differences are represented by * symbol above the linkers. *, ** and *** indicate, respectively, p ≤ 0.01, p ≤ 0.001 p ≤ 0.0001 by ANOVA One Way followed by the Tukey test.

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction

    Global effects of RG108 on DNA modifications and DNMTs and TETs enzymatic activities in hBMSCs. hBMSCs were treated with 50 μM RG108 for 3 days and compared to DMEM and DMSO controls. (A) Global methylation was assessed using Imprint Methylated DNA Quantification Kit showing a significant decrease in the RG108 group. (B) Global hydroxymethylation was analyzed using Quest 5-hmC DNA Elisa kit; no statistical differences are observed amongst groups. DNMTs (C) and TETs (D) enzymatic activities were assessed using colorimetric assays. Global methylation and hydroxymethylation experiments were performed in biological triplicates and DNMTs and TETs activities were performed in biological duplicates. For all graphics, ** or *** above the bars represent significant inter-group differences when compared to DMEM. Other significant differences are represented by * symbol above the linkers. ** and *** indicate, respectively, p ≤ 0.001 and p ≤ 0.0001 by ANOVA One Way followed by the Tukey test.
    Figure Legend Snippet: Global effects of RG108 on DNA modifications and DNMTs and TETs enzymatic activities in hBMSCs. hBMSCs were treated with 50 μM RG108 for 3 days and compared to DMEM and DMSO controls. (A) Global methylation was assessed using Imprint Methylated DNA Quantification Kit showing a significant decrease in the RG108 group. (B) Global hydroxymethylation was analyzed using Quest 5-hmC DNA Elisa kit; no statistical differences are observed amongst groups. DNMTs (C) and TETs (D) enzymatic activities were assessed using colorimetric assays. Global methylation and hydroxymethylation experiments were performed in biological triplicates and DNMTs and TETs activities were performed in biological duplicates. For all graphics, ** or *** above the bars represent significant inter-group differences when compared to DMEM. Other significant differences are represented by * symbol above the linkers. ** and *** indicate, respectively, p ≤ 0.001 and p ≤ 0.0001 by ANOVA One Way followed by the Tukey test.

    Techniques Used: Methylation, Enzyme-linked Immunosorbent Assay

    2) Product Images from "Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9"

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    Journal: mBio

    doi: 10.1128/mBio.00648-15

    DNA modification in phage T4 showing C-containing DNA (left), HMC-containing DNA (middle), and glc-HMC DNA (right).
    Figure Legend Snippet: DNA modification in phage T4 showing C-containing DNA (left), HMC-containing DNA (middle), and glc-HMC DNA (right).

    Techniques Used: Modification, Gas Chromatography

    Glc-HMC and HMC modifications inhibit attack by the CRISPR-Cas9 system on phage T4. (A) Diagram of the strategy used to validate CRISPR spacers in a transformation assay. Bacteria containing the type II CRISPR system were transformed with a pUC19 plasmid containing either a T4 protospacer and PAM sequence or a nonspecific DNA sequence. Antibiotic selection for the pUC19 plasmid and quantification of the efficiency of transformation reveal the efficacy of CRISPR system cleavage of unmodified DNA containing a protospacer and PAM. (B) Results of plasmid challenge tests. The efficiency of transformation is the ratio of colony counts of cells transformed with equal amounts of pUC19 that contain a protospacer targeting the plasmid (numerator) to the colony counts of cells transformed with pUC19 (denominator). (C) Diagram of plaque assays to assess inhibition of T4 infection with CRISPR-Cas9. (D to F) Results of plaque assays in which the E. coli strains indicated were infected with up to 1 × 10 4 PFU of T4(C) (panel D), T4(glc-HMC) (panel E), or T4(HMC) (panel F). E. coli strains expressed Cas9 and crRNAs targeting T4 or controls. Starting from the left in each panel, None indicates no crRNA or Cas9, non-sp indicates nonspecific crRNA, 1 contained the maximum number of cytosines in the target strand and seed sequence, 2 contained the maximum number of cytosines in the target and complementary strands, 3 contained no cytosines in the target strand and seven cytosines in the complementary strand, and 4 contained the fewest cytosines in the target and complementary strands. Mean values were compared with the Kruskal-Wallis test. *, P
    Figure Legend Snippet: Glc-HMC and HMC modifications inhibit attack by the CRISPR-Cas9 system on phage T4. (A) Diagram of the strategy used to validate CRISPR spacers in a transformation assay. Bacteria containing the type II CRISPR system were transformed with a pUC19 plasmid containing either a T4 protospacer and PAM sequence or a nonspecific DNA sequence. Antibiotic selection for the pUC19 plasmid and quantification of the efficiency of transformation reveal the efficacy of CRISPR system cleavage of unmodified DNA containing a protospacer and PAM. (B) Results of plasmid challenge tests. The efficiency of transformation is the ratio of colony counts of cells transformed with equal amounts of pUC19 that contain a protospacer targeting the plasmid (numerator) to the colony counts of cells transformed with pUC19 (denominator). (C) Diagram of plaque assays to assess inhibition of T4 infection with CRISPR-Cas9. (D to F) Results of plaque assays in which the E. coli strains indicated were infected with up to 1 × 10 4 PFU of T4(C) (panel D), T4(glc-HMC) (panel E), or T4(HMC) (panel F). E. coli strains expressed Cas9 and crRNAs targeting T4 or controls. Starting from the left in each panel, None indicates no crRNA or Cas9, non-sp indicates nonspecific crRNA, 1 contained the maximum number of cytosines in the target strand and seed sequence, 2 contained the maximum number of cytosines in the target and complementary strands, 3 contained no cytosines in the target strand and seven cytosines in the complementary strand, and 4 contained the fewest cytosines in the target and complementary strands. Mean values were compared with the Kruskal-Wallis test. *, P

    Techniques Used: Gas Chromatography, CRISPR, Transformation Assay, Plasmid Preparation, Sequencing, Selection, Inhibition, Infection

    3) Product Images from "A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD"

    Article Title: A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0185-9

    DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p
    Figure Legend Snippet: DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p

    Techniques Used: Polymerase Chain Reaction, BAC Assay, Mouse Assay, Standard Deviation, Methylation

    4) Product Images from "Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes"

    Article Title: Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes

    Journal: Genome Research

    doi: 10.1101/gr.126417.111

    Marked inter-tissue differences in global 5hmC levels. ( A ) Dot-blots of decreasing amounts of a PCR product of the mouse Tex19 promoter sequence in which all cytosines are either unmodified ( C ), methylated (5mC), or hydroxymethylated (5hmC), were probed with α-5hmC and α-5mC antibodies. The α-5hmC and α-5mC antibodies are specific for their respective marks. ( B ) Duplicate dot-blots of DNA from human tissues and ES cells probed with antibodies specific to 5hmC or 5mC show that global 5hmC levels vary markedly between normal human tissues. An α-ssDNA antibody was used to control for loading; 500 ng and 100 ng were loaded in the upper and lower lanes, respectively. Ten nanograms of amplified mouse Tex19.1 promoter was used as a control; (solid circle) unmethylated DNA; (dotted circle) methylated DNA; (dashed circle) hydroxymethylated DNA. ( C ) Inter-tissue differences in global 5hmC levels as determined by densitometric analysis of dot-blots shown in panel B . 5hmC values were normalized to an ssDNA loading control and scaled relative to brain. Values are the means of two independent biological replicates. Spleen and ES values represent single measurements. ( D ). ( E ) Duplicate dot-blots of DNA from mouse tissues and ES cells probed with antibodies specific to 5hmC or 5mC show that global 5hmC levels vary markedly between normal mouse tissues. An α-ssDNA antibody was used to control for loading. Ten nanograms of amplified mouse Tex19.1 promoter was used as a control; (solid circle) unmethylated DNA; (dotted circle) methylated DNA; (dashed circle) hydroxymethylated DNA.
    Figure Legend Snippet: Marked inter-tissue differences in global 5hmC levels. ( A ) Dot-blots of decreasing amounts of a PCR product of the mouse Tex19 promoter sequence in which all cytosines are either unmodified ( C ), methylated (5mC), or hydroxymethylated (5hmC), were probed with α-5hmC and α-5mC antibodies. The α-5hmC and α-5mC antibodies are specific for their respective marks. ( B ) Duplicate dot-blots of DNA from human tissues and ES cells probed with antibodies specific to 5hmC or 5mC show that global 5hmC levels vary markedly between normal human tissues. An α-ssDNA antibody was used to control for loading; 500 ng and 100 ng were loaded in the upper and lower lanes, respectively. Ten nanograms of amplified mouse Tex19.1 promoter was used as a control; (solid circle) unmethylated DNA; (dotted circle) methylated DNA; (dashed circle) hydroxymethylated DNA. ( C ) Inter-tissue differences in global 5hmC levels as determined by densitometric analysis of dot-blots shown in panel B . 5hmC values were normalized to an ssDNA loading control and scaled relative to brain. Values are the means of two independent biological replicates. Spleen and ES values represent single measurements. ( D ). ( E ) Duplicate dot-blots of DNA from mouse tissues and ES cells probed with antibodies specific to 5hmC or 5mC show that global 5hmC levels vary markedly between normal mouse tissues. An α-ssDNA antibody was used to control for loading. Ten nanograms of amplified mouse Tex19.1 promoter was used as a control; (solid circle) unmethylated DNA; (dotted circle) methylated DNA; (dashed circle) hydroxymethylated DNA.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Methylation, Amplification

    Reduced global 5hmC levels and TET1/2/3 gene expression in human cell lines. ( A ) Duplicate dot-blots of DNA (500 ng) from normal human breast tissue, a primary mammary epithelial cell line from non-cancerous tissue, and eight breast cancer cell lines probed with antibodies specific to 5hmC or ssDNA illustrate that global 5hmC levels are reduced in the DNA of primary and cancer cell lines relative to normal breast. ( B ) Inter-sample differences in global 5hmC levels by densitometric analysis of dot-blots shown in panel A . 5hmC values were normalized to the ssDNA loading control and scaled relative to normal breast DNA. Values are the means of two technical replicates. ( C ) RT-qPCR analysis of expression levels of TET1 , TET2 , and TET3 in normal human breast tissue, a primary mammary epithelial cell line, and eight breast cancer cell lines. Expression levels were normalized to GAPDH expression, and expression levels in normal human breast set to 1. Error bars represent the SD of two technical replicates. ( D ) Duplicate dot-blots of DNA (500 ng) from normal human colon and liver tissue and six cancer cell lines probed with antibodies specific to 5hmC or ssDNA illustrate that global 5hmC levels are also reduced in the DNA of colon and liver cancer cell lines relative to normal tissue. ( E ) Duplicate dot-blots of DNA (500 ng) from normal human breast tissue, and the primary mammary epithelial cell line derived from that tissue probed with antibodies specific to 5hmC, 5mC, or ssDNA show that global levels of 5hmC, but not 5mC, are gradually reduced upon transformation of normal breast tissue to cell culture. Ten nanograms of amplified mouse Tex19.1 promoter in which all cytosines were either unmodified (C), methylated (mC), or hydroxymethylated (hmC), was used as a control.
    Figure Legend Snippet: Reduced global 5hmC levels and TET1/2/3 gene expression in human cell lines. ( A ) Duplicate dot-blots of DNA (500 ng) from normal human breast tissue, a primary mammary epithelial cell line from non-cancerous tissue, and eight breast cancer cell lines probed with antibodies specific to 5hmC or ssDNA illustrate that global 5hmC levels are reduced in the DNA of primary and cancer cell lines relative to normal breast. ( B ) Inter-sample differences in global 5hmC levels by densitometric analysis of dot-blots shown in panel A . 5hmC values were normalized to the ssDNA loading control and scaled relative to normal breast DNA. Values are the means of two technical replicates. ( C ) RT-qPCR analysis of expression levels of TET1 , TET2 , and TET3 in normal human breast tissue, a primary mammary epithelial cell line, and eight breast cancer cell lines. Expression levels were normalized to GAPDH expression, and expression levels in normal human breast set to 1. Error bars represent the SD of two technical replicates. ( D ) Duplicate dot-blots of DNA (500 ng) from normal human colon and liver tissue and six cancer cell lines probed with antibodies specific to 5hmC or ssDNA illustrate that global 5hmC levels are also reduced in the DNA of colon and liver cancer cell lines relative to normal tissue. ( E ) Duplicate dot-blots of DNA (500 ng) from normal human breast tissue, and the primary mammary epithelial cell line derived from that tissue probed with antibodies specific to 5hmC, 5mC, or ssDNA show that global levels of 5hmC, but not 5mC, are gradually reduced upon transformation of normal breast tissue to cell culture. Ten nanograms of amplified mouse Tex19.1 promoter in which all cytosines were either unmodified (C), methylated (mC), or hydroxymethylated (hmC), was used as a control.

    Techniques Used: Expressing, Quantitative RT-PCR, Derivative Assay, Transformation Assay, Cell Culture, Amplification, Methylation

    5) Product Images from "Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes"

    Article Title: Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddv198

    Quantification by enzymatic assay of 5hmC and 5mC at selected CCGG sites in the MYOD1 neighborhood and in MYF6. The average percentages of 5hmC ( A ) and 5mC ( B ) at the indicated CCGG sites in biological replicate samples were determined by an enzymatic
    Figure Legend Snippet: Quantification by enzymatic assay of 5hmC and 5mC at selected CCGG sites in the MYOD1 neighborhood and in MYF6. The average percentages of 5hmC ( A ) and 5mC ( B ) at the indicated CCGG sites in biological replicate samples were determined by an enzymatic

    Techniques Used: Enzymatic Assay

    6) Product Images from "Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays"

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays

    Journal: ACS Nano

    doi: 10.1021/acsnano.8b03023

    Global correlation between 5-hmC profiles produced by optical 5-hmC mapping and hMeDIP-seq. (A) Coverage as a function of distance from TSS. (B) Coverage as a function of distance from H3K4me1 histone modification peaks. (C) Coverage as a function of distance from H3K27Ac histone modification peaks. (D) Comparison of coverage produced by both methods in a representative 500 kbp region from chromosome 1. hMeDIP-seq results are presented in 1 kbp resolution.
    Figure Legend Snippet: Global correlation between 5-hmC profiles produced by optical 5-hmC mapping and hMeDIP-seq. (A) Coverage as a function of distance from TSS. (B) Coverage as a function of distance from H3K4me1 histone modification peaks. (C) Coverage as a function of distance from H3K27Ac histone modification peaks. (D) Comparison of coverage produced by both methods in a representative 500 kbp region from chromosome 1. hMeDIP-seq results are presented in 1 kbp resolution.

    Techniques Used: Produced, Modification

    5-hmC coverage across gene bodies, in correlation with gene expression level. Gene lengths were normalized to 15 kbp, and 3 kbp was added to each gene upstream of the TSS and downstream of the TES. Left: optical mapping data. Right: hMeDIP-seq data.
    Figure Legend Snippet: 5-hmC coverage across gene bodies, in correlation with gene expression level. Gene lengths were normalized to 15 kbp, and 3 kbp was added to each gene upstream of the TSS and downstream of the TES. Left: optical mapping data. Right: hMeDIP-seq data.

    Techniques Used: Expressing

    Epigenetic characterization of variable regions by optical 5-hmC mapping “long reads”. Light blue: hMeDIP-seq. Black: optical 5-hmC mapping. Blue: gene symbol. (A) 5-hmC coverage of 23 kbp around the HLA-A gene. (B) 5-hmC coverage of 111 kbp containing the histone gene cluster. (C) 5-hmC coverage of 103 kbp around TLR7 and TLR8-AS1. (D) 5-hmC coverage of 98 kbp containing the TLR cluster located on chromosome 4.
    Figure Legend Snippet: Epigenetic characterization of variable regions by optical 5-hmC mapping “long reads”. Light blue: hMeDIP-seq. Black: optical 5-hmC mapping. Blue: gene symbol. (A) 5-hmC coverage of 23 kbp around the HLA-A gene. (B) 5-hmC coverage of 111 kbp containing the histone gene cluster. (C) 5-hmC coverage of 103 kbp around TLR7 and TLR8-AS1. (D) 5-hmC coverage of 98 kbp containing the TLR cluster located on chromosome 4.

    Techniques Used:

    Optical 5-hmC mapping experimental scheme. (A) Left: Scanning electron microscope (SEM) image of a silicon nanochannel array. Right: Stretched DNA molecules (gray) fluorescently labeled in two colors. Green: Sequence specific genetic barcode. Red: 5-hmC labels. (B) Fluorescently labeled molecules are extended in nanochannel arrays by electrophoresis. (C) Fluorescence intensity of genetic labels (green) and 5-hmC labels (red) along a single molecule. (D) Genetic labels (green) are used to align a digital representation of the molecule in part C (yellow) to an in silico generated reference (gray) of chromosome 5, highlighting large structural variations such as the 7 kbp deletion in the midright part of the molecule, denoted by the diagonal alignment marks. 5-hmC labels (red) are mapped on the basis of genetic alignment.
    Figure Legend Snippet: Optical 5-hmC mapping experimental scheme. (A) Left: Scanning electron microscope (SEM) image of a silicon nanochannel array. Right: Stretched DNA molecules (gray) fluorescently labeled in two colors. Green: Sequence specific genetic barcode. Red: 5-hmC labels. (B) Fluorescently labeled molecules are extended in nanochannel arrays by electrophoresis. (C) Fluorescence intensity of genetic labels (green) and 5-hmC labels (red) along a single molecule. (D) Genetic labels (green) are used to align a digital representation of the molecule in part C (yellow) to an in silico generated reference (gray) of chromosome 5, highlighting large structural variations such as the 7 kbp deletion in the midright part of the molecule, denoted by the diagonal alignment marks. 5-hmC labels (red) are mapped on the basis of genetic alignment.

    Techniques Used: Microscopy, Labeling, Sequencing, Electrophoresis, Fluorescence, In Silico, Generated

    Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).
    Figure Legend Snippet: Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).

    Techniques Used: Labeling, Lambda DNA Preparation

    7) Product Images from "Biochemical Characterization of Recombinant ?-Glucosyltransferase and Analysis of Global 5-Hydroxymethylcytosine in Unique Genomes"

    Article Title: Biochemical Characterization of Recombinant ?-Glucosyltransferase and Analysis of Global 5-Hydroxymethylcytosine in Unique Genomes

    Journal: Biochemistry

    doi: 10.1021/bi2014739

    Purity and initial characterization of the β-GT enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by MfeI. A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.
    Figure Legend Snippet: Purity and initial characterization of the β-GT enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by MfeI. A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.

    Techniques Used: Staining, SDS Page, Recombinant

    8) Product Images from "Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression"

    Article Title: Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression

    Journal: Epigenetics

    doi: 10.1080/15592294.2016.1265713

    Average levels of 5hmC and 5mC at 12 of the 21 tested CCGG sites and RNA-seq RPKM values for the associated genes. (a) The average levels of 5hmC/total C at 12 of the 21 CCGG sites that were determined by Epimark assays on biological replicates for four of the 14 examined sample types. (b) The average levels of 5mC/total C from these assays. (c) RPKM (reads per kilobase per million mapped reads), median values from 430, 218, 125, and 393 samples of generic SkM, left ventricle, cerebellum, and whole blood samples (data for leukocytes are not available), respectively. 30 RPKM values are shown on a log scale for 11 of the examined genes. Tables S3–S5 give the Epimark and RPKM data for all studied sites and samples.
    Figure Legend Snippet: Average levels of 5hmC and 5mC at 12 of the 21 tested CCGG sites and RNA-seq RPKM values for the associated genes. (a) The average levels of 5hmC/total C at 12 of the 21 CCGG sites that were determined by Epimark assays on biological replicates for four of the 14 examined sample types. (b) The average levels of 5mC/total C from these assays. (c) RPKM (reads per kilobase per million mapped reads), median values from 430, 218, 125, and 393 samples of generic SkM, left ventricle, cerebellum, and whole blood samples (data for leukocytes are not available), respectively. 30 RPKM values are shown on a log scale for 11 of the examined genes. Tables S3–S5 give the Epimark and RPKM data for all studied sites and samples.

    Techniques Used: RNA Sequencing Assay

    SIX2 , a developmental TF gene, exhibits three hypermethylated DMRs in SkM and aorta that correlate with specific gene expression in these tissues. (a) SIX2 , the LINC01121 ncRNA gene (dashed line) and RNA-seq for the minus-strand of cell cultures and non-strand-specific RNA-seq for tissues (chr2:45,224,940–45,243,926). (b) Chromatin state segmentation; dashed box, the SIX2 gene body for reference. (c) Significant hyper- or hypo-methylated CpG sites from RRBS profiles. 23 (d) and (e) Bisulfite-seq and TAB-seq. Dashed boxes, Mb/Mt- and SkM-hypermethylated region; purple box, subregion in osteoblasts, SkM, and aorta lacking DNA hypermethylation observed in Mb and Mt. Arrowhead, the CCGG site analyzed for by Epimark assay and shown to have the highest 5hmC in SkM but only with an average of 7% of all C as 5hmC ( Fig. 2a ).
    Figure Legend Snippet: SIX2 , a developmental TF gene, exhibits three hypermethylated DMRs in SkM and aorta that correlate with specific gene expression in these tissues. (a) SIX2 , the LINC01121 ncRNA gene (dashed line) and RNA-seq for the minus-strand of cell cultures and non-strand-specific RNA-seq for tissues (chr2:45,224,940–45,243,926). (b) Chromatin state segmentation; dashed box, the SIX2 gene body for reference. (c) Significant hyper- or hypo-methylated CpG sites from RRBS profiles. 23 (d) and (e) Bisulfite-seq and TAB-seq. Dashed boxes, Mb/Mt- and SkM-hypermethylated region; purple box, subregion in osteoblasts, SkM, and aorta lacking DNA hypermethylation observed in Mb and Mt. Arrowhead, the CCGG site analyzed for by Epimark assay and shown to have the highest 5hmC in SkM but only with an average of 7% of all C as 5hmC ( Fig. 2a ).

    Techniques Used: Expressing, RNA Sequencing Assay, Methylation, Bisulfite Sequencing

    Both hypermethylated and hypomethylated DMRs within the gene body of CDH15 are associated with gene expression. (a) RefSeq structure for CDH15 and adjacent genes (chr16:89,232,229–89,271,355) and RNA-seq data for cell cultures as in Fig. 1 . Gray box, an apparent ncRNA seen in lung fibroblasts and ESC; red dashed line, the region of the SLC22A31 transcript. (b) DNase-seq mapping of DNaseI hypersensitive sites. Dashed rectangle CDH15 3′DMR sequence that was cloned and used for reporter gene assays; horizontal blue bar, epigenetic marks at the myogenic enhancer-like region. (c) Chromatin state segmentation. Note 0.2- and 0.4-kb subregions of active promoter chromatin in the cloned region of lung fibroblasts and HepG2 cells, respectively as compared with poised promoter chromatin in the other non-myogenic samples. (d) Significant hyper- or hypomethylated sites from RRBS. 23 (e) Bisulfite-seq. Dashed box, cloned DMR sequences. Epimark-tested CCGG sites: arrowhead, site which had high 5 hmC in SkM, heart, and cerebellum; purple line, site with negligible 5 hmC in all tested samples; lollipop, site with high 5 hmC only in SkM ( Fig. 2a ).
    Figure Legend Snippet: Both hypermethylated and hypomethylated DMRs within the gene body of CDH15 are associated with gene expression. (a) RefSeq structure for CDH15 and adjacent genes (chr16:89,232,229–89,271,355) and RNA-seq data for cell cultures as in Fig. 1 . Gray box, an apparent ncRNA seen in lung fibroblasts and ESC; red dashed line, the region of the SLC22A31 transcript. (b) DNase-seq mapping of DNaseI hypersensitive sites. Dashed rectangle CDH15 3′DMR sequence that was cloned and used for reporter gene assays; horizontal blue bar, epigenetic marks at the myogenic enhancer-like region. (c) Chromatin state segmentation. Note 0.2- and 0.4-kb subregions of active promoter chromatin in the cloned region of lung fibroblasts and HepG2 cells, respectively as compared with poised promoter chromatin in the other non-myogenic samples. (d) Significant hyper- or hypomethylated sites from RRBS. 23 (e) Bisulfite-seq. Dashed box, cloned DMR sequences. Epimark-tested CCGG sites: arrowhead, site which had high 5 hmC in SkM, heart, and cerebellum; purple line, site with negligible 5 hmC in all tested samples; lollipop, site with high 5 hmC only in SkM ( Fig. 2a ).

    Techniques Used: Expressing, RNA Sequencing Assay, Sequencing, Clone Assay, Bisulfite Sequencing

    Intragenic Mb/Mt DNA hypermethylation, decreased CTCF binding, and loss of poised promoter chromatin in PITX3 correlated with gene expression in Mb and Mt. (a) RefSeq gene structure for PITX3 , a developmental gene, at chr10:103,989,638–104,003,464 (all coordinates for figures are in hg19 and all tracks are aligned) and ENCODE RNA-seq data. The sequence-specific minus-strand RNA-seq profile is shown for cell cultures and the not strand-specific RNA-seq data for tissues. 37 (b) CTCF binding from ENCODE data (dot, predicted insulator; green box, preferential Mb/Mt CTCF binding site. (c) Chromatin state segmentation from RoadMap data 37 with the indicated color code; Pr, promoter; Enh, enhancer; Enh/Pr, both active promoter-type and enhancer-type histone modification; Repressed, enriched in H3K27me3 (weak, light gray; strong, dark gray) or H3K9me3 (violet). (d) Statistically significant hypermethylated sites as determined by RRBS for comparison of the set of Mb and Mt vs. 16 types of non-muscle cell cultures 23 and CGIs from the UCSC Genome Browser. 37 (e) Bisulfite-seq profiles 37 with blue bars indicating regions with significantly lower methylation compared with most of the given genome. 28,72 (f) TAB-seq profile of the distribution of 5hmC in the same prefrontal cortex (PFC) DNA sample from brain used for bisulfite-seq. Mb, myoblasts; LCL, GM12868 lymphoblastoid cell line; HMEC, human mammary epithelial cells; ESC, H1 embryonic stem cells; Sk muscle #1, psoas muscle; Sk muscle #2, unknown type of skeletal muscle; Lung fib, IMR-90, fetal lung fibroblast cell line; heart, left ventricle. Dashed box, cloned DMR sequences; arrowhead, Epimark-assayed CCGG, which had high 5hmC in SkM ( Fig. 2a ).
    Figure Legend Snippet: Intragenic Mb/Mt DNA hypermethylation, decreased CTCF binding, and loss of poised promoter chromatin in PITX3 correlated with gene expression in Mb and Mt. (a) RefSeq gene structure for PITX3 , a developmental gene, at chr10:103,989,638–104,003,464 (all coordinates for figures are in hg19 and all tracks are aligned) and ENCODE RNA-seq data. The sequence-specific minus-strand RNA-seq profile is shown for cell cultures and the not strand-specific RNA-seq data for tissues. 37 (b) CTCF binding from ENCODE data (dot, predicted insulator; green box, preferential Mb/Mt CTCF binding site. (c) Chromatin state segmentation from RoadMap data 37 with the indicated color code; Pr, promoter; Enh, enhancer; Enh/Pr, both active promoter-type and enhancer-type histone modification; Repressed, enriched in H3K27me3 (weak, light gray; strong, dark gray) or H3K9me3 (violet). (d) Statistically significant hypermethylated sites as determined by RRBS for comparison of the set of Mb and Mt vs. 16 types of non-muscle cell cultures 23 and CGIs from the UCSC Genome Browser. 37 (e) Bisulfite-seq profiles 37 with blue bars indicating regions with significantly lower methylation compared with most of the given genome. 28,72 (f) TAB-seq profile of the distribution of 5hmC in the same prefrontal cortex (PFC) DNA sample from brain used for bisulfite-seq. Mb, myoblasts; LCL, GM12868 lymphoblastoid cell line; HMEC, human mammary epithelial cells; ESC, H1 embryonic stem cells; Sk muscle #1, psoas muscle; Sk muscle #2, unknown type of skeletal muscle; Lung fib, IMR-90, fetal lung fibroblast cell line; heart, left ventricle. Dashed box, cloned DMR sequences; arrowhead, Epimark-assayed CCGG, which had high 5hmC in SkM ( Fig. 2a ).

    Techniques Used: Binding Assay, Expressing, RNA Sequencing Assay, Sequencing, Modification, Bisulfite Sequencing, Methylation, Clone Assay

    NRXN2 , a neuronal gene, displays a Mb/Mt-specific alternative promoter whose DNA hypomethylation persists in SkM despite the loss of promoter activity. (a) RefSeq gene isoforms structures for NRXN2 (chr11:64,371,048–64,493,639) and RNA-seq as in Fig. 3 but also with the ENCODE profile of 5′ cap mapping (CAGE). 37 Purple broken arrow on left, TSS for the Mb-associated transcript. (b) Chromatin state segmentation. (c) Significant hyper- or hypomethylated DMRs from 33 RRBS profiles. 24 (d) and (e) Bisulfite-seq and TAB-seq. Highlighted green region, Mb/Mt-specific promoter region within NRXN2 . Arrowhead, Epimark-tested site with high 5hmC in cerebellum ( Fig. 2a ).
    Figure Legend Snippet: NRXN2 , a neuronal gene, displays a Mb/Mt-specific alternative promoter whose DNA hypomethylation persists in SkM despite the loss of promoter activity. (a) RefSeq gene isoforms structures for NRXN2 (chr11:64,371,048–64,493,639) and RNA-seq as in Fig. 3 but also with the ENCODE profile of 5′ cap mapping (CAGE). 37 Purple broken arrow on left, TSS for the Mb-associated transcript. (b) Chromatin state segmentation. (c) Significant hyper- or hypomethylated DMRs from 33 RRBS profiles. 24 (d) and (e) Bisulfite-seq and TAB-seq. Highlighted green region, Mb/Mt-specific promoter region within NRXN2 . Arrowhead, Epimark-tested site with high 5hmC in cerebellum ( Fig. 2a ).

    Techniques Used: Activity Assay, RNA Sequencing Assay, Bisulfite Sequencing

    9) Product Images from "Biochemical Characterization of Recombinant ?-Glucosyltransferase and Analysis of Global 5-Hydroxymethylcytosine in Unique Genomes"

    Article Title: Biochemical Characterization of Recombinant ?-Glucosyltransferase and Analysis of Global 5-Hydroxymethylcytosine in Unique Genomes

    Journal: Biochemistry

    doi: 10.1021/bi2014739

    Substrate–velocity curves of recombinant β - GT. (A) Recombinant β-GT activity with UDP-[ 3 H]glucose substrate. Glucosylation reactions were conducted at UDP-[ 3 H]glucose substrate concentrations of 0, 2.5, 5, 10, 25, and 50 μM and fixed enzyme and 5-hmC DNA concentrations of 0.01 and 2.5 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m UDP-glucose values. (B) Recombinant β-GT activity with a 5-hmC DNA substrate. Glucosylation reactions were conducted with 5-hmC DNA substrate concentrations of 0, 0.125, 0.25, 0.5, 1, 3.6, and 7.2 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m 5-hmC values.
    Figure Legend Snippet: Substrate–velocity curves of recombinant β - GT. (A) Recombinant β-GT activity with UDP-[ 3 H]glucose substrate. Glucosylation reactions were conducted at UDP-[ 3 H]glucose substrate concentrations of 0, 2.5, 5, 10, 25, and 50 μM and fixed enzyme and 5-hmC DNA concentrations of 0.01 and 2.5 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m UDP-glucose values. (B) Recombinant β-GT activity with a 5-hmC DNA substrate. Glucosylation reactions were conducted with 5-hmC DNA substrate concentrations of 0, 0.125, 0.25, 0.5, 1, 3.6, and 7.2 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m 5-hmC values.

    Techniques Used: Recombinant, Activity Assay, Concentration Assay

    Linearity of glucosylation by recombinant β-GT. (A) Linearity of glucosylase reaction as a function of time with four different concentrations of UDP-[ 3 H]glucose: 50 (◇), 25 (▼), 10 (△), and 5 μM (■). All reaction mixtures contained 0.01 μM β-GT and 2.5 μM 5-hmC and were incubated at 25 °C. Twenty-five microliters of the reaction mixture was spotted on DE81 at 1 min time intervals and processed as described in Experimental Procedures . (B) Linearity of the glucosylase reaction as a function of recombinant β-GT enzyme concentration (0.00025–0.02 μM) with 50 (△) or 25 μM UDP-[ 3 H]glucose (■). Twenty-five microliters of the reaction mixture was spotted on DE81 after incubation with the appropriate concentration of the β-GT enzyme for 2 min.
    Figure Legend Snippet: Linearity of glucosylation by recombinant β-GT. (A) Linearity of glucosylase reaction as a function of time with four different concentrations of UDP-[ 3 H]glucose: 50 (◇), 25 (▼), 10 (△), and 5 μM (■). All reaction mixtures contained 0.01 μM β-GT and 2.5 μM 5-hmC and were incubated at 25 °C. Twenty-five microliters of the reaction mixture was spotted on DE81 at 1 min time intervals and processed as described in Experimental Procedures . (B) Linearity of the glucosylase reaction as a function of recombinant β-GT enzyme concentration (0.00025–0.02 μM) with 50 (△) or 25 μM UDP-[ 3 H]glucose (■). Twenty-five microliters of the reaction mixture was spotted on DE81 after incubation with the appropriate concentration of the β-GT enzyme for 2 min.

    Techniques Used: Recombinant, Incubation, Concentration Assay

    Global 5-hmC levels determined in the genomes of various tissues, and corresponding tumor samples, by recombinant β-GT. (A) Calibration curve using different amounts of 0, 0.5, 1, 2, 5, 10, and 20 μM 5-hmC with 5 units of recombinant β-GT and 25 μM UDP-[ 3 H]glucose in a total reaction volume of 25 μL. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured. The linear relationship between [ 3 H]glucose incorporation and the molarity of 5-hmC is 0.9997. (B) Glucosylation is utilized to measure global 5-hmC levels in matched normal and tumor genomic DNA samples, as described in Experimental Procedures . Note that all tumor samples have significantly lower levels of 5-hmC when compared to the matched normal sample ( p
    Figure Legend Snippet: Global 5-hmC levels determined in the genomes of various tissues, and corresponding tumor samples, by recombinant β-GT. (A) Calibration curve using different amounts of 0, 0.5, 1, 2, 5, 10, and 20 μM 5-hmC with 5 units of recombinant β-GT and 25 μM UDP-[ 3 H]glucose in a total reaction volume of 25 μL. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured. The linear relationship between [ 3 H]glucose incorporation and the molarity of 5-hmC is 0.9997. (B) Glucosylation is utilized to measure global 5-hmC levels in matched normal and tumor genomic DNA samples, as described in Experimental Procedures . Note that all tumor samples have significantly lower levels of 5-hmC when compared to the matched normal sample ( p

    Techniques Used: Recombinant

    Nonprocessive glucosylation catalyzed by recombinant β-GT. Incorporation of UDP-[ 3 H]glucose after the enzyme was preincubated on ice with 50 μM UDP-[ 3 H]glucose and 1.35 μM 5-hmC DNA. Three minutes after the start of the reaction the mixture was divided into two equal portions, one chased with nonbiotinylated 5-hmC DNA (▼) and the other with an equal volume of water (○) as described in Experimental Procedures . After the chase, both reaction mixtures were incubated at (25 °C) for 1 min and then monitored at 30 s intervals by processing 25 μL of the reaction mixture in duplicate. Measurements were obtained from streptavidin magnetic beads with captured glucosylated [ 3 H]-5-hmC.
    Figure Legend Snippet: Nonprocessive glucosylation catalyzed by recombinant β-GT. Incorporation of UDP-[ 3 H]glucose after the enzyme was preincubated on ice with 50 μM UDP-[ 3 H]glucose and 1.35 μM 5-hmC DNA. Three minutes after the start of the reaction the mixture was divided into two equal portions, one chased with nonbiotinylated 5-hmC DNA (▼) and the other with an equal volume of water (○) as described in Experimental Procedures . After the chase, both reaction mixtures were incubated at (25 °C) for 1 min and then monitored at 30 s intervals by processing 25 μL of the reaction mixture in duplicate. Measurements were obtained from streptavidin magnetic beads with captured glucosylated [ 3 H]-5-hmC.

    Techniques Used: Recombinant, Incubation, Magnetic Beads

    Glucosylation of different 5-hmC-containing substrates by recombinant β-GT. (A) Recombinant β-GT activity with 100 bp double-stranded DNA substrates containing 2 (●), 6 (△), 12 (■), or 24 cytosines (○) that are hydroxymethylated. Glucosylation reactions were performed at 5-hmC concentrations of 0, 0.125, 0.25, 0.5, 1, and 2.5 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively, in a total volume of 25 μL. (B) End point analysis of recombinant β-GT activity with 55 bp double-stranded substrates containing hemi-5-hmC pair A, hemi-5-hmC pair B, or symmetrical 5-hmC. Glucosylation reactions were performed with 1 μM 5-hmC, 50 μM UDP-[ 3 H]glucose, and 5 units of recombinant β-GT in a 25 μL reaction mixture for 2 h. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured.
    Figure Legend Snippet: Glucosylation of different 5-hmC-containing substrates by recombinant β-GT. (A) Recombinant β-GT activity with 100 bp double-stranded DNA substrates containing 2 (●), 6 (△), 12 (■), or 24 cytosines (○) that are hydroxymethylated. Glucosylation reactions were performed at 5-hmC concentrations of 0, 0.125, 0.25, 0.5, 1, and 2.5 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively, in a total volume of 25 μL. (B) End point analysis of recombinant β-GT activity with 55 bp double-stranded substrates containing hemi-5-hmC pair A, hemi-5-hmC pair B, or symmetrical 5-hmC. Glucosylation reactions were performed with 1 μM 5-hmC, 50 μM UDP-[ 3 H]glucose, and 5 units of recombinant β-GT in a 25 μL reaction mixture for 2 h. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured.

    Techniques Used: Recombinant, Activity Assay

    Purity and initial characterization of the β-GT enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by MfeI. A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.
    Figure Legend Snippet: Purity and initial characterization of the β-GT enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by MfeI. A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.

    Techniques Used: Staining, SDS Page, Recombinant

    Reaction mechanism of recombinant β-GT and double-reciprocal plots of the initial velocity vs substrate concentration. (A) Scheme detailing the turnover of recombinant β-GT enzyme in the presence or absence of product inhibitors. The substrate 5-hmC or UDP-glucose is S. The product glucosylated 5-hmC is I. The inhibitor constant is K i . The binary inhibitor constant is α K i . The degree of modification the first substrate exerts on binding of the second substrate is α. (B) Double-reciprocal plots for fixed 5-hmC substrate and variable UDP concentrations of 0 (■), 2 (○), 8 (◆), 12 (△), and 16 μM (▼). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of K m UDP-glucose vs UDP was used to calculate the K i value of UDP as a competitive inhibitor with respect to the formation of a β-GT–UDP-glucose complex. (C) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable UDP concentrations of 0 (■), 20 (△), 30 (▼), and 40 μM (◊). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC]. The inset replot of slope vs UDP was used to calculate the α K i of UDP as a mixed inhibitor with respect to the formation of a β-GT–5-hmC complex. (D) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable glucosylated 5-hmC concentrations of 0 (■), 1 (△), 2.5 (▼), and 5 (○). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC DNA]. The inset replot of K m 5-hmC vs 5-ghmC was utilized to calculate the K i value of 5-ghmC as a competitive inhibitor with respect to the formation of a β-GT–5-hmC complex. (E) Double-reciprocal plots for 5-hmC concentration and variable glucosylated 5-hmC concentrations of 0 (○), 1 (■), 2.5 (▽), and 5 μM (◆). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of slope vs 5-ghmC was used to calculate the α K i of 5-ghmC as a mixed inhibitor with respect to formation of a β-GT–UDP-glucose complex.
    Figure Legend Snippet: Reaction mechanism of recombinant β-GT and double-reciprocal plots of the initial velocity vs substrate concentration. (A) Scheme detailing the turnover of recombinant β-GT enzyme in the presence or absence of product inhibitors. The substrate 5-hmC or UDP-glucose is S. The product glucosylated 5-hmC is I. The inhibitor constant is K i . The binary inhibitor constant is α K i . The degree of modification the first substrate exerts on binding of the second substrate is α. (B) Double-reciprocal plots for fixed 5-hmC substrate and variable UDP concentrations of 0 (■), 2 (○), 8 (◆), 12 (△), and 16 μM (▼). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of K m UDP-glucose vs UDP was used to calculate the K i value of UDP as a competitive inhibitor with respect to the formation of a β-GT–UDP-glucose complex. (C) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable UDP concentrations of 0 (■), 20 (△), 30 (▼), and 40 μM (◊). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC]. The inset replot of slope vs UDP was used to calculate the α K i of UDP as a mixed inhibitor with respect to the formation of a β-GT–5-hmC complex. (D) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable glucosylated 5-hmC concentrations of 0 (■), 1 (△), 2.5 (▼), and 5 (○). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC DNA]. The inset replot of K m 5-hmC vs 5-ghmC was utilized to calculate the K i value of 5-ghmC as a competitive inhibitor with respect to the formation of a β-GT–5-hmC complex. (E) Double-reciprocal plots for 5-hmC concentration and variable glucosylated 5-hmC concentrations of 0 (○), 1 (■), 2.5 (▽), and 5 μM (◆). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of slope vs 5-ghmC was used to calculate the α K i of 5-ghmC as a mixed inhibitor with respect to formation of a β-GT–UDP-glucose complex.

    Techniques Used: Recombinant, Concentration Assay, Modification, Binding Assay

    10) Product Images from "A method for the efficient and selective identification of 5-hydroxymethyluracil in genomic DNA"

    Article Title: A method for the efficient and selective identification of 5-hydroxymethyluracil in genomic DNA

    Journal: Biology methods and protocols

    doi: 10.1093/biomethods/bpw006

    DNA substrate specificity of JGT. ( A ) Recombinant JGT or ( B ) T4 β GT and UDP-[ 3 H]glucose incubated with 36 nt-long dsDNA substrate containing one 5hmU or 5hmC residue, as described in the “Materials and methods” section. The modified base within the dsDNA substrate was present in the context of a matched base pair (5hmU:A, 5hmC:G) or mismatched base pair (5hmU:G). CPM were measured and converted into micromolar glucose transferred. All experiments were performed in triplicate, error bars represent standard deviations. ( C ) UDP-Glo assay of glucosyltransferase activity of JGT for dsDNA substrates used above as described in the “Materials and methods” section. The amount of UDP Cleaved, indicative of the transfer of glucose to DNA, was estimated from a standard curve of UDP. All experiments were performed in triplicate and error bars are representative of standard deviation.
    Figure Legend Snippet: DNA substrate specificity of JGT. ( A ) Recombinant JGT or ( B ) T4 β GT and UDP-[ 3 H]glucose incubated with 36 nt-long dsDNA substrate containing one 5hmU or 5hmC residue, as described in the “Materials and methods” section. The modified base within the dsDNA substrate was present in the context of a matched base pair (5hmU:A, 5hmC:G) or mismatched base pair (5hmU:G). CPM were measured and converted into micromolar glucose transferred. All experiments were performed in triplicate, error bars represent standard deviations. ( C ) UDP-Glo assay of glucosyltransferase activity of JGT for dsDNA substrates used above as described in the “Materials and methods” section. The amount of UDP Cleaved, indicative of the transfer of glucose to DNA, was estimated from a standard curve of UDP. All experiments were performed in triplicate and error bars are representative of standard deviation.

    Techniques Used: Recombinant, Incubation, Modification, Glo Assay, Activity Assay, Standard Deviation

    JGT is unable to utilize UDP-6-N 3 -glucose. Denaturing PAGE for monitoring the reaction mixtures of DNA substrates treated with ( A ) T4 β GT or ( B ) JGT and UDP-glucose or UDP-6-N 3 -glucose (azido-UDP-glucose). The 15-nt-long 32 P labeled dsDNA substrate containing either a 5hmU or 5hmC were incubated with the indicated GT enzyme and nucleotide sugar as described in the “Materials and methods” section. The addition of a glucose moiety to the DNA substrate results in a visible shift on PAGE, with an even greater shift upon subsequent addition of biotin. No enzyme control indicates DNA substrate incubated without the addition of the corresponding GT enzyme. The band indicated with an asterisk is a 14-nt-long glucosylated degradation product.
    Figure Legend Snippet: JGT is unable to utilize UDP-6-N 3 -glucose. Denaturing PAGE for monitoring the reaction mixtures of DNA substrates treated with ( A ) T4 β GT or ( B ) JGT and UDP-glucose or UDP-6-N 3 -glucose (azido-UDP-glucose). The 15-nt-long 32 P labeled dsDNA substrate containing either a 5hmU or 5hmC were incubated with the indicated GT enzyme and nucleotide sugar as described in the “Materials and methods” section. The addition of a glucose moiety to the DNA substrate results in a visible shift on PAGE, with an even greater shift upon subsequent addition of biotin. No enzyme control indicates DNA substrate incubated without the addition of the corresponding GT enzyme. The band indicated with an asterisk is a 14-nt-long glucosylated degradation product.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Labeling, Incubation

    11) Product Images from "Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes"

    Article Title: Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes

    Journal: Genome Research

    doi: 10.1101/gr.126417.111

    Marked inter-tissue differences in global 5hmC levels. ( A ) Dot-blots of decreasing amounts of a PCR product of the mouse Tex19 promoter sequence in which all cytosines are either unmodified ( C ), methylated (5mC), or hydroxymethylated (5hmC), were probed with α-5hmC and α-5mC antibodies. The α-5hmC and α-5mC antibodies are specific for their respective marks. ( B ) Duplicate dot-blots of DNA from human tissues and ES cells probed with antibodies specific to 5hmC or 5mC show that global 5hmC levels vary markedly between normal human tissues. An α-ssDNA antibody was used to control for loading; 500 ng and 100 ng were loaded in the upper and lower lanes, respectively. Ten nanograms of amplified mouse Tex19.1 promoter was used as a control; (solid circle) unmethylated DNA; (dotted circle) methylated DNA; (dashed circle) hydroxymethylated DNA. ( C ) Inter-tissue differences in global 5hmC levels as determined by densitometric analysis of dot-blots shown in panel B . 5hmC values were normalized to an ssDNA loading control and scaled relative to brain. Values are the means of two independent biological replicates. Spleen and ES values represent single measurements. ( D ). ( E ) Duplicate dot-blots of DNA from mouse tissues and ES cells probed with antibodies specific to 5hmC or 5mC show that global 5hmC levels vary markedly between normal mouse tissues. An α-ssDNA antibody was used to control for loading. Ten nanograms of amplified mouse Tex19.1 promoter was used as a control; (solid circle) unmethylated DNA; (dotted circle) methylated DNA; (dashed circle) hydroxymethylated DNA.
    Figure Legend Snippet: Marked inter-tissue differences in global 5hmC levels. ( A ) Dot-blots of decreasing amounts of a PCR product of the mouse Tex19 promoter sequence in which all cytosines are either unmodified ( C ), methylated (5mC), or hydroxymethylated (5hmC), were probed with α-5hmC and α-5mC antibodies. The α-5hmC and α-5mC antibodies are specific for their respective marks. ( B ) Duplicate dot-blots of DNA from human tissues and ES cells probed with antibodies specific to 5hmC or 5mC show that global 5hmC levels vary markedly between normal human tissues. An α-ssDNA antibody was used to control for loading; 500 ng and 100 ng were loaded in the upper and lower lanes, respectively. Ten nanograms of amplified mouse Tex19.1 promoter was used as a control; (solid circle) unmethylated DNA; (dotted circle) methylated DNA; (dashed circle) hydroxymethylated DNA. ( C ) Inter-tissue differences in global 5hmC levels as determined by densitometric analysis of dot-blots shown in panel B . 5hmC values were normalized to an ssDNA loading control and scaled relative to brain. Values are the means of two independent biological replicates. Spleen and ES values represent single measurements. ( D ). ( E ) Duplicate dot-blots of DNA from mouse tissues and ES cells probed with antibodies specific to 5hmC or 5mC show that global 5hmC levels vary markedly between normal mouse tissues. An α-ssDNA antibody was used to control for loading. Ten nanograms of amplified mouse Tex19.1 promoter was used as a control; (solid circle) unmethylated DNA; (dotted circle) methylated DNA; (dashed circle) hydroxymethylated DNA.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Methylation, Amplification

    Genomic patterns of 5hmC enrichment are tissue-specific. ( A ) Shown are the patterns of 5hmC enrichment [log 2 (input/IP)] across the HOXA cluster for multiple replicates of multiple tissues. ( B ) A dendrogram derived from unsupervised hierarchical consensus clustering of 14 human DNA samples based on 5hmC enrichment levels for all 72,000 probes on each tiling microarray. Samples cluster by tissue type. AU (approximately unbiased) P -value of robustness of each cluster; (**) P > 0.01; (***) P > 0.001.
    Figure Legend Snippet: Genomic patterns of 5hmC enrichment are tissue-specific. ( A ) Shown are the patterns of 5hmC enrichment [log 2 (input/IP)] across the HOXA cluster for multiple replicates of multiple tissues. ( B ) A dendrogram derived from unsupervised hierarchical consensus clustering of 14 human DNA samples based on 5hmC enrichment levels for all 72,000 probes on each tiling microarray. Samples cluster by tissue type. AU (approximately unbiased) P -value of robustness of each cluster; (**) P > 0.01; (***) P > 0.001.

    Techniques Used: Derivative Assay, Microarray

    Reduced global 5hmC levels and TET1/2/3 gene expression in human cell lines. ( A ) Duplicate dot-blots of DNA (500 ng) from normal human breast tissue, a primary mammary epithelial cell line from non-cancerous tissue, and eight breast cancer cell lines probed with antibodies specific to 5hmC or ssDNA illustrate that global 5hmC levels are reduced in the DNA of primary and cancer cell lines relative to normal breast. ( B ) Inter-sample differences in global 5hmC levels by densitometric analysis of dot-blots shown in panel A . 5hmC values were normalized to the ssDNA loading control and scaled relative to normal breast DNA. Values are the means of two technical replicates. ( C ) RT-qPCR analysis of expression levels of TET1 , TET2 , and TET3 in normal human breast tissue, a primary mammary epithelial cell line, and eight breast cancer cell lines. Expression levels were normalized to GAPDH expression, and expression levels in normal human breast set to 1. Error bars represent the SD of two technical replicates. ( D ) Duplicate dot-blots of DNA (500 ng) from normal human colon and liver tissue and six cancer cell lines probed with antibodies specific to 5hmC or ssDNA illustrate that global 5hmC levels are also reduced in the DNA of colon and liver cancer cell lines relative to normal tissue. ( E ) Duplicate dot-blots of DNA (500 ng) from normal human breast tissue, and the primary mammary epithelial cell line derived from that tissue probed with antibodies specific to 5hmC, 5mC, or ssDNA show that global levels of 5hmC, but not 5mC, are gradually reduced upon transformation of normal breast tissue to cell culture. Ten nanograms of amplified mouse Tex19.1 promoter in which all cytosines were either unmodified (C), methylated (mC), or hydroxymethylated (hmC), was used as a control.
    Figure Legend Snippet: Reduced global 5hmC levels and TET1/2/3 gene expression in human cell lines. ( A ) Duplicate dot-blots of DNA (500 ng) from normal human breast tissue, a primary mammary epithelial cell line from non-cancerous tissue, and eight breast cancer cell lines probed with antibodies specific to 5hmC or ssDNA illustrate that global 5hmC levels are reduced in the DNA of primary and cancer cell lines relative to normal breast. ( B ) Inter-sample differences in global 5hmC levels by densitometric analysis of dot-blots shown in panel A . 5hmC values were normalized to the ssDNA loading control and scaled relative to normal breast DNA. Values are the means of two technical replicates. ( C ) RT-qPCR analysis of expression levels of TET1 , TET2 , and TET3 in normal human breast tissue, a primary mammary epithelial cell line, and eight breast cancer cell lines. Expression levels were normalized to GAPDH expression, and expression levels in normal human breast set to 1. Error bars represent the SD of two technical replicates. ( D ) Duplicate dot-blots of DNA (500 ng) from normal human colon and liver tissue and six cancer cell lines probed with antibodies specific to 5hmC or ssDNA illustrate that global 5hmC levels are also reduced in the DNA of colon and liver cancer cell lines relative to normal tissue. ( E ) Duplicate dot-blots of DNA (500 ng) from normal human breast tissue, and the primary mammary epithelial cell line derived from that tissue probed with antibodies specific to 5hmC, 5mC, or ssDNA show that global levels of 5hmC, but not 5mC, are gradually reduced upon transformation of normal breast tissue to cell culture. Ten nanograms of amplified mouse Tex19.1 promoter in which all cytosines were either unmodified (C), methylated (mC), or hydroxymethylated (hmC), was used as a control.

    Techniques Used: Expressing, Quantitative RT-PCR, Derivative Assay, Transformation Assay, Cell Culture, Amplification, Methylation

    Tissue type, not transcription level, is the major modifier of 5hmC levels in genes
    Figure Legend Snippet: Tissue type, not transcription level, is the major modifier of 5hmC levels in genes

    Techniques Used:

    12) Product Images from "Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation"

    Article Title: Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation

    Journal: Epigenetics

    doi: 10.4161/epi.24280

    Figure 1. Schematic presentation of the HMST-Seq method. For (A) “C + mC” library, the genomic DNA was first glucosylated, and then digested with MspI. For (B) “C” library and (C) “C + mC + hmC”
    Figure Legend Snippet: Figure 1. Schematic presentation of the HMST-Seq method. For (A) “C + mC” library, the genomic DNA was first glucosylated, and then digested with MspI. For (B) “C” library and (C) “C + mC + hmC”

    Techniques Used:

    13) Product Images from "Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays"

    Article Title: Epigenetic Optical Mapping of 5-Hydroxymethylcytosine in Nanochannel Arrays

    Journal: ACS Nano

    doi: 10.1021/acsnano.8b03023

    Optical 5-hmC mapping experimental scheme. (A) Left: Scanning electron microscope (SEM) image of a silicon nanochannel array. Right: Stretched DNA molecules (gray) fluorescently labeled in two colors. Green: Sequence specific genetic barcode. Red: 5-hmC labels. (B) Fluorescently labeled molecules are extended in nanochannel arrays by electrophoresis. (C) Fluorescence intensity of genetic labels (green) and 5-hmC labels (red) along a single molecule. (D) Genetic labels (green) are used to align a digital representation of the molecule in part C (yellow) to an in silico generated reference (gray) of chromosome 5, highlighting large structural variations such as the 7 kbp deletion in the midright part of the molecule, denoted by the diagonal alignment marks. 5-hmC labels (red) are mapped on the basis of genetic alignment.
    Figure Legend Snippet: Optical 5-hmC mapping experimental scheme. (A) Left: Scanning electron microscope (SEM) image of a silicon nanochannel array. Right: Stretched DNA molecules (gray) fluorescently labeled in two colors. Green: Sequence specific genetic barcode. Red: 5-hmC labels. (B) Fluorescently labeled molecules are extended in nanochannel arrays by electrophoresis. (C) Fluorescence intensity of genetic labels (green) and 5-hmC labels (red) along a single molecule. (D) Genetic labels (green) are used to align a digital representation of the molecule in part C (yellow) to an in silico generated reference (gray) of chromosome 5, highlighting large structural variations such as the 7 kbp deletion in the midright part of the molecule, denoted by the diagonal alignment marks. 5-hmC labels (red) are mapped on the basis of genetic alignment.

    Techniques Used: Microscopy, Labeling, Sequencing, Electrophoresis, Fluorescence, In Silico, Generated

    Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).
    Figure Legend Snippet: Assessment of 5-hmC labeling efficiency. Lambda DNA was nicked with Nt.BspQI (nine expected labeling spots) and labeled with either 5-hmC or fluorescent dUTP. 5-hmC was labeled according to our labeling scheme, and the samples were mixed and imaged together in order to evaluate the labeling efficiency. (A) Representative field of view showing a mixed population of green (nicking) and red (5-hmC) labeled molecules. (B) Histograms showing the number of labels per molecule for 5-hmC labeling (top) and nicking (bottom).

    Techniques Used: Labeling, Lambda DNA Preparation

    14) Product Images from "Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation"

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation

    Journal: Cancer Medicine

    doi: 10.1002/cam4.830

    DNA demethylation occurs at hypermethylated promoters in cell lines expressing MBD‐TET1‐CDwt. Bisulfite genomic sequencing of TRH (A) and MAL (B) promoter regions is shown in parental cell line HEK293T, along with its derivatives, TET1‐CDwt #2, MBD‐TET1‐CDmut #9, and MBD‐TET1‐CDwt #10. Analyzed regions are located within the first intron of TRH and MAL genes. Note that both promoters are demethylated only in the MBD‐TET1‐CDwt cell line. Closed and open circles indicate the methylated and unmethylated CpG sites, respectively. (C) The differences in methylation status within a specific locus of the MAL promoter were analyzed and quantitated using EpiMark 5‐hmC and 5‐mC Analysis Kit. Note that C and 5‐hmC were seen only in the MBD‐TET1‐CDwt cell line.
    Figure Legend Snippet: DNA demethylation occurs at hypermethylated promoters in cell lines expressing MBD‐TET1‐CDwt. Bisulfite genomic sequencing of TRH (A) and MAL (B) promoter regions is shown in parental cell line HEK293T, along with its derivatives, TET1‐CDwt #2, MBD‐TET1‐CDmut #9, and MBD‐TET1‐CDwt #10. Analyzed regions are located within the first intron of TRH and MAL genes. Note that both promoters are demethylated only in the MBD‐TET1‐CDwt cell line. Closed and open circles indicate the methylated and unmethylated CpG sites, respectively. (C) The differences in methylation status within a specific locus of the MAL promoter were analyzed and quantitated using EpiMark 5‐hmC and 5‐mC Analysis Kit. Note that C and 5‐hmC were seen only in the MBD‐TET1‐CDwt cell line.

    Techniques Used: Expressing, Genomic Sequencing, Methylation

    15) Product Images from "Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes *"

    Article Title: Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.217083

    MspI and HpaII isoschizomer can distinguish between 5-hmC and 5-ghmC at the internal cytosine residue. Cleavage specificity is shown for MspI and HpaII on FAM-end-labeled oligonucleotide duplex with internal CG being symmetrically hydroxymethylated or hemihydroxymethylated in the presence or absence of β-GT. Complete cleavage products are labeled as 24 and 19 nt, respectively, on a non-denaturing acrylamide gel for double-hydroxymethylated ( upper panel ) and denaturing acrylamide gel for hemi-hydroxymethylated DNA ( lower panel ). The arrow at the right indicates small amounts of 24-nt-long product.
    Figure Legend Snippet: MspI and HpaII isoschizomer can distinguish between 5-hmC and 5-ghmC at the internal cytosine residue. Cleavage specificity is shown for MspI and HpaII on FAM-end-labeled oligonucleotide duplex with internal CG being symmetrically hydroxymethylated or hemihydroxymethylated in the presence or absence of β-GT. Complete cleavage products are labeled as 24 and 19 nt, respectively, on a non-denaturing acrylamide gel for double-hydroxymethylated ( upper panel ) and denaturing acrylamide gel for hemi-hydroxymethylated DNA ( lower panel ). The arrow at the right indicates small amounts of 24-nt-long product.

    Techniques Used: Labeling, Acrylamide Gel Assay

    Methylation and hydroxymethylation analysis across gene body. A , VANGL1. B , EGFR . The genes are depicted with exons shown as black rectangles . The interrogated CCGG site numbers are indicated on the top along with the primer sets on the bottom . Diagrams are not drawn to scale. Total methylated cytosines (5-mC plus 5-hmC) obtained from β-GT-treated ( BGT ) HpaII-digested DNA and β-GT-treated MspI-digested DNA show the percentage of 5-hmC ( bottom panels ) at each specific site.
    Figure Legend Snippet: Methylation and hydroxymethylation analysis across gene body. A , VANGL1. B , EGFR . The genes are depicted with exons shown as black rectangles . The interrogated CCGG site numbers are indicated on the top along with the primer sets on the bottom . Diagrams are not drawn to scale. Total methylated cytosines (5-mC plus 5-hmC) obtained from β-GT-treated ( BGT ) HpaII-digested DNA and β-GT-treated MspI-digested DNA show the percentage of 5-hmC ( bottom panels ) at each specific site.

    Techniques Used: Methylation

    Cloning, sequence identification, and tissue-specific distribution of 5-hydroxymethylcytosine. A , a scheme for cloning and identification of 5-hmC containing DNA fragments using MspI restriction enzyme and β-GT ( Beta-GT ) is shown. B , a scheme for identification of 5-hmC containing CCGG locus using MspI ( M ) and HpaII ( H ) isoschizomers using glucosylation reaction is shown along with control ( C ). C , locus-specific end point PCR to interrogate and detect 5-hmC at CCGG sites in mouse genomic DNA is shown. The loci are discovered based on cloning scheme as shown in A and in supplemental Table 1 . Mouse brain, liver, heart, and spleen along with NIH3T3 cultured cell DNAs were interrogated for the 4 loci ( supplemental Table 3 : loci 2 and 3 are 5′ and 3′ MspI sites of chromosome 10, respectively, bp 34,574,152; locus 4 is the 5′ MspI site of chromosome 12, bp 17,432,255; locus 12 is the 5′ MspI site of Lpr1 intron, bp 2,372,508) as shown. D , shown is the locus-specific end point PCR to interrogate and detect 5-hmC at CCGG sites in human genomic DNA The loci are discovered based on the cloning scheme as shown in A and in supplemental Table 2 . Human brain (pons, occipital lobe ( OL )), liver, heart, and spleen along with HeLa-cultured cell DNAs were interrogated for both of the VANGL1 loci as shown. The control DNA interrogated fragment without CCGG sequence is miR17A.
    Figure Legend Snippet: Cloning, sequence identification, and tissue-specific distribution of 5-hydroxymethylcytosine. A , a scheme for cloning and identification of 5-hmC containing DNA fragments using MspI restriction enzyme and β-GT ( Beta-GT ) is shown. B , a scheme for identification of 5-hmC containing CCGG locus using MspI ( M ) and HpaII ( H ) isoschizomers using glucosylation reaction is shown along with control ( C ). C , locus-specific end point PCR to interrogate and detect 5-hmC at CCGG sites in mouse genomic DNA is shown. The loci are discovered based on cloning scheme as shown in A and in supplemental Table 1 . Mouse brain, liver, heart, and spleen along with NIH3T3 cultured cell DNAs were interrogated for the 4 loci ( supplemental Table 3 : loci 2 and 3 are 5′ and 3′ MspI sites of chromosome 10, respectively, bp 34,574,152; locus 4 is the 5′ MspI site of chromosome 12, bp 17,432,255; locus 12 is the 5′ MspI site of Lpr1 intron, bp 2,372,508) as shown. D , shown is the locus-specific end point PCR to interrogate and detect 5-hmC at CCGG sites in human genomic DNA The loci are discovered based on the cloning scheme as shown in A and in supplemental Table 2 . Human brain (pons, occipital lobe ( OL )), liver, heart, and spleen along with HeLa-cultured cell DNAs were interrogated for both of the VANGL1 loci as shown. The control DNA interrogated fragment without CCGG sequence is miR17A.

    Techniques Used: Clone Assay, Sequencing, Polymerase Chain Reaction, Cell Culture

    16) Product Images from "MicroRNA-494 is a master epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors"

    Article Title: MicroRNA-494 is a master epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.27816

    miR-494 is associated with HCC EMT and vascular invasion in human HCC tumors. (A) Analysis of miR-494 expression of 91 VI − and 81 VI + HCC tumors by qRT-PCR. (B) Vascular invasion is associated with higher miR-494 levels in human HCC tumors. Human HCC tumor samples were classified into two groups according to low and high miR-494 expression levels by median division of qRT-PCR results. (C) Representative Images of immunohistochemical staining for vimentin, E-cadherin, 5hmC, and TET1 in HCC tumors with low (miR-494 low ) and high miR-494 (miR-494 high ) expression levels. Scale bars, 100 μm. (D) Bioluminescent images showed a suppression of tumor formation in nude mice implanted with miR-494 knockdown SNU449-Luc cells (n = 5 for each group). (E) Suppression of lung metastasis in nude mice implanted with miR-494 knockdown SNU449-Luc cells was monitored by ex vivo bioluminescent imaging. Luciferase activity of lung metastasis was quantified for anti-miR-494 or anti-miR control group (n = 7 for each group). (F) Schematic presentation of miR-494 action in HCC EMT and vascular invasion. miR-494 suppresses multiple invasion-suppressor miRNAs though epigenetic repression by targeting TET methylcytosine dioxygenase in invasive human hepatocarcinoma tumors.
    Figure Legend Snippet: miR-494 is associated with HCC EMT and vascular invasion in human HCC tumors. (A) Analysis of miR-494 expression of 91 VI − and 81 VI + HCC tumors by qRT-PCR. (B) Vascular invasion is associated with higher miR-494 levels in human HCC tumors. Human HCC tumor samples were classified into two groups according to low and high miR-494 expression levels by median division of qRT-PCR results. (C) Representative Images of immunohistochemical staining for vimentin, E-cadherin, 5hmC, and TET1 in HCC tumors with low (miR-494 low ) and high miR-494 (miR-494 high ) expression levels. Scale bars, 100 μm. (D) Bioluminescent images showed a suppression of tumor formation in nude mice implanted with miR-494 knockdown SNU449-Luc cells (n = 5 for each group). (E) Suppression of lung metastasis in nude mice implanted with miR-494 knockdown SNU449-Luc cells was monitored by ex vivo bioluminescent imaging. Luciferase activity of lung metastasis was quantified for anti-miR-494 or anti-miR control group (n = 7 for each group). (F) Schematic presentation of miR-494 action in HCC EMT and vascular invasion. miR-494 suppresses multiple invasion-suppressor miRNAs though epigenetic repression by targeting TET methylcytosine dioxygenase in invasive human hepatocarcinoma tumors.

    Techniques Used: Expressing, Quantitative RT-PCR, Immunohistochemistry, Staining, Mouse Assay, Ex Vivo, Imaging, Luciferase, Activity Assay

    TET methylcytosine dioxygenase is essential for inhibition of miR-494-mediated HCC invasion/EMT and suppression of multiple invasion-suppressor miRNAs. (A) SNU449 cells transduced with the combination of anti-miR-494, TET1 (wild-type), or negative controls (anti-miR control and catalytic-dead TET1m mutant) and (B) HepG2 cells transduced with the combination of miR-494, TET1, or negative controls (anti-miR control and catalytic-dead TET1m mutant) were subjected to cell invasion assay. Migrated cells in fields were quantified and representative photographs are shown. Data are represented as mean ± standard deviation (SD) from four independent experiments. (C) Cell lysates from HepG2 cells infected with the combination of the miR-494 with TET1 or the control vector were subjected to western blotting for the indicated proteins. (D) qRT-PCR analysis of invasion-suppressor miRNAs with total RNAs isolated from HepG2 cells transduced with the combination of miR-494 with TET1 or the empty vector. Data are represented as mean ± SD from five independent experiments. (E) gDNA isolated from HepG2 cells transduced with the combination of miR-494 with TET1 or TET3 expression vector was denatured and neutralized. 5hmC levels were determined by the dot blot assay using anti-5hmC antibody. (F and G) SNU449 cells expressing a combination of the control and anti-miR-494 and the TET1 short hairpin RNA (shRNA) expression vector were subjected to either (F) western blotting analysis of TET1 or (G) cell invasion assay. Data are represented as mean ± SD from five independent experiments. * P
    Figure Legend Snippet: TET methylcytosine dioxygenase is essential for inhibition of miR-494-mediated HCC invasion/EMT and suppression of multiple invasion-suppressor miRNAs. (A) SNU449 cells transduced with the combination of anti-miR-494, TET1 (wild-type), or negative controls (anti-miR control and catalytic-dead TET1m mutant) and (B) HepG2 cells transduced with the combination of miR-494, TET1, or negative controls (anti-miR control and catalytic-dead TET1m mutant) were subjected to cell invasion assay. Migrated cells in fields were quantified and representative photographs are shown. Data are represented as mean ± standard deviation (SD) from four independent experiments. (C) Cell lysates from HepG2 cells infected with the combination of the miR-494 with TET1 or the control vector were subjected to western blotting for the indicated proteins. (D) qRT-PCR analysis of invasion-suppressor miRNAs with total RNAs isolated from HepG2 cells transduced with the combination of miR-494 with TET1 or the empty vector. Data are represented as mean ± SD from five independent experiments. (E) gDNA isolated from HepG2 cells transduced with the combination of miR-494 with TET1 or TET3 expression vector was denatured and neutralized. 5hmC levels were determined by the dot blot assay using anti-5hmC antibody. (F and G) SNU449 cells expressing a combination of the control and anti-miR-494 and the TET1 short hairpin RNA (shRNA) expression vector were subjected to either (F) western blotting analysis of TET1 or (G) cell invasion assay. Data are represented as mean ± SD from five independent experiments. * P

    Techniques Used: Inhibition, Transduction, Mutagenesis, Invasion Assay, Standard Deviation, Infection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Isolation, Expressing, Dot Blot, shRNA

    miR-494 triggers gene inactivation of multiple invasion-suppressor microRNAs by targeting TET methylcytosine dioxygenase. (A) A list of selected high-scoring predicted miR-494 targets was produced by various programs using different algorithms. (B) Cell lysates isolated from five HCC cell lines were subjected to western blotting analysis for TET1, TET2, and TET3 proteins. (C and D) Total RNAs or cell lysates isolated from HepG2 cells transfected with miR-494 mimic or (C) SNU449 cells transfected with miR-494 inhibitor (D) were subjected to qRT-PCR or western blotting analysis for TET1, TET2, and TET3 mRNA. Data are represented as mean ± standard deviation (SD) from five independent experiments. (E) Putative binding sites of miR-494 in TET1 3′ UTR. Predicted 8- or 7-mer binding seeds of miR-494 to TET1 3′ UTR are indicated with vertical lines. (F) Luciferase assay of the TET1 3′ UTR luciferase plasmid. CV-1 cells were transiently cotransfected with the wild-type or miR-494-binding mutant of human TET1 3′ UTR luciferase plasmid with Renilla luciferase reporter for normalization. Data are represented as mean ± SD from four independent experiments. Putative miR-494-binding site on the 242-248 base-pair region of TET1 3′ UTR was mutated as indicated. (G) gDNA purified from HepG2 cells transfected with miR-494 or TET1 short hairpin RNA-expressing vector or the negative control was denatured and neutralized. Global 5hmC levels were then determined using a dot blot assay with anti-5hmC antibody. * P
    Figure Legend Snippet: miR-494 triggers gene inactivation of multiple invasion-suppressor microRNAs by targeting TET methylcytosine dioxygenase. (A) A list of selected high-scoring predicted miR-494 targets was produced by various programs using different algorithms. (B) Cell lysates isolated from five HCC cell lines were subjected to western blotting analysis for TET1, TET2, and TET3 proteins. (C and D) Total RNAs or cell lysates isolated from HepG2 cells transfected with miR-494 mimic or (C) SNU449 cells transfected with miR-494 inhibitor (D) were subjected to qRT-PCR or western blotting analysis for TET1, TET2, and TET3 mRNA. Data are represented as mean ± standard deviation (SD) from five independent experiments. (E) Putative binding sites of miR-494 in TET1 3′ UTR. Predicted 8- or 7-mer binding seeds of miR-494 to TET1 3′ UTR are indicated with vertical lines. (F) Luciferase assay of the TET1 3′ UTR luciferase plasmid. CV-1 cells were transiently cotransfected with the wild-type or miR-494-binding mutant of human TET1 3′ UTR luciferase plasmid with Renilla luciferase reporter for normalization. Data are represented as mean ± SD from four independent experiments. Putative miR-494-binding site on the 242-248 base-pair region of TET1 3′ UTR was mutated as indicated. (G) gDNA purified from HepG2 cells transfected with miR-494 or TET1 short hairpin RNA-expressing vector or the negative control was denatured and neutralized. Global 5hmC levels were then determined using a dot blot assay with anti-5hmC antibody. * P

    Techniques Used: Produced, Isolation, Western Blot, Transfection, Quantitative RT-PCR, Standard Deviation, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Purification, shRNA, Expressing, Negative Control, Dot Blot

    miR-494 promotes HCC cell migration/invasion and suppresses multiple invasion-suppressor microRNAs by inhibiting DNA demethylation of their proximal CpG islands. (A, B, and C) (A) HepG2 cells, (B) Hep3B cells, or (C) SNU398 cells, transduced with miR-494 or the nontarget control vector, were subjected to cell migration and invasion assays. Migrated/invaded cells in fields were quantified and representative photographs are shown. Data are represented as mean ± standard deviation (SD) from four independent experiments. (D and E) qRT-PCR analysis of RNAs from HepG2 cells transduced with miR-494-expressing vector or the nontarget control vector for (D) indicated biomarkers of EMT and (E) indicated microRNAs. (F) Methylation-specific PCR analysis of the proximal CpG island regions of the indicated miRNA genes with gDNAs purified from HepG2 cells transduced with the miR-494 expression vector. PBGD served as the control. (G) DNA methylation status of the proximal CpG regions of the indicated miRNA genes in HepG2 cells transduced with either miR-494-expressing vector or the nontarget control vector. DNA methylation status was determined using methylation-sensitive/dependent restriction digestion followed by qRT-PCR analysis. Data are represented as mean ± SD from three independent experiments. (H) Restored expression of miR-200c upon adding DNA demethylating agent 5′-Aza in HepG2 cells transduced with the miR-494-expressing vector. Data are represented as mean ± SD from five independent experiments. (I) GlucMS-qPCR analysis of proximal CpG islands within the indicated miRNA gene’s upstream regions enriched for 5hmC in HepG2 cells transduced with the miR-494-expressing vector. Data are represented as mean ± SD from five independent experiments. ** P
    Figure Legend Snippet: miR-494 promotes HCC cell migration/invasion and suppresses multiple invasion-suppressor microRNAs by inhibiting DNA demethylation of their proximal CpG islands. (A, B, and C) (A) HepG2 cells, (B) Hep3B cells, or (C) SNU398 cells, transduced with miR-494 or the nontarget control vector, were subjected to cell migration and invasion assays. Migrated/invaded cells in fields were quantified and representative photographs are shown. Data are represented as mean ± standard deviation (SD) from four independent experiments. (D and E) qRT-PCR analysis of RNAs from HepG2 cells transduced with miR-494-expressing vector or the nontarget control vector for (D) indicated biomarkers of EMT and (E) indicated microRNAs. (F) Methylation-specific PCR analysis of the proximal CpG island regions of the indicated miRNA genes with gDNAs purified from HepG2 cells transduced with the miR-494 expression vector. PBGD served as the control. (G) DNA methylation status of the proximal CpG regions of the indicated miRNA genes in HepG2 cells transduced with either miR-494-expressing vector or the nontarget control vector. DNA methylation status was determined using methylation-sensitive/dependent restriction digestion followed by qRT-PCR analysis. Data are represented as mean ± SD from three independent experiments. (H) Restored expression of miR-200c upon adding DNA demethylating agent 5′-Aza in HepG2 cells transduced with the miR-494-expressing vector. Data are represented as mean ± SD from five independent experiments. (I) GlucMS-qPCR analysis of proximal CpG islands within the indicated miRNA gene’s upstream regions enriched for 5hmC in HepG2 cells transduced with the miR-494-expressing vector. Data are represented as mean ± SD from five independent experiments. ** P

    Techniques Used: Migration, Transduction, Plasmid Preparation, Standard Deviation, Quantitative RT-PCR, Expressing, Methylation, Polymerase Chain Reaction, Purification, DNA Methylation Assay, Real-time Polymerase Chain Reaction

    17) Product Images from "GADD45a physically and functionally interacts with TET1"

    Article Title: GADD45a physically and functionally interacts with TET1

    Journal: Differentiation; Research in Biological Diversity

    doi: 10.1016/j.diff.2015.10.003

    Synergistic target gene activation by GADD45a-TET1 is accompanied by increase in hmC and reduction of fC/caC in promoter CpGs. (A, B) TCEAL7 , DHRS2 , MAGEB2 expression in HEK293T cells upon transfection of empty vector (Ctrl, control), GADD45a (G45a) alone or with increasing doses of TET1, catalytic domain only (TET1 CD ), or catalytically inactive TET1 (TET CI ) as indicated. Relative expression was monitored by qPCR. Bar graphs represent the mean of n =4 (A) or n =3 (B) experiments with error bars as±SD. (C, D) Kinetics of hmC (C) and fC/caC (D) level changes in the TCEAL7 locus upon GADD45a and TET expression. HEK293T cells were transfected with empty vector (Ctrl) or GADD45a (G45a) or TET1 as indicated. Genomic DNA was harvested 14 h or 24 h after transfection. hmC and fC/caC were analyzed at positions −2648, −78, +34 and +457 relative to the transcription start site (TSS). Analysis was by modification-sensitive qPCR following MspI restriction on T4 β-glucosyltransferase (β-GT) treated or control treated plasmid DNA. % of MspI resistance following β-GT treatment is displayed as % hmC. % of MspI resistance in control treated DNA (−β-GT) is displayed as % fC/caC. Bar graphs represent the mean of biological triplicates ( n =3) with error bars as±SD. p -Values: (*) p
    Figure Legend Snippet: Synergistic target gene activation by GADD45a-TET1 is accompanied by increase in hmC and reduction of fC/caC in promoter CpGs. (A, B) TCEAL7 , DHRS2 , MAGEB2 expression in HEK293T cells upon transfection of empty vector (Ctrl, control), GADD45a (G45a) alone or with increasing doses of TET1, catalytic domain only (TET1 CD ), or catalytically inactive TET1 (TET CI ) as indicated. Relative expression was monitored by qPCR. Bar graphs represent the mean of n =4 (A) or n =3 (B) experiments with error bars as±SD. (C, D) Kinetics of hmC (C) and fC/caC (D) level changes in the TCEAL7 locus upon GADD45a and TET expression. HEK293T cells were transfected with empty vector (Ctrl) or GADD45a (G45a) or TET1 as indicated. Genomic DNA was harvested 14 h or 24 h after transfection. hmC and fC/caC were analyzed at positions −2648, −78, +34 and +457 relative to the transcription start site (TSS). Analysis was by modification-sensitive qPCR following MspI restriction on T4 β-glucosyltransferase (β-GT) treated or control treated plasmid DNA. % of MspI resistance following β-GT treatment is displayed as % hmC. % of MspI resistance in control treated DNA (−β-GT) is displayed as % fC/caC. Bar graphs represent the mean of biological triplicates ( n =3) with error bars as±SD. p -Values: (*) p

    Techniques Used: Activation Assay, Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Modification

    GADD45a promotes TET1-mediated mC oxidation and reporter DNA demethylation. (A–G) Methylation analysis of in vitro methylated oct4TK-GFP reporter by methylation sensitive PCR. (A) HEK293T cells were transfected with oct4TK-GFP along with empty vector (Ctrl), GADD45a (G45a) or TET1 expression constructs as indicated. Plasmid DNA was recovered 48 h after transfection and subjected to HpaII restriction digest and qPCR. HpaII resistance reflecting the fraction of modified C is displayed. Ctrl, control; TET1 CI , TET1 catalytically inactive mutant. Bar graphs represent the mean of biological triplicates ( n =3) with error bars±SD. (B–D) Kinetics of mC and its oxidized derivatives during oct4TK-GFP reporter demethylation analyzed by modification-sensitive qPCR. Cells were treated as in (A) and plasmid DNA was recovered at indicated time points after transfection. mC and its oxidized derivatives were analyzed using qPCR following HpaII or MspI restriction digest on T4 β-glucosyltransferase (β-GT) treated or control treated plasmid DNA. In (B), % modified cytosine (mC+hmC+fC+caC) is displayed as % HpaII resistance. (C) and (D) display % hmC and % fC/caC determined by MspI resistance following β-GT treatment and MspI resistance in control treated DNA (without β-GT), respectively. Error bars indicate±SD ( n =3). (E, F) HEK293T cells were treated as in (A), but pre-transfected with the indicated siRNAs 24 h before DNA transfection. HpaII resistance reflecting the fraction of modified C is displayed. (G) HEK293T cells were transfected with oct4TK-GFP without any effector protein with the indicated siRNAs, whereby “mock” represents transfection reagent only. oct4TK-GFP plasmid was recovered 72 h after siRNA and 48 h after oct4TK-GFP transfection. HpaII cleavage is displayed as % demethylation, reflecting formation of unmodified C. Bar graphs represent the mean of biological triplicates ( n =3) with error bars±SD. p -Values: (*) p
    Figure Legend Snippet: GADD45a promotes TET1-mediated mC oxidation and reporter DNA demethylation. (A–G) Methylation analysis of in vitro methylated oct4TK-GFP reporter by methylation sensitive PCR. (A) HEK293T cells were transfected with oct4TK-GFP along with empty vector (Ctrl), GADD45a (G45a) or TET1 expression constructs as indicated. Plasmid DNA was recovered 48 h after transfection and subjected to HpaII restriction digest and qPCR. HpaII resistance reflecting the fraction of modified C is displayed. Ctrl, control; TET1 CI , TET1 catalytically inactive mutant. Bar graphs represent the mean of biological triplicates ( n =3) with error bars±SD. (B–D) Kinetics of mC and its oxidized derivatives during oct4TK-GFP reporter demethylation analyzed by modification-sensitive qPCR. Cells were treated as in (A) and plasmid DNA was recovered at indicated time points after transfection. mC and its oxidized derivatives were analyzed using qPCR following HpaII or MspI restriction digest on T4 β-glucosyltransferase (β-GT) treated or control treated plasmid DNA. In (B), % modified cytosine (mC+hmC+fC+caC) is displayed as % HpaII resistance. (C) and (D) display % hmC and % fC/caC determined by MspI resistance following β-GT treatment and MspI resistance in control treated DNA (without β-GT), respectively. Error bars indicate±SD ( n =3). (E, F) HEK293T cells were treated as in (A), but pre-transfected with the indicated siRNAs 24 h before DNA transfection. HpaII resistance reflecting the fraction of modified C is displayed. (G) HEK293T cells were transfected with oct4TK-GFP without any effector protein with the indicated siRNAs, whereby “mock” represents transfection reagent only. oct4TK-GFP plasmid was recovered 72 h after siRNA and 48 h after oct4TK-GFP transfection. HpaII cleavage is displayed as % demethylation, reflecting formation of unmodified C. Bar graphs represent the mean of biological triplicates ( n =3) with error bars±SD. p -Values: (*) p

    Techniques Used: Methylation, In Vitro, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing, Construct, Real-time Polymerase Chain Reaction, Modification, Mutagenesis

    18) Product Images from "Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine"

    Article Title: Comparative characterization of the PvuRts1I family of restriction enzymes and their application in mapping genomic 5-hydroxymethylcytosine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr607

    Activity of AbaSDFI on synthetic oligonucleotides with different modified recognition sites. ( A ) Expected digested fragments from AbaSDFI digestion. Sequences of the oligonucleotide can be found in Supplementary Table S4 ; ( B ) Activity of AbaSDFI on the synthetic oligonucleotide with two 5ghmC, separated by 21 nt; 5 pmol of DNA substrate was digested using a titration of AbaSDFI and resolved on a 20% polyacrylamide PAGE. The gel was stained with SYBR Gold. ( C ) Activity of AbaSDFI on the synthetic oligonucleotide with one 5ghmC and one 5mC; ( D ) activity of AbaSDFI on the synthetic oligonucleotide with one 5ghmC and one C; ( E ) activity of AbaSDFI on the synthetic oligonucleotide with only one 5ghmC and no cytosine in the region 20–25 nt away ( Supplementary Table S4 ); ( F ) activity of AbaSDFI on the synthetic oligonucleotide with two unmodified C [compare with substrate in (D)].
    Figure Legend Snippet: Activity of AbaSDFI on synthetic oligonucleotides with different modified recognition sites. ( A ) Expected digested fragments from AbaSDFI digestion. Sequences of the oligonucleotide can be found in Supplementary Table S4 ; ( B ) Activity of AbaSDFI on the synthetic oligonucleotide with two 5ghmC, separated by 21 nt; 5 pmol of DNA substrate was digested using a titration of AbaSDFI and resolved on a 20% polyacrylamide PAGE. The gel was stained with SYBR Gold. ( C ) Activity of AbaSDFI on the synthetic oligonucleotide with one 5ghmC and one 5mC; ( D ) activity of AbaSDFI on the synthetic oligonucleotide with one 5ghmC and one C; ( E ) activity of AbaSDFI on the synthetic oligonucleotide with only one 5ghmC and no cytosine in the region 20–25 nt away ( Supplementary Table S4 ); ( F ) activity of AbaSDFI on the synthetic oligonucleotide with two unmodified C [compare with substrate in (D)].

    Techniques Used: Activity Assay, Modification, Titration, Polyacrylamide Gel Electrophoresis, Staining

    Cleavage position of AbaSDFI near a hemi-5ghmC site. ( A ) The structure of the oligonucleotide used. The modified cytosine and the positions of the synthetic markers are indicated. ( B ) Digested labeled oligonucleotides resolved in a 20% polyacrylamide 7 M urea denaturing gel. Left panel: lane 1, bottom strand 5′-labeled; lane 2, top strand 5′-labeled; M1, synthetic markers indicated on the bottom strand in (A). Right panel: lane 3, 3′-labeled on both strands; M2, synthetic markers indicated on the top strand in (A). Schematic drawings of the digested products are shown on the side of the gels corresponding to digested bands.
    Figure Legend Snippet: Cleavage position of AbaSDFI near a hemi-5ghmC site. ( A ) The structure of the oligonucleotide used. The modified cytosine and the positions of the synthetic markers are indicated. ( B ) Digested labeled oligonucleotides resolved in a 20% polyacrylamide 7 M urea denaturing gel. Left panel: lane 1, bottom strand 5′-labeled; lane 2, top strand 5′-labeled; M1, synthetic markers indicated on the bottom strand in (A). Right panel: lane 3, 3′-labeled on both strands; M2, synthetic markers indicated on the top strand in (A). Schematic drawings of the digested products are shown on the side of the gels corresponding to digested bands.

    Techniques Used: Modification, Labeling

    Relative selectivity of PvuRts1I, PpeHI and AbaSDFI on unmodified cytosine ( C ), 5mC, 5hmC and 5ghmC (see ‘Materials and Methods’ section for description of methods). In each gel, the amount of enzyme is titrated from left (high) to right (low). All DNA substrates were made by PCR. ( A ) PvuRts1I, the approximate relative selectivity is 5hmC:5ghmC:5mC:C = 2000:2000:8:1; ( B ) PpeHI, the approximate relative selectivity is 5hmC:5ghmC:5mC:C = 128:256:2:1; ( C ) AbaSDFI, the approximate relative selectivity is 5hmC:5ghmC:5mC:C = 500:8000:1:ND (none detected). ( D ) Comparison of the relative selectivity on 5ghmC, 5hmC, 5mC and C among PvuRts1I, PpeHI and AbaSDFI. The relative selectivity is plotted in log scale and normalized based on the 5mC activity.
    Figure Legend Snippet: Relative selectivity of PvuRts1I, PpeHI and AbaSDFI on unmodified cytosine ( C ), 5mC, 5hmC and 5ghmC (see ‘Materials and Methods’ section for description of methods). In each gel, the amount of enzyme is titrated from left (high) to right (low). All DNA substrates were made by PCR. ( A ) PvuRts1I, the approximate relative selectivity is 5hmC:5ghmC:5mC:C = 2000:2000:8:1; ( B ) PpeHI, the approximate relative selectivity is 5hmC:5ghmC:5mC:C = 128:256:2:1; ( C ) AbaSDFI, the approximate relative selectivity is 5hmC:5ghmC:5mC:C = 500:8000:1:ND (none detected). ( D ) Comparison of the relative selectivity on 5ghmC, 5hmC, 5mC and C among PvuRts1I, PpeHI and AbaSDFI. The relative selectivity is plotted in log scale and normalized based on the 5mC activity.

    Techniques Used: Polymerase Chain Reaction, Activity Assay

    19) Product Images from "A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD"

    Article Title: A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0185-9

    DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls ( grey circles on right ) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% ( a ). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR ( b ). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice ( n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals ( c ). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals ( open symbols ) and representative unmethylated samples ( filled symbols ) from 17, 30 and 36 week old C9-BAC mice ( n = 3 per age group) ( d )
    Figure Legend Snippet: DNA hypermethylation at the expanded C9ORF72 promoter appears in a fraction of adult mice. Site-specific DNA methylation sensitive PCR assessment of the human C9ORF72 promoter in the cortex of C9-BAC mice at seven time points, indicated in weeks (wks) of age. Two HhaI restriction sites located at −215 and −109 base pairs from the transcriptional start site were interrogated; three hypermethylated animals are indicated by open shapes (17wks square, 30wks triangle and 36wks circle). Assay controls ( grey circles on right ) include DNA isolated from post mortem brain tissues of ALS patients with the hexanucleotide repeat expansion (C9+) with (me+) or without (me-) promoter hypermethylation, an unaffected healthy control (C9-) individual, and synthetic DNA enriched (CTL Me 100%) or depleted of 5mC (CTL Me 0%). Values are plotted relative to the synthetic high control, which is set to 100% ( a ). C9ORF72 promoter methylation assessment from brain cortex, cerebellum, blood and tail clippings of a 30 week old hypermethylated mouse using HhaI methylation sensitive PCR ( b ). Bisulfite pyrosequencing of brain cortex from 17, 30 and 36 weeks old C9-BAC mice ( n = 1 per age group per methylation status) across 8 CpG dinucleotides within the human C9ORF72 promoter, positions relative to TSS are shown on the x-axis. Open symbols indicate samples from hypermethylated (me+) animals, filled symbols are samples from unmethylated (me-) animals ( c ). Glycine-Proline DPR assessment of whole brain tissue samples from three hypermethylated animals ( open symbols ) and representative unmethylated samples ( filled symbols ) from 17, 30 and 36 week old C9-BAC mice ( n = 3 per age group) ( d )

    Techniques Used: Mouse Assay, DNA Methylation Assay, Polymerase Chain Reaction, BAC Assay, Isolation, CTL Assay, Methylation

    DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p
    Figure Legend Snippet: DNA demethylation is observed at the expanded C9ORF72 promoter distinctively in the brain. Two CpG dinucleotides located within MspI/HpaII restriction sites at positions −313 and +104 base pairs from the C9ORF72 transcriptional start site were interrogated by 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) sensitive PCR. The y-axis indicates percent 5hmC ( black ) and 5mC ( grey ) from brain cortex samples for a subset of C9-BAC mice ( a , b ), error bars represent standard deviation, experiments were performed in duplicates ( N = 2 from a single biological sample for each age and methylation status). Assessment of 5hmC enrichment at two restriction sites across tissue types of a 30 week old hypermethylated mouse are illustrated in c and d . Student’s t-test was performed to determine significance, indicated by p

    Techniques Used: Polymerase Chain Reaction, BAC Assay, Mouse Assay, Standard Deviation, Methylation

    20) Product Images from "Effect of valproic acid on mitochondrial epigenetics"

    Article Title: Effect of valproic acid on mitochondrial epigenetics

    Journal: European journal of pharmacology

    doi: 10.1016/j.ejphar.2012.06.019

    Representation of the selected mouse mitochondrial DNA regions analyzed with the beta-glu-5hmC-sensitive restriction enzymes CviAII and CviQI for 5hmC modifications. The presentation is based on the Mus musculus mitochondrion, complete genome 16299 residue.
    Figure Legend Snippet: Representation of the selected mouse mitochondrial DNA regions analyzed with the beta-glu-5hmC-sensitive restriction enzymes CviAII and CviQI for 5hmC modifications. The presentation is based on the Mus musculus mitochondrion, complete genome 16299 residue.

    Techniques Used:

    Effect of VPA treatment on global 5mC and 5hmC content in mitochondrial DNA (time course). Mitochondrial samples were collected after 1 day (panels in row A) and 3 days (panels in row B) of treatment with 1 mM VPA (striped bars) and its vehicle (open
    Figure Legend Snippet: Effect of VPA treatment on global 5mC and 5hmC content in mitochondrial DNA (time course). Mitochondrial samples were collected after 1 day (panels in row A) and 3 days (panels in row B) of treatment with 1 mM VPA (striped bars) and its vehicle (open

    Techniques Used:

    Effect of a 3-day VPA treatment on sequence-specific 5hmC content in mitochondrial DNA. Mitochondria were isolated and their DNA extracted after 3 days of treatment with 1 mM VPA (striped bars) and its vehicle (open bars). The content of 5hmC in the sequences
    Figure Legend Snippet: Effect of a 3-day VPA treatment on sequence-specific 5hmC content in mitochondrial DNA. Mitochondria were isolated and their DNA extracted after 3 days of treatment with 1 mM VPA (striped bars) and its vehicle (open bars). The content of 5hmC in the sequences

    Techniques Used: Sequencing, Isolation

    Effect of a 3-day treatment with a HDAC inhibitor MS-275 on sequence-specific 5hmC content in mitochondrial DNA. Mitochondria were isolated and their DNA extracted after 3 days of treatment with 5 µM MS-275 (striped bars) and its vehicle (open
    Figure Legend Snippet: Effect of a 3-day treatment with a HDAC inhibitor MS-275 on sequence-specific 5hmC content in mitochondrial DNA. Mitochondria were isolated and their DNA extracted after 3 days of treatment with 5 µM MS-275 (striped bars) and its vehicle (open

    Techniques Used: Mass Spectrometry, Sequencing, Isolation

    21) Product Images from "WT1 recruits TET2 to regulate its target gene expression and suppress leukemia cell proliferation"

    Article Title: WT1 recruits TET2 to regulate its target gene expression and suppress leukemia cell proliferation

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2014.12.023

    AML-derived mutations in TET2 disrupt WT1 binding
    Figure Legend Snippet: AML-derived mutations in TET2 disrupt WT1 binding

    Techniques Used: Derivative Assay, Binding Assay

    TET2 directly binds to WT1
    Figure Legend Snippet: TET2 directly binds to WT1

    Techniques Used:

    TET2 is recruited by WT1 to its target genes
    Figure Legend Snippet: TET2 is recruited by WT1 to its target genes

    Techniques Used:

    TET2 inhibits leukemia cell proliferation in a WT1-dependent manner
    Figure Legend Snippet: TET2 inhibits leukemia cell proliferation in a WT1-dependent manner

    Techniques Used:

    AML-derived WT1 binding-defective TET2 mutants fail to suppress leukemia cell proliferation
    Figure Legend Snippet: AML-derived WT1 binding-defective TET2 mutants fail to suppress leukemia cell proliferation

    Techniques Used: Derivative Assay, Binding Assay

    TET2 activates WT1 target genes
    Figure Legend Snippet: TET2 activates WT1 target genes

    Techniques Used:

    22) Product Images from "Two novel DXZ4-associated long noncoding RNAs show developmental changes in expression coincident with heterochromatin formation at the human (Homo sapiens) macrosatellite repeat"

    Article Title: Two novel DXZ4-associated long noncoding RNAs show developmental changes in expression coincident with heterochromatin formation at the human (Homo sapiens) macrosatellite repeat

    Journal: Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology

    doi: 10.1007/s10577-015-9479-3

    Related promoters flanking DXZ4 drive transcription toward the array
    Figure Legend Snippet: Related promoters flanking DXZ4 drive transcription toward the array

    Techniques Used:

    RNA FISH Images showing allele-specific expression and spatial arrangement of DXZ4-associated lncRNAs
    Figure Legend Snippet: RNA FISH Images showing allele-specific expression and spatial arrangement of DXZ4-associated lncRNAs

    Techniques Used: Fluorescence In Situ Hybridization, Expressing

    23) Product Images from "Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes *"

    Article Title: Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.217083

    Validation of MspI and HpaII isoschizomer- and β-glucosyltransferase-mediated 5-hmC detection and quantitation. A , shown is unmethylated, methylated, and hydroxymethylated duplex DNAs with centrally located CCGG ( boxed ), where the internal C is either unmethylated C or 5-mC or 5-hmC (shown in gray ). B , shown is validation for locus-specific 5-hmC detection in a fixed amount of pre-mixed DNAs, as shown in A , using MspI in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected 5-hmC is plotted. C , shown is similar validation for locus-specific methylcytosine ( 5-mC + 5-hmC ) detection in fixed amount of pre-mixed DNAs, as shown in A , using HpaII in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected total methyl cytosine ( 5-mC + 5-hmC ) is plotted.
    Figure Legend Snippet: Validation of MspI and HpaII isoschizomer- and β-glucosyltransferase-mediated 5-hmC detection and quantitation. A , shown is unmethylated, methylated, and hydroxymethylated duplex DNAs with centrally located CCGG ( boxed ), where the internal C is either unmethylated C or 5-mC or 5-hmC (shown in gray ). B , shown is validation for locus-specific 5-hmC detection in a fixed amount of pre-mixed DNAs, as shown in A , using MspI in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected 5-hmC is plotted. C , shown is similar validation for locus-specific methylcytosine ( 5-mC + 5-hmC ) detection in fixed amount of pre-mixed DNAs, as shown in A , using HpaII in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected total methyl cytosine ( 5-mC + 5-hmC ) is plotted.

    Techniques Used: Quantitation Assay, Methylation, Gas Chromatography, Real-time Polymerase Chain Reaction

    MspI and HpaII isoschizomer can distinguish between 5-hmC and 5-ghmC at the internal cytosine residue. Cleavage specificity is shown for MspI and HpaII on FAM-end-labeled oligonucleotide duplex with internal CG being symmetrically hydroxymethylated or hemihydroxymethylated in the presence or absence of β-GT. Complete cleavage products are labeled as 24 and 19 nt, respectively, on a non-denaturing acrylamide gel for double-hydroxymethylated ( upper panel ) and denaturing acrylamide gel for hemi-hydroxymethylated DNA ( lower panel ). The arrow at the right indicates small amounts of 24-nt-long product.
    Figure Legend Snippet: MspI and HpaII isoschizomer can distinguish between 5-hmC and 5-ghmC at the internal cytosine residue. Cleavage specificity is shown for MspI and HpaII on FAM-end-labeled oligonucleotide duplex with internal CG being symmetrically hydroxymethylated or hemihydroxymethylated in the presence or absence of β-GT. Complete cleavage products are labeled as 24 and 19 nt, respectively, on a non-denaturing acrylamide gel for double-hydroxymethylated ( upper panel ) and denaturing acrylamide gel for hemi-hydroxymethylated DNA ( lower panel ). The arrow at the right indicates small amounts of 24-nt-long product.

    Techniques Used: Labeling, Acrylamide Gel Assay

    Methylation and hydroxymethylation analysis across gene body. A , VANGL1. B , EGFR . The genes are depicted with exons shown as black rectangles . The interrogated CCGG site numbers are indicated on the top along with the primer sets on the bottom . Diagrams are not drawn to scale. Total methylated cytosines (5-mC plus 5-hmC) obtained from β-GT-treated ( BGT ) HpaII-digested DNA and β-GT-treated MspI-digested DNA show the percentage of 5-hmC ( bottom panels ) at each specific site.
    Figure Legend Snippet: Methylation and hydroxymethylation analysis across gene body. A , VANGL1. B , EGFR . The genes are depicted with exons shown as black rectangles . The interrogated CCGG site numbers are indicated on the top along with the primer sets on the bottom . Diagrams are not drawn to scale. Total methylated cytosines (5-mC plus 5-hmC) obtained from β-GT-treated ( BGT ) HpaII-digested DNA and β-GT-treated MspI-digested DNA show the percentage of 5-hmC ( bottom panels ) at each specific site.

    Techniques Used: Methylation

    Analysis of 5-mC and 5-hmC during embryonic stem cell differentiation to embryoid body. A , LIF withdrawal leading to embryonic stem cell marker Oct4 and Nanog repression is shown. Shown is a Western blot of ES cell extract after LIF withdrawal along with NIH3T3 (3T3), as shown on the top , with the indicated antibodies on the left . Gapdh is the loading control. B , Oct4 and Nanog gene are methylated upon embryoid body formation. Day post-LIF withdrawal is on the left along with NIH3T3. UDP-Glc and β-GT are shown on the top along with restriction enzyme MspI ( M ) and HpaII ( H ) cleavage along with control ( C ). C , 5-hmC detection during embryoid body formation is shown. Day post-LIF withdrawal is shown on the left. D , global methylation analysis using LC-MS during embryoid body formation is shown. Each analysis point was performed in duplicate. Deoxycytosine (C), 5-methyldeoxycytosine (5-mC or mC ) and 5-hydroxymethyldeoxycytosine (5-hmC or hmC ) are shown.
    Figure Legend Snippet: Analysis of 5-mC and 5-hmC during embryonic stem cell differentiation to embryoid body. A , LIF withdrawal leading to embryonic stem cell marker Oct4 and Nanog repression is shown. Shown is a Western blot of ES cell extract after LIF withdrawal along with NIH3T3 (3T3), as shown on the top , with the indicated antibodies on the left . Gapdh is the loading control. B , Oct4 and Nanog gene are methylated upon embryoid body formation. Day post-LIF withdrawal is on the left along with NIH3T3. UDP-Glc and β-GT are shown on the top along with restriction enzyme MspI ( M ) and HpaII ( H ) cleavage along with control ( C ). C , 5-hmC detection during embryoid body formation is shown. Day post-LIF withdrawal is shown on the left. D , global methylation analysis using LC-MS during embryoid body formation is shown. Each analysis point was performed in duplicate. Deoxycytosine (C), 5-methyldeoxycytosine (5-mC or mC ) and 5-hydroxymethyldeoxycytosine (5-hmC or hmC ) are shown.

    Techniques Used: Cell Differentiation, Marker, Western Blot, Methylation, Gas Chromatography, Liquid Chromatography with Mass Spectroscopy

    Cloning, sequence identification, and tissue-specific distribution of 5-hydroxymethylcytosine. A , a scheme for cloning and identification of 5-hmC containing DNA fragments using MspI restriction enzyme and β-GT ( Beta-GT ) is shown. B , a scheme for identification of 5-hmC containing CCGG locus using MspI ( M ) and HpaII ( H ) isoschizomers using glucosylation reaction is shown along with control ( C ). C , locus-specific end point PCR to interrogate and detect 5-hmC at CCGG sites in mouse genomic DNA is shown. The loci are discovered based on cloning scheme as shown in A and in supplemental Table 1 . Mouse brain, liver, heart, and spleen along with NIH3T3 cultured cell DNAs were interrogated for the 4 loci ( supplemental Table 3 : loci 2 and 3 are 5′ and 3′ MspI sites of chromosome 10, respectively, bp 34,574,152; locus 4 is the 5′ MspI site of chromosome 12, bp 17,432,255; locus 12 is the 5′ MspI site of Lpr1 intron, bp 2,372,508) as shown. D , shown is the locus-specific end point PCR to interrogate and detect 5-hmC at CCGG sites in human genomic DNA The loci are discovered based on the cloning scheme as shown in A and in supplemental Table 2 . Human brain (pons, occipital lobe ( OL )), liver, heart, and spleen along with HeLa-cultured cell DNAs were interrogated for both of the VANGL1 loci as shown. The control DNA interrogated fragment without CCGG sequence is miR17A.
    Figure Legend Snippet: Cloning, sequence identification, and tissue-specific distribution of 5-hydroxymethylcytosine. A , a scheme for cloning and identification of 5-hmC containing DNA fragments using MspI restriction enzyme and β-GT ( Beta-GT ) is shown. B , a scheme for identification of 5-hmC containing CCGG locus using MspI ( M ) and HpaII ( H ) isoschizomers using glucosylation reaction is shown along with control ( C ). C , locus-specific end point PCR to interrogate and detect 5-hmC at CCGG sites in mouse genomic DNA is shown. The loci are discovered based on cloning scheme as shown in A and in supplemental Table 1 . Mouse brain, liver, heart, and spleen along with NIH3T3 cultured cell DNAs were interrogated for the 4 loci ( supplemental Table 3 : loci 2 and 3 are 5′ and 3′ MspI sites of chromosome 10, respectively, bp 34,574,152; locus 4 is the 5′ MspI site of chromosome 12, bp 17,432,255; locus 12 is the 5′ MspI site of Lpr1 intron, bp 2,372,508) as shown. D , shown is the locus-specific end point PCR to interrogate and detect 5-hmC at CCGG sites in human genomic DNA The loci are discovered based on the cloning scheme as shown in A and in supplemental Table 2 . Human brain (pons, occipital lobe ( OL )), liver, heart, and spleen along with HeLa-cultured cell DNAs were interrogated for both of the VANGL1 loci as shown. The control DNA interrogated fragment without CCGG sequence is miR17A.

    Techniques Used: Clone Assay, Sequencing, Polymerase Chain Reaction, Cell Culture

    24) Product Images from "Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase"

    Article Title: Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase

    Journal: Nature biotechnology

    doi: 10.1038/nbt.4204

    Development and validation of ACE-Seq. ( a-c ) 1 ng of T4 phage genomic DNAs with homogeneous modifications ( a : T4-C; b : T4-hmC; c : T4-ghmC) were heated, snap frozen, and incubated with A3A before amplification of a genomic segment, TA cloning, and Sanger sequencing of individual clones. Illustrative sequencing traces from individual clones are shown below the reference genome. Arrows denote deamination events (C > T transitions). Deamination events are quantified as the number of cytosines that were deaminated across the sum of all clones (93 cytosines per clone; T4-C 5 clones, T4-hmC 3 clones, T4-ghmC 4 clones). ( d,f ) Rates of non-conversion for enzymatically-methylated λ phage gDNA (5mCG, CH) and T4-hmC phage gDNA in ACE-Seq as determined by Illumina sequencing, using inputs of either ( d ) 1 ng of each alone or ( f ) 100 pg each as spike-ins averaged across six mammalian DNA samples (see Supplementary Table 1 ). Mean values listed above each bar, and error bars represent standard deviations. Individual data points are overlaid on the plot. ( e ) Representative LC-MS/MS traces of C, 5mC, 5hmC, and 5ghmC nucleosides after a 1:1 mix of methylated λ gDNA and T4-hmC gDNA was subjected to ACE-Seq treatment (compared to untreated control sample). Percentages denote amount detected after ACE-Seq treatment, averaged across three independent replicates.
    Figure Legend Snippet: Development and validation of ACE-Seq. ( a-c ) 1 ng of T4 phage genomic DNAs with homogeneous modifications ( a : T4-C; b : T4-hmC; c : T4-ghmC) were heated, snap frozen, and incubated with A3A before amplification of a genomic segment, TA cloning, and Sanger sequencing of individual clones. Illustrative sequencing traces from individual clones are shown below the reference genome. Arrows denote deamination events (C > T transitions). Deamination events are quantified as the number of cytosines that were deaminated across the sum of all clones (93 cytosines per clone; T4-C 5 clones, T4-hmC 3 clones, T4-ghmC 4 clones). ( d,f ) Rates of non-conversion for enzymatically-methylated λ phage gDNA (5mCG, CH) and T4-hmC phage gDNA in ACE-Seq as determined by Illumina sequencing, using inputs of either ( d ) 1 ng of each alone or ( f ) 100 pg each as spike-ins averaged across six mammalian DNA samples (see Supplementary Table 1 ). Mean values listed above each bar, and error bars represent standard deviations. Individual data points are overlaid on the plot. ( e ) Representative LC-MS/MS traces of C, 5mC, 5hmC, and 5ghmC nucleosides after a 1:1 mix of methylated λ gDNA and T4-hmC gDNA was subjected to ACE-Seq treatment (compared to untreated control sample). Percentages denote amount detected after ACE-Seq treatment, averaged across three independent replicates.

    Techniques Used: Incubation, Amplification, TA Cloning, Sequencing, Clone Assay, Methylation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    25) Product Images from "Genome-wide comparison of DNA hydroxymethylation in mouse embryonic stem cells and neural progenitor cells by a new comparative hMeDIP-seq method"

    Article Title: Genome-wide comparison of DNA hydroxymethylation in mouse embryonic stem cells and neural progenitor cells by a new comparative hMeDIP-seq method

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt091

    Relationship between the changes in DNA hydroxymethylation and gene expression during neural differentiation. ( a ) Down- and up-regulated genes during neural differentiation were sorted (NPC versus ESC, log 2 mRNA expression ratio ≤ −1 or ≥1). The changes in 5hmC peak density at promoters (TSS ± 1 kb) in down- and up-regulated genes were compared during neural differentiation ( P = 0). * P
    Figure Legend Snippet: Relationship between the changes in DNA hydroxymethylation and gene expression during neural differentiation. ( a ) Down- and up-regulated genes during neural differentiation were sorted (NPC versus ESC, log 2 mRNA expression ratio ≤ −1 or ≥1). The changes in 5hmC peak density at promoters (TSS ± 1 kb) in down- and up-regulated genes were compared during neural differentiation ( P = 0). * P

    Techniques Used: Expressing

    Correlation between DNA hydroxymethylation and gene expression in ESCs and NPCs. ( a ) Genes were separated into five groups (from high to low) according to their expression levels in ESCs: top 20%; 20–40%; 40–60%; 60–80%; bottom 20%. The average 5hmC densities of the five groups of genes were plotted across the promoter or gene body regions. Left: TSS ± 5 kb regions. Right: gene body regions. ( b ) Genes were also separated into five groups according to their expression levels in NPCs. The average 5hmC densities of the five groups of genes were plotted across the promoter or gene body regions. Left: TSS ± 5 kb regions. Right: gene body regions. ( c ) Genes were classified into two groups with respect to the presence (5hmC+) or absence (5hmC−) of 5hmC peak(s) at their promoters (TSS ± 1 kb) or in gene body regions (from TSS + 1 kb to TES) in ESCs. Gene expression levels were compared between ‘5hmC+’ and ‘5hmC−’ groups. Left: promoter, P = 0; Right: gene body region, P = 1.08358e-13. * P
    Figure Legend Snippet: Correlation between DNA hydroxymethylation and gene expression in ESCs and NPCs. ( a ) Genes were separated into five groups (from high to low) according to their expression levels in ESCs: top 20%; 20–40%; 40–60%; 60–80%; bottom 20%. The average 5hmC densities of the five groups of genes were plotted across the promoter or gene body regions. Left: TSS ± 5 kb regions. Right: gene body regions. ( b ) Genes were also separated into five groups according to their expression levels in NPCs. The average 5hmC densities of the five groups of genes were plotted across the promoter or gene body regions. Left: TSS ± 5 kb regions. Right: gene body regions. ( c ) Genes were classified into two groups with respect to the presence (5hmC+) or absence (5hmC−) of 5hmC peak(s) at their promoters (TSS ± 1 kb) or in gene body regions (from TSS + 1 kb to TES) in ESCs. Gene expression levels were compared between ‘5hmC+’ and ‘5hmC−’ groups. Left: promoter, P = 0; Right: gene body region, P = 1.08358e-13. * P

    Techniques Used: Expressing

    Identification of DHMRs between ESCs and NPCs. ( a ) All 5hmC peaks called from the pooled hMeDIP-seq data were separated into three groups: down-regulated (log 2 5hmC density ratio ≤ −1), up-regulated (log 2 5hmC density ratio ≥ 1) and no significant change (−1
    Figure Legend Snippet: Identification of DHMRs between ESCs and NPCs. ( a ) All 5hmC peaks called from the pooled hMeDIP-seq data were separated into three groups: down-regulated (log 2 5hmC density ratio ≤ −1), up-regulated (log 2 5hmC density ratio ≥ 1) and no significant change (−1

    Techniques Used:

    Differential Tet1/2/3 gene expression and global 5hmC levels in ESCs and NPCs. ( a ) RT-qPCR analysis of Tet1 , Tet2 and Tet3 mRNA levels in ESCs and NPCs (mean values ± SD, n = 3). ( b ) Immunofluorescence analysis of 5hmC levels in ESCs and NPCs. Bar: 50 μm. ( c ) Dot blot analysis of the global 5hmC levels in the gDNA of ESCs and ESC-derived NPCs. One hundred fifty nanogram gDNA for each dot. ( d ) The relative 5hmC signal of dot blot and comparative hMeDIP-seq in ESCs and NPCs. *The ratio of hMeDIP/Input reads numbers in ESCs was set as 100%. ( e ) Distribution of 5hmC in TSS ± 5 kb and gene body regions in ESCs. ( f ) Distribution of 5hmC in TSS ± 5 kb and gene body regions in NPCs.
    Figure Legend Snippet: Differential Tet1/2/3 gene expression and global 5hmC levels in ESCs and NPCs. ( a ) RT-qPCR analysis of Tet1 , Tet2 and Tet3 mRNA levels in ESCs and NPCs (mean values ± SD, n = 3). ( b ) Immunofluorescence analysis of 5hmC levels in ESCs and NPCs. Bar: 50 μm. ( c ) Dot blot analysis of the global 5hmC levels in the gDNA of ESCs and ESC-derived NPCs. One hundred fifty nanogram gDNA for each dot. ( d ) The relative 5hmC signal of dot blot and comparative hMeDIP-seq in ESCs and NPCs. *The ratio of hMeDIP/Input reads numbers in ESCs was set as 100%. ( e ) Distribution of 5hmC in TSS ± 5 kb and gene body regions in ESCs. ( f ) Distribution of 5hmC in TSS ± 5 kb and gene body regions in NPCs.

    Techniques Used: Expressing, Quantitative RT-PCR, Immunofluorescence, Dot Blot, Derivative Assay

    26) Product Images from "Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes"

    Article Title: Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddv198

    Quantification by enzymatic assay of 5hmC and 5mC at selected CCGG sites in the MYOD1 neighborhood and in MYF6. The average percentages of 5hmC ( A ) and 5mC ( B ) at the indicated CCGG sites in biological replicate samples were determined by an enzymatic
    Figure Legend Snippet: Quantification by enzymatic assay of 5hmC and 5mC at selected CCGG sites in the MYOD1 neighborhood and in MYF6. The average percentages of 5hmC ( A ) and 5mC ( B ) at the indicated CCGG sites in biological replicate samples were determined by an enzymatic

    Techniques Used: Enzymatic Assay

    27) Product Images from "Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes"

    Article Title: Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddv198

    Quantification by enzymatic assay of 5hmC and 5mC at selected CCGG sites in the MYOD1 neighborhood and in MYF6. The average percentages of 5hmC ( A ) and 5mC ( B ) at the indicated CCGG sites in biological replicate samples were determined by an enzymatic
    Figure Legend Snippet: Quantification by enzymatic assay of 5hmC and 5mC at selected CCGG sites in the MYOD1 neighborhood and in MYF6. The average percentages of 5hmC ( A ) and 5mC ( B ) at the indicated CCGG sites in biological replicate samples were determined by an enzymatic

    Techniques Used: Enzymatic Assay

    28) Product Images from "Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression"

    Article Title: Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression

    Journal: Epigenetics

    doi: 10.1080/15592294.2016.1265713

    Average levels of 5hmC and 5mC at 12 of the 21 tested CCGG sites and RNA-seq RPKM values for the associated genes. (a) The average levels of 5hmC/total C at 12 of the 21 CCGG sites that were determined by Epimark assays on biological replicates for four of the 14 examined sample types. (b) The average levels of 5mC/total C from these assays. (c) RPKM (reads per kilobase per million mapped reads), median values from 430, 218, 125, and 393 samples of generic SkM, left ventricle, cerebellum, and whole blood samples (data for leukocytes are not available), respectively. 30 RPKM values are shown on a log scale for 11 of the examined genes. Tables S3–S5 give the Epimark and RPKM data for all studied sites and samples.
    Figure Legend Snippet: Average levels of 5hmC and 5mC at 12 of the 21 tested CCGG sites and RNA-seq RPKM values for the associated genes. (a) The average levels of 5hmC/total C at 12 of the 21 CCGG sites that were determined by Epimark assays on biological replicates for four of the 14 examined sample types. (b) The average levels of 5mC/total C from these assays. (c) RPKM (reads per kilobase per million mapped reads), median values from 430, 218, 125, and 393 samples of generic SkM, left ventricle, cerebellum, and whole blood samples (data for leukocytes are not available), respectively. 30 RPKM values are shown on a log scale for 11 of the examined genes. Tables S3–S5 give the Epimark and RPKM data for all studied sites and samples.

    Techniques Used: RNA Sequencing Assay

    SIX2 , a developmental TF gene, exhibits three hypermethylated DMRs in SkM and aorta that correlate with specific gene expression in these tissues. (a) SIX2 , the LINC01121 ncRNA gene (dashed line) and RNA-seq for the minus-strand of cell cultures and non-strand-specific RNA-seq for tissues (chr2:45,224,940–45,243,926). (b) Chromatin state segmentation; dashed box, the SIX2 gene body for reference. (c) Significant hyper- or hypo-methylated CpG sites from RRBS profiles. 23 (d) and (e) Bisulfite-seq and TAB-seq. Dashed boxes, Mb/Mt- and SkM-hypermethylated region; purple box, subregion in osteoblasts, SkM, and aorta lacking DNA hypermethylation observed in Mb and Mt. Arrowhead, the CCGG site analyzed for by Epimark assay and shown to have the highest 5hmC in SkM but only with an average of 7% of all C as 5hmC ( Fig. 2a ).
    Figure Legend Snippet: SIX2 , a developmental TF gene, exhibits three hypermethylated DMRs in SkM and aorta that correlate with specific gene expression in these tissues. (a) SIX2 , the LINC01121 ncRNA gene (dashed line) and RNA-seq for the minus-strand of cell cultures and non-strand-specific RNA-seq for tissues (chr2:45,224,940–45,243,926). (b) Chromatin state segmentation; dashed box, the SIX2 gene body for reference. (c) Significant hyper- or hypo-methylated CpG sites from RRBS profiles. 23 (d) and (e) Bisulfite-seq and TAB-seq. Dashed boxes, Mb/Mt- and SkM-hypermethylated region; purple box, subregion in osteoblasts, SkM, and aorta lacking DNA hypermethylation observed in Mb and Mt. Arrowhead, the CCGG site analyzed for by Epimark assay and shown to have the highest 5hmC in SkM but only with an average of 7% of all C as 5hmC ( Fig. 2a ).

    Techniques Used: Expressing, RNA Sequencing Assay, Methylation, Bisulfite Sequencing

    Skeletal muscle, brain, and heart group together upon hierarchical clustering of 5 hmC levels at the analyzed sites in diverse samples. Vectors of aggregate values of 5hmC (a) and 5mC levels (b) for each analyzed tissue at analyzed CpG sites (Tables S1–S4) were clustered as described in Methods. Average values for modified C/total C are shaded on the blue-orange scale with missing values shown in white. The names of the sites that were significantly hypermethylated or hypomethylated in myogenic progenitor cells (Mb and Mt) vs. 16 types of non-muscle cell cultures as previously determined by RRBS 23,24 are shown in pink or blue, respectively. No RRBS data were available for the IRS1 −1.7 site; the STX16 in8 site was constitutively methylated in cell cultures but the STX16 gene had a Mb/Mt hypermethylated DMR upstream of the promoter; these sites are shown in black).
    Figure Legend Snippet: Skeletal muscle, brain, and heart group together upon hierarchical clustering of 5 hmC levels at the analyzed sites in diverse samples. Vectors of aggregate values of 5hmC (a) and 5mC levels (b) for each analyzed tissue at analyzed CpG sites (Tables S1–S4) were clustered as described in Methods. Average values for modified C/total C are shaded on the blue-orange scale with missing values shown in white. The names of the sites that were significantly hypermethylated or hypomethylated in myogenic progenitor cells (Mb and Mt) vs. 16 types of non-muscle cell cultures as previously determined by RRBS 23,24 are shown in pink or blue, respectively. No RRBS data were available for the IRS1 −1.7 site; the STX16 in8 site was constitutively methylated in cell cultures but the STX16 gene had a Mb/Mt hypermethylated DMR upstream of the promoter; these sites are shown in black).

    Techniques Used: Modification, Methylation

    Intragenic Mb/Mt DNA hypermethylation, decreased CTCF binding, and loss of poised promoter chromatin in PITX3 correlated with gene expression in Mb and Mt. (a) RefSeq gene structure for PITX3 , a developmental gene, at chr10:103,989,638–104,003,464 (all coordinates for figures are in hg19 and all tracks are aligned) and ENCODE RNA-seq data. The sequence-specific minus-strand RNA-seq profile is shown for cell cultures and the not strand-specific RNA-seq data for tissues. 37 (b) CTCF binding from ENCODE data (dot, predicted insulator; green box, preferential Mb/Mt CTCF binding site. (c) Chromatin state segmentation from RoadMap data 37 with the indicated color code; Pr, promoter; Enh, enhancer; Enh/Pr, both active promoter-type and enhancer-type histone modification; Repressed, enriched in H3K27me3 (weak, light gray; strong, dark gray) or H3K9me3 (violet). (d) Statistically significant hypermethylated sites as determined by RRBS for comparison of the set of Mb and Mt vs. 16 types of non-muscle cell cultures 23 and CGIs from the UCSC Genome Browser. 37 (e) Bisulfite-seq profiles 37 with blue bars indicating regions with significantly lower methylation compared with most of the given genome. 28,72 (f) TAB-seq profile of the distribution of 5hmC in the same prefrontal cortex (PFC) DNA sample from brain used for bisulfite-seq. Mb, myoblasts; LCL, GM12868 lymphoblastoid cell line; HMEC, human mammary epithelial cells; ESC, H1 embryonic stem cells; Sk muscle #1, psoas muscle; Sk muscle #2, unknown type of skeletal muscle; Lung fib, IMR-90, fetal lung fibroblast cell line; heart, left ventricle. Dashed box, cloned DMR sequences; arrowhead, Epimark-assayed CCGG, which had high 5hmC in SkM ( Fig. 2a ).
    Figure Legend Snippet: Intragenic Mb/Mt DNA hypermethylation, decreased CTCF binding, and loss of poised promoter chromatin in PITX3 correlated with gene expression in Mb and Mt. (a) RefSeq gene structure for PITX3 , a developmental gene, at chr10:103,989,638–104,003,464 (all coordinates for figures are in hg19 and all tracks are aligned) and ENCODE RNA-seq data. The sequence-specific minus-strand RNA-seq profile is shown for cell cultures and the not strand-specific RNA-seq data for tissues. 37 (b) CTCF binding from ENCODE data (dot, predicted insulator; green box, preferential Mb/Mt CTCF binding site. (c) Chromatin state segmentation from RoadMap data 37 with the indicated color code; Pr, promoter; Enh, enhancer; Enh/Pr, both active promoter-type and enhancer-type histone modification; Repressed, enriched in H3K27me3 (weak, light gray; strong, dark gray) or H3K9me3 (violet). (d) Statistically significant hypermethylated sites as determined by RRBS for comparison of the set of Mb and Mt vs. 16 types of non-muscle cell cultures 23 and CGIs from the UCSC Genome Browser. 37 (e) Bisulfite-seq profiles 37 with blue bars indicating regions with significantly lower methylation compared with most of the given genome. 28,72 (f) TAB-seq profile of the distribution of 5hmC in the same prefrontal cortex (PFC) DNA sample from brain used for bisulfite-seq. Mb, myoblasts; LCL, GM12868 lymphoblastoid cell line; HMEC, human mammary epithelial cells; ESC, H1 embryonic stem cells; Sk muscle #1, psoas muscle; Sk muscle #2, unknown type of skeletal muscle; Lung fib, IMR-90, fetal lung fibroblast cell line; heart, left ventricle. Dashed box, cloned DMR sequences; arrowhead, Epimark-assayed CCGG, which had high 5hmC in SkM ( Fig. 2a ).

    Techniques Used: Binding Assay, Expressing, RNA Sequencing Assay, Sequencing, Modification, Bisulfite Sequencing, Methylation, Clone Assay

    NRXN2 , a neuronal gene, displays a Mb/Mt-specific alternative promoter whose DNA hypomethylation persists in SkM despite the loss of promoter activity. (a) RefSeq gene isoforms structures for NRXN2 (chr11:64,371,048–64,493,639) and RNA-seq as in Fig. 3 but also with the ENCODE profile of 5′ cap mapping (CAGE). 37 Purple broken arrow on left, TSS for the Mb-associated transcript. (b) Chromatin state segmentation. (c) Significant hyper- or hypomethylated DMRs from 33 RRBS profiles. 24 (d) and (e) Bisulfite-seq and TAB-seq. Highlighted green region, Mb/Mt-specific promoter region within NRXN2 . Arrowhead, Epimark-tested site with high 5hmC in cerebellum ( Fig. 2a ).
    Figure Legend Snippet: NRXN2 , a neuronal gene, displays a Mb/Mt-specific alternative promoter whose DNA hypomethylation persists in SkM despite the loss of promoter activity. (a) RefSeq gene isoforms structures for NRXN2 (chr11:64,371,048–64,493,639) and RNA-seq as in Fig. 3 but also with the ENCODE profile of 5′ cap mapping (CAGE). 37 Purple broken arrow on left, TSS for the Mb-associated transcript. (b) Chromatin state segmentation. (c) Significant hyper- or hypomethylated DMRs from 33 RRBS profiles. 24 (d) and (e) Bisulfite-seq and TAB-seq. Highlighted green region, Mb/Mt-specific promoter region within NRXN2 . Arrowhead, Epimark-tested site with high 5hmC in cerebellum ( Fig. 2a ).

    Techniques Used: Activity Assay, RNA Sequencing Assay, Bisulfite Sequencing

    29) Product Images from "Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression"

    Article Title: Association of 5-hydroxymethylation and 5-methylation of DNA cytosine with tissue-specific gene expression

    Journal: Epigenetics

    doi: 10.1080/15592294.2016.1265713

    Average levels of 5hmC and 5mC at 12 of the 21 tested CCGG sites and RNA-seq RPKM values for the associated genes. (a) The average levels of 5hmC/total C at 12 of the 21 CCGG sites that were determined by Epimark assays on biological replicates for four of the 14 examined sample types. (b) The average levels of 5mC/total C from these assays. (c) RPKM (reads per kilobase per million mapped reads), median values from 430, 218, 125, and 393 samples of generic SkM, left ventricle, cerebellum, and whole blood samples (data for leukocytes are not available), respectively. 30 RPKM values are shown on a log scale for 11 of the examined genes. Tables S3–S5 give the Epimark and RPKM data for all studied sites and samples.
    Figure Legend Snippet: Average levels of 5hmC and 5mC at 12 of the 21 tested CCGG sites and RNA-seq RPKM values for the associated genes. (a) The average levels of 5hmC/total C at 12 of the 21 CCGG sites that were determined by Epimark assays on biological replicates for four of the 14 examined sample types. (b) The average levels of 5mC/total C from these assays. (c) RPKM (reads per kilobase per million mapped reads), median values from 430, 218, 125, and 393 samples of generic SkM, left ventricle, cerebellum, and whole blood samples (data for leukocytes are not available), respectively. 30 RPKM values are shown on a log scale for 11 of the examined genes. Tables S3–S5 give the Epimark and RPKM data for all studied sites and samples.

    Techniques Used: RNA Sequencing Assay

    Skeletal muscle, brain, and heart group together upon hierarchical clustering of 5 hmC levels at the analyzed sites in diverse samples. Vectors of aggregate values of 5hmC (a) and 5mC levels (b) for each analyzed tissue at analyzed CpG sites (Tables S1–S4) were clustered as described in Methods. Average values for modified C/total C are shaded on the blue-orange scale with missing values shown in white. The names of the sites that were significantly hypermethylated or hypomethylated in myogenic progenitor cells (Mb and Mt) vs. 16 types of non-muscle cell cultures as previously determined by RRBS 23,24 are shown in pink or blue, respectively. No RRBS data were available for the IRS1 −1.7 site; the STX16 in8 site was constitutively methylated in cell cultures but the STX16 gene had a Mb/Mt hypermethylated DMR upstream of the promoter; these sites are shown in black).
    Figure Legend Snippet: Skeletal muscle, brain, and heart group together upon hierarchical clustering of 5 hmC levels at the analyzed sites in diverse samples. Vectors of aggregate values of 5hmC (a) and 5mC levels (b) for each analyzed tissue at analyzed CpG sites (Tables S1–S4) were clustered as described in Methods. Average values for modified C/total C are shaded on the blue-orange scale with missing values shown in white. The names of the sites that were significantly hypermethylated or hypomethylated in myogenic progenitor cells (Mb and Mt) vs. 16 types of non-muscle cell cultures as previously determined by RRBS 23,24 are shown in pink or blue, respectively. No RRBS data were available for the IRS1 −1.7 site; the STX16 in8 site was constitutively methylated in cell cultures but the STX16 gene had a Mb/Mt hypermethylated DMR upstream of the promoter; these sites are shown in black).

    Techniques Used: Modification, Methylation

    30) Product Images from "Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes"

    Article Title: Tissue-specific epigenetics in gene neighborhoods: myogenic transcription factor genes

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddv198

    Quantification by enzymatic assay of 5hmC and 5mC at selected CCGG sites in the MYOD1 neighborhood and in MYF6. The average percentages of 5hmC ( A ) and 5mC ( B ) at the indicated CCGG sites in biological replicate samples were determined by an enzymatic
    Figure Legend Snippet: Quantification by enzymatic assay of 5hmC and 5mC at selected CCGG sites in the MYOD1 neighborhood and in MYF6. The average percentages of 5hmC ( A ) and 5mC ( B ) at the indicated CCGG sites in biological replicate samples were determined by an enzymatic

    Techniques Used: Enzymatic Assay

    31) Product Images from "Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9"

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    Journal: mBio

    doi: 10.1128/mBio.00648-15

    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.
    Figure Legend Snippet: Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Techniques Used: Modification, Mobility Shift, Sequencing, Methylation

    32) Product Images from "Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion"

    Article Title: Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2013.01.032

    Evidence that 5hmC Levels Increase at ICRs in Somatic Cells after Fusion with EGCs (A) Detection of 5hmC ( I ) and unmodified cytosine ( II ) in human heterokaryon samples. Genomic DNA was divided and was either treated with T4-β-glucosyltransferase, which binds glucose groups selectively at 5hmC sites (red asterisk) and creates 5hgmC (open hexagon, left), or left untreated (H 2 O). Samples were digested with MspI (which does not digest 5hgmC) or left undigested (H 2 O), and the abundance of locus-specific DNA in each was compared by qPCR. In strategy II , unmodified (C) and modified CpG (5mC and 5hmC) levels were evaluated by HpaII digestion (right); DNA samples were treated with HpaII (which does not cut 5mC and 5hmC), left undigested (H 2 O), or treated with MspI (which cuts both and provides a positive control). The abundance of locus-specific DNA within each of these samples was estimated by qPCR and used to calculate the percentage of HpaII resistance. (B) Levels of 5hmC at OCT4 in hB cells before (0 hr), and 48 hr and 72 hr after fusion with mouse EGCs (black bars) or ESCs (white bars) are shown as the mean and SE of three to five independent experiments. (C) HpaII digestion analysis of OCT4 in hB lymphocytes before (0 hr) and 48 hr and 72 hr after fusion with EGCs (closed circles) or ESCs (open circles) are shown. Red bars mark the position of primer-amplified PCR products derived from the promoter (right) and downstream of the TSS (left), and values represent the mean and SE of three to five independent experiments. ∗∗ , p value
    Figure Legend Snippet: Evidence that 5hmC Levels Increase at ICRs in Somatic Cells after Fusion with EGCs (A) Detection of 5hmC ( I ) and unmodified cytosine ( II ) in human heterokaryon samples. Genomic DNA was divided and was either treated with T4-β-glucosyltransferase, which binds glucose groups selectively at 5hmC sites (red asterisk) and creates 5hgmC (open hexagon, left), or left untreated (H 2 O). Samples were digested with MspI (which does not digest 5hgmC) or left undigested (H 2 O), and the abundance of locus-specific DNA in each was compared by qPCR. In strategy II , unmodified (C) and modified CpG (5mC and 5hmC) levels were evaluated by HpaII digestion (right); DNA samples were treated with HpaII (which does not cut 5mC and 5hmC), left undigested (H 2 O), or treated with MspI (which cuts both and provides a positive control). The abundance of locus-specific DNA within each of these samples was estimated by qPCR and used to calculate the percentage of HpaII resistance. (B) Levels of 5hmC at OCT4 in hB cells before (0 hr), and 48 hr and 72 hr after fusion with mouse EGCs (black bars) or ESCs (white bars) are shown as the mean and SE of three to five independent experiments. (C) HpaII digestion analysis of OCT4 in hB lymphocytes before (0 hr) and 48 hr and 72 hr after fusion with EGCs (closed circles) or ESCs (open circles) are shown. Red bars mark the position of primer-amplified PCR products derived from the promoter (right) and downstream of the TSS (left), and values represent the mean and SE of three to five independent experiments. ∗∗ , p value

    Techniques Used: Real-time Polymerase Chain Reaction, Modification, Positive Control, Amplification, Polymerase Chain Reaction, Derivative Assay

    33) Product Images from "MicroRNA-494 is a master epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors"

    Article Title: MicroRNA-494 is a master epigenetic regulator of multiple invasion-suppressor microRNAs by targeting ten eleven translocation 1 in invasive human hepatocellular carcinoma tumors

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.27816

    miR-494 promotes HCC cell migration/invasion and suppresses multiple invasion-suppressor microRNAs by inhibiting DNA demethylation of their proximal CpG islands. (A, B, and C) (A) HepG2 cells, (B) Hep3B cells, or (C) SNU398 cells, transduced with miR-494 or the nontarget control vector, were subjected to cell migration and invasion assays. Migrated/invaded cells in fields were quantified and representative photographs are shown. Data are represented as mean ± standard deviation (SD) from four independent experiments. (D and E) qRT-PCR analysis of RNAs from HepG2 cells transduced with miR-494-expressing vector or the nontarget control vector for (D) indicated biomarkers of EMT and (E) indicated microRNAs. (F) Methylation-specific PCR analysis of the proximal CpG island regions of the indicated miRNA genes with gDNAs purified from HepG2 cells transduced with the miR-494 expression vector. PBGD served as the control. (G) DNA methylation status of the proximal CpG regions of the indicated miRNA genes in HepG2 cells transduced with either miR-494-expressing vector or the nontarget control vector. DNA methylation status was determined using methylation-sensitive/dependent restriction digestion followed by qRT-PCR analysis. Data are represented as mean ± SD from three independent experiments. (H) Restored expression of miR-200c upon adding DNA demethylating agent 5′-Aza in HepG2 cells transduced with the miR-494-expressing vector. Data are represented as mean ± SD from five independent experiments. (I) GlucMS-qPCR analysis of proximal CpG islands within the indicated miRNA gene’s upstream regions enriched for 5hmC in HepG2 cells transduced with the miR-494-expressing vector. Data are represented as mean ± SD from five independent experiments. ** P
    Figure Legend Snippet: miR-494 promotes HCC cell migration/invasion and suppresses multiple invasion-suppressor microRNAs by inhibiting DNA demethylation of their proximal CpG islands. (A, B, and C) (A) HepG2 cells, (B) Hep3B cells, or (C) SNU398 cells, transduced with miR-494 or the nontarget control vector, were subjected to cell migration and invasion assays. Migrated/invaded cells in fields were quantified and representative photographs are shown. Data are represented as mean ± standard deviation (SD) from four independent experiments. (D and E) qRT-PCR analysis of RNAs from HepG2 cells transduced with miR-494-expressing vector or the nontarget control vector for (D) indicated biomarkers of EMT and (E) indicated microRNAs. (F) Methylation-specific PCR analysis of the proximal CpG island regions of the indicated miRNA genes with gDNAs purified from HepG2 cells transduced with the miR-494 expression vector. PBGD served as the control. (G) DNA methylation status of the proximal CpG regions of the indicated miRNA genes in HepG2 cells transduced with either miR-494-expressing vector or the nontarget control vector. DNA methylation status was determined using methylation-sensitive/dependent restriction digestion followed by qRT-PCR analysis. Data are represented as mean ± SD from three independent experiments. (H) Restored expression of miR-200c upon adding DNA demethylating agent 5′-Aza in HepG2 cells transduced with the miR-494-expressing vector. Data are represented as mean ± SD from five independent experiments. (I) GlucMS-qPCR analysis of proximal CpG islands within the indicated miRNA gene’s upstream regions enriched for 5hmC in HepG2 cells transduced with the miR-494-expressing vector. Data are represented as mean ± SD from five independent experiments. ** P

    Techniques Used: Migration, Transduction, Plasmid Preparation, Standard Deviation, Quantitative RT-PCR, Expressing, Methylation, Polymerase Chain Reaction, Purification, DNA Methylation Assay, Real-time Polymerase Chain Reaction

    34) Product Images from "DNA methylation and differentiation: HOX genes in muscle cells"

    Article Title: DNA methylation and differentiation: HOX genes in muscle cells

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-6-25

    Cell type–specific differences in DNA methylation and transcription in the region containing HOXB5 , HOXB6 and HOXB-AS3 variant genes. (a) 42 MbMt-hypermethylated sites in a subregion of HOXB (chr17:46,665,998–46,684,371). (b) Chromatin segmentation state maps. (c) Strand-specific RNA-seq as in Figure 4 . The pink boxes indicate the RNA-seq evidence for HOXB-AS3 variant 3 as the predominant variant expressed in Mb. (d) RRBS data for two control Mb cell strains and Mt preparations derived from them, as well as two fetal lung fibroblast cell strains analyzed as technical duplicates. Arrows and highlighted subregions are described in the text.
    Figure Legend Snippet: Cell type–specific differences in DNA methylation and transcription in the region containing HOXB5 , HOXB6 and HOXB-AS3 variant genes. (a) 42 MbMt-hypermethylated sites in a subregion of HOXB (chr17:46,665,998–46,684,371). (b) Chromatin segmentation state maps. (c) Strand-specific RNA-seq as in Figure 4 . The pink boxes indicate the RNA-seq evidence for HOXB-AS3 variant 3 as the predominant variant expressed in Mb. (d) RRBS data for two control Mb cell strains and Mt preparations derived from them, as well as two fetal lung fibroblast cell strains analyzed as technical duplicates. Arrows and highlighted subregions are described in the text.

    Techniques Used: DNA Methylation Assay, Variant Assay, RNA Sequencing Assay, Derivative Assay

    Myogenic hypermethylation, enrichment in CpG islands and extensive myogenesis-associated transcription localized to the 151-kb HOXC cluster. (a) MyoD binding profiles show that inferred MYOD binding sites form a distant border on both sides of the HOXC cluster. MYOD binding sites were extrapolated and are depicted as in Figure 1 . The visualized chromosomal region from the UCSC Genome Browser for this figure is chr12:54,052,006–54,706,150 (654 kb). (b) 119 MbMt-hypermethylated sites and the distribution of CpG islands. (c) Layered RNA-seq track as in Figure 2 with additional layered tracks for H3K4me3, H3K4me1 and H3K27Ac by ChIP-seq (ENCODE/Broad Institute). (d) Chromatin state segmentation analysis as in Figure 1 . The pink-highlighted region is the HOXC gene cluster shown in Figure 2 .
    Figure Legend Snippet: Myogenic hypermethylation, enrichment in CpG islands and extensive myogenesis-associated transcription localized to the 151-kb HOXC cluster. (a) MyoD binding profiles show that inferred MYOD binding sites form a distant border on both sides of the HOXC cluster. MYOD binding sites were extrapolated and are depicted as in Figure 1 . The visualized chromosomal region from the UCSC Genome Browser for this figure is chr12:54,052,006–54,706,150 (654 kb). (b) 119 MbMt-hypermethylated sites and the distribution of CpG islands. (c) Layered RNA-seq track as in Figure 2 with additional layered tracks for H3K4me3, H3K4me1 and H3K27Ac by ChIP-seq (ENCODE/Broad Institute). (d) Chromatin state segmentation analysis as in Figure 1 . The pink-highlighted region is the HOXC gene cluster shown in Figure 2 .

    Techniques Used: Binding Assay, RNA Sequencing Assay, Chromatin Immunoprecipitation

    Myogenic hypermethylation in the central region of the HOXB gene cluster, which is preferentially transcribed in myogenic cells. (a) 88 MbMt-hypermethylated sites in the chr17:46,602,904–46,814,469 region. (b) Examples of RRBS data. (c) Strand-specific RNA-seq as in Figure 1 , except that the vertical viewing ranges were 1–10 for the plus strand was and 1–100 for the minus strand. (d) Chromatin state segmentation analysis. (e) The MyoD binding site track shows no C2C12-extrapolated MYOD sites in this region. Arrows, empty boxes and the triangle denote features mentioned in the text.
    Figure Legend Snippet: Myogenic hypermethylation in the central region of the HOXB gene cluster, which is preferentially transcribed in myogenic cells. (a) 88 MbMt-hypermethylated sites in the chr17:46,602,904–46,814,469 region. (b) Examples of RRBS data. (c) Strand-specific RNA-seq as in Figure 1 , except that the vertical viewing ranges were 1–10 for the plus strand was and 1–100 for the minus strand. (d) Chromatin state segmentation analysis. (e) The MyoD binding site track shows no C2C12-extrapolated MYOD sites in this region. Arrows, empty boxes and the triangle denote features mentioned in the text.

    Techniques Used: RNA Sequencing Assay, Binding Assay

    Peripheral myogenic hypermethylation and a central myogenic hypomethylated site in the HOXA gene cluster. (a) 187 MbMt-hypermethylated and 20 muscle-hypermethylated sites as well as one MbMt-hypomethylated site in the chr7:27,116,782–27,273,459 region. (b) Examples of RRBS data. (c) RNA-seq profiles as in Figure 1 . (d) Chromatin state segmentation analysis. (e) MyoD binding sites from C2C12.
    Figure Legend Snippet: Peripheral myogenic hypermethylation and a central myogenic hypomethylated site in the HOXA gene cluster. (a) 187 MbMt-hypermethylated and 20 muscle-hypermethylated sites as well as one MbMt-hypomethylated site in the chr7:27,116,782–27,273,459 region. (b) Examples of RRBS data. (c) RNA-seq profiles as in Figure 1 . (d) Chromatin state segmentation analysis. (e) MyoD binding sites from C2C12.

    Techniques Used: RNA Sequencing Assay, Binding Assay

    35) Product Images from "Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome"

    Article Title: Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome

    Journal: Genome Biology

    doi: 10.1186/gb-2012-13-10-r93

    5hmC profiling of mouse liver DNA . (a) An 11 kb promoter array region split into six indicated regions for epigenetic mapping analysis. (b) 5hmC and 5mC enrichment peaks in liver DNA map largely to intra-genic regions: left, distribution of all array probes; right, 5hmC and 5mC enrichment peaks. Chi 2 values indicate significance of the peak distributions compared to distribution of all array probes. (c) EpiMark qPCR of hmCpG (purple), 5mCpG (red) and non-modified CpG (green) levels over loci in control livers (n = 2). Percentage scores represent frequency of each CpG state over a single Msp I site. '5hmC +ve', 5hmC-positive regions; '5hmC -ve', 5hmC-negative regions. Error bars represent standard errors. (d) Box plot showing levels of 5hmC (purple) and 5mC (red) over 1 kb long enhancer and promoter regions. Asterisk denotes significant difference in signal levels ( P
    Figure Legend Snippet: 5hmC profiling of mouse liver DNA . (a) An 11 kb promoter array region split into six indicated regions for epigenetic mapping analysis. (b) 5hmC and 5mC enrichment peaks in liver DNA map largely to intra-genic regions: left, distribution of all array probes; right, 5hmC and 5mC enrichment peaks. Chi 2 values indicate significance of the peak distributions compared to distribution of all array probes. (c) EpiMark qPCR of hmCpG (purple), 5mCpG (red) and non-modified CpG (green) levels over loci in control livers (n = 2). Percentage scores represent frequency of each CpG state over a single Msp I site. '5hmC +ve', 5hmC-positive regions; '5hmC -ve', 5hmC-negative regions. Error bars represent standard errors. (d) Box plot showing levels of 5hmC (purple) and 5mC (red) over 1 kb long enhancer and promoter regions. Asterisk denotes significant difference in signal levels ( P

    Techniques Used: Real-time Polymerase Chain Reaction, Modification

    36) Product Images from "Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation"

    Article Title: Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation

    Journal: Epigenetics

    doi: 10.4161/epi.24280

    Figure 1. Schematic presentation of the HMST-Seq method. For (A) “C + mC” library, the genomic DNA was first glucosylated, and then digested with MspI. For (B) “C” library and (C) “C + mC + hmC”
    Figure Legend Snippet: Figure 1. Schematic presentation of the HMST-Seq method. For (A) “C + mC” library, the genomic DNA was first glucosylated, and then digested with MspI. For (B) “C” library and (C) “C + mC + hmC”

    Techniques Used:

    37) Product Images from "Effect of valproic acid on mitochondrial epigenetics"

    Article Title: Effect of valproic acid on mitochondrial epigenetics

    Journal: European journal of pharmacology

    doi: 10.1016/j.ejphar.2012.06.019

    Representation of the selected mouse mitochondrial DNA regions analyzed with the beta-glu-5hmC-sensitive restriction enzymes CviAII and CviQI for 5hmC modifications. The presentation is based on the Mus musculus mitochondrion, complete genome 16299 residue.
    Figure Legend Snippet: Representation of the selected mouse mitochondrial DNA regions analyzed with the beta-glu-5hmC-sensitive restriction enzymes CviAII and CviQI for 5hmC modifications. The presentation is based on the Mus musculus mitochondrion, complete genome 16299 residue.

    Techniques Used:

    Effect of VPA treatment on global 5mC and 5hmC content in mitochondrial DNA (time course). Mitochondrial samples were collected after 1 day (panels in row A) and 3 days (panels in row B) of treatment with 1 mM VPA (striped bars) and its vehicle (open
    Figure Legend Snippet: Effect of VPA treatment on global 5mC and 5hmC content in mitochondrial DNA (time course). Mitochondrial samples were collected after 1 day (panels in row A) and 3 days (panels in row B) of treatment with 1 mM VPA (striped bars) and its vehicle (open

    Techniques Used:

    Effect of VPA treatment on global 5mC and 5hmC content in nuclear DNA. Nuclear samples were collected after 1 day (panels in row A) and 3 days (panels in row B) of treatment with 1 mM VPA (striped bars) and its vehicle (open bars). ELISA assays were used
    Figure Legend Snippet: Effect of VPA treatment on global 5mC and 5hmC content in nuclear DNA. Nuclear samples were collected after 1 day (panels in row A) and 3 days (panels in row B) of treatment with 1 mM VPA (striped bars) and its vehicle (open bars). ELISA assays were used

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Effect of a 3-day VPA treatment on sequence-specific 5hmC content in mitochondrial DNA. Mitochondria were isolated and their DNA extracted after 3 days of treatment with 1 mM VPA (striped bars) and its vehicle (open bars). The content of 5hmC in the sequences
    Figure Legend Snippet: Effect of a 3-day VPA treatment on sequence-specific 5hmC content in mitochondrial DNA. Mitochondria were isolated and their DNA extracted after 3 days of treatment with 1 mM VPA (striped bars) and its vehicle (open bars). The content of 5hmC in the sequences

    Techniques Used: Sequencing, Isolation

    Effect of a 3-day treatment with a HDAC inhibitor MS-275 on sequence-specific 5hmC content in mitochondrial DNA. Mitochondria were isolated and their DNA extracted after 3 days of treatment with 5 µM MS-275 (striped bars) and its vehicle (open
    Figure Legend Snippet: Effect of a 3-day treatment with a HDAC inhibitor MS-275 on sequence-specific 5hmC content in mitochondrial DNA. Mitochondria were isolated and their DNA extracted after 3 days of treatment with 5 µM MS-275 (striped bars) and its vehicle (open

    Techniques Used: Mass Spectrometry, Sequencing, Isolation

    38) Product Images from "Genome-wide comparison of DNA hydroxymethylation in mouse embryonic stem cells and neural progenitor cells by a new comparative hMeDIP-seq method"

    Article Title: Genome-wide comparison of DNA hydroxymethylation in mouse embryonic stem cells and neural progenitor cells by a new comparative hMeDIP-seq method

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt091

    Differential Tet1/2/3 gene expression and global 5hmC levels in ESCs and NPCs. ( a ) RT-qPCR analysis of Tet1 , Tet2 and Tet3 mRNA levels in ESCs and NPCs (mean values ± SD, n = 3). ( b ) Immunofluorescence analysis of 5hmC levels in ESCs and NPCs. Bar: 50 μm. ( c ) Dot blot analysis of the global 5hmC levels in the gDNA of ESCs and ESC-derived NPCs. One hundred fifty nanogram gDNA for each dot. ( d ) The relative 5hmC signal of dot blot and comparative hMeDIP-seq in ESCs and NPCs. *The ratio of hMeDIP/Input reads numbers in ESCs was set as 100%. ( e ) Distribution of 5hmC in TSS ± 5 kb and gene body regions in ESCs. ( f ) Distribution of 5hmC in TSS ± 5 kb and gene body regions in NPCs.
    Figure Legend Snippet: Differential Tet1/2/3 gene expression and global 5hmC levels in ESCs and NPCs. ( a ) RT-qPCR analysis of Tet1 , Tet2 and Tet3 mRNA levels in ESCs and NPCs (mean values ± SD, n = 3). ( b ) Immunofluorescence analysis of 5hmC levels in ESCs and NPCs. Bar: 50 μm. ( c ) Dot blot analysis of the global 5hmC levels in the gDNA of ESCs and ESC-derived NPCs. One hundred fifty nanogram gDNA for each dot. ( d ) The relative 5hmC signal of dot blot and comparative hMeDIP-seq in ESCs and NPCs. *The ratio of hMeDIP/Input reads numbers in ESCs was set as 100%. ( e ) Distribution of 5hmC in TSS ± 5 kb and gene body regions in ESCs. ( f ) Distribution of 5hmC in TSS ± 5 kb and gene body regions in NPCs.

    Techniques Used: Expressing, Quantitative RT-PCR, Immunofluorescence, Dot Blot, Derivative Assay

    39) Product Images from "Biochemical Characterization of Recombinant ?-Glucosyltransferase and Analysis of Global 5-Hydroxymethylcytosine in Unique Genomes"

    Article Title: Biochemical Characterization of Recombinant ?-Glucosyltransferase and Analysis of Global 5-Hydroxymethylcytosine in Unique Genomes

    Journal: Biochemistry

    doi: 10.1021/bi2014739

    Substrate–velocity curves of recombinant β - GT. (A) Recombinant β-GT activity with UDP-[ 3 H]glucose substrate. Glucosylation reactions were conducted at UDP-[ 3 H]glucose substrate concentrations of 0, 2.5, 5, 10, 25, and 50 μM and fixed enzyme and 5-hmC DNA concentrations of 0.01 and 2.5 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m UDP-glucose values. (B) Recombinant β-GT activity with a 5-hmC DNA substrate. Glucosylation reactions were conducted with 5-hmC DNA substrate concentrations of 0, 0.125, 0.25, 0.5, 1, 3.6, and 7.2 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m 5-hmC values.
    Figure Legend Snippet: Substrate–velocity curves of recombinant β - GT. (A) Recombinant β-GT activity with UDP-[ 3 H]glucose substrate. Glucosylation reactions were conducted at UDP-[ 3 H]glucose substrate concentrations of 0, 2.5, 5, 10, 25, and 50 μM and fixed enzyme and 5-hmC DNA concentrations of 0.01 and 2.5 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m UDP-glucose values. (B) Recombinant β-GT activity with a 5-hmC DNA substrate. Glucosylation reactions were conducted with 5-hmC DNA substrate concentrations of 0, 0.125, 0.25, 0.5, 1, 3.6, and 7.2 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m 5-hmC values.

    Techniques Used: Recombinant, Activity Assay, Concentration Assay

    Linearity of glucosylation by recombinant β-GT. (A) Linearity of glucosylase reaction as a function of time with four different concentrations of UDP-[ 3 H]glucose: 50 (◇), 25 (▼), 10 (△), and 5 μM (■). All reaction mixtures contained 0.01 μM β-GT and 2.5 μM 5-hmC and were incubated at 25 °C. Twenty-five microliters of the reaction mixture was spotted on DE81 at 1 min time intervals and processed as described in Experimental Procedures . (B) Linearity of the glucosylase reaction as a function of recombinant β-GT enzyme concentration (0.00025–0.02 μM) with 50 (△) or 25 μM UDP-[ 3 H]glucose (■). Twenty-five microliters of the reaction mixture was spotted on DE81 after incubation with the appropriate concentration of the β-GT enzyme for 2 min.
    Figure Legend Snippet: Linearity of glucosylation by recombinant β-GT. (A) Linearity of glucosylase reaction as a function of time with four different concentrations of UDP-[ 3 H]glucose: 50 (◇), 25 (▼), 10 (△), and 5 μM (■). All reaction mixtures contained 0.01 μM β-GT and 2.5 μM 5-hmC and were incubated at 25 °C. Twenty-five microliters of the reaction mixture was spotted on DE81 at 1 min time intervals and processed as described in Experimental Procedures . (B) Linearity of the glucosylase reaction as a function of recombinant β-GT enzyme concentration (0.00025–0.02 μM) with 50 (△) or 25 μM UDP-[ 3 H]glucose (■). Twenty-five microliters of the reaction mixture was spotted on DE81 after incubation with the appropriate concentration of the β-GT enzyme for 2 min.

    Techniques Used: Recombinant, Incubation, Concentration Assay

    Global 5-hmC levels determined in the genomes of various tissues, and corresponding tumor samples, by recombinant β-GT. (A) Calibration curve using different amounts of 0, 0.5, 1, 2, 5, 10, and 20 μM 5-hmC with 5 units of recombinant β-GT and 25 μM UDP-[ 3 H]glucose in a total reaction volume of 25 μL. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured. The linear relationship between [ 3 H]glucose incorporation and the molarity of 5-hmC is 0.9997. (B) Glucosylation is utilized to measure global 5-hmC levels in matched normal and tumor genomic DNA samples, as described in Experimental Procedures . Note that all tumor samples have significantly lower levels of 5-hmC when compared to the matched normal sample ( p
    Figure Legend Snippet: Global 5-hmC levels determined in the genomes of various tissues, and corresponding tumor samples, by recombinant β-GT. (A) Calibration curve using different amounts of 0, 0.5, 1, 2, 5, 10, and 20 μM 5-hmC with 5 units of recombinant β-GT and 25 μM UDP-[ 3 H]glucose in a total reaction volume of 25 μL. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured. The linear relationship between [ 3 H]glucose incorporation and the molarity of 5-hmC is 0.9997. (B) Glucosylation is utilized to measure global 5-hmC levels in matched normal and tumor genomic DNA samples, as described in Experimental Procedures . Note that all tumor samples have significantly lower levels of 5-hmC when compared to the matched normal sample ( p

    Techniques Used: Recombinant

    Nonprocessive glucosylation catalyzed by recombinant β-GT. Incorporation of UDP-[ 3 H]glucose after the enzyme was preincubated on ice with 50 μM UDP-[ 3 H]glucose and 1.35 μM 5-hmC DNA. Three minutes after the start of the reaction the mixture was divided into two equal portions, one chased with nonbiotinylated 5-hmC DNA (▼) and the other with an equal volume of water (○) as described in Experimental Procedures . After the chase, both reaction mixtures were incubated at (25 °C) for 1 min and then monitored at 30 s intervals by processing 25 μL of the reaction mixture in duplicate. Measurements were obtained from streptavidin magnetic beads with captured glucosylated [ 3 H]-5-hmC.
    Figure Legend Snippet: Nonprocessive glucosylation catalyzed by recombinant β-GT. Incorporation of UDP-[ 3 H]glucose after the enzyme was preincubated on ice with 50 μM UDP-[ 3 H]glucose and 1.35 μM 5-hmC DNA. Three minutes after the start of the reaction the mixture was divided into two equal portions, one chased with nonbiotinylated 5-hmC DNA (▼) and the other with an equal volume of water (○) as described in Experimental Procedures . After the chase, both reaction mixtures were incubated at (25 °C) for 1 min and then monitored at 30 s intervals by processing 25 μL of the reaction mixture in duplicate. Measurements were obtained from streptavidin magnetic beads with captured glucosylated [ 3 H]-5-hmC.

    Techniques Used: Recombinant, Incubation, Magnetic Beads

    Glucosylation of different 5-hmC-containing substrates by recombinant β-GT. (A) Recombinant β-GT activity with 100 bp double-stranded DNA substrates containing 2 (●), 6 (△), 12 (■), or 24 cytosines (○) that are hydroxymethylated. Glucosylation reactions were performed at 5-hmC concentrations of 0, 0.125, 0.25, 0.5, 1, and 2.5 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively, in a total volume of 25 μL. (B) End point analysis of recombinant β-GT activity with 55 bp double-stranded substrates containing hemi-5-hmC pair A, hemi-5-hmC pair B, or symmetrical 5-hmC. Glucosylation reactions were performed with 1 μM 5-hmC, 50 μM UDP-[ 3 H]glucose, and 5 units of recombinant β-GT in a 25 μL reaction mixture for 2 h. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured.
    Figure Legend Snippet: Glucosylation of different 5-hmC-containing substrates by recombinant β-GT. (A) Recombinant β-GT activity with 100 bp double-stranded DNA substrates containing 2 (●), 6 (△), 12 (■), or 24 cytosines (○) that are hydroxymethylated. Glucosylation reactions were performed at 5-hmC concentrations of 0, 0.125, 0.25, 0.5, 1, and 2.5 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively, in a total volume of 25 μL. (B) End point analysis of recombinant β-GT activity with 55 bp double-stranded substrates containing hemi-5-hmC pair A, hemi-5-hmC pair B, or symmetrical 5-hmC. Glucosylation reactions were performed with 1 μM 5-hmC, 50 μM UDP-[ 3 H]glucose, and 5 units of recombinant β-GT in a 25 μL reaction mixture for 2 h. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured.

    Techniques Used: Recombinant, Activity Assay

    Purity and initial characterization of the β-GT enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by MfeI. A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.
    Figure Legend Snippet: Purity and initial characterization of the β-GT enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by MfeI. A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.

    Techniques Used: Staining, SDS Page, Recombinant

    Reaction mechanism of recombinant β-GT and double-reciprocal plots of the initial velocity vs substrate concentration. (A) Scheme detailing the turnover of recombinant β-GT enzyme in the presence or absence of product inhibitors. The substrate 5-hmC or UDP-glucose is S. The product glucosylated 5-hmC is I. The inhibitor constant is K i . The binary inhibitor constant is α K i . The degree of modification the first substrate exerts on binding of the second substrate is α. (B) Double-reciprocal plots for fixed 5-hmC substrate and variable UDP concentrations of 0 (■), 2 (○), 8 (◆), 12 (△), and 16 μM (▼). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of K m UDP-glucose vs UDP was used to calculate the K i value of UDP as a competitive inhibitor with respect to the formation of a β-GT–UDP-glucose complex. (C) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable UDP concentrations of 0 (■), 20 (△), 30 (▼), and 40 μM (◊). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC]. The inset replot of slope vs UDP was used to calculate the α K i of UDP as a mixed inhibitor with respect to the formation of a β-GT–5-hmC complex. (D) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable glucosylated 5-hmC concentrations of 0 (■), 1 (△), 2.5 (▼), and 5 (○). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC DNA]. The inset replot of K m 5-hmC vs 5-ghmC was utilized to calculate the K i value of 5-ghmC as a competitive inhibitor with respect to the formation of a β-GT–5-hmC complex. (E) Double-reciprocal plots for 5-hmC concentration and variable glucosylated 5-hmC concentrations of 0 (○), 1 (■), 2.5 (▽), and 5 μM (◆). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of slope vs 5-ghmC was used to calculate the α K i of 5-ghmC as a mixed inhibitor with respect to formation of a β-GT–UDP-glucose complex.
    Figure Legend Snippet: Reaction mechanism of recombinant β-GT and double-reciprocal plots of the initial velocity vs substrate concentration. (A) Scheme detailing the turnover of recombinant β-GT enzyme in the presence or absence of product inhibitors. The substrate 5-hmC or UDP-glucose is S. The product glucosylated 5-hmC is I. The inhibitor constant is K i . The binary inhibitor constant is α K i . The degree of modification the first substrate exerts on binding of the second substrate is α. (B) Double-reciprocal plots for fixed 5-hmC substrate and variable UDP concentrations of 0 (■), 2 (○), 8 (◆), 12 (△), and 16 μM (▼). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of K m UDP-glucose vs UDP was used to calculate the K i value of UDP as a competitive inhibitor with respect to the formation of a β-GT–UDP-glucose complex. (C) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable UDP concentrations of 0 (■), 20 (△), 30 (▼), and 40 μM (◊). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC]. The inset replot of slope vs UDP was used to calculate the α K i of UDP as a mixed inhibitor with respect to the formation of a β-GT–5-hmC complex. (D) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable glucosylated 5-hmC concentrations of 0 (■), 1 (△), 2.5 (▼), and 5 (○). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC DNA]. The inset replot of K m 5-hmC vs 5-ghmC was utilized to calculate the K i value of 5-ghmC as a competitive inhibitor with respect to the formation of a β-GT–5-hmC complex. (E) Double-reciprocal plots for 5-hmC concentration and variable glucosylated 5-hmC concentrations of 0 (○), 1 (■), 2.5 (▽), and 5 μM (◆). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of slope vs 5-ghmC was used to calculate the α K i of 5-ghmC as a mixed inhibitor with respect to formation of a β-GT–UDP-glucose complex.

    Techniques Used: Recombinant, Concentration Assay, Modification, Binding Assay

    40) Product Images from "Biochemical Characterization of Recombinant ?-Glucosyltransferase and Analysis of Global 5-Hydroxymethylcytosine in Unique Genomes"

    Article Title: Biochemical Characterization of Recombinant ?-Glucosyltransferase and Analysis of Global 5-Hydroxymethylcytosine in Unique Genomes

    Journal: Biochemistry

    doi: 10.1021/bi2014739

    Substrate–velocity curves of recombinant β - GT. (A) Recombinant β-GT activity with UDP-[ 3 H]glucose substrate. Glucosylation reactions were conducted at UDP-[ 3 H]glucose substrate concentrations of 0, 2.5, 5, 10, 25, and 50 μM and fixed enzyme and 5-hmC DNA concentrations of 0.01 and 2.5 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m UDP-glucose values. (B) Recombinant β-GT activity with a 5-hmC DNA substrate. Glucosylation reactions were conducted with 5-hmC DNA substrate concentrations of 0, 0.125, 0.25, 0.5, 1, 3.6, and 7.2 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m 5-hmC values.
    Figure Legend Snippet: Substrate–velocity curves of recombinant β - GT. (A) Recombinant β-GT activity with UDP-[ 3 H]glucose substrate. Glucosylation reactions were conducted at UDP-[ 3 H]glucose substrate concentrations of 0, 2.5, 5, 10, 25, and 50 μM and fixed enzyme and 5-hmC DNA concentrations of 0.01 and 2.5 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m UDP-glucose values. (B) Recombinant β-GT activity with a 5-hmC DNA substrate. Glucosylation reactions were conducted with 5-hmC DNA substrate concentrations of 0, 0.125, 0.25, 0.5, 1, 3.6, and 7.2 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively. Product formation is plotted vs substrate concentration, and nonlinear regression was performed to determine K m 5-hmC values.

    Techniques Used: Recombinant, Activity Assay, Concentration Assay

    Linearity of glucosylation by recombinant β-GT. (A) Linearity of glucosylase reaction as a function of time with four different concentrations of UDP-[ 3 H]glucose: 50 (◇), 25 (▼), 10 (△), and 5 μM (■). All reaction mixtures contained 0.01 μM β-GT and 2.5 μM 5-hmC and were incubated at 25 °C. Twenty-five microliters of the reaction mixture was spotted on DE81 at 1 min time intervals and processed as described in Experimental Procedures . (B) Linearity of the glucosylase reaction as a function of recombinant β-GT enzyme concentration (0.00025–0.02 μM) with 50 (△) or 25 μM UDP-[ 3 H]glucose (■). Twenty-five microliters of the reaction mixture was spotted on DE81 after incubation with the appropriate concentration of the β-GT enzyme for 2 min.
    Figure Legend Snippet: Linearity of glucosylation by recombinant β-GT. (A) Linearity of glucosylase reaction as a function of time with four different concentrations of UDP-[ 3 H]glucose: 50 (◇), 25 (▼), 10 (△), and 5 μM (■). All reaction mixtures contained 0.01 μM β-GT and 2.5 μM 5-hmC and were incubated at 25 °C. Twenty-five microliters of the reaction mixture was spotted on DE81 at 1 min time intervals and processed as described in Experimental Procedures . (B) Linearity of the glucosylase reaction as a function of recombinant β-GT enzyme concentration (0.00025–0.02 μM) with 50 (△) or 25 μM UDP-[ 3 H]glucose (■). Twenty-five microliters of the reaction mixture was spotted on DE81 after incubation with the appropriate concentration of the β-GT enzyme for 2 min.

    Techniques Used: Recombinant, Incubation, Concentration Assay

    Global 5-hmC levels determined in the genomes of various tissues, and corresponding tumor samples, by recombinant β-GT. (A) Calibration curve using different amounts of 0, 0.5, 1, 2, 5, 10, and 20 μM 5-hmC with 5 units of recombinant β-GT and 25 μM UDP-[ 3 H]glucose in a total reaction volume of 25 μL. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured. The linear relationship between [ 3 H]glucose incorporation and the molarity of 5-hmC is 0.9997. (B) Glucosylation is utilized to measure global 5-hmC levels in matched normal and tumor genomic DNA samples, as described in Experimental Procedures . Note that all tumor samples have significantly lower levels of 5-hmC when compared to the matched normal sample ( p
    Figure Legend Snippet: Global 5-hmC levels determined in the genomes of various tissues, and corresponding tumor samples, by recombinant β-GT. (A) Calibration curve using different amounts of 0, 0.5, 1, 2, 5, 10, and 20 μM 5-hmC with 5 units of recombinant β-GT and 25 μM UDP-[ 3 H]glucose in a total reaction volume of 25 μL. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured. The linear relationship between [ 3 H]glucose incorporation and the molarity of 5-hmC is 0.9997. (B) Glucosylation is utilized to measure global 5-hmC levels in matched normal and tumor genomic DNA samples, as described in Experimental Procedures . Note that all tumor samples have significantly lower levels of 5-hmC when compared to the matched normal sample ( p

    Techniques Used: Recombinant

    Nonprocessive glucosylation catalyzed by recombinant β-GT. Incorporation of UDP-[ 3 H]glucose after the enzyme was preincubated on ice with 50 μM UDP-[ 3 H]glucose and 1.35 μM 5-hmC DNA. Three minutes after the start of the reaction the mixture was divided into two equal portions, one chased with nonbiotinylated 5-hmC DNA (▼) and the other with an equal volume of water (○) as described in Experimental Procedures . After the chase, both reaction mixtures were incubated at (25 °C) for 1 min and then monitored at 30 s intervals by processing 25 μL of the reaction mixture in duplicate. Measurements were obtained from streptavidin magnetic beads with captured glucosylated [ 3 H]-5-hmC.
    Figure Legend Snippet: Nonprocessive glucosylation catalyzed by recombinant β-GT. Incorporation of UDP-[ 3 H]glucose after the enzyme was preincubated on ice with 50 μM UDP-[ 3 H]glucose and 1.35 μM 5-hmC DNA. Three minutes after the start of the reaction the mixture was divided into two equal portions, one chased with nonbiotinylated 5-hmC DNA (▼) and the other with an equal volume of water (○) as described in Experimental Procedures . After the chase, both reaction mixtures were incubated at (25 °C) for 1 min and then monitored at 30 s intervals by processing 25 μL of the reaction mixture in duplicate. Measurements were obtained from streptavidin magnetic beads with captured glucosylated [ 3 H]-5-hmC.

    Techniques Used: Recombinant, Incubation, Magnetic Beads

    Glucosylation of different 5-hmC-containing substrates by recombinant β-GT. (A) Recombinant β-GT activity with 100 bp double-stranded DNA substrates containing 2 (●), 6 (△), 12 (■), or 24 cytosines (○) that are hydroxymethylated. Glucosylation reactions were performed at 5-hmC concentrations of 0, 0.125, 0.25, 0.5, 1, and 2.5 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively, in a total volume of 25 μL. (B) End point analysis of recombinant β-GT activity with 55 bp double-stranded substrates containing hemi-5-hmC pair A, hemi-5-hmC pair B, or symmetrical 5-hmC. Glucosylation reactions were performed with 1 μM 5-hmC, 50 μM UDP-[ 3 H]glucose, and 5 units of recombinant β-GT in a 25 μL reaction mixture for 2 h. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured.
    Figure Legend Snippet: Glucosylation of different 5-hmC-containing substrates by recombinant β-GT. (A) Recombinant β-GT activity with 100 bp double-stranded DNA substrates containing 2 (●), 6 (△), 12 (■), or 24 cytosines (○) that are hydroxymethylated. Glucosylation reactions were performed at 5-hmC concentrations of 0, 0.125, 0.25, 0.5, 1, and 2.5 μM and fixed enzyme and UDP-[ 3 H]glucose concentrations of 0.01 and 50 μM, respectively, in a total volume of 25 μL. (B) End point analysis of recombinant β-GT activity with 55 bp double-stranded substrates containing hemi-5-hmC pair A, hemi-5-hmC pair B, or symmetrical 5-hmC. Glucosylation reactions were performed with 1 μM 5-hmC, 50 μM UDP-[ 3 H]glucose, and 5 units of recombinant β-GT in a 25 μL reaction mixture for 2 h. The reaction mixture was spotted in duplicate on DE81 membranes, and glucosyl group incorporation was measured.

    Techniques Used: Recombinant, Activity Assay

    Purity and initial characterization of the β-GT enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by MfeI. A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.
    Figure Legend Snippet: Purity and initial characterization of the β-GT enzyme. (A) Coomassie blue-stained SDS–PAGE gel showing 8 μg (80 units) of recombinant β-GT enzyme. (B) Glucosylation of 5-hmC DNA (T4 phage gt −/– DNA) with recombinant β-GT protects it from cleavage by MfeI. A time course of the glucosylation reaction followed by MfeI digestion is shown. The arrow indicates T4 phage gt −/– DNA.

    Techniques Used: Staining, SDS Page, Recombinant

    Reaction mechanism of recombinant β-GT and double-reciprocal plots of the initial velocity vs substrate concentration. (A) Scheme detailing the turnover of recombinant β-GT enzyme in the presence or absence of product inhibitors. The substrate 5-hmC or UDP-glucose is S. The product glucosylated 5-hmC is I. The inhibitor constant is K i . The binary inhibitor constant is α K i . The degree of modification the first substrate exerts on binding of the second substrate is α. (B) Double-reciprocal plots for fixed 5-hmC substrate and variable UDP concentrations of 0 (■), 2 (○), 8 (◆), 12 (△), and 16 μM (▼). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of K m UDP-glucose vs UDP was used to calculate the K i value of UDP as a competitive inhibitor with respect to the formation of a β-GT–UDP-glucose complex. (C) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable UDP concentrations of 0 (■), 20 (△), 30 (▼), and 40 μM (◊). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC]. The inset replot of slope vs UDP was used to calculate the α K i of UDP as a mixed inhibitor with respect to the formation of a β-GT–5-hmC complex. (D) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable glucosylated 5-hmC concentrations of 0 (■), 1 (△), 2.5 (▼), and 5 (○). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC DNA]. The inset replot of K m 5-hmC vs 5-ghmC was utilized to calculate the K i value of 5-ghmC as a competitive inhibitor with respect to the formation of a β-GT–5-hmC complex. (E) Double-reciprocal plots for 5-hmC concentration and variable glucosylated 5-hmC concentrations of 0 (○), 1 (■), 2.5 (▽), and 5 μM (◆). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of slope vs 5-ghmC was used to calculate the α K i of 5-ghmC as a mixed inhibitor with respect to formation of a β-GT–UDP-glucose complex.
    Figure Legend Snippet: Reaction mechanism of recombinant β-GT and double-reciprocal plots of the initial velocity vs substrate concentration. (A) Scheme detailing the turnover of recombinant β-GT enzyme in the presence or absence of product inhibitors. The substrate 5-hmC or UDP-glucose is S. The product glucosylated 5-hmC is I. The inhibitor constant is K i . The binary inhibitor constant is α K i . The degree of modification the first substrate exerts on binding of the second substrate is α. (B) Double-reciprocal plots for fixed 5-hmC substrate and variable UDP concentrations of 0 (■), 2 (○), 8 (◆), 12 (△), and 16 μM (▼). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of K m UDP-glucose vs UDP was used to calculate the K i value of UDP as a competitive inhibitor with respect to the formation of a β-GT–UDP-glucose complex. (C) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable UDP concentrations of 0 (■), 20 (△), 30 (▼), and 40 μM (◊). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC]. The inset replot of slope vs UDP was used to calculate the α K i of UDP as a mixed inhibitor with respect to the formation of a β-GT–5-hmC complex. (D) Double-reciprocal plots for fixed UDP-[ 3 H]glucose substrate and variable glucosylated 5-hmC concentrations of 0 (■), 1 (△), 2.5 (▼), and 5 (○). Linear regression was performed, and 1/ v was plotted vs 1/[5-hmC DNA]. The inset replot of K m 5-hmC vs 5-ghmC was utilized to calculate the K i value of 5-ghmC as a competitive inhibitor with respect to the formation of a β-GT–5-hmC complex. (E) Double-reciprocal plots for 5-hmC concentration and variable glucosylated 5-hmC concentrations of 0 (○), 1 (■), 2.5 (▽), and 5 μM (◆). Linear regression was performed, and 1/ v was plotted vs 1/[UDP-[ 3 H]glucose]. The inset replot of slope vs 5-ghmC was used to calculate the α K i of 5-ghmC as a mixed inhibitor with respect to formation of a β-GT–UDP-glucose complex.

    Techniques Used: Recombinant, Concentration Assay, Modification, Binding Assay

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome
    Article Snippet: .. To quantify the absolute levels of both 5hmC as well as 5mC over these regions, we used the EpiMark™ 5hmC and 5-mC Analysis Kit (New England BioLabs) followed by qPCR (Figure ; Additional file ; see Materials and methods). ..

    Article Title: Single base resolution analysis of 5-hydroxymethylcytosine in 188 human genes: implications for hepatic gene expression
    Article Snippet: .. Validation of hmC values by qPCR Validation of BS and hmC calls at selected CCGG sites was done by EpiMark 5-hmC and 5-mC Analysis Kit (New England Biolabs, MA, USA). .. Three aliquots per sample were processed from 1–1.5 μg of input DNA: (i) fully untreated (positive control); (ii) βGT/MspI-treated (hmC signal) and (iii) MspI-treated (negative control).

    Quantitation Assay:

    Article Title: Targeted TET oxidase activity through methyl‐CpG‐binding domain extensively suppresses cancer cell proliferation
    Article Snippet: .. Quantitation of 5‐mC and 5‐hmC EpiMark 5‐hmC and 5‐mC Analysis Kit (New England Biolabs, Ipswich, MA, USA) was used to quantitate the amounts of C, 5‐mC, and 5‐hmC in the MAL promoter region. ..

    DNA Sequencing:

    Article Title: Stable 5-hydroxymethylcytosine (5hmC) acquisition marks gene activation during chondrogenic differentiation
    Article Snippet: .. Validation of the enriched DNA sequencing results was performed using the EpiMark 5hmC and 5mC Analysis Kit (NEB) as per suppliers’ protocol. .. The EpiMark treated DNA was subjected to quantitative PCR using site-specific primers and the percentage of 5hmC was calculated using the EpiMark comparative Ct method ( , ).

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    New England Biolabs t4 β glucosyltransferase β gt
    Validation of MspI and HpaII isoschizomer- and <t>β-glucosyltransferase-mediated</t> 5-hmC detection and quantitation. A , shown is unmethylated, methylated, and hydroxymethylated duplex DNAs with centrally located CCGG ( boxed ), where the internal C is either unmethylated C or 5-mC or 5-hmC (shown in gray ). B , shown is validation for locus-specific 5-hmC detection in a fixed amount of pre-mixed DNAs, as shown in A , using MspI in the presence of <t>β-GT</t> and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected 5-hmC is plotted. C , shown is similar validation for locus-specific methylcytosine ( 5-mC + 5-hmC ) detection in fixed amount of pre-mixed DNAs, as shown in A , using HpaII in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected total methyl cytosine ( 5-mC + 5-hmC ) is plotted.
    T4 β Glucosyltransferase β Gt, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 β glucosyltransferase β gt treatment
    Synergistic target gene activation by GADD45a-TET1 is accompanied by increase in hmC and reduction of fC/caC in promoter CpGs. (A, B) TCEAL7 , DHRS2 , MAGEB2 expression in HEK293T cells upon transfection of empty vector (Ctrl, control), GADD45a (G45a) alone or with increasing doses of TET1, catalytic domain only (TET1 CD ), or catalytically inactive TET1 (TET CI ) as indicated. Relative expression was monitored by qPCR. Bar graphs represent the mean of n =4 (A) or n =3 (B) experiments with error bars as±SD. (C, D) Kinetics of hmC (C) and fC/caC (D) level changes in the TCEAL7 locus upon GADD45a and TET expression. HEK293T cells were transfected with empty vector (Ctrl) or GADD45a (G45a) or TET1 as indicated. Genomic DNA was harvested 14 h or 24 h after transfection. hmC and fC/caC were analyzed at positions −2648, −78, +34 and +457 relative to the transcription start site (TSS). Analysis was by modification-sensitive qPCR following MspI restriction on <t>T4</t> <t>β-glucosyltransferase</t> (β-GT) treated or control treated plasmid DNA. % of MspI resistance following β-GT treatment is displayed as % hmC. % of MspI resistance in control treated DNA (−β-GT) is displayed as % fC/caC. Bar graphs represent the mean of biological triplicates ( n =3) with error bars as±SD. p -Values: (*) p
    T4 β Glucosyltransferase β Gt Treatment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 β glucosyltransferase βgt
    Synergistic target gene activation by GADD45a-TET1 is accompanied by increase in hmC and reduction of fC/caC in promoter CpGs. (A, B) TCEAL7 , DHRS2 , MAGEB2 expression in HEK293T cells upon transfection of empty vector (Ctrl, control), GADD45a (G45a) alone or with increasing doses of TET1, catalytic domain only (TET1 CD ), or catalytically inactive TET1 (TET CI ) as indicated. Relative expression was monitored by qPCR. Bar graphs represent the mean of n =4 (A) or n =3 (B) experiments with error bars as±SD. (C, D) Kinetics of hmC (C) and fC/caC (D) level changes in the TCEAL7 locus upon GADD45a and TET expression. HEK293T cells were transfected with empty vector (Ctrl) or GADD45a (G45a) or TET1 as indicated. Genomic DNA was harvested 14 h or 24 h after transfection. hmC and fC/caC were analyzed at positions −2648, −78, +34 and +457 relative to the transcription start site (TSS). Analysis was by modification-sensitive qPCR following MspI restriction on <t>T4</t> <t>β-glucosyltransferase</t> (β-GT) treated or control treated plasmid DNA. % of MspI resistance following β-GT treatment is displayed as % hmC. % of MspI resistance in control treated DNA (−β-GT) is displayed as % fC/caC. Bar graphs represent the mean of biological triplicates ( n =3) with error bars as±SD. p -Values: (*) p
    T4 β Glucosyltransferase βgt, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Validation of MspI and HpaII isoschizomer- and β-glucosyltransferase-mediated 5-hmC detection and quantitation. A , shown is unmethylated, methylated, and hydroxymethylated duplex DNAs with centrally located CCGG ( boxed ), where the internal C is either unmethylated C or 5-mC or 5-hmC (shown in gray ). B , shown is validation for locus-specific 5-hmC detection in a fixed amount of pre-mixed DNAs, as shown in A , using MspI in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected 5-hmC is plotted. C , shown is similar validation for locus-specific methylcytosine ( 5-mC + 5-hmC ) detection in fixed amount of pre-mixed DNAs, as shown in A , using HpaII in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected total methyl cytosine ( 5-mC + 5-hmC ) is plotted.

    Journal: The Journal of Biological Chemistry

    Article Title: Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes *

    doi: 10.1074/jbc.M110.217083

    Figure Lengend Snippet: Validation of MspI and HpaII isoschizomer- and β-glucosyltransferase-mediated 5-hmC detection and quantitation. A , shown is unmethylated, methylated, and hydroxymethylated duplex DNAs with centrally located CCGG ( boxed ), where the internal C is either unmethylated C or 5-mC or 5-hmC (shown in gray ). B , shown is validation for locus-specific 5-hmC detection in a fixed amount of pre-mixed DNAs, as shown in A , using MspI in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected 5-hmC is plotted. C , shown is similar validation for locus-specific methylcytosine ( 5-mC + 5-hmC ) detection in fixed amount of pre-mixed DNAs, as shown in A , using HpaII in the presence of β-GT and cofactor UDP-Glc. The reaction products were subjected to qPCR, and the ratio between observed and expected total methyl cytosine ( 5-mC + 5-hmC ) is plotted.

    Article Snippet: Glucosylation of 5-hmC-containing Oligonucleotide Substrates with T4 β-Glucosyltransferase The hmC residues within the MspI site were glucosylated by incubating 200 pmol of DNA substrates with 1 μl (10 units) of T4 β-glucosyltransferase (β-GT) (NEB) for 1 h at 37 °C in a total 50-μl reaction containing 1× NEBuffer 4 supplemented with 0.1 mm UDP-Glc.

    Techniques: Quantitation Assay, Methylation, Gas Chromatography, Real-time Polymerase Chain Reaction

    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Journal: mBio

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    doi: 10.1128/mBio.00648-15

    Figure Lengend Snippet: Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Article Snippet: One microgram of T4(C), T4(HMC), or T4(glc-HMC) was digested with AluI (R0137s; NEB), MspJI (R0661S; NEB), or T4 phage β-glucosyltransferase (M0357S; NEB) in accordance with NEB-specified protocols.

    Techniques: Modification, Mobility Shift, Sequencing, Methylation

    Synergistic target gene activation by GADD45a-TET1 is accompanied by increase in hmC and reduction of fC/caC in promoter CpGs. (A, B) TCEAL7 , DHRS2 , MAGEB2 expression in HEK293T cells upon transfection of empty vector (Ctrl, control), GADD45a (G45a) alone or with increasing doses of TET1, catalytic domain only (TET1 CD ), or catalytically inactive TET1 (TET CI ) as indicated. Relative expression was monitored by qPCR. Bar graphs represent the mean of n =4 (A) or n =3 (B) experiments with error bars as±SD. (C, D) Kinetics of hmC (C) and fC/caC (D) level changes in the TCEAL7 locus upon GADD45a and TET expression. HEK293T cells were transfected with empty vector (Ctrl) or GADD45a (G45a) or TET1 as indicated. Genomic DNA was harvested 14 h or 24 h after transfection. hmC and fC/caC were analyzed at positions −2648, −78, +34 and +457 relative to the transcription start site (TSS). Analysis was by modification-sensitive qPCR following MspI restriction on T4 β-glucosyltransferase (β-GT) treated or control treated plasmid DNA. % of MspI resistance following β-GT treatment is displayed as % hmC. % of MspI resistance in control treated DNA (−β-GT) is displayed as % fC/caC. Bar graphs represent the mean of biological triplicates ( n =3) with error bars as±SD. p -Values: (*) p

    Journal: Differentiation; Research in Biological Diversity

    Article Title: GADD45a physically and functionally interacts with TET1

    doi: 10.1016/j.diff.2015.10.003

    Figure Lengend Snippet: Synergistic target gene activation by GADD45a-TET1 is accompanied by increase in hmC and reduction of fC/caC in promoter CpGs. (A, B) TCEAL7 , DHRS2 , MAGEB2 expression in HEK293T cells upon transfection of empty vector (Ctrl, control), GADD45a (G45a) alone or with increasing doses of TET1, catalytic domain only (TET1 CD ), or catalytically inactive TET1 (TET CI ) as indicated. Relative expression was monitored by qPCR. Bar graphs represent the mean of n =4 (A) or n =3 (B) experiments with error bars as±SD. (C, D) Kinetics of hmC (C) and fC/caC (D) level changes in the TCEAL7 locus upon GADD45a and TET expression. HEK293T cells were transfected with empty vector (Ctrl) or GADD45a (G45a) or TET1 as indicated. Genomic DNA was harvested 14 h or 24 h after transfection. hmC and fC/caC were analyzed at positions −2648, −78, +34 and +457 relative to the transcription start site (TSS). Analysis was by modification-sensitive qPCR following MspI restriction on T4 β-glucosyltransferase (β-GT) treated or control treated plasmid DNA. % of MspI resistance following β-GT treatment is displayed as % hmC. % of MspI resistance in control treated DNA (−β-GT) is displayed as % fC/caC. Bar graphs represent the mean of biological triplicates ( n =3) with error bars as±SD. p -Values: (*) p

    Article Snippet: T4 β-glucosyltransferase (β-GT) treatment was performed according to manufacturer's instructions (NEB EpiMark Kit).

    Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Modification

    GADD45a promotes TET1-mediated mC oxidation and reporter DNA demethylation. (A–G) Methylation analysis of in vitro methylated oct4TK-GFP reporter by methylation sensitive PCR. (A) HEK293T cells were transfected with oct4TK-GFP along with empty vector (Ctrl), GADD45a (G45a) or TET1 expression constructs as indicated. Plasmid DNA was recovered 48 h after transfection and subjected to HpaII restriction digest and qPCR. HpaII resistance reflecting the fraction of modified C is displayed. Ctrl, control; TET1 CI , TET1 catalytically inactive mutant. Bar graphs represent the mean of biological triplicates ( n =3) with error bars±SD. (B–D) Kinetics of mC and its oxidized derivatives during oct4TK-GFP reporter demethylation analyzed by modification-sensitive qPCR. Cells were treated as in (A) and plasmid DNA was recovered at indicated time points after transfection. mC and its oxidized derivatives were analyzed using qPCR following HpaII or MspI restriction digest on T4 β-glucosyltransferase (β-GT) treated or control treated plasmid DNA. In (B), % modified cytosine (mC+hmC+fC+caC) is displayed as % HpaII resistance. (C) and (D) display % hmC and % fC/caC determined by MspI resistance following β-GT treatment and MspI resistance in control treated DNA (without β-GT), respectively. Error bars indicate±SD ( n =3). (E, F) HEK293T cells were treated as in (A), but pre-transfected with the indicated siRNAs 24 h before DNA transfection. HpaII resistance reflecting the fraction of modified C is displayed. (G) HEK293T cells were transfected with oct4TK-GFP without any effector protein with the indicated siRNAs, whereby “mock” represents transfection reagent only. oct4TK-GFP plasmid was recovered 72 h after siRNA and 48 h after oct4TK-GFP transfection. HpaII cleavage is displayed as % demethylation, reflecting formation of unmodified C. Bar graphs represent the mean of biological triplicates ( n =3) with error bars±SD. p -Values: (*) p

    Journal: Differentiation; Research in Biological Diversity

    Article Title: GADD45a physically and functionally interacts with TET1

    doi: 10.1016/j.diff.2015.10.003

    Figure Lengend Snippet: GADD45a promotes TET1-mediated mC oxidation and reporter DNA demethylation. (A–G) Methylation analysis of in vitro methylated oct4TK-GFP reporter by methylation sensitive PCR. (A) HEK293T cells were transfected with oct4TK-GFP along with empty vector (Ctrl), GADD45a (G45a) or TET1 expression constructs as indicated. Plasmid DNA was recovered 48 h after transfection and subjected to HpaII restriction digest and qPCR. HpaII resistance reflecting the fraction of modified C is displayed. Ctrl, control; TET1 CI , TET1 catalytically inactive mutant. Bar graphs represent the mean of biological triplicates ( n =3) with error bars±SD. (B–D) Kinetics of mC and its oxidized derivatives during oct4TK-GFP reporter demethylation analyzed by modification-sensitive qPCR. Cells were treated as in (A) and plasmid DNA was recovered at indicated time points after transfection. mC and its oxidized derivatives were analyzed using qPCR following HpaII or MspI restriction digest on T4 β-glucosyltransferase (β-GT) treated or control treated plasmid DNA. In (B), % modified cytosine (mC+hmC+fC+caC) is displayed as % HpaII resistance. (C) and (D) display % hmC and % fC/caC determined by MspI resistance following β-GT treatment and MspI resistance in control treated DNA (without β-GT), respectively. Error bars indicate±SD ( n =3). (E, F) HEK293T cells were treated as in (A), but pre-transfected with the indicated siRNAs 24 h before DNA transfection. HpaII resistance reflecting the fraction of modified C is displayed. (G) HEK293T cells were transfected with oct4TK-GFP without any effector protein with the indicated siRNAs, whereby “mock” represents transfection reagent only. oct4TK-GFP plasmid was recovered 72 h after siRNA and 48 h after oct4TK-GFP transfection. HpaII cleavage is displayed as % demethylation, reflecting formation of unmodified C. Bar graphs represent the mean of biological triplicates ( n =3) with error bars±SD. p -Values: (*) p

    Article Snippet: T4 β-glucosyltransferase (β-GT) treatment was performed according to manufacturer's instructions (NEB EpiMark Kit).

    Techniques: Methylation, In Vitro, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Expressing, Construct, Real-time Polymerase Chain Reaction, Modification, Mutagenesis