dna methylation treatment  (New England Biolabs)


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    New England Biolabs dna methylation treatment
    mtDNA methylation identified by pyro-sequencing. Levels of <t>DNA</t> methylation at <t>CpG</t> sites in a HSP, b LSP, and c ND6 regions of 143B cells, 143B 143B early and 143B 143B late tumors were determined by pyro-sequencing. Statistical significance was determined by One-way ANOVA. Bars represent the mean of the percentage of DNA methylation (mean ± SEM; n = 3). * and ** indicate p values of
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    1) Product Images from "The degree of mitochondrial DNA methylation in tumor models of glioblastoma and osteosarcoma"

    Article Title: The degree of mitochondrial DNA methylation in tumor models of glioblastoma and osteosarcoma

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-018-0590-0

    mtDNA methylation identified by pyro-sequencing. Levels of DNA methylation at CpG sites in a HSP, b LSP, and c ND6 regions of 143B cells, 143B 143B early and 143B 143B late tumors were determined by pyro-sequencing. Statistical significance was determined by One-way ANOVA. Bars represent the mean of the percentage of DNA methylation (mean ± SEM; n = 3). * and ** indicate p values of
    Figure Legend Snippet: mtDNA methylation identified by pyro-sequencing. Levels of DNA methylation at CpG sites in a HSP, b LSP, and c ND6 regions of 143B cells, 143B 143B early and 143B 143B late tumors were determined by pyro-sequencing. Statistical significance was determined by One-way ANOVA. Bars represent the mean of the percentage of DNA methylation (mean ± SEM; n = 3). * and ** indicate p values of

    Techniques Used: Methylation, Sequencing, DNA Methylation Assay

    2) Product Images from "Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule"

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

    Journal: Bioconjugate Chemistry

    doi: 10.1021/bc1000388

    Effect of TFO-141S treatment on plasmid conformation. (A) Agarose gel electrophoresis of plasmids p39E and p11-1 treated with the TFO, WT M.SssI, C141S, or the TFO−C141S conjugate: lane 1, untreated; lane 2, without TFO−C141S; lane 3, TFO; lane 4, M.SssI; lane 5, C141S; lane 6, TFO−C141S; lane 7, TFO- C141S without SAM; lane 8, 100-fold excess of TFO and TFO−C141S; lane 9, marker; lane 10, without TFO−C141S; lane 11, C141S; lane 12, TFO−C141S. (B) Agarose gel electrophoresis of plasmid p39E treated with active and heat-inactivated C141S and TFO−C141S. The supercoiled plasmid was incubated at 30 °C for different time points as indicated above the lanes (hours). Then the samples were deproteinized before electrophoresis as described in Experimental Procedures . (C) Agarose gel electrophoresis of plasmid p39C and p39E treated with the TFO−C141S conjugate only or in the presence of 100-fold excess of TFO. Plasmids were incubated as in part B. Lane a is purified plasmid.
    Figure Legend Snippet: Effect of TFO-141S treatment on plasmid conformation. (A) Agarose gel electrophoresis of plasmids p39E and p11-1 treated with the TFO, WT M.SssI, C141S, or the TFO−C141S conjugate: lane 1, untreated; lane 2, without TFO−C141S; lane 3, TFO; lane 4, M.SssI; lane 5, C141S; lane 6, TFO−C141S; lane 7, TFO- C141S without SAM; lane 8, 100-fold excess of TFO and TFO−C141S; lane 9, marker; lane 10, without TFO−C141S; lane 11, C141S; lane 12, TFO−C141S. (B) Agarose gel electrophoresis of plasmid p39E treated with active and heat-inactivated C141S and TFO−C141S. The supercoiled plasmid was incubated at 30 °C for different time points as indicated above the lanes (hours). Then the samples were deproteinized before electrophoresis as described in Experimental Procedures . (C) Agarose gel electrophoresis of plasmid p39C and p39E treated with the TFO−C141S conjugate only or in the presence of 100-fold excess of TFO. Plasmids were incubated as in part B. Lane a is purified plasmid.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Marker, Incubation, Electrophoresis, Purification

    Effect of TFO−C141S treatment on GFP expression in EpCAM positive SKOV3 cells. (A) Relative GFP expression measured 48 h after transfection of pretreated p39E. Plasmid p39E was treated as indicated: p39E = treatment without TFO−C141S, treated with TFO only, with untargeted M.SssI or C141S, with the TFO−C141S conjugate or with 100-fold excess of TFO and TFO−C141S (=competition). The value obtained with p39E without TFO−C141S was taken as 100%. Shown is the average GFP expression (±SD) of one representative transfection performed in triplicate. (B) Relative GFP expression measured 48 h after transfection of pretreated deletion derivatives p7-2 and p4-1. For each derivative, the values obtained with samples treated without TFO−C141S were taken as 100%. Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate. (C) Relative GFP expression was measured 48 h after transfection of pretreated p39E or p39C. Treatments were as indicated: (+) or (−) indicates the presence or absence of the methyl donor (SAM). Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate.
    Figure Legend Snippet: Effect of TFO−C141S treatment on GFP expression in EpCAM positive SKOV3 cells. (A) Relative GFP expression measured 48 h after transfection of pretreated p39E. Plasmid p39E was treated as indicated: p39E = treatment without TFO−C141S, treated with TFO only, with untargeted M.SssI or C141S, with the TFO−C141S conjugate or with 100-fold excess of TFO and TFO−C141S (=competition). The value obtained with p39E without TFO−C141S was taken as 100%. Shown is the average GFP expression (±SD) of one representative transfection performed in triplicate. (B) Relative GFP expression measured 48 h after transfection of pretreated deletion derivatives p7-2 and p4-1. For each derivative, the values obtained with samples treated without TFO−C141S were taken as 100%. Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate. (C) Relative GFP expression was measured 48 h after transfection of pretreated p39E or p39C. Treatments were as indicated: (+) or (−) indicates the presence or absence of the methyl donor (SAM). Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate.

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    3) Product Images from "Alterations of sorbin and SH3 domain containing 3 (SORBS3) in human skeletal muscle following Roux-en-Y gastric bypass surgery"

    Article Title: Alterations of sorbin and SH3 domain containing 3 (SORBS3) in human skeletal muscle following Roux-en-Y gastric bypass surgery

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-017-0396-5

    In vitro DNA methylation of the SORBS3 human promoter is associated with decreased gene expression. Data presented as mean ± SD. The mean represents four independent experiments with five replicates per experiment. * P
    Figure Legend Snippet: In vitro DNA methylation of the SORBS3 human promoter is associated with decreased gene expression. Data presented as mean ± SD. The mean represents four independent experiments with five replicates per experiment. * P

    Techniques Used: In Vitro, DNA Methylation Assay, Expressing

    Average methylation levels of SORBS3 DMCs from lean and obese participants in a previous study and the present study pre- and post-surgery levels. The average methylation was assessed with all DMCs, regardless of methylation direction ( a ) and of only the DMCs that were consistent in the direction of methylation ( b ). Data presented as mean ± SD
    Figure Legend Snippet: Average methylation levels of SORBS3 DMCs from lean and obese participants in a previous study and the present study pre- and post-surgery levels. The average methylation was assessed with all DMCs, regardless of methylation direction ( a ) and of only the DMCs that were consistent in the direction of methylation ( b ). Data presented as mean ± SD

    Techniques Used: Methylation

    Differentially methylated cytosine (DMC) distribution among the promoter and 5′ untranslated regions of sorbin and SH3 domain containing 3 ( SORBS3 ) variants 1 and 2. The DMCs are derived from a previous study in obesity (Ln = lean vs Ob = obese) and the present RYGB cohort (bariatric)
    Figure Legend Snippet: Differentially methylated cytosine (DMC) distribution among the promoter and 5′ untranslated regions of sorbin and SH3 domain containing 3 ( SORBS3 ) variants 1 and 2. The DMCs are derived from a previous study in obesity (Ln = lean vs Ob = obese) and the present RYGB cohort (bariatric)

    Techniques Used: Methylation, Derivative Assay

    Differentially methylated cytosines (DMCs) associated with SORBS3 detected using pyrosequencing on the sense strand ( a ) and antisense strand ( b ) pre- and post-surgery. Data presented as mean ± SD
    Figure Legend Snippet: Differentially methylated cytosines (DMCs) associated with SORBS3 detected using pyrosequencing on the sense strand ( a ) and antisense strand ( b ) pre- and post-surgery. Data presented as mean ± SD

    Techniques Used: Methylation

    4) Product Images from "Measuring quantitative effects of methylation on transcription factor–DNA binding affinity"

    Article Title: Measuring quantitative effects of methylation on transcription factor–DNA binding affinity

    Journal: Science Advances

    doi: 10.1126/sciadv.aao1799

    Overview of Methyl-Spec-seq. ( A ) Schematic representation of the general workflow of Methyl-Spec-seq (see Materials and Methods). Briefly, differentially barcoded DNA libraries with variable regions are mixed and used in protein-DNA binding reactions. The DNA libraries are either treated with M.SssI methyltransferase enzyme to incorporate methyl-CpGs or left untreated and can also have synthetic 5′-methyl cytidine (mC). The letters “M” and “W” in red represent mC and mC on the complementary strand opposing a G, respectively. The protein-DNA complex is separated from the unbound DNA, following the binding reaction, in 9% polyacrylamide gel. The bound and unbound fractions are then polymerase chain reaction (PCR)–amplified (eight cycles) using Illumina-specific primers (text S1), and the resulting indexed samples are sequenced to generate energy logos for the binding sites. ( B ) Randomized double-stranded DNA (dsDNA) library used to measure the binding specificity of ZFP57 and the effect of methylation on binding. The full-length DNA libraries are shown in text S1. The regions highlighted in blue are the unique barcodes to distinguish the libraries during sequencing, whereas “N” in bold represent variable regions within the libraries. ( C ) Relative binding energy for all 64 variants in R2 libraries with different types of methylation, ranked from low to high binding energies of the unmethylated DNA. The relative binding energies are represented in units of kT , where k is the Boltzmann constant and T is the temperature used in the binding experiments. The 64 sequences of R2 libraries are listed vertically, and the relative binding energies depending on the methylation status are plotted. ( D ) Meth-eLogo based on the regression of the ZFP57 reference site and all its single variants. The significant effect of methylation at positions 4 and 5, which is the binding site for finger 3 (F3), is also shown. The effect of CpG methylation (mCPG) on binding specificity was calculated from the ePWM listed in fig. S2.
    Figure Legend Snippet: Overview of Methyl-Spec-seq. ( A ) Schematic representation of the general workflow of Methyl-Spec-seq (see Materials and Methods). Briefly, differentially barcoded DNA libraries with variable regions are mixed and used in protein-DNA binding reactions. The DNA libraries are either treated with M.SssI methyltransferase enzyme to incorporate methyl-CpGs or left untreated and can also have synthetic 5′-methyl cytidine (mC). The letters “M” and “W” in red represent mC and mC on the complementary strand opposing a G, respectively. The protein-DNA complex is separated from the unbound DNA, following the binding reaction, in 9% polyacrylamide gel. The bound and unbound fractions are then polymerase chain reaction (PCR)–amplified (eight cycles) using Illumina-specific primers (text S1), and the resulting indexed samples are sequenced to generate energy logos for the binding sites. ( B ) Randomized double-stranded DNA (dsDNA) library used to measure the binding specificity of ZFP57 and the effect of methylation on binding. The full-length DNA libraries are shown in text S1. The regions highlighted in blue are the unique barcodes to distinguish the libraries during sequencing, whereas “N” in bold represent variable regions within the libraries. ( C ) Relative binding energy for all 64 variants in R2 libraries with different types of methylation, ranked from low to high binding energies of the unmethylated DNA. The relative binding energies are represented in units of kT , where k is the Boltzmann constant and T is the temperature used in the binding experiments. The 64 sequences of R2 libraries are listed vertically, and the relative binding energies depending on the methylation status are plotted. ( D ) Meth-eLogo based on the regression of the ZFP57 reference site and all its single variants. The significant effect of methylation at positions 4 and 5, which is the binding site for finger 3 (F3), is also shown. The effect of CpG methylation (mCPG) on binding specificity was calculated from the ePWM listed in fig. S2.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Amplification, Methylation, Sequencing, CpG Methylation Assay

    5) Product Images from "Epigenetic Regulation of ZBTB18 Promotes Glioblastoma Progression"

    Article Title: Epigenetic Regulation of ZBTB18 Promotes Glioblastoma Progression

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-16-0494

    The core promoter region of ZBTB18 is essential for promoter activity and is sensitive to DNA methylation. (A) Schematic representation of the ZBTB18 promoter regions cloned in the pGL3 luciferase reporter vector. (B) Analysis of ZBTB18 promoter constructs activity by dual luciferase assay. (C) In vitro methylation assay of ZBTB18 promoter 4. The plasmid DNA was methylated with SssI or HpaII methylase. (D) Control restriction digestion of the methylase reaction using the methylation sensitive (HpaII) and the methylation insensitive (McrBC) restriction enzymes.
    Figure Legend Snippet: The core promoter region of ZBTB18 is essential for promoter activity and is sensitive to DNA methylation. (A) Schematic representation of the ZBTB18 promoter regions cloned in the pGL3 luciferase reporter vector. (B) Analysis of ZBTB18 promoter constructs activity by dual luciferase assay. (C) In vitro methylation assay of ZBTB18 promoter 4. The plasmid DNA was methylated with SssI or HpaII methylase. (D) Control restriction digestion of the methylase reaction using the methylation sensitive (HpaII) and the methylation insensitive (McrBC) restriction enzymes.

    Techniques Used: Activity Assay, DNA Methylation Assay, Clone Assay, Luciferase, Plasmid Preparation, Construct, In Vitro, Methylation

    6) Product Images from "Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation"

    Article Title: Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.808451

    Human METTL12 is a protein-specific MTase. A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.
    Figure Legend Snippet: Human METTL12 is a protein-specific MTase. A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.

    Techniques Used: CRISPR, Generated, Methylation, Incubation, Recombinant, SDS Page, Staining, Mutagenesis, Activity Assay

    7) Product Images from "Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation"

    Article Title: Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00683

    Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535
    Figure Legend Snippet: Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535

    Techniques Used: Binding Assay, Methylation

    8) Product Images from "Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™"

    Article Title: Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0173186

    (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.
    Figure Legend Snippet: (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.

    Techniques Used:

    (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.
    Figure Legend Snippet: (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.

    Techniques Used:

    A. Transcript coverage (FPKM) of Wolbachia genes reveals over 88% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample. B. Transcript coverage (FPKM) of Wolbachia genes reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample.
    Figure Legend Snippet: A. Transcript coverage (FPKM) of Wolbachia genes reveals over 88% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample. B. Transcript coverage (FPKM) of Wolbachia genes reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample.

    Techniques Used:

    9) Product Images from "The use of Multiple Displacement Amplified DNA as a control for Methylation Specific PCR, Pyrosequencing, Bisulfite Sequencing and Methylation-Sensitive Restriction Enzyme PCR"

    Article Title: The use of Multiple Displacement Amplified DNA as a control for Methylation Specific PCR, Pyrosequencing, Bisulfite Sequencing and Methylation-Sensitive Restriction Enzyme PCR

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-91

    Bisulfite sequencing results for MMP-14 . a) When genomic DNA (lane 1) and bisulfite treated mDNA (lane 2) and uDNA (lane 3) were used as template for sequencing in combination with primers for MMP-14 a PCR product was generated for all samples but not the negative control (lane 4). Sequencing results for b) non-amplified genomic DNA, c) uDNA and d) mDNA demonstrate that MDA treatment generates DNA (uDNA) free of all methylation as when it is bisulfite treated all cytosine are converted to thymine [indicated by asterix (*)]. In addition, sequencing also demonstrates that M.SssI treatment (mDNA) methylates CpG motifs as cytosines are retained when present as part of a CpG dinucleotide (indicated by *).
    Figure Legend Snippet: Bisulfite sequencing results for MMP-14 . a) When genomic DNA (lane 1) and bisulfite treated mDNA (lane 2) and uDNA (lane 3) were used as template for sequencing in combination with primers for MMP-14 a PCR product was generated for all samples but not the negative control (lane 4). Sequencing results for b) non-amplified genomic DNA, c) uDNA and d) mDNA demonstrate that MDA treatment generates DNA (uDNA) free of all methylation as when it is bisulfite treated all cytosine are converted to thymine [indicated by asterix (*)]. In addition, sequencing also demonstrates that M.SssI treatment (mDNA) methylates CpG motifs as cytosines are retained when present as part of a CpG dinucleotide (indicated by *).

    Techniques Used: Methylation Sequencing, Sequencing, Polymerase Chain Reaction, Generated, Negative Control, Amplification, Multiple Displacement Amplification, Methylation

    Methylation-Sensitive Restriction Enzyme PCR for MMP-1 and MMP-3 . a) PCR using primers spanning the restriction site for MMP-1 and MMP-3 gave a PCR product with mDNA but not with uDNA. In contrast, undigested samples gave PCR products for both mDNA and uDNA. b) PCR using digested DNA from MDA-MB231 (231), MDA-MB468 (468) and HFFF2 identified that the CpG motif is methylated for all three cell lines in the MMP-3 amplicon, but only for MDA-MB468 (468) and HFFF2 for the MMP-1 amplicon, with the MDA-MB231 (231) being unmethylated. However, the undigested DNA gave a PCR product with all three cells lines.
    Figure Legend Snippet: Methylation-Sensitive Restriction Enzyme PCR for MMP-1 and MMP-3 . a) PCR using primers spanning the restriction site for MMP-1 and MMP-3 gave a PCR product with mDNA but not with uDNA. In contrast, undigested samples gave PCR products for both mDNA and uDNA. b) PCR using digested DNA from MDA-MB231 (231), MDA-MB468 (468) and HFFF2 identified that the CpG motif is methylated for all three cell lines in the MMP-3 amplicon, but only for MDA-MB468 (468) and HFFF2 for the MMP-1 amplicon, with the MDA-MB231 (231) being unmethylated. However, the undigested DNA gave a PCR product with all three cells lines.

    Techniques Used: Methylation, Polymerase Chain Reaction, Multiple Displacement Amplification, Amplification

    10) Product Images from "The IGF1 P2 promoter is an epigenetic QTL for circulating IGF1 and human growth"

    Article Title: The IGF1 P2 promoter is an epigenetic QTL for circulating IGF1 and human growth

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-015-0062-8

    . The demethylation of the IGF1 -P2 region increases luciferase activity. Results of luciferase assays in transiently transfected HEK293 cells. Forty-eight hours after transfection, basal activity of unmethylated and methylated, Firefly luciferase reporter plasmids containing IGF1 promoter 2 CG fragment, and empty Firefly luciferase reporter plasmid, pCpGL-Basic, was measured, and normalized to the activity of co-transfected Renilla luciferase plasmid. Results were analyzed by paired t -test, ** P
    Figure Legend Snippet: . The demethylation of the IGF1 -P2 region increases luciferase activity. Results of luciferase assays in transiently transfected HEK293 cells. Forty-eight hours after transfection, basal activity of unmethylated and methylated, Firefly luciferase reporter plasmids containing IGF1 promoter 2 CG fragment, and empty Firefly luciferase reporter plasmid, pCpGL-Basic, was measured, and normalized to the activity of co-transfected Renilla luciferase plasmid. Results were analyzed by paired t -test, ** P

    Techniques Used: Luciferase, Activity Assay, Transfection, Methylation, Plasmid Preparation

    11) Product Images from "Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule"

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

    Journal: Bioconjugate Chemistry

    doi: 10.1021/bc1000388

    Effect of TFO-141S treatment on plasmid conformation. (A) Agarose gel electrophoresis of plasmids p39E and p11-1 treated with the TFO, WT M.SssI, C141S, or the TFO−C141S conjugate: lane 1, untreated; lane 2, without TFO−C141S; lane 3, TFO; lane 4, M.SssI; lane 5, C141S; lane 6, TFO−C141S; lane 7, TFO- C141S without SAM; lane 8, 100-fold excess of TFO and TFO−C141S; lane 9, marker; lane 10, without TFO−C141S; lane 11, C141S; lane 12, TFO−C141S. (B) Agarose gel electrophoresis of plasmid p39E treated with active and heat-inactivated C141S and TFO−C141S. The supercoiled plasmid was incubated at 30 °C for different time points as indicated above the lanes (hours). Then the samples were deproteinized before electrophoresis as described in Experimental Procedures . (C) Agarose gel electrophoresis of plasmid p39C and p39E treated with the TFO−C141S conjugate only or in the presence of 100-fold excess of TFO. Plasmids were incubated as in part B. Lane a is purified plasmid.
    Figure Legend Snippet: Effect of TFO-141S treatment on plasmid conformation. (A) Agarose gel electrophoresis of plasmids p39E and p11-1 treated with the TFO, WT M.SssI, C141S, or the TFO−C141S conjugate: lane 1, untreated; lane 2, without TFO−C141S; lane 3, TFO; lane 4, M.SssI; lane 5, C141S; lane 6, TFO−C141S; lane 7, TFO- C141S without SAM; lane 8, 100-fold excess of TFO and TFO−C141S; lane 9, marker; lane 10, without TFO−C141S; lane 11, C141S; lane 12, TFO−C141S. (B) Agarose gel electrophoresis of plasmid p39E treated with active and heat-inactivated C141S and TFO−C141S. The supercoiled plasmid was incubated at 30 °C for different time points as indicated above the lanes (hours). Then the samples were deproteinized before electrophoresis as described in Experimental Procedures . (C) Agarose gel electrophoresis of plasmid p39C and p39E treated with the TFO−C141S conjugate only or in the presence of 100-fold excess of TFO. Plasmids were incubated as in part B. Lane a is purified plasmid.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Marker, Incubation, Electrophoresis, Purification

    Effect of TFO−C141S treatment on GFP expression in EpCAM positive SKOV3 cells. (A) Relative GFP expression measured 48 h after transfection of pretreated p39E. Plasmid p39E was treated as indicated: p39E = treatment without TFO−C141S, treated with TFO only, with untargeted M.SssI or C141S, with the TFO−C141S conjugate or with 100-fold excess of TFO and TFO−C141S (=competition). The value obtained with p39E without TFO−C141S was taken as 100%. Shown is the average GFP expression (±SD) of one representative transfection performed in triplicate. (B) Relative GFP expression measured 48 h after transfection of pretreated deletion derivatives p7-2 and p4-1. For each derivative, the values obtained with samples treated without TFO−C141S were taken as 100%. Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate. (C) Relative GFP expression was measured 48 h after transfection of pretreated p39E or p39C. Treatments were as indicated: (+) or (−) indicates the presence or absence of the methyl donor (SAM). Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate.
    Figure Legend Snippet: Effect of TFO−C141S treatment on GFP expression in EpCAM positive SKOV3 cells. (A) Relative GFP expression measured 48 h after transfection of pretreated p39E. Plasmid p39E was treated as indicated: p39E = treatment without TFO−C141S, treated with TFO only, with untargeted M.SssI or C141S, with the TFO−C141S conjugate or with 100-fold excess of TFO and TFO−C141S (=competition). The value obtained with p39E without TFO−C141S was taken as 100%. Shown is the average GFP expression (±SD) of one representative transfection performed in triplicate. (B) Relative GFP expression measured 48 h after transfection of pretreated deletion derivatives p7-2 and p4-1. For each derivative, the values obtained with samples treated without TFO−C141S were taken as 100%. Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate. (C) Relative GFP expression was measured 48 h after transfection of pretreated p39E or p39C. Treatments were as indicated: (+) or (−) indicates the presence or absence of the methyl donor (SAM). Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate.

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    12) Product Images from "DNA METHYLATION INHIBITION INCREASES T CELL KIR EXPRESSION THROUGH EFFECTS ON BOTH PROMOTER METHYLATION AND TRANSCRIPTION FACTORS"

    Article Title: DNA METHYLATION INHIBITION INCREASES T CELL KIR EXPRESSION THROUGH EFFECTS ON BOTH PROMOTER METHYLATION AND TRANSCRIPTION FACTORS

    Journal: Clinical immunology (Orlando, Fla.)

    doi: 10.1016/j.clim.2008.08.009

    Effect of 5-azaC on KIR2DL2 and KIR2DL4 promoter methylation
    Figure Legend Snippet: Effect of 5-azaC on KIR2DL2 and KIR2DL4 promoter methylation

    Techniques Used: Methylation

    KIR2DL4 induced by 5-azaC is functional
    Figure Legend Snippet: KIR2DL4 induced by 5-azaC is functional

    Techniques Used: Functional Assay

    13) Product Images from "Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations"

    Article Title: Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1612444114

    IFIT1 PPP- and m7Gppp- binding mechanism. ( A ) Simulated annealing 2 F o – F c omit map of the PPP moiety contoured at 1σ. ( B ) Cartoon/stick representation of residues making specific contacts with the triphosphate group from PPP-RNA–bound
    Figure Legend Snippet: IFIT1 PPP- and m7Gppp- binding mechanism. ( A ) Simulated annealing 2 F o – F c omit map of the PPP moiety contoured at 1σ. ( B ) Cartoon/stick representation of residues making specific contacts with the triphosphate group from PPP-RNA–bound

    Techniques Used: Binding Assay

    IFIT1 senses N1 and N2 ribose 2′-O methylation ( A and B ) EMSAs between 1 µM or 2.5 µM IFIT1 and differentially methylated m7Gppp-RNA ( C ) Comparison of IFIT1 binding to 35 nM Cap0-MHV, Cap1-MHV, or N2Me-MHV. Cap1 methylation reduces
    Figure Legend Snippet: IFIT1 senses N1 and N2 ribose 2′-O methylation ( A and B ) EMSAs between 1 µM or 2.5 µM IFIT1 and differentially methylated m7Gppp-RNA ( C ) Comparison of IFIT1 binding to 35 nM Cap0-MHV, Cap1-MHV, or N2Me-MHV. Cap1 methylation reduces

    Techniques Used: Methylation, Binding Assay

    14) Product Images from "Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations"

    Article Title: Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1612444114

    IFIT1 PPP- and m7Gppp- binding mechanism. ( A ) Simulated annealing 2 F o – F c omit map of the PPP moiety contoured at 1σ. ( B ) Cartoon/stick representation of residues making specific contacts with the triphosphate group from PPP-RNA–bound
    Figure Legend Snippet: IFIT1 PPP- and m7Gppp- binding mechanism. ( A ) Simulated annealing 2 F o – F c omit map of the PPP moiety contoured at 1σ. ( B ) Cartoon/stick representation of residues making specific contacts with the triphosphate group from PPP-RNA–bound

    Techniques Used: Binding Assay

    IFIT1 mRNA cap-binding mechanism. ( A ) The IFIT1 PPP (blue) adopts an extended conformation compared with the “bent” IFIT5 PPP (pink). The γ-phosphate from PPP–RNA-bound IFIT1 points toward the nearby unoccupied cap-binding
    Figure Legend Snippet: IFIT1 mRNA cap-binding mechanism. ( A ) The IFIT1 PPP (blue) adopts an extended conformation compared with the “bent” IFIT5 PPP (pink). The γ-phosphate from PPP–RNA-bound IFIT1 points toward the nearby unoccupied cap-binding

    Techniques Used: Binding Assay

    15) Product Images from "Cytosine methylation determines hot spots of DNA damage in the human P53 gene"

    Article Title: Cytosine methylation determines hot spots of DNA damage in the human P53 gene

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Distribution of BPDE adducts along P53 sequences in plasmid DNA differing in methylation status. DNA was methylated (+SAM) or mock-methylated (−SAM) with the CpG-specific methylase Sss I, modified with BPDE (0.2 μM), and the distribution of adducts was analyzed by UvrABC incision and LMPCR. ( A ) Exon 5, nontranscribed strand. ( B ) Exon 7, nontranscribed strand. ( C ) Exon 8, nontranscribed strand. Stars mark codons containing CpG dinucleotides.
    Figure Legend Snippet: Distribution of BPDE adducts along P53 sequences in plasmid DNA differing in methylation status. DNA was methylated (+SAM) or mock-methylated (−SAM) with the CpG-specific methylase Sss I, modified with BPDE (0.2 μM), and the distribution of adducts was analyzed by UvrABC incision and LMPCR. ( A ) Exon 5, nontranscribed strand. ( B ) Exon 7, nontranscribed strand. ( C ) Exon 8, nontranscribed strand. Stars mark codons containing CpG dinucleotides.

    Techniques Used: Plasmid Preparation, Methylation, Modification

    Mapping of UvrABC-induced 5′ break positions in BPDE-modified methylated and unmethylated DNA fragments. A 5′ end-labeled Ava II– Ssp I DNA fragment containing sequences of exon 8 of the P53 gene was methylated with Sss I or mock-methylated, treated with BPDE, and then reacted with UvrABC nucleases. ( A ) Autoradiogram. Lanes: 1–4, no BPDE treatment; 9 and 10, BPDE treatment of unmethylated and methylated DNA, respectively; 5–8, Maxam–Gilbert sequencing reactions. *C marks methylated cytosines at CpG sites indicated by a missing band in the C-specific reaction. ( B ) Quantitation. The intensities of BPDE adduct-induced UvrABC incisions at different sequences from methylated DNA ( Upper ) or unmethylated DNA ( Lower ) were quantitated by phosphorimaging.
    Figure Legend Snippet: Mapping of UvrABC-induced 5′ break positions in BPDE-modified methylated and unmethylated DNA fragments. A 5′ end-labeled Ava II– Ssp I DNA fragment containing sequences of exon 8 of the P53 gene was methylated with Sss I or mock-methylated, treated with BPDE, and then reacted with UvrABC nucleases. ( A ) Autoradiogram. Lanes: 1–4, no BPDE treatment; 9 and 10, BPDE treatment of unmethylated and methylated DNA, respectively; 5–8, Maxam–Gilbert sequencing reactions. *C marks methylated cytosines at CpG sites indicated by a missing band in the C-specific reaction. ( B ) Quantitation. The intensities of BPDE adduct-induced UvrABC incisions at different sequences from methylated DNA ( Upper ) or unmethylated DNA ( Lower ) were quantitated by phosphorimaging.

    Techniques Used: Modification, Methylation, Labeling, Antiviral Assay, Sequencing, Quantitation Assay

    Distribution of BPDE adducts along the methylated and unmethylated CpG island of the human PGK1 gene. DNA containing sequences from the Xa (unmethylated; lanes 3 and 4) or from the Xi (methylated; lanes 5 and 6) was treated with BPDE (1 μM), and the distribution of adducts in the PGK1 promoter and CpG island was determined after UvrABC incision and LMPCR. Lanes 1 and 2 are Maxam–Gilbert sequencing controls. Stars indicate positions where an increase of modification was noticed with the Xi DNA containing methylated PGK1 sequences; ○, other modified G positions.
    Figure Legend Snippet: Distribution of BPDE adducts along the methylated and unmethylated CpG island of the human PGK1 gene. DNA containing sequences from the Xa (unmethylated; lanes 3 and 4) or from the Xi (methylated; lanes 5 and 6) was treated with BPDE (1 μM), and the distribution of adducts in the PGK1 promoter and CpG island was determined after UvrABC incision and LMPCR. Lanes 1 and 2 are Maxam–Gilbert sequencing controls. Stars indicate positions where an increase of modification was noticed with the Xi DNA containing methylated PGK1 sequences; ○, other modified G positions.

    Techniques Used: Methylation, Sequencing, Modification

    16) Product Images from "mRNA maturation in giant viruses: variation on a theme"

    Article Title: mRNA maturation in giant viruses: variation on a theme

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv224

    Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN 13 ), N7- or 2′O-methylated capped RNA substrates ( 7Me GpppAN 13 or GpppA 2′OMe N 13 ), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.
    Figure Legend Snippet: Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN 13 ), N7- or 2′O-methylated capped RNA substrates ( 7Me GpppAN 13 or GpppA 2′OMe N 13 ), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.

    Techniques Used: Activity Assay, Methylation

    17) Product Images from "MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells"

    Article Title: MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.11.027

    Expression of M.EcoGII-Lamin B1 to Target M.EcoGII to the Nuclear Envelope (A–C) Representative dot blot of genomic DNA from HeLa 1.2.11 cells induced (+) or induced not (−) to express M.EcoGII-v5-Lamin B1 (A) or v5-M.EcoGII (B). The membrane was probed with a m6A antibody. The normalized intensities are shown. (B and D) Example of immunofluorescence of HeLa 1.2.11 cells induced (+) or not induced (–) to express M.EcoGII-v5-Lamin B1 (B) or v5-M.EcoGII (D). V5 tag (left panel) or m6A (right panel) (red), Lamin A/C (green), DNA (blue), and merge. Scale bar, 10 μm. The enlarged part of the nucleus of m6A and Lamin A/C staining is shown. Scale bar, 1 μm.
    Figure Legend Snippet: Expression of M.EcoGII-Lamin B1 to Target M.EcoGII to the Nuclear Envelope (A–C) Representative dot blot of genomic DNA from HeLa 1.2.11 cells induced (+) or induced not (−) to express M.EcoGII-v5-Lamin B1 (A) or v5-M.EcoGII (B). The membrane was probed with a m6A antibody. The normalized intensities are shown. (B and D) Example of immunofluorescence of HeLa 1.2.11 cells induced (+) or not induced (–) to express M.EcoGII-v5-Lamin B1 (B) or v5-M.EcoGII (D). V5 tag (left panel) or m6A (right panel) (red), Lamin A/C (green), DNA (blue), and merge. Scale bar, 10 μm. The enlarged part of the nucleus of m6A and Lamin A/C staining is shown. Scale bar, 1 μm.

    Techniques Used: Expressing, Dot Blot, Immunofluorescence, Staining

    Activity of M.EcoGII and Detection of m6A-DNA (A) Dot blot of fragmented genomic DNA (input) or DNA immunoprecipitated with a m6A antibody (m6A-IP) from E. coli ER2796, E. coli MG1655, or U2OS cells expressing or not expressing M.EcoGII. The membranes were probed with a m6A antibody. Quantification of m6A enrichment in the immunoprecipitated fractions relative to the input material is shown from three independent replicates (R1, R2, and R3; mean ± SD). (B) Dot blot with increasing amounts of sheared genomic DNA extracted from U2OS cells expressing or not expressing M.EcoGII. The membrane was probed with a m6A antibody. The graph represents the mean intensity ± SD of the m6A signal from two independent experiments (R1 and R2). (C) Dot blot of plasmid DNA in vitro methylated with increasing amounts of M.EcoGII recombinant enzyme. 500 ng of DNA from each reaction was loaded on a membrane probed with a m6A antibody. The normalized intensity relative to the unmethylated plasmid is shown.
    Figure Legend Snippet: Activity of M.EcoGII and Detection of m6A-DNA (A) Dot blot of fragmented genomic DNA (input) or DNA immunoprecipitated with a m6A antibody (m6A-IP) from E. coli ER2796, E. coli MG1655, or U2OS cells expressing or not expressing M.EcoGII. The membranes were probed with a m6A antibody. Quantification of m6A enrichment in the immunoprecipitated fractions relative to the input material is shown from three independent replicates (R1, R2, and R3; mean ± SD). (B) Dot blot with increasing amounts of sheared genomic DNA extracted from U2OS cells expressing or not expressing M.EcoGII. The membrane was probed with a m6A antibody. The graph represents the mean intensity ± SD of the m6A signal from two independent experiments (R1 and R2). (C) Dot blot of plasmid DNA in vitro methylated with increasing amounts of M.EcoGII recombinant enzyme. 500 ng of DNA from each reaction was loaded on a membrane probed with a m6A antibody. The normalized intensity relative to the unmethylated plasmid is shown.

    Techniques Used: Activity Assay, Dot Blot, Immunoprecipitation, Expressing, Plasmid Preparation, In Vitro, Methylation, Recombinant

    M.EcoGII Targeted to the Nuclear Envelope Can Contact and Methylate Telomeric DNA (A–C) Representative dot blot of captured telomeric DNA from asynchronous HeLa 1.2.11 induced to express the indicated vectors for 24 hr (A) or G1/S-arrested cells induced for 8 hr (C). DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents the normalized intensities relative to input (mean ± SD). (B) Heatmap of the number of reads per million obtained at individual chromosome ends in HeLa 1.2.11 cells expressing M.EcoGII-Lamin B1 (M-LB1). The log2 M.EcoGII-LB1/M.EcoGII ratio is shown. Chromosome ends with positive enrichment are highlighted in red. A box with a cross represents a bin without an associated DNA sequence and therefore excluded from the analysis. (D) Representative DNA-IF of G1/S-arrested HeLa 1.2.11 cells induced to express the indicated vectors for 8 hr. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm.
    Figure Legend Snippet: M.EcoGII Targeted to the Nuclear Envelope Can Contact and Methylate Telomeric DNA (A–C) Representative dot blot of captured telomeric DNA from asynchronous HeLa 1.2.11 induced to express the indicated vectors for 24 hr (A) or G1/S-arrested cells induced for 8 hr (C). DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents the normalized intensities relative to input (mean ± SD). (B) Heatmap of the number of reads per million obtained at individual chromosome ends in HeLa 1.2.11 cells expressing M.EcoGII-Lamin B1 (M-LB1). The log2 M.EcoGII-LB1/M.EcoGII ratio is shown. Chromosome ends with positive enrichment are highlighted in red. A box with a cross represents a bin without an associated DNA sequence and therefore excluded from the analysis. (D) Representative DNA-IF of G1/S-arrested HeLa 1.2.11 cells induced to express the indicated vectors for 8 hr. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm.

    Techniques Used: Dot Blot, Expressing, Sequencing

    M.EcoGII Can Methylate GATC-Free DNA Regions and Is Specific to Its Region of Targeting (A) Dot blot with 500 ng of oligonucleotides corresponding to the C-rich strand of telomeric repeats (TelC), the G-rich strand of telomeric repeats (TelG), and scramble (Scr TelC and Scr TelG) before or after in vitro methylation using recombinant M.EcoGII. The number of adenine present in the sequences is indicated. The membrane was probed with a m6A antibody, and the relative signal intensity was measured as indicated. (B) Representative immunostaining and FISH of HeLa 1.2.11 cells transduced with the indicated vectors. V5 tag (green); TelC, telomeres (magenta); CEN, centromeres (red), DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (C) Representative dot blot of captured telomeric DNA (T) from HeLa 1.2.11 cells expressing the indicated vectors. DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents normalized intensities relative to input (mean ± SD). (D) DNA immunofluorescence (DNA-IF) of HeLa 1.2.11 cells expressing M-TRF1. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (E) Total number of telomeric reads per million from whole-genome sequencing data obtained from HeLa 1.2.11 transduced with the indicated vectors. (F) Number of reads per million from whole-genome sequencing data obtained in M-TRF1 cells relative to M.EcoGII along chromosome ends (mean ± SEM).
    Figure Legend Snippet: M.EcoGII Can Methylate GATC-Free DNA Regions and Is Specific to Its Region of Targeting (A) Dot blot with 500 ng of oligonucleotides corresponding to the C-rich strand of telomeric repeats (TelC), the G-rich strand of telomeric repeats (TelG), and scramble (Scr TelC and Scr TelG) before or after in vitro methylation using recombinant M.EcoGII. The number of adenine present in the sequences is indicated. The membrane was probed with a m6A antibody, and the relative signal intensity was measured as indicated. (B) Representative immunostaining and FISH of HeLa 1.2.11 cells transduced with the indicated vectors. V5 tag (green); TelC, telomeres (magenta); CEN, centromeres (red), DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (C) Representative dot blot of captured telomeric DNA (T) from HeLa 1.2.11 cells expressing the indicated vectors. DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents normalized intensities relative to input (mean ± SD). (D) DNA immunofluorescence (DNA-IF) of HeLa 1.2.11 cells expressing M-TRF1. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (E) Total number of telomeric reads per million from whole-genome sequencing data obtained from HeLa 1.2.11 transduced with the indicated vectors. (F) Number of reads per million from whole-genome sequencing data obtained in M-TRF1 cells relative to M.EcoGII along chromosome ends (mean ± SEM).

    Techniques Used: Dot Blot, In Vitro, Methylation, Recombinant, Immunostaining, Fluorescence In Situ Hybridization, Transduction, Expressing, Immunofluorescence, Sequencing

    Schematic Overview of MadID (A) Left, GATC distribution on human chromosome 1 (hg38 assembly). The blue gradient represents the score for the GATC site within 1 kb genome segments. Magnification from 1 to 0.1 Mb is shown. Green line, telomere T 2 AG 3 sequence; red dashed line, 1 Mb of centromere DNA. Right, smooth scatter graphs of the A+T nucleotide and GATC motif count per 1 kb genome segment (hg38, chromosomes [chr] 1–22, X, and Y). (B) Experimental setup and detection. (1a) DNA methyltransferases catalyze the transfer of a methyl group to DNA. (1b) M.EcoGII is fused to a destabilization domain (DD) for proteasome degradation unless the compound Shield1 is added to stabilize the protein. M.EcoGII is targeted to the nuclear envelope by fusion with Lamin B1, to telomeres by fusion with telomeric repeat binding factor 1 (TRF1), or to centromeres by fusion with CENP-C. Precise targeting of M.EcoGII causes methylation of DNA in the surrounding regions (m6A). (2a) m6A detection in situ by immunostaining with a m6A antibody. (2b) Genome-wide m6A detection by m6A-specific immunoprecipitation (m6A-IP), followed by whole-genome sequencing. (2c) DNA regions of interest can be purified by chromatin immunoprecipitation or probe-based capture techniques, and m6A can be detected on dot blots using the m6A-specific antibody.
    Figure Legend Snippet: Schematic Overview of MadID (A) Left, GATC distribution on human chromosome 1 (hg38 assembly). The blue gradient represents the score for the GATC site within 1 kb genome segments. Magnification from 1 to 0.1 Mb is shown. Green line, telomere T 2 AG 3 sequence; red dashed line, 1 Mb of centromere DNA. Right, smooth scatter graphs of the A+T nucleotide and GATC motif count per 1 kb genome segment (hg38, chromosomes [chr] 1–22, X, and Y). (B) Experimental setup and detection. (1a) DNA methyltransferases catalyze the transfer of a methyl group to DNA. (1b) M.EcoGII is fused to a destabilization domain (DD) for proteasome degradation unless the compound Shield1 is added to stabilize the protein. M.EcoGII is targeted to the nuclear envelope by fusion with Lamin B1, to telomeres by fusion with telomeric repeat binding factor 1 (TRF1), or to centromeres by fusion with CENP-C. Precise targeting of M.EcoGII causes methylation of DNA in the surrounding regions (m6A). (2a) m6A detection in situ by immunostaining with a m6A antibody. (2b) Genome-wide m6A detection by m6A-specific immunoprecipitation (m6A-IP), followed by whole-genome sequencing. (2c) DNA regions of interest can be purified by chromatin immunoprecipitation or probe-based capture techniques, and m6A can be detected on dot blots using the m6A-specific antibody.

    Techniques Used: Sequencing, Binding Assay, Methylation, In Situ, Immunostaining, Genome Wide, Immunoprecipitation, Purification, Chromatin Immunoprecipitation

    18) Product Images from "Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™"

    Article Title: Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0173186

    (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.
    Figure Legend Snippet: (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.

    Techniques Used:

    (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.
    Figure Legend Snippet: (A) Transcript abundance of all 940 annotated Wolbachia genes (listed by geneID number) reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. Each transcript is indicated by in red (the FPKM value in the total RNA) and blue (the FPKM value in capped-RNA sample)(B) A closer view (note difference in y-axis scales between panel A and B) of transcript abundance reveals an enrichment in Wolbachia transcripts in the capped RNA sample.

    Techniques Used:

    A. Transcript coverage (FPKM) of Wolbachia genes reveals over 88% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample. B. Transcript coverage (FPKM) of Wolbachia genes reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample.
    Figure Legend Snippet: A. Transcript coverage (FPKM) of Wolbachia genes reveals over 88% of Wolbachia transcripts from B . malayi adult male RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample. B. Transcript coverage (FPKM) of Wolbachia genes reveals over 95% of Wolbachia transcripts from B . malayi MF RNA were enriched using the Cappable-seq technique. A closer view of transcript abundance (inset) reveals most Wolbachia transcripts in total RNA are present in very low abundance, whereas the Wolbachia transcripts are more abundant in the capped RNA sample. Points along the y-axis are indicative of Wolbachia transcripts that were undetectable in total RNA that were detected in the capped RNA sample.

    Techniques Used:

    19) Product Images from "The dual methyltransferase METTL13 targets N terminus and Lys55 of eEF1A and modulates codon-specific translation rates"

    Article Title: The dual methyltransferase METTL13 targets N terminus and Lys55 of eEF1A and modulates codon-specific translation rates

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05646-y

    Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 H]-AdoMet and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading
    Figure Legend Snippet: Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 H]-AdoMet and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading

    Techniques Used: Mass Spectrometry, Construct, Activity Assay, Incubation, Methylation, Staining

    20) Product Images from "Single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc02215a"

    Article Title: Single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase †Electronic supplementary information (ESI) available. See DOI: 10.1039/c8sc02215a

    Journal: Chemical Science

    doi: 10.1039/c8sc02215a

    Schematic illustration of DNA MTase assay based on single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification. This assay involves three reaction steps: (1) Dam MTase-directed cleavage of hairpin substrates, (2) single-ribonucleotide repair-mediated cyclic ligation-dependent SDA, and (3) RNase HII-catalyzed cyclic cleavage of signal probes for the generation of a distinct fluorescence signal.
    Figure Legend Snippet: Schematic illustration of DNA MTase assay based on single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification. This assay involves three reaction steps: (1) Dam MTase-directed cleavage of hairpin substrates, (2) single-ribonucleotide repair-mediated cyclic ligation-dependent SDA, and (3) RNase HII-catalyzed cyclic cleavage of signal probes for the generation of a distinct fluorescence signal.

    Techniques Used: Ligation, Amplification, Fluorescence

    Mechanism of RNase HII-catalyzed single-ribonucleotide repairing. The single ribonucleotide misincorporated within the 5′-DNA-RNA-DNA-3′/3′-DNA-5′duplexes can be specifically recognized and excised by RNase HII through hydrolyzing the phosphodiester bonds 5′ to the ribonucleotide at the DNA–RNA junction, leaving a single nucleotide gap with 5′ PO 4 and 3′ OH ends.
    Figure Legend Snippet: Mechanism of RNase HII-catalyzed single-ribonucleotide repairing. The single ribonucleotide misincorporated within the 5′-DNA-RNA-DNA-3′/3′-DNA-5′duplexes can be specifically recognized and excised by RNase HII through hydrolyzing the phosphodiester bonds 5′ to the ribonucleotide at the DNA–RNA junction, leaving a single nucleotide gap with 5′ PO 4 and 3′ OH ends.

    Techniques Used:

    21) Product Images from "Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿"

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00108-07

    Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.
    Figure Legend Snippet: Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Techniques Used: Purification, Incubation, Polymerase Chain Reaction, Concentration Assay, In Vitro

    22) Product Images from "Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule"

    Article Title: Targeted DNA Methylation by a DNA Methyltransferase Coupled to a Triple Helix Forming Oligonucleotide To Down-Regulate the Epithelial Cell Adhesion Molecule

    Journal: Bioconjugate Chemistry

    doi: 10.1021/bc1000388

    Effect of TFO-141S treatment on plasmid conformation. (A) Agarose gel electrophoresis of plasmids p39E and p11-1 treated with the TFO, WT M.SssI, C141S, or the TFO−C141S conjugate: lane 1, untreated; lane 2, without TFO−C141S; lane 3, TFO; lane 4, M.SssI; lane 5, C141S; lane 6, TFO−C141S; lane 7, TFO- C141S without SAM; lane 8, 100-fold excess of TFO and TFO−C141S; lane 9, marker; lane 10, without TFO−C141S; lane 11, C141S; lane 12, TFO−C141S. (B) Agarose gel electrophoresis of plasmid p39E treated with active and heat-inactivated C141S and TFO−C141S. The supercoiled plasmid was incubated at 30 °C for different time points as indicated above the lanes (hours). Then the samples were deproteinized before electrophoresis as described in Experimental Procedures . (C) Agarose gel electrophoresis of plasmid p39C and p39E treated with the TFO−C141S conjugate only or in the presence of 100-fold excess of TFO. Plasmids were incubated as in part B. Lane a is purified plasmid.
    Figure Legend Snippet: Effect of TFO-141S treatment on plasmid conformation. (A) Agarose gel electrophoresis of plasmids p39E and p11-1 treated with the TFO, WT M.SssI, C141S, or the TFO−C141S conjugate: lane 1, untreated; lane 2, without TFO−C141S; lane 3, TFO; lane 4, M.SssI; lane 5, C141S; lane 6, TFO−C141S; lane 7, TFO- C141S without SAM; lane 8, 100-fold excess of TFO and TFO−C141S; lane 9, marker; lane 10, without TFO−C141S; lane 11, C141S; lane 12, TFO−C141S. (B) Agarose gel electrophoresis of plasmid p39E treated with active and heat-inactivated C141S and TFO−C141S. The supercoiled plasmid was incubated at 30 °C for different time points as indicated above the lanes (hours). Then the samples were deproteinized before electrophoresis as described in Experimental Procedures . (C) Agarose gel electrophoresis of plasmid p39C and p39E treated with the TFO−C141S conjugate only or in the presence of 100-fold excess of TFO. Plasmids were incubated as in part B. Lane a is purified plasmid.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Marker, Incubation, Electrophoresis, Purification

    Effect of TFO−C141S treatment on GFP expression in EpCAM positive SKOV3 cells. (A) Relative GFP expression measured 48 h after transfection of pretreated p39E. Plasmid p39E was treated as indicated: p39E = treatment without TFO−C141S, treated with TFO only, with untargeted M.SssI or C141S, with the TFO−C141S conjugate or with 100-fold excess of TFO and TFO−C141S (=competition). The value obtained with p39E without TFO−C141S was taken as 100%. Shown is the average GFP expression (±SD) of one representative transfection performed in triplicate. (B) Relative GFP expression measured 48 h after transfection of pretreated deletion derivatives p7-2 and p4-1. For each derivative, the values obtained with samples treated without TFO−C141S were taken as 100%. Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate. (C) Relative GFP expression was measured 48 h after transfection of pretreated p39E or p39C. Treatments were as indicated: (+) or (−) indicates the presence or absence of the methyl donor (SAM). Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate.
    Figure Legend Snippet: Effect of TFO−C141S treatment on GFP expression in EpCAM positive SKOV3 cells. (A) Relative GFP expression measured 48 h after transfection of pretreated p39E. Plasmid p39E was treated as indicated: p39E = treatment without TFO−C141S, treated with TFO only, with untargeted M.SssI or C141S, with the TFO−C141S conjugate or with 100-fold excess of TFO and TFO−C141S (=competition). The value obtained with p39E without TFO−C141S was taken as 100%. Shown is the average GFP expression (±SD) of one representative transfection performed in triplicate. (B) Relative GFP expression measured 48 h after transfection of pretreated deletion derivatives p7-2 and p4-1. For each derivative, the values obtained with samples treated without TFO−C141S were taken as 100%. Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate. (C) Relative GFP expression was measured 48 h after transfection of pretreated p39E or p39C. Treatments were as indicated: (+) or (−) indicates the presence or absence of the methyl donor (SAM). Shown is the average GFP expression (±SEM) of the mean of three independent transfections performed in triplicate.

    Techniques Used: Expressing, Transfection, Plasmid Preparation

    23) Product Images from "Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells"

    Article Title: Uncovering the Role of Hypermethylation by CTG Expansion in Myotonic Dystrophy Type 1 Using Mutant Human Embryonic Stem Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2015.06.003

    Functional Assay for the Regulatory Role of the DMR (A) Schematic illustration describing the different inserts tested in this study. Fragments I, II, and III designate the DMR sequence in both orientations, the SIX5 promoter, and the entire region that spans from the DMR to the SIX5 promoter (includes 12 repeats), respectively. The size of each fragment is indicated in parentheses. (B) Zebrafish enhancer assay for the DMR region. GFP expression was observed in the epidermis and branchial arches of zebrafish embryos (marked by arrows) at 48 and 72 hr post-fertilization (hpf). Scale bars, 100 μm. (C) Relative luciferase activity from transiently transfected 293T, COS-7, and HepG2 cells. Luciferase activity was measured 48 hr after transfection. Renilla expression levels were used to normalize cell number, transfection efficiency, and general effects on gene transcription. Data were normalized to luciferase levels of the empty vector pGL3 Basic. Bar graphs stand for mean values (indicated on top of each bar) and SD from three independent experiments with three technical replicates each (paired t test, ∗∗ p
    Figure Legend Snippet: Functional Assay for the Regulatory Role of the DMR (A) Schematic illustration describing the different inserts tested in this study. Fragments I, II, and III designate the DMR sequence in both orientations, the SIX5 promoter, and the entire region that spans from the DMR to the SIX5 promoter (includes 12 repeats), respectively. The size of each fragment is indicated in parentheses. (B) Zebrafish enhancer assay for the DMR region. GFP expression was observed in the epidermis and branchial arches of zebrafish embryos (marked by arrows) at 48 and 72 hr post-fertilization (hpf). Scale bars, 100 μm. (C) Relative luciferase activity from transiently transfected 293T, COS-7, and HepG2 cells. Luciferase activity was measured 48 hr after transfection. Renilla expression levels were used to normalize cell number, transfection efficiency, and general effects on gene transcription. Data were normalized to luciferase levels of the empty vector pGL3 Basic. Bar graphs stand for mean values (indicated on top of each bar) and SD from three independent experiments with three technical replicates each (paired t test, ∗∗ p

    Techniques Used: Functional Assay, Sequencing, Expressing, Luciferase, Activity Assay, Transfection, Plasmid Preparation

    24) Product Images from "Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos"

    Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054801

    LTR methylation and gag expression of HIV-1 in the plasmid-transfected spermatozoa and sperm-derived zygotes and 2-cell embryos. A: The detection of LTR methylation by BSP and Thymine/Adenine cloning while transfection with unmethylated plasmid. The methylation status of 15 clones for each sample is presented; each column represents one CpG position in the U3-R region, with each circle in the column indicating either cytosine (open circles) or methyl cytosine (filled circles), the same below. (A 1 ) plasmid; (A 2 ) spermatozoa; (A3) zygotes; (A 4 ) 2-cell embryos. B: The detection of gag transcription while transfection with unmethylated plasmid (B 1 ) the results of RT-PCR. M: Marker; 1: positive control; 2: spermatozoa; 3: -RT; 4: -T; 5: human β-actin. (B 2 ) the results of first-round of nested RT-PCR. M: Marker; 1: positive control; 2∶2-cell embryos; 3: -RT; 4: -T; 5: hamster β-actin. (B 3 ) the results of second-round of nested RT-PCR. M: Marker; 1: positive control; 2: the first round product; 3: -RT; 4: -T. The results showed a clear correlation between LTR methylation and gag transcription of HIV-1 either in spermatozoa or in 2-cell embryos. C: The detection of LTR methylation while transfection with methylated plasmid. (C 1 ) plasmid; (C 2 ) spermatozoa; (C 3 ) zygotes; (C 4 ) 2-cell embryos. D: The detection of gag transcription while transfection with methylated plasmid. (D 1 ) the results of first-round of nested RT-PCR. M: Marker; 1: positive control; 2: spermatozoa; 3: -RT; 4: -T; 5: human β-actin. (D 2 ) The results of second-round. M: Marker; 1: positive control; 2: the first round product; 3: -RT; 4: -T. (D 3 ) the results of first-round of nested RT-PCR M: Marker; 1: positive control; 2∶2-cell embryos; 3: -RT; 4: -T; 5: hamster β-actin. (D 4 ) the results of second-round. M: Marker; 1: positive control; 2: the first-round product; 3: -RT; 4: -T. The results showed a clear correlation between LTR methylation and gag transcription of HIV-1 either in spermatozoa or in 2-cell embryos. E: CMV methylation in the spermatozoa transfected with methylated pIRES2-EGFP and sperm-derived zygotes and 2-cell embryos. The detection of CpG methylation by BSP and Thymine/Adenine cloning. (E 1 ) m-pIRES2-EGFP; (E 2 ) spermatozoa; (E3) zygotes; (E4) 2-cell embryos. The results showed that demethylation of some CpG sites in CMV promoter has already occurred in the spermatozoa and almost all CpG sites were demethylated in zygotes and 2-cell embryos. F: The effects of SAM on methylation of CpG sites in HIV-1 LTR in the 2-cell embryos. (F 1 ) SAM treatment; (F 2 ) SAM-free treatment. The results showed that the methylation rate of HIV-1 LTR in 2-cell embryos markedly increased after treatment with SAM. G: The detection of HIV-1 Gag translation in spermatozoa by IF assay. (G 1 ) transfection with unmethylated plasmid; (G 2 ) transfection with methylated plasmid; (G3) negative control; (G 4 ) positive control. No positive signal for HIV-1 P24 Gag protein was observed in (G 1 ), (G 2 ) and (G 3 ), and the strong signals for sperm protein were visible in (G 4 ). H: The detection of Gag translation in 2-cell embryos by IF assay. (H 1 ) negative control; (H 2 ) derived from unmethylated sperm; (H 3 ) derived from methylated sperm. The positive signal for HIV-1 P24 Gag protein was observed in (H 2 ) and (H 3 ). The results showed that HIV-1 gag was able to express its protein in the 2-cell embryos and not in the spermatozoa.
    Figure Legend Snippet: LTR methylation and gag expression of HIV-1 in the plasmid-transfected spermatozoa and sperm-derived zygotes and 2-cell embryos. A: The detection of LTR methylation by BSP and Thymine/Adenine cloning while transfection with unmethylated plasmid. The methylation status of 15 clones for each sample is presented; each column represents one CpG position in the U3-R region, with each circle in the column indicating either cytosine (open circles) or methyl cytosine (filled circles), the same below. (A 1 ) plasmid; (A 2 ) spermatozoa; (A3) zygotes; (A 4 ) 2-cell embryos. B: The detection of gag transcription while transfection with unmethylated plasmid (B 1 ) the results of RT-PCR. M: Marker; 1: positive control; 2: spermatozoa; 3: -RT; 4: -T; 5: human β-actin. (B 2 ) the results of first-round of nested RT-PCR. M: Marker; 1: positive control; 2∶2-cell embryos; 3: -RT; 4: -T; 5: hamster β-actin. (B 3 ) the results of second-round of nested RT-PCR. M: Marker; 1: positive control; 2: the first round product; 3: -RT; 4: -T. The results showed a clear correlation between LTR methylation and gag transcription of HIV-1 either in spermatozoa or in 2-cell embryos. C: The detection of LTR methylation while transfection with methylated plasmid. (C 1 ) plasmid; (C 2 ) spermatozoa; (C 3 ) zygotes; (C 4 ) 2-cell embryos. D: The detection of gag transcription while transfection with methylated plasmid. (D 1 ) the results of first-round of nested RT-PCR. M: Marker; 1: positive control; 2: spermatozoa; 3: -RT; 4: -T; 5: human β-actin. (D 2 ) The results of second-round. M: Marker; 1: positive control; 2: the first round product; 3: -RT; 4: -T. (D 3 ) the results of first-round of nested RT-PCR M: Marker; 1: positive control; 2∶2-cell embryos; 3: -RT; 4: -T; 5: hamster β-actin. (D 4 ) the results of second-round. M: Marker; 1: positive control; 2: the first-round product; 3: -RT; 4: -T. The results showed a clear correlation between LTR methylation and gag transcription of HIV-1 either in spermatozoa or in 2-cell embryos. E: CMV methylation in the spermatozoa transfected with methylated pIRES2-EGFP and sperm-derived zygotes and 2-cell embryos. The detection of CpG methylation by BSP and Thymine/Adenine cloning. (E 1 ) m-pIRES2-EGFP; (E 2 ) spermatozoa; (E3) zygotes; (E4) 2-cell embryos. The results showed that demethylation of some CpG sites in CMV promoter has already occurred in the spermatozoa and almost all CpG sites were demethylated in zygotes and 2-cell embryos. F: The effects of SAM on methylation of CpG sites in HIV-1 LTR in the 2-cell embryos. (F 1 ) SAM treatment; (F 2 ) SAM-free treatment. The results showed that the methylation rate of HIV-1 LTR in 2-cell embryos markedly increased after treatment with SAM. G: The detection of HIV-1 Gag translation in spermatozoa by IF assay. (G 1 ) transfection with unmethylated plasmid; (G 2 ) transfection with methylated plasmid; (G3) negative control; (G 4 ) positive control. No positive signal for HIV-1 P24 Gag protein was observed in (G 1 ), (G 2 ) and (G 3 ), and the strong signals for sperm protein were visible in (G 4 ). H: The detection of Gag translation in 2-cell embryos by IF assay. (H 1 ) negative control; (H 2 ) derived from unmethylated sperm; (H 3 ) derived from methylated sperm. The positive signal for HIV-1 P24 Gag protein was observed in (H 2 ) and (H 3 ). The results showed that HIV-1 gag was able to express its protein in the 2-cell embryos and not in the spermatozoa.

    Techniques Used: Methylation, Expressing, Plasmid Preparation, Transfection, Derivative Assay, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Positive Control, CpG Methylation Assay, Negative Control

    25) Product Images from "Methyltransferase-directed covalent coupling of fluorophores to DNA covalent coupling of fluorophores to DNA †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04229eClick here for additional data file."

    Article Title: Methyltransferase-directed covalent coupling of fluorophores to DNA covalent coupling of fluorophores to DNA †Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc04229eClick here for additional data file.

    Journal: Chemical Science

    doi: 10.1039/c6sc04229e

    Two step labelling scheme. In the first step, the DNA is transalkylated using a DNA MTase and an AdoMet analogue which results in the DNA molecules carrying functional groups at known loci. Amine (R1) functionalized DNA can be coupled to a fluorophore using NHS ester chemistry, whereas for azide (R2) functionalized DNA this can be done using CuAAC and strain-promoted azide–alkyne cycloaddition (SPAAC) reactions.
    Figure Legend Snippet: Two step labelling scheme. In the first step, the DNA is transalkylated using a DNA MTase and an AdoMet analogue which results in the DNA molecules carrying functional groups at known loci. Amine (R1) functionalized DNA can be coupled to a fluorophore using NHS ester chemistry, whereas for azide (R2) functionalized DNA this can be done using CuAAC and strain-promoted azide–alkyne cycloaddition (SPAAC) reactions.

    Techniques Used: Functional Assay

    26) Product Images from "Epigenetic Regulation of PLIN1 in Obese Women and its Relation to Lipolysis"

    Article Title: Epigenetic Regulation of PLIN1 in Obese Women and its Relation to Lipolysis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09232-y

    PLIN1 promoter methylation inhibits promoter activity. PLIN1 promoter activity is decreased after (hatched bar) versus without (black bar) DNA methylation by SssI methyltransferase. hMSCs were transfected with methylated and unmethylated pCpGL- PLIN1 plasmid. As negative control, cells were transfected with empty vector, pCpGL-basic. Each sample was prepared in quadruplicates and the experiment was repeated three times. Y axis is the ratio between firefly and renilla luciferase. Renilla luciferase is expressed from a second plasmid as an internal control. RLU = Relative luciferase units. *** P
    Figure Legend Snippet: PLIN1 promoter methylation inhibits promoter activity. PLIN1 promoter activity is decreased after (hatched bar) versus without (black bar) DNA methylation by SssI methyltransferase. hMSCs were transfected with methylated and unmethylated pCpGL- PLIN1 plasmid. As negative control, cells were transfected with empty vector, pCpGL-basic. Each sample was prepared in quadruplicates and the experiment was repeated three times. Y axis is the ratio between firefly and renilla luciferase. Renilla luciferase is expressed from a second plasmid as an internal control. RLU = Relative luciferase units. *** P

    Techniques Used: Methylation, Activity Assay, DNA Methylation Assay, Transfection, Plasmid Preparation, Negative Control, Luciferase

    27) Product Images from "HP1 reshapes the nucleosome core to promote phase separation of heterochromatin"

    Article Title: HP1 reshapes the nucleosome core to promote phase separation of heterochromatin

    Journal: Nature

    doi: 10.1038/s41586-019-1669-2

    Characterization of H3 Enzymatic Methylation by Dim5. Methylation of Histone H3 was followed by online LC-MS-ETD-MS of the intact proteins. (a) Charge state envelope of untreated (left panel) and methylated (right panel) H3. The lower panel, focused on a single charge state, shows disappearance of the starting material and the formation of higher mass species, spaced 14 Da apart. (b) ETD-MS of precursor ions corresponding to unmethylated H3 (top), H3-K9me3 (middle) and H3-K9me3-K18me3 (bottom). The rationale for these assignments is based on the precursor mass values and by the product ions. Z-ions (purple) do not change between the three spectra, while the pattern of c-ions (blue) are mass shifted by 42 Da and charge-state shifted by 1+ at C9 between the top and middle/bottom panels, and again at C18 between the middle and lower panels. These assignments are further validated by bottom-up proteomics analysis of Lys-C digested samples (not shown). (c) The precursor ion spectra in (a) were deconvoluted using Xtract, which models both the charge states and the isotope distributions. Deconvoluted MH+ values are consistent with multiple methylation states (due to the difficulty of modeling isotope distributions from large proteins, particularly as there is some underlying oxidation, Xtract sometimes picks the wrong monoisotope). Deconvoluted intensities show that the enzymatically treated sample (right) contains no significant unmethylated H3 or mono- and di-methylated H3K9. While, 100% of analyzed sample is tri-methylated at H3K9, additional methylations occur at H3K18 as noted above. The sample analyzed here was used to carry out all the experiments to minimize variability.
    Figure Legend Snippet: Characterization of H3 Enzymatic Methylation by Dim5. Methylation of Histone H3 was followed by online LC-MS-ETD-MS of the intact proteins. (a) Charge state envelope of untreated (left panel) and methylated (right panel) H3. The lower panel, focused on a single charge state, shows disappearance of the starting material and the formation of higher mass species, spaced 14 Da apart. (b) ETD-MS of precursor ions corresponding to unmethylated H3 (top), H3-K9me3 (middle) and H3-K9me3-K18me3 (bottom). The rationale for these assignments is based on the precursor mass values and by the product ions. Z-ions (purple) do not change between the three spectra, while the pattern of c-ions (blue) are mass shifted by 42 Da and charge-state shifted by 1+ at C9 between the top and middle/bottom panels, and again at C18 between the middle and lower panels. These assignments are further validated by bottom-up proteomics analysis of Lys-C digested samples (not shown). (c) The precursor ion spectra in (a) were deconvoluted using Xtract, which models both the charge states and the isotope distributions. Deconvoluted MH+ values are consistent with multiple methylation states (due to the difficulty of modeling isotope distributions from large proteins, particularly as there is some underlying oxidation, Xtract sometimes picks the wrong monoisotope). Deconvoluted intensities show that the enzymatically treated sample (right) contains no significant unmethylated H3 or mono- and di-methylated H3K9. While, 100% of analyzed sample is tri-methylated at H3K9, additional methylations occur at H3K18 as noted above. The sample analyzed here was used to carry out all the experiments to minimize variability.

    Techniques Used: Methylation, Liquid Chromatography with Mass Spectroscopy

    28) Product Images from "Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells"

    Article Title: Simultaneous measurement of chromatin accessibility, DNA methylation, and nucleosome phasing in single cells

    Journal: eLife

    doi: 10.7554/eLife.23203

    Heatmaps of average GpC and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates MTase treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009
    Figure Legend Snippet: Heatmaps of average GpC and CpG methylation across DHS regions in GM12878 cells. Each row represents data from an individual cell, both treated and control samples are plotted together. Cells were grouped using hierarchical clustering based on GpC methylation (left) and CpG methylation (right) within 2 kb regions around DHSs. As expected GpC methylation clearly separates MTase treated and untreated samples. Endogenous CpG methylation does not differ systematically between MTase treated and untreated samples. DOI: http://dx.doi.org/10.7554/eLife.23203.009

    Techniques Used: Gel Permeation Chromatography, CpG Methylation Assay, Methylation

    Schematic of experimental set up. A total of 19 individual cells from GM12878 were profiled in this study, 12 of these cells were exposed to GpC MTase and seven were subjected to the same process without exposure to MTase. For K562 11 cells were profiled all of which were subjected to GpC MTase treatment. DOI: http://dx.doi.org/10.7554/eLife.23203.005
    Figure Legend Snippet: Schematic of experimental set up. A total of 19 individual cells from GM12878 were profiled in this study, 12 of these cells were exposed to GpC MTase and seven were subjected to the same process without exposure to MTase. For K562 11 cells were profiled all of which were subjected to GpC MTase treatment. DOI: http://dx.doi.org/10.7554/eLife.23203.005

    Techniques Used: Gel Permeation Chromatography

    Cumulative distribution of average GpC methylation in DHSs in GM12878 and K562 cells. Plot of cumulative distribution of GpC methylation for individual GM12878 and K562 cells at DHSs with at least four covered GpC. GM12878 and K562 cells exposed to GpC MTase show similar distributions. About 50% of all cells show no or low methylation (
    Figure Legend Snippet: Cumulative distribution of average GpC methylation in DHSs in GM12878 and K562 cells. Plot of cumulative distribution of GpC methylation for individual GM12878 and K562 cells at DHSs with at least four covered GpC. GM12878 and K562 cells exposed to GpC MTase show similar distributions. About 50% of all cells show no or low methylation (

    Techniques Used: Gel Permeation Chromatography, Methylation

    Average CpG and GpC methylation levels in single cells. Boxplots representing the methylation level at CpG and GpC dinucleotides for groups of cells (GM12878 w/ and w/o MTase,K562 w/ MTase). GM12878 and K562 cells show different levels of CpG methylation. The difference in CpG methylation between GM12878 w/o MTase and GM12878 w/ MTase treatment was largely driven by two cells. These cells were kept as no other criterion suggested their removal. GpC MTase treated cells shows a clear enrichment of GpC methylation while GM12878 cells not exposed to MTase do not show levels above 1%. These might reflect incomplete conversion, minimal cross-contamination during the parallel preparation, or activity of endogenous methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.23203.008
    Figure Legend Snippet: Average CpG and GpC methylation levels in single cells. Boxplots representing the methylation level at CpG and GpC dinucleotides for groups of cells (GM12878 w/ and w/o MTase,K562 w/ MTase). GM12878 and K562 cells show different levels of CpG methylation. The difference in CpG methylation between GM12878 w/o MTase and GM12878 w/ MTase treatment was largely driven by two cells. These cells were kept as no other criterion suggested their removal. GpC MTase treated cells shows a clear enrichment of GpC methylation while GM12878 cells not exposed to MTase do not show levels above 1%. These might reflect incomplete conversion, minimal cross-contamination during the parallel preparation, or activity of endogenous methyltransferases. DOI: http://dx.doi.org/10.7554/eLife.23203.008

    Techniques Used: Gel Permeation Chromatography, Methylation, CpG Methylation Assay, Activity Assay

    29) Product Images from "RNA matchmaking remodels lncRNA structure and promotes PRC2 activity"

    Article Title: RNA matchmaking remodels lncRNA structure and promotes PRC2 activity

    Journal: bioRxiv

    doi: 10.1101/2020.04.13.040071

    Duplex RNA promotes PRC2 activity. a , Native gel of di-nucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40-bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. b , Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4 and AEBP2, analyzed by SDS-PAGE and stained with Coomassie blue. c , Histone methyltransferase assay (HMTase assay) was performed with recombinant PRC2 complex, di-nucleosomes, S-Adenosylmethionine (SAM) with and without the co-factor JARID2 (amino acids 119-574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. d , Native 0.5X TBE gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration.
    Figure Legend Snippet: Duplex RNA promotes PRC2 activity. a , Native gel of di-nucleosomes reconstituted via salt dialysis using a DNA template containing two 601 sequences surrounding 40-bp of linker DNA. DNA and nucleosome samples were run on a 5% native polyacrylamide gel and stained with SYBR Gold. b , Recombinant human PRC2 complex includes SUZ12, EZH2, EED, RBBP4 and AEBP2, analyzed by SDS-PAGE and stained with Coomassie blue. c , Histone methyltransferase assay (HMTase assay) was performed with recombinant PRC2 complex, di-nucleosomes, S-Adenosylmethionine (SAM) with and without the co-factor JARID2 (amino acids 119-574). PRC2 activity was determined by SDS-PAGE followed by H3K27me3 and total H3 Western blot analysis. d , Native 0.5X TBE gel of RNA annealing titration with HOTAIR forward and reverse fragments to show formation of dsRNA. HMTase assay with annealed HOTAIR dsRNA titration.

    Techniques Used: Activity Assay, Staining, Recombinant, SDS Page, HMT Assay, Western Blot, Titration

    30) Product Images from "In situ structure of rotavirus VP1 RNA-dependent RNA polymerase"

    Article Title: In situ structure of rotavirus VP1 RNA-dependent RNA polymerase

    Journal: bioRxiv

    doi: 10.1101/605063

    Cryo-EM images of rotavirus particles in vitreous ice after incubation with nucleotides. The images shown are low-pass filtered at 15 Å resolution. The scale bar is 100 nm. (a) TLPs only occasionally showed RNA strands emerging from viruses (indicated by arrowheads) and the RdRp reconstruction did not show any RNA density in the active site. (b) DLPs with emerging RNA strands. Released transcripts are also visible in the background.
    Figure Legend Snippet: Cryo-EM images of rotavirus particles in vitreous ice after incubation with nucleotides. The images shown are low-pass filtered at 15 Å resolution. The scale bar is 100 nm. (a) TLPs only occasionally showed RNA strands emerging from viruses (indicated by arrowheads) and the RdRp reconstruction did not show any RNA density in the active site. (b) DLPs with emerging RNA strands. Released transcripts are also visible in the background.

    Techniques Used: Incubation

    31) Product Images from "Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations"

    Article Title: Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2′-O methylations

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1612444114

    IFIT1 PPP- and m7Gppp- binding mechanism. ( A ) Simulated annealing 2 F o – F c omit map of the PPP moiety contoured at 1σ. ( B ) Cartoon/stick representation of residues making specific contacts with the triphosphate group from PPP-RNA–bound
    Figure Legend Snippet: IFIT1 PPP- and m7Gppp- binding mechanism. ( A ) Simulated annealing 2 F o – F c omit map of the PPP moiety contoured at 1σ. ( B ) Cartoon/stick representation of residues making specific contacts with the triphosphate group from PPP-RNA–bound

    Techniques Used: Binding Assay

    IFIT1 mRNA cap-binding mechanism. ( A ) The IFIT1 PPP (blue) adopts an extended conformation compared with the “bent” IFIT5 PPP (pink). The γ-phosphate from PPP–RNA-bound IFIT1 points toward the nearby unoccupied cap-binding
    Figure Legend Snippet: IFIT1 mRNA cap-binding mechanism. ( A ) The IFIT1 PPP (blue) adopts an extended conformation compared with the “bent” IFIT5 PPP (pink). The γ-phosphate from PPP–RNA-bound IFIT1 points toward the nearby unoccupied cap-binding

    Techniques Used: Binding Assay

    32) Product Images from "Hypermethylated LTR retrotransposon exhibits enhancer activity"

    Article Title: Hypermethylated LTR retrotransposon exhibits enhancer activity

    Journal: Epigenetics

    doi: 10.1080/15592294.2017.1289300

    The ERV-9 LTR retrotransposon in the human β -globin gene locus is hypermethylated in erythroid and non-erythroid cells. (a) Map of the ERV-9 LTR and the human β -globin gene locus. Hatched box, ERV-9 LTR. Solid bars, hypersensitive sites of the β -globin (b) Upper panel: locations of the 65 CpGs, numbered 1–65 in CGI (#1–40), E and P (#41–45), and R-U5 (46–65). Lower panels: Methylation status of the LTR in K562, Day 13 erythroid progenitor and neuronal progenitor cells determined by BS-Sseq and BS-seq. Solid circles: methylated mCpGs; unfilled circles: unmethylated CpGs. In NeurP cells, the height of each bar represented the extent of mC in each CpG. The enhancer CpGs #41–43 were mapped by BS-seq in NeurP cells but not in K562 and EryP cells by BS-Sseq (see text). Percentages: Percentages of methylated mCpGs in CGI, E+P, and R-U5 regions.
    Figure Legend Snippet: The ERV-9 LTR retrotransposon in the human β -globin gene locus is hypermethylated in erythroid and non-erythroid cells. (a) Map of the ERV-9 LTR and the human β -globin gene locus. Hatched box, ERV-9 LTR. Solid bars, hypersensitive sites of the β -globin (b) Upper panel: locations of the 65 CpGs, numbered 1–65 in CGI (#1–40), E and P (#41–45), and R-U5 (46–65). Lower panels: Methylation status of the LTR in K562, Day 13 erythroid progenitor and neuronal progenitor cells determined by BS-Sseq and BS-seq. Solid circles: methylated mCpGs; unfilled circles: unmethylated CpGs. In NeurP cells, the height of each bar represented the extent of mC in each CpG. The enhancer CpGs #41–43 were mapped by BS-seq in NeurP cells but not in K562 and EryP cells by BS-Sseq (see text). Percentages: Percentages of methylated mCpGs in CGI, E+P, and R-U5 regions.

    Techniques Used: Methylation

    Hypermethylated ERV-9 bound cognate TFs to assemble the LTR-pol II transcription complex. (a) Wt LTR-dmGFP and SssI LTR-dmGFP transfected into K562 cells bound key TFs, pol II, H3K4me1 and MeCP2 as shown by ChIP-qPCR. CGI and E+P (left and right panels): ChIP-qPCR with primers specific for CGI and E+P regions. Values were averages of 2 independent antibody pull-down reactions; the error bars represent range of the values. (b) Top: Map of the short arm of chromosome 11, chr11p; left to right direction: telomere to centromere. Vertical bar: location of the 100 kb β -globin gene locus in 11p15.4 on the minus strand. Coordinates in megabases (Mbs) increase from telomere to centromere. Middle: ChIP-seq of β -globin gene locus in Day 5 human erythroid progenitor cells from GSE 36985 and 52924, and in K562 cells from ENCODE viewed with UCSC Genome Browser. Double vertical lines mark the ERV-9 LTR region in each track. Bottom: RNA-seq of β -globin and K562 and neuronal progenitor cells from ENCODE.
    Figure Legend Snippet: Hypermethylated ERV-9 bound cognate TFs to assemble the LTR-pol II transcription complex. (a) Wt LTR-dmGFP and SssI LTR-dmGFP transfected into K562 cells bound key TFs, pol II, H3K4me1 and MeCP2 as shown by ChIP-qPCR. CGI and E+P (left and right panels): ChIP-qPCR with primers specific for CGI and E+P regions. Values were averages of 2 independent antibody pull-down reactions; the error bars represent range of the values. (b) Top: Map of the short arm of chromosome 11, chr11p; left to right direction: telomere to centromere. Vertical bar: location of the 100 kb β -globin gene locus in 11p15.4 on the minus strand. Coordinates in megabases (Mbs) increase from telomere to centromere. Middle: ChIP-seq of β -globin gene locus in Day 5 human erythroid progenitor cells from GSE 36985 and 52924, and in K562 cells from ENCODE viewed with UCSC Genome Browser. Double vertical lines mark the ERV-9 LTR region in each track. Bottom: RNA-seq of β -globin and K562 and neuronal progenitor cells from ENCODE.

    Techniques Used: Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, RNA Sequencing Assay

    33) Product Images from "The use of Multiple Displacement Amplified DNA as a control for Methylation Specific PCR, Pyrosequencing, Bisulfite Sequencing and Methylation-Sensitive Restriction Enzyme PCR"

    Article Title: The use of Multiple Displacement Amplified DNA as a control for Methylation Specific PCR, Pyrosequencing, Bisulfite Sequencing and Methylation-Sensitive Restriction Enzyme PCR

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-91

    Bisulfite sequencing results for MMP-14 . a) When genomic DNA (lane 1) and bisulfite treated mDNA (lane 2) and uDNA (lane 3) were used as template for sequencing in combination with primers for MMP-14 a PCR product was generated for all samples but not the negative control (lane 4). Sequencing results for b) non-amplified genomic DNA, c) uDNA and d) mDNA demonstrate that MDA treatment generates DNA (uDNA) free of all methylation as when it is bisulfite treated all cytosine are converted to thymine [indicated by asterix (*)]. In addition, sequencing also demonstrates that M.SssI treatment (mDNA) methylates CpG motifs as cytosines are retained when present as part of a CpG dinucleotide (indicated by *).
    Figure Legend Snippet: Bisulfite sequencing results for MMP-14 . a) When genomic DNA (lane 1) and bisulfite treated mDNA (lane 2) and uDNA (lane 3) were used as template for sequencing in combination with primers for MMP-14 a PCR product was generated for all samples but not the negative control (lane 4). Sequencing results for b) non-amplified genomic DNA, c) uDNA and d) mDNA demonstrate that MDA treatment generates DNA (uDNA) free of all methylation as when it is bisulfite treated all cytosine are converted to thymine [indicated by asterix (*)]. In addition, sequencing also demonstrates that M.SssI treatment (mDNA) methylates CpG motifs as cytosines are retained when present as part of a CpG dinucleotide (indicated by *).

    Techniques Used: Methylation Sequencing, Sequencing, Polymerase Chain Reaction, Generated, Negative Control, Amplification, Multiple Displacement Amplification, Methylation

    Methylation-Sensitive Restriction Enzyme PCR for MMP-1 and MMP-3 . a) PCR using primers spanning the restriction site for MMP-1 and MMP-3 gave a PCR product with mDNA but not with uDNA. In contrast, undigested samples gave PCR products for both mDNA and uDNA. b) PCR using digested DNA from MDA-MB231 (231), MDA-MB468 (468) and HFFF2 identified that the CpG motif is methylated for all three cell lines in the MMP-3 amplicon, but only for MDA-MB468 (468) and HFFF2 for the MMP-1 amplicon, with the MDA-MB231 (231) being unmethylated. However, the undigested DNA gave a PCR product with all three cells lines.
    Figure Legend Snippet: Methylation-Sensitive Restriction Enzyme PCR for MMP-1 and MMP-3 . a) PCR using primers spanning the restriction site for MMP-1 and MMP-3 gave a PCR product with mDNA but not with uDNA. In contrast, undigested samples gave PCR products for both mDNA and uDNA. b) PCR using digested DNA from MDA-MB231 (231), MDA-MB468 (468) and HFFF2 identified that the CpG motif is methylated for all three cell lines in the MMP-3 amplicon, but only for MDA-MB468 (468) and HFFF2 for the MMP-1 amplicon, with the MDA-MB231 (231) being unmethylated. However, the undigested DNA gave a PCR product with all three cells lines.

    Techniques Used: Methylation, Polymerase Chain Reaction, Multiple Displacement Amplification, Amplification

    34) Product Images from "Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation"

    Article Title: Methylation of human eukaryotic elongation factor alpha (eEF1A) by a member of a novel protein lysine methyltransferase family modulates mRNA translation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx432

    Gene structure and targeting strategy for the endothelin converting enzyme 2 ( ECE2 ) /EEF1AKMT4 locus. ( A ) Topology diagram of the 7BS MTase fold. α-helices and β-strands are depicted as grey boxes (denoted Z and A–E) and black arrows (denoted 1–7), respectively, and annotated according to previously established nomenclature ( 50 ). ( B ) Organization and annotation of the human ECE2 locus. Top, current annotation of the ECE2 locus. Regions in the genomic DNA corresponding to annotated exons of ECE2 (located on the forward stand) and CAMK2N2 (located on the reverse strand) are indicated in blue and green, respectively (based on the annotated gene structure according to Ensembl (assembly GRCh37)). Middle, tracks representing spliced expressed sequence tags (ESTs) exported from the UCSC genome browser. Bottom, alternative gene model supported by the EST data. ( C ) Gene targeting strategy and disruption of the EEF1AKMT4 locus by CRISPR/Cas9 technology. Top, schematic representation of the EEF1AKMT4 gene. Exons and introns are represented by boxes (gray, coding region; white, untranslated regions) and lines, respectively. An arrow indicates the region targeted by CRISPR. Bottom, DNA and protein sequence of targeted region of EEF1AKMT4 locus in HAP-1 wild-type (WT) and EEF1AKMT4 knockout (KO) cells. The conserved motif Post I is indicated by a rectangle and the last residue of the motif, D88, is shown in red. ( D ) Structural support for an involvement of D88 in coordinating AdoMet binding. The figure is based on a previously published structure (PDB ID: 2PXX) of eEF1A-KMT4 in complex with S -adenosylhomocysteine (AdoHcy; the unmethylated counterpart of AdoMet) ( 32 ). AdoHcy and D88 are represented as sticks and the second β-strand (β2) is represented as a cartoon. Possible hydrogen bonds between the carboxyl group of D88 and the ribose moiety of AdoHcy are shown (dashed lines). The figure was generated using the PyMOL Molecular Graphics System, Version 1.3 (Schrodinger, LLC).
    Figure Legend Snippet: Gene structure and targeting strategy for the endothelin converting enzyme 2 ( ECE2 ) /EEF1AKMT4 locus. ( A ) Topology diagram of the 7BS MTase fold. α-helices and β-strands are depicted as grey boxes (denoted Z and A–E) and black arrows (denoted 1–7), respectively, and annotated according to previously established nomenclature ( 50 ). ( B ) Organization and annotation of the human ECE2 locus. Top, current annotation of the ECE2 locus. Regions in the genomic DNA corresponding to annotated exons of ECE2 (located on the forward stand) and CAMK2N2 (located on the reverse strand) are indicated in blue and green, respectively (based on the annotated gene structure according to Ensembl (assembly GRCh37)). Middle, tracks representing spliced expressed sequence tags (ESTs) exported from the UCSC genome browser. Bottom, alternative gene model supported by the EST data. ( C ) Gene targeting strategy and disruption of the EEF1AKMT4 locus by CRISPR/Cas9 technology. Top, schematic representation of the EEF1AKMT4 gene. Exons and introns are represented by boxes (gray, coding region; white, untranslated regions) and lines, respectively. An arrow indicates the region targeted by CRISPR. Bottom, DNA and protein sequence of targeted region of EEF1AKMT4 locus in HAP-1 wild-type (WT) and EEF1AKMT4 knockout (KO) cells. The conserved motif Post I is indicated by a rectangle and the last residue of the motif, D88, is shown in red. ( D ) Structural support for an involvement of D88 in coordinating AdoMet binding. The figure is based on a previously published structure (PDB ID: 2PXX) of eEF1A-KMT4 in complex with S -adenosylhomocysteine (AdoHcy; the unmethylated counterpart of AdoMet) ( 32 ). AdoHcy and D88 are represented as sticks and the second β-strand (β2) is represented as a cartoon. Possible hydrogen bonds between the carboxyl group of D88 and the ribose moiety of AdoHcy are shown (dashed lines). The figure was generated using the PyMOL Molecular Graphics System, Version 1.3 (Schrodinger, LLC).

    Techniques Used: Sequencing, CRISPR, Knock-Out, Binding Assay, Generated

    35) Product Images from "MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells"

    Article Title: MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.11.027

    Expression of M.EcoGII-Lamin B1 to Target M.EcoGII to the Nuclear Envelope (A–C) Representative dot blot of genomic DNA from HeLa 1.2.11 cells induced (+) or induced not (−) to express M.EcoGII-v5-Lamin B1 (A) or v5-M.EcoGII (B). The membrane was probed with a m6A antibody. The normalized intensities are shown. (B and D) Example of immunofluorescence of HeLa 1.2.11 cells induced (+) or not induced (–) to express M.EcoGII-v5-Lamin B1 (B) or v5-M.EcoGII (D). V5 tag (left panel) or m6A (right panel) (red), Lamin A/C (green), DNA (blue), and merge. Scale bar, 10 μm. The enlarged part of the nucleus of m6A and Lamin A/C staining is shown. Scale bar, 1 μm.
    Figure Legend Snippet: Expression of M.EcoGII-Lamin B1 to Target M.EcoGII to the Nuclear Envelope (A–C) Representative dot blot of genomic DNA from HeLa 1.2.11 cells induced (+) or induced not (−) to express M.EcoGII-v5-Lamin B1 (A) or v5-M.EcoGII (B). The membrane was probed with a m6A antibody. The normalized intensities are shown. (B and D) Example of immunofluorescence of HeLa 1.2.11 cells induced (+) or not induced (–) to express M.EcoGII-v5-Lamin B1 (B) or v5-M.EcoGII (D). V5 tag (left panel) or m6A (right panel) (red), Lamin A/C (green), DNA (blue), and merge. Scale bar, 10 μm. The enlarged part of the nucleus of m6A and Lamin A/C staining is shown. Scale bar, 1 μm.

    Techniques Used: Expressing, Dot Blot, Immunofluorescence, Staining

    Activity of M.EcoGII and Detection of m6A-DNA (A) Dot blot of fragmented genomic DNA (input) or DNA immunoprecipitated with a m6A antibody (m6A-IP) from E. coli ER2796, E. coli MG1655, or U2OS cells expressing or not expressing M.EcoGII. The membranes were probed with a m6A antibody. Quantification of m6A enrichment in the immunoprecipitated fractions relative to the input material is shown from three independent replicates (R1, R2, and R3; mean ± SD). (B) Dot blot with increasing amounts of sheared genomic DNA extracted from U2OS cells expressing or not expressing M.EcoGII. The membrane was probed with a m6A antibody. The graph represents the mean intensity ± SD of the m6A signal from two independent experiments (R1 and R2). (C) Dot blot of plasmid DNA in vitro methylated with increasing amounts of M.EcoGII recombinant enzyme. 500 ng of DNA from each reaction was loaded on a membrane probed with a m6A antibody. The normalized intensity relative to the unmethylated plasmid is shown.
    Figure Legend Snippet: Activity of M.EcoGII and Detection of m6A-DNA (A) Dot blot of fragmented genomic DNA (input) or DNA immunoprecipitated with a m6A antibody (m6A-IP) from E. coli ER2796, E. coli MG1655, or U2OS cells expressing or not expressing M.EcoGII. The membranes were probed with a m6A antibody. Quantification of m6A enrichment in the immunoprecipitated fractions relative to the input material is shown from three independent replicates (R1, R2, and R3; mean ± SD). (B) Dot blot with increasing amounts of sheared genomic DNA extracted from U2OS cells expressing or not expressing M.EcoGII. The membrane was probed with a m6A antibody. The graph represents the mean intensity ± SD of the m6A signal from two independent experiments (R1 and R2). (C) Dot blot of plasmid DNA in vitro methylated with increasing amounts of M.EcoGII recombinant enzyme. 500 ng of DNA from each reaction was loaded on a membrane probed with a m6A antibody. The normalized intensity relative to the unmethylated plasmid is shown.

    Techniques Used: Activity Assay, Dot Blot, Immunoprecipitation, Expressing, Plasmid Preparation, In Vitro, Methylation, Recombinant

    M.EcoGII Targeted to the Nuclear Envelope Can Contact and Methylate Telomeric DNA (A–C) Representative dot blot of captured telomeric DNA from asynchronous HeLa 1.2.11 induced to express the indicated vectors for 24 hr (A) or G1/S-arrested cells induced for 8 hr (C). DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents the normalized intensities relative to input (mean ± SD). (B) Heatmap of the number of reads per million obtained at individual chromosome ends in HeLa 1.2.11 cells expressing M.EcoGII-Lamin B1 (M-LB1). The log2 M.EcoGII-LB1/M.EcoGII ratio is shown. Chromosome ends with positive enrichment are highlighted in red. A box with a cross represents a bin without an associated DNA sequence and therefore excluded from the analysis. (D) Representative DNA-IF of G1/S-arrested HeLa 1.2.11 cells induced to express the indicated vectors for 8 hr. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm.
    Figure Legend Snippet: M.EcoGII Targeted to the Nuclear Envelope Can Contact and Methylate Telomeric DNA (A–C) Representative dot blot of captured telomeric DNA from asynchronous HeLa 1.2.11 induced to express the indicated vectors for 24 hr (A) or G1/S-arrested cells induced for 8 hr (C). DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents the normalized intensities relative to input (mean ± SD). (B) Heatmap of the number of reads per million obtained at individual chromosome ends in HeLa 1.2.11 cells expressing M.EcoGII-Lamin B1 (M-LB1). The log2 M.EcoGII-LB1/M.EcoGII ratio is shown. Chromosome ends with positive enrichment are highlighted in red. A box with a cross represents a bin without an associated DNA sequence and therefore excluded from the analysis. (D) Representative DNA-IF of G1/S-arrested HeLa 1.2.11 cells induced to express the indicated vectors for 8 hr. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm.

    Techniques Used: Dot Blot, Expressing, Sequencing

    M.EcoGII Can Methylate GATC-Free DNA Regions and Is Specific to Its Region of Targeting (A) Dot blot with 500 ng of oligonucleotides corresponding to the C-rich strand of telomeric repeats (TelC), the G-rich strand of telomeric repeats (TelG), and scramble (Scr TelC and Scr TelG) before or after in vitro methylation using recombinant M.EcoGII. The number of adenine present in the sequences is indicated. The membrane was probed with a m6A antibody, and the relative signal intensity was measured as indicated. (B) Representative immunostaining and FISH of HeLa 1.2.11 cells transduced with the indicated vectors. V5 tag (green); TelC, telomeres (magenta); CEN, centromeres (red), DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (C) Representative dot blot of captured telomeric DNA (T) from HeLa 1.2.11 cells expressing the indicated vectors. DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents normalized intensities relative to input (mean ± SD). (D) DNA immunofluorescence (DNA-IF) of HeLa 1.2.11 cells expressing M-TRF1. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (E) Total number of telomeric reads per million from whole-genome sequencing data obtained from HeLa 1.2.11 transduced with the indicated vectors. (F) Number of reads per million from whole-genome sequencing data obtained in M-TRF1 cells relative to M.EcoGII along chromosome ends (mean ± SEM).
    Figure Legend Snippet: M.EcoGII Can Methylate GATC-Free DNA Regions and Is Specific to Its Region of Targeting (A) Dot blot with 500 ng of oligonucleotides corresponding to the C-rich strand of telomeric repeats (TelC), the G-rich strand of telomeric repeats (TelG), and scramble (Scr TelC and Scr TelG) before or after in vitro methylation using recombinant M.EcoGII. The number of adenine present in the sequences is indicated. The membrane was probed with a m6A antibody, and the relative signal intensity was measured as indicated. (B) Representative immunostaining and FISH of HeLa 1.2.11 cells transduced with the indicated vectors. V5 tag (green); TelC, telomeres (magenta); CEN, centromeres (red), DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (C) Representative dot blot of captured telomeric DNA (T) from HeLa 1.2.11 cells expressing the indicated vectors. DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents normalized intensities relative to input (mean ± SD). (D) DNA immunofluorescence (DNA-IF) of HeLa 1.2.11 cells expressing M-TRF1. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (E) Total number of telomeric reads per million from whole-genome sequencing data obtained from HeLa 1.2.11 transduced with the indicated vectors. (F) Number of reads per million from whole-genome sequencing data obtained in M-TRF1 cells relative to M.EcoGII along chromosome ends (mean ± SEM).

    Techniques Used: Dot Blot, In Vitro, Methylation, Recombinant, Immunostaining, Fluorescence In Situ Hybridization, Transduction, Expressing, Immunofluorescence, Sequencing

    Schematic Overview of MadID (A) Left, GATC distribution on human chromosome 1 (hg38 assembly). The blue gradient represents the score for the GATC site within 1 kb genome segments. Magnification from 1 to 0.1 Mb is shown. Green line, telomere T 2 AG 3 sequence; red dashed line, 1 Mb of centromere DNA. Right, smooth scatter graphs of the A+T nucleotide and GATC motif count per 1 kb genome segment (hg38, chromosomes [chr] 1–22, X, and Y). (B) Experimental setup and detection. (1a) DNA methyltransferases catalyze the transfer of a methyl group to DNA. (1b) M.EcoGII is fused to a destabilization domain (DD) for proteasome degradation unless the compound Shield1 is added to stabilize the protein. M.EcoGII is targeted to the nuclear envelope by fusion with Lamin B1, to telomeres by fusion with telomeric repeat binding factor 1 (TRF1), or to centromeres by fusion with CENP-C. Precise targeting of M.EcoGII causes methylation of DNA in the surrounding regions (m6A). (2a) m6A detection in situ by immunostaining with a m6A antibody. (2b) Genome-wide m6A detection by m6A-specific immunoprecipitation (m6A-IP), followed by whole-genome sequencing. (2c) DNA regions of interest can be purified by chromatin immunoprecipitation or probe-based capture techniques, and m6A can be detected on dot blots using the m6A-specific antibody.
    Figure Legend Snippet: Schematic Overview of MadID (A) Left, GATC distribution on human chromosome 1 (hg38 assembly). The blue gradient represents the score for the GATC site within 1 kb genome segments. Magnification from 1 to 0.1 Mb is shown. Green line, telomere T 2 AG 3 sequence; red dashed line, 1 Mb of centromere DNA. Right, smooth scatter graphs of the A+T nucleotide and GATC motif count per 1 kb genome segment (hg38, chromosomes [chr] 1–22, X, and Y). (B) Experimental setup and detection. (1a) DNA methyltransferases catalyze the transfer of a methyl group to DNA. (1b) M.EcoGII is fused to a destabilization domain (DD) for proteasome degradation unless the compound Shield1 is added to stabilize the protein. M.EcoGII is targeted to the nuclear envelope by fusion with Lamin B1, to telomeres by fusion with telomeric repeat binding factor 1 (TRF1), or to centromeres by fusion with CENP-C. Precise targeting of M.EcoGII causes methylation of DNA in the surrounding regions (m6A). (2a) m6A detection in situ by immunostaining with a m6A antibody. (2b) Genome-wide m6A detection by m6A-specific immunoprecipitation (m6A-IP), followed by whole-genome sequencing. (2c) DNA regions of interest can be purified by chromatin immunoprecipitation or probe-based capture techniques, and m6A can be detected on dot blots using the m6A-specific antibody.

    Techniques Used: Sequencing, Binding Assay, Methylation, In Situ, Immunostaining, Genome Wide, Immunoprecipitation, Purification, Chromatin Immunoprecipitation

    36) Product Images from "MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells"

    Article Title: MadID, a Versatile Approach to Map Protein-DNA Interactions, Highlights Telomere-Nuclear Envelope Contact Sites in Human Cells

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2018.11.027

    Expression of M.EcoGII-Lamin B1 to Target M.EcoGII to the Nuclear Envelope (A–C) Representative dot blot of genomic DNA from HeLa 1.2.11 cells induced (+) or induced not (−) to express M.EcoGII-v5-Lamin B1 (A) or v5-M.EcoGII (B). The membrane was probed with a m6A antibody. The normalized intensities are shown. (B and D) Example of immunofluorescence of HeLa 1.2.11 cells induced (+) or not induced (–) to express M.EcoGII-v5-Lamin B1 (B) or v5-M.EcoGII (D). V5 tag (left panel) or m6A (right panel) (red), Lamin A/C (green), DNA (blue), and merge. Scale bar, 10 μm. The enlarged part of the nucleus of m6A and Lamin A/C staining is shown. Scale bar, 1 μm.
    Figure Legend Snippet: Expression of M.EcoGII-Lamin B1 to Target M.EcoGII to the Nuclear Envelope (A–C) Representative dot blot of genomic DNA from HeLa 1.2.11 cells induced (+) or induced not (−) to express M.EcoGII-v5-Lamin B1 (A) or v5-M.EcoGII (B). The membrane was probed with a m6A antibody. The normalized intensities are shown. (B and D) Example of immunofluorescence of HeLa 1.2.11 cells induced (+) or not induced (–) to express M.EcoGII-v5-Lamin B1 (B) or v5-M.EcoGII (D). V5 tag (left panel) or m6A (right panel) (red), Lamin A/C (green), DNA (blue), and merge. Scale bar, 10 μm. The enlarged part of the nucleus of m6A and Lamin A/C staining is shown. Scale bar, 1 μm.

    Techniques Used: Expressing, Dot Blot, Immunofluorescence, Staining

    Activity of M.EcoGII and Detection of m6A-DNA (A) Dot blot of fragmented genomic DNA (input) or DNA immunoprecipitated with a m6A antibody (m6A-IP) from E. coli ER2796, E. coli MG1655, or U2OS cells expressing or not expressing M.EcoGII. The membranes were probed with a m6A antibody. Quantification of m6A enrichment in the immunoprecipitated fractions relative to the input material is shown from three independent replicates (R1, R2, and R3; mean ± SD). (B) Dot blot with increasing amounts of sheared genomic DNA extracted from U2OS cells expressing or not expressing M.EcoGII. The membrane was probed with a m6A antibody. The graph represents the mean intensity ± SD of the m6A signal from two independent experiments (R1 and R2). (C) Dot blot of plasmid DNA in vitro methylated with increasing amounts of M.EcoGII recombinant enzyme. 500 ng of DNA from each reaction was loaded on a membrane probed with a m6A antibody. The normalized intensity relative to the unmethylated plasmid is shown.
    Figure Legend Snippet: Activity of M.EcoGII and Detection of m6A-DNA (A) Dot blot of fragmented genomic DNA (input) or DNA immunoprecipitated with a m6A antibody (m6A-IP) from E. coli ER2796, E. coli MG1655, or U2OS cells expressing or not expressing M.EcoGII. The membranes were probed with a m6A antibody. Quantification of m6A enrichment in the immunoprecipitated fractions relative to the input material is shown from three independent replicates (R1, R2, and R3; mean ± SD). (B) Dot blot with increasing amounts of sheared genomic DNA extracted from U2OS cells expressing or not expressing M.EcoGII. The membrane was probed with a m6A antibody. The graph represents the mean intensity ± SD of the m6A signal from two independent experiments (R1 and R2). (C) Dot blot of plasmid DNA in vitro methylated with increasing amounts of M.EcoGII recombinant enzyme. 500 ng of DNA from each reaction was loaded on a membrane probed with a m6A antibody. The normalized intensity relative to the unmethylated plasmid is shown.

    Techniques Used: Activity Assay, Dot Blot, Immunoprecipitation, Expressing, Plasmid Preparation, In Vitro, Methylation, Recombinant

    M.EcoGII Targeted to the Nuclear Envelope Can Contact and Methylate Telomeric DNA (A–C) Representative dot blot of captured telomeric DNA from asynchronous HeLa 1.2.11 induced to express the indicated vectors for 24 hr (A) or G1/S-arrested cells induced for 8 hr (C). DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents the normalized intensities relative to input (mean ± SD). (B) Heatmap of the number of reads per million obtained at individual chromosome ends in HeLa 1.2.11 cells expressing M.EcoGII-Lamin B1 (M-LB1). The log2 M.EcoGII-LB1/M.EcoGII ratio is shown. Chromosome ends with positive enrichment are highlighted in red. A box with a cross represents a bin without an associated DNA sequence and therefore excluded from the analysis. (D) Representative DNA-IF of G1/S-arrested HeLa 1.2.11 cells induced to express the indicated vectors for 8 hr. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm.
    Figure Legend Snippet: M.EcoGII Targeted to the Nuclear Envelope Can Contact and Methylate Telomeric DNA (A–C) Representative dot blot of captured telomeric DNA from asynchronous HeLa 1.2.11 induced to express the indicated vectors for 24 hr (A) or G1/S-arrested cells induced for 8 hr (C). DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents the normalized intensities relative to input (mean ± SD). (B) Heatmap of the number of reads per million obtained at individual chromosome ends in HeLa 1.2.11 cells expressing M.EcoGII-Lamin B1 (M-LB1). The log2 M.EcoGII-LB1/M.EcoGII ratio is shown. Chromosome ends with positive enrichment are highlighted in red. A box with a cross represents a bin without an associated DNA sequence and therefore excluded from the analysis. (D) Representative DNA-IF of G1/S-arrested HeLa 1.2.11 cells induced to express the indicated vectors for 8 hr. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm.

    Techniques Used: Dot Blot, Expressing, Sequencing

    M.EcoGII Can Methylate GATC-Free DNA Regions and Is Specific to Its Region of Targeting (A) Dot blot with 500 ng of oligonucleotides corresponding to the C-rich strand of telomeric repeats (TelC), the G-rich strand of telomeric repeats (TelG), and scramble (Scr TelC and Scr TelG) before or after in vitro methylation using recombinant M.EcoGII. The number of adenine present in the sequences is indicated. The membrane was probed with a m6A antibody, and the relative signal intensity was measured as indicated. (B) Representative immunostaining and FISH of HeLa 1.2.11 cells transduced with the indicated vectors. V5 tag (green); TelC, telomeres (magenta); CEN, centromeres (red), DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (C) Representative dot blot of captured telomeric DNA (T) from HeLa 1.2.11 cells expressing the indicated vectors. DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents normalized intensities relative to input (mean ± SD). (D) DNA immunofluorescence (DNA-IF) of HeLa 1.2.11 cells expressing M-TRF1. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (E) Total number of telomeric reads per million from whole-genome sequencing data obtained from HeLa 1.2.11 transduced with the indicated vectors. (F) Number of reads per million from whole-genome sequencing data obtained in M-TRF1 cells relative to M.EcoGII along chromosome ends (mean ± SEM).
    Figure Legend Snippet: M.EcoGII Can Methylate GATC-Free DNA Regions and Is Specific to Its Region of Targeting (A) Dot blot with 500 ng of oligonucleotides corresponding to the C-rich strand of telomeric repeats (TelC), the G-rich strand of telomeric repeats (TelG), and scramble (Scr TelC and Scr TelG) before or after in vitro methylation using recombinant M.EcoGII. The number of adenine present in the sequences is indicated. The membrane was probed with a m6A antibody, and the relative signal intensity was measured as indicated. (B) Representative immunostaining and FISH of HeLa 1.2.11 cells transduced with the indicated vectors. V5 tag (green); TelC, telomeres (magenta); CEN, centromeres (red), DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (C) Representative dot blot of captured telomeric DNA (T) from HeLa 1.2.11 cells expressing the indicated vectors. DNA was probed with a m6A antibody (m6A) and a telomeric probe (T 2 AG 3 ). The graph represents normalized intensities relative to input (mean ± SD). (D) DNA immunofluorescence (DNA-IF) of HeLa 1.2.11 cells expressing M-TRF1. m6A (green); TelC, telomeres (magenta); CEN, centromeres (red); DNA (blue); and merge. Scale bar, 10 μm. The percentage of colocalization is shown. (E) Total number of telomeric reads per million from whole-genome sequencing data obtained from HeLa 1.2.11 transduced with the indicated vectors. (F) Number of reads per million from whole-genome sequencing data obtained in M-TRF1 cells relative to M.EcoGII along chromosome ends (mean ± SEM).

    Techniques Used: Dot Blot, In Vitro, Methylation, Recombinant, Immunostaining, Fluorescence In Situ Hybridization, Transduction, Expressing, Immunofluorescence, Sequencing

    Schematic Overview of MadID (A) Left, GATC distribution on human chromosome 1 (hg38 assembly). The blue gradient represents the score for the GATC site within 1 kb genome segments. Magnification from 1 to 0.1 Mb is shown. Green line, telomere T 2 AG 3 sequence; red dashed line, 1 Mb of centromere DNA. Right, smooth scatter graphs of the A+T nucleotide and GATC motif count per 1 kb genome segment (hg38, chromosomes [chr] 1–22, X, and Y). (B) Experimental setup and detection. (1a) DNA methyltransferases catalyze the transfer of a methyl group to DNA. (1b) M.EcoGII is fused to a destabilization domain (DD) for proteasome degradation unless the compound Shield1 is added to stabilize the protein. M.EcoGII is targeted to the nuclear envelope by fusion with Lamin B1, to telomeres by fusion with telomeric repeat binding factor 1 (TRF1), or to centromeres by fusion with CENP-C. Precise targeting of M.EcoGII causes methylation of DNA in the surrounding regions (m6A). (2a) m6A detection in situ by immunostaining with a m6A antibody. (2b) Genome-wide m6A detection by m6A-specific immunoprecipitation (m6A-IP), followed by whole-genome sequencing. (2c) DNA regions of interest can be purified by chromatin immunoprecipitation or probe-based capture techniques, and m6A can be detected on dot blots using the m6A-specific antibody.
    Figure Legend Snippet: Schematic Overview of MadID (A) Left, GATC distribution on human chromosome 1 (hg38 assembly). The blue gradient represents the score for the GATC site within 1 kb genome segments. Magnification from 1 to 0.1 Mb is shown. Green line, telomere T 2 AG 3 sequence; red dashed line, 1 Mb of centromere DNA. Right, smooth scatter graphs of the A+T nucleotide and GATC motif count per 1 kb genome segment (hg38, chromosomes [chr] 1–22, X, and Y). (B) Experimental setup and detection. (1a) DNA methyltransferases catalyze the transfer of a methyl group to DNA. (1b) M.EcoGII is fused to a destabilization domain (DD) for proteasome degradation unless the compound Shield1 is added to stabilize the protein. M.EcoGII is targeted to the nuclear envelope by fusion with Lamin B1, to telomeres by fusion with telomeric repeat binding factor 1 (TRF1), or to centromeres by fusion with CENP-C. Precise targeting of M.EcoGII causes methylation of DNA in the surrounding regions (m6A). (2a) m6A detection in situ by immunostaining with a m6A antibody. (2b) Genome-wide m6A detection by m6A-specific immunoprecipitation (m6A-IP), followed by whole-genome sequencing. (2c) DNA regions of interest can be purified by chromatin immunoprecipitation or probe-based capture techniques, and m6A can be detected on dot blots using the m6A-specific antibody.

    Techniques Used: Sequencing, Binding Assay, Methylation, In Situ, Immunostaining, Genome Wide, Immunoprecipitation, Purification, Chromatin Immunoprecipitation

    37) Product Images from "The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41"

    Article Title: The LspC3–41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3–41

    Journal: BMC Microbiology

    doi: 10.1186/s12866-017-1014-6

    The transformation frequencies of pBU4 into L. sphaericus C3–41 and its derivate mutants. Light gray column: pBU4 untreated; deep gray column: pBU4 methylated with C3–41 CFE; black column: pBU4 methylated with MTase M. Hae III. *, no transformant observed
    Figure Legend Snippet: The transformation frequencies of pBU4 into L. sphaericus C3–41 and its derivate mutants. Light gray column: pBU4 untreated; deep gray column: pBU4 methylated with C3–41 CFE; black column: pBU4 methylated with MTase M. Hae III. *, no transformant observed

    Techniques Used: Transformation Assay, Methylation

    The effect of Bsph_0498 (encoding LspC3–41I) on the restriction role of L. sphaericus C3–41 CFE. Untreated and pre-treated plasmid pBU4 was incubated with CFE and then subjected to restriction assays, and the reaction mixture was analyzed by agarose gel electrophoresis as show in the left three pictures (L) and Southern blot analysis as show in the right three pictures (R). a untreated pBU4. Lane 1, C3–41; lane 2, Δ0498; lane 3, RC0498; lane 4, Hae III digested; lane 5, Untreated. b pBU4 methylated with C3–41 CFE. Lane 1, C3–41; lane 2, Δ0498; lane 3, RC0498; lane 4, Hae III digested; lane 5, Untreated. c pBU4 methylated with MTase M. Hae III. Lane 1, C3–41; lane 2, Δ0498; lane 3, RC0498; lane 4, Hae III digested; lane 5, Untreated. M: DNA marker
    Figure Legend Snippet: The effect of Bsph_0498 (encoding LspC3–41I) on the restriction role of L. sphaericus C3–41 CFE. Untreated and pre-treated plasmid pBU4 was incubated with CFE and then subjected to restriction assays, and the reaction mixture was analyzed by agarose gel electrophoresis as show in the left three pictures (L) and Southern blot analysis as show in the right three pictures (R). a untreated pBU4. Lane 1, C3–41; lane 2, Δ0498; lane 3, RC0498; lane 4, Hae III digested; lane 5, Untreated. b pBU4 methylated with C3–41 CFE. Lane 1, C3–41; lane 2, Δ0498; lane 3, RC0498; lane 4, Hae III digested; lane 5, Untreated. c pBU4 methylated with MTase M. Hae III. Lane 1, C3–41; lane 2, Δ0498; lane 3, RC0498; lane 4, Hae III digested; lane 5, Untreated. M: DNA marker

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Southern Blot, Methylation, Marker

    38) Product Images from "Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation"

    Article Title: Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.8b00683

    Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535
    Figure Legend Snippet: Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535

    Techniques Used: Binding Assay, Methylation

    39) Product Images from "Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line"

    Article Title: Identification and Characterization of Germ Cell Genes Expressed in the F9 Testicular Teratoma Stem Cell Line

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103837

    Methylation status of individual CpG sites in the Tex13 promoter. A. The CpG islands of mouse Tex13 were predicted with the MethPrimer program ( http://www.urogene.org/ methprimer) and are indicated in gray. B. The DNA methylation status of individual CpG sites on the Tex13 promoter was assessed by sodium bisulfite sequencing analysis. Genomic DNA was prepared from F9 and NIH3T3 cells. The black and white circles represent methylated and unmethylated CpGs, respectively. C. Effect of methylation on Tex13 transcriptional activity. Luciferase constructs [pGL3-Basic and Tex13 (−402/+20)] were in vitro methylated or mock-methylated with Sss I methyltransferase, and transfected into F9 cells. Firefly luciferase activity was assessed and normalized with respect to that of Renilla luciferase. Data are shown as relative fold increases compared with the results from mock-methylated pGL3-Basic. The presented values represent the mean ± SD of three independent experiments; ** p
    Figure Legend Snippet: Methylation status of individual CpG sites in the Tex13 promoter. A. The CpG islands of mouse Tex13 were predicted with the MethPrimer program ( http://www.urogene.org/ methprimer) and are indicated in gray. B. The DNA methylation status of individual CpG sites on the Tex13 promoter was assessed by sodium bisulfite sequencing analysis. Genomic DNA was prepared from F9 and NIH3T3 cells. The black and white circles represent methylated and unmethylated CpGs, respectively. C. Effect of methylation on Tex13 transcriptional activity. Luciferase constructs [pGL3-Basic and Tex13 (−402/+20)] were in vitro methylated or mock-methylated with Sss I methyltransferase, and transfected into F9 cells. Firefly luciferase activity was assessed and normalized with respect to that of Renilla luciferase. Data are shown as relative fold increases compared with the results from mock-methylated pGL3-Basic. The presented values represent the mean ± SD of three independent experiments; ** p

    Techniques Used: Methylation, DNA Methylation Assay, Methylation Sequencing, Activity Assay, Luciferase, Construct, In Vitro, Transfection

    Tissue distribution and developmental expression patterns of germ cell-specific genes expressed in F9 cells. A. The expression patterns of nine genes predicted to be testis-specific or -predominant, as assessed in testis, F9 cells and NIH3T3 cells. Glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) was included as a loading control. Except for Cpvl , all of the genes were detected in both testis and F9 cells. It should be noted that the two bands for Dmrt1 in NIH3T3 cells are non-specific, based on in silico investigation and an additional PCR analysis (data not shown). B. The tissue distributions of transcripts were assessed by RT-PCR analysis in various tissues of adult male mice. Complementary DNAs from various mouse tissues were amplified by PCR, with Gapdh included as a loading control. Seven genes were found to be testis-specific or -predominant. C. Developmental expression patterns during spermatogenesis. The stage-specific expression of the genes was determined from mouse testes on different days after birth (days 8, 10, 12, 14, 16, 20, 30, and 56). Abbreviations: PL , preleptotene; L , leptotene; Z , zygotene; P , pachytene; D , diplotene; MI , meiotic division I; and MII , meiotic division II. Complementary DNA from germ cell-lacking testes from W/W v mutant mice was also examined. Consistent with our microarray analysis, Tex13 , Stra8 , Fancd2os , 1700061G19Rik and Triml1 were found to be germ cell-specific.
    Figure Legend Snippet: Tissue distribution and developmental expression patterns of germ cell-specific genes expressed in F9 cells. A. The expression patterns of nine genes predicted to be testis-specific or -predominant, as assessed in testis, F9 cells and NIH3T3 cells. Glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) was included as a loading control. Except for Cpvl , all of the genes were detected in both testis and F9 cells. It should be noted that the two bands for Dmrt1 in NIH3T3 cells are non-specific, based on in silico investigation and an additional PCR analysis (data not shown). B. The tissue distributions of transcripts were assessed by RT-PCR analysis in various tissues of adult male mice. Complementary DNAs from various mouse tissues were amplified by PCR, with Gapdh included as a loading control. Seven genes were found to be testis-specific or -predominant. C. Developmental expression patterns during spermatogenesis. The stage-specific expression of the genes was determined from mouse testes on different days after birth (days 8, 10, 12, 14, 16, 20, 30, and 56). Abbreviations: PL , preleptotene; L , leptotene; Z , zygotene; P , pachytene; D , diplotene; MI , meiotic division I; and MII , meiotic division II. Complementary DNA from germ cell-lacking testes from W/W v mutant mice was also examined. Consistent with our microarray analysis, Tex13 , Stra8 , Fancd2os , 1700061G19Rik and Triml1 were found to be germ cell-specific.

    Techniques Used: Expressing, In Silico, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Amplification, Mutagenesis, Microarray

    Promoter analysis of five germ cell-specific genes in F9 cells. A. Promoter-luciferase reporter analysis of five germ cell genes identified as being expressed in F9 cells. F9 cells were transfected with luciferase constructs containing 1.5-kb upstream regions spanning the transcription start site (TSS) (−1500 to +20 bp) of the indicated genes. The relative promoter activities are represented as the fold increase versus the expression from the promoter-less pGL3-Basic vector. The presented values represent the mean ± SD of three independent experiments. B. Promoter-luciferase reporter analysis of Dmrt1 , Stra8 and Tex13 in NIH3T3 cells. Reporter constructs containing promoters were transiently transfected into NIH3T3 cells. The presented values represent the mean ± SD of three independent experiments. Statistical significance in A and B was determined by the Student's t-test; * p
    Figure Legend Snippet: Promoter analysis of five germ cell-specific genes in F9 cells. A. Promoter-luciferase reporter analysis of five germ cell genes identified as being expressed in F9 cells. F9 cells were transfected with luciferase constructs containing 1.5-kb upstream regions spanning the transcription start site (TSS) (−1500 to +20 bp) of the indicated genes. The relative promoter activities are represented as the fold increase versus the expression from the promoter-less pGL3-Basic vector. The presented values represent the mean ± SD of three independent experiments. B. Promoter-luciferase reporter analysis of Dmrt1 , Stra8 and Tex13 in NIH3T3 cells. Reporter constructs containing promoters were transiently transfected into NIH3T3 cells. The presented values represent the mean ± SD of three independent experiments. Statistical significance in A and B was determined by the Student's t-test; * p

    Techniques Used: Luciferase, Transfection, Construct, Expressing, Plasmid Preparation

    Localization of GFP-TEX13 in F9 cells and NIH 3T3 cells. A. Cells transiently expressing GFP-TEX13 fusion proteins were visualized under fluorescent light, and the protein location was determined. Hoechst 33258 dye (blue) was used to stain nuclei. GFP-TEX13 localization was heterogeneous. Scale bar, 20 µm. B. Quantification of the different patterns of TEX13 sub-cellular localization in F9 cells and NIH 3T3 cells. Scale bar, 20 µm. Nu, nuclear; Cy = Nu, diffuse; Cy, cytoplasmic.
    Figure Legend Snippet: Localization of GFP-TEX13 in F9 cells and NIH 3T3 cells. A. Cells transiently expressing GFP-TEX13 fusion proteins were visualized under fluorescent light, and the protein location was determined. Hoechst 33258 dye (blue) was used to stain nuclei. GFP-TEX13 localization was heterogeneous. Scale bar, 20 µm. B. Quantification of the different patterns of TEX13 sub-cellular localization in F9 cells and NIH 3T3 cells. Scale bar, 20 µm. Nu, nuclear; Cy = Nu, diffuse; Cy, cytoplasmic.

    Techniques Used: Expressing, Staining

    Promoter analysis of the sequence upstream of the mouse Tex13 gene. A. Promoter activity of the murine Tex13 5′-flanking sequences. The Tex13 upstream region used for the reporter assay is shown on the left. Various genomic regions were introduced into the promoter-less pGL3-Basic vector, and the reporter constructs were transiently transfected into F9 and NIH3T3 cells. Strong promoter activities were observed in F9 cells. The relative promoter activities are represented as the fold increase versus expression from pGL3-Basic. The presented values represent the mean ± SD of three independent experiments. Statistical significance was determined by the Student's t-test; * p
    Figure Legend Snippet: Promoter analysis of the sequence upstream of the mouse Tex13 gene. A. Promoter activity of the murine Tex13 5′-flanking sequences. The Tex13 upstream region used for the reporter assay is shown on the left. Various genomic regions were introduced into the promoter-less pGL3-Basic vector, and the reporter constructs were transiently transfected into F9 and NIH3T3 cells. Strong promoter activities were observed in F9 cells. The relative promoter activities are represented as the fold increase versus expression from pGL3-Basic. The presented values represent the mean ± SD of three independent experiments. Statistical significance was determined by the Student's t-test; * p

    Techniques Used: Sequencing, Activity Assay, Reporter Assay, Plasmid Preparation, Construct, Transfection, Expressing

    40) Product Images from "Epigenetic Regulation of PLIN1 in Obese Women and its Relation to Lipolysis"

    Article Title: Epigenetic Regulation of PLIN1 in Obese Women and its Relation to Lipolysis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-09232-y

    PLIN1 promoter methylation inhibits promoter activity. PLIN1 promoter activity is decreased after (hatched bar) versus without (black bar) DNA methylation by SssI methyltransferase. hMSCs were transfected with methylated and unmethylated pCpGL- PLIN1 plasmid. As negative control, cells were transfected with empty vector, pCpGL-basic. Each sample was prepared in quadruplicates and the experiment was repeated three times. Y axis is the ratio between firefly and renilla luciferase. Renilla luciferase is expressed from a second plasmid as an internal control. RLU = Relative luciferase units. *** P
    Figure Legend Snippet: PLIN1 promoter methylation inhibits promoter activity. PLIN1 promoter activity is decreased after (hatched bar) versus without (black bar) DNA methylation by SssI methyltransferase. hMSCs were transfected with methylated and unmethylated pCpGL- PLIN1 plasmid. As negative control, cells were transfected with empty vector, pCpGL-basic. Each sample was prepared in quadruplicates and the experiment was repeated three times. Y axis is the ratio between firefly and renilla luciferase. Renilla luciferase is expressed from a second plasmid as an internal control. RLU = Relative luciferase units. *** P

    Techniques Used: Methylation, Activity Assay, DNA Methylation Assay, Transfection, Plasmid Preparation, Negative Control, Luciferase

    Global demethylation affects Perilipin levels in adipocytes. ( a ) PLIN1 mRNA as determined by RT-qPCR and ( b,c ) Perilipin protein as determined by Western blot were increased after treating hMSCs with DNA methyltransferase inhibitor RG108 (50 µM) (hatched bars) as compared to vehicle (black bars). The experiment was repeated three times. Representative Western blots are shown. These blot pictures were cropped and the full-length blot pictures are presented in Supplementary Fig. S1 . Results are presented as relative fold change ± SD vs. vehicle-treated cells. ( d ) Global DNA methylation in adipocytes was decreased after 24 h treatment with DNA methyltransferase inhibitor RG108 at a concentration of 50 µM (hatched bar) or 200 µM (white bar) compared to non-treated control cells (black bar). The experiment was repeated twice. ( e ) The methyltransferase inhibitor RG108 decreased methylation of specific CpG-sites in the PLIN1 promoter in adipocytes. Methylation of cg08749443 and cg04998447 was determined by Pyrosequencing after adipocytes were treated with 50 µM RG108 (hatched bar) compared to non-treated control cells (black bar). The analysis was repeated twice, n > 3. *** P
    Figure Legend Snippet: Global demethylation affects Perilipin levels in adipocytes. ( a ) PLIN1 mRNA as determined by RT-qPCR and ( b,c ) Perilipin protein as determined by Western blot were increased after treating hMSCs with DNA methyltransferase inhibitor RG108 (50 µM) (hatched bars) as compared to vehicle (black bars). The experiment was repeated three times. Representative Western blots are shown. These blot pictures were cropped and the full-length blot pictures are presented in Supplementary Fig. S1 . Results are presented as relative fold change ± SD vs. vehicle-treated cells. ( d ) Global DNA methylation in adipocytes was decreased after 24 h treatment with DNA methyltransferase inhibitor RG108 at a concentration of 50 µM (hatched bar) or 200 µM (white bar) compared to non-treated control cells (black bar). The experiment was repeated twice. ( e ) The methyltransferase inhibitor RG108 decreased methylation of specific CpG-sites in the PLIN1 promoter in adipocytes. Methylation of cg08749443 and cg04998447 was determined by Pyrosequencing after adipocytes were treated with 50 µM RG108 (hatched bar) compared to non-treated control cells (black bar). The analysis was repeated twice, n > 3. *** P

    Techniques Used: Quantitative RT-PCR, Western Blot, DNA Methylation Assay, Concentration Assay, Methylation

    Related Articles

    Gel Permeation Chromatography:

    Article Title: Single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase †Electronic supplementary
    Article Snippet: .. DNA adenine methyltransferase (Dam MTase), 10× Dam MTase reaction buffer (500 mM trizma hydrochloride (Tris–HCl), 50 mM 2-mercaptoethanol (β-ME), 100 mM ethylenediaminetetraacetic acid (EDTA), pH 7.5), CpG methyltransferase (M.SssI), GpC methyltransferase (M.CviPI), Dpn I, 10× CutSmart buffer (500 mM potassium acetate (KAc), 200 mM tris-acetate, 100 mM magnesium acetate (Mg(Ac)2 ), 1 mg mL–1 bovine serum albumin (BSA), pH 7.9), S -adenosylmethionine (SAM), Taq DNA ligase, 10× Taq DNA ligase reaction buffer (200 mM Tris–HCl, 250 mM KAc, 100 mM Mg(Ac)2 , 100 mM DL-dithiothreitol (DTT), 10 mM nicotinamide adenine dinucleotide (NAD), 1% triton X-100, pH 7.6), Bst DNA polymerase (large fragment), 10× ThermoPol reaction buffer (200 mM Tris–HCl, 100 mM potassium chloride (KCl), 100 mM ammonium sulfate (NH4 )2 SO4 ), 20 mM magnesium sulfate (MgSO4 ), 1% triton X-100, pH 8.8), ribonuclease HII (RNase HII), deoxyadenosine triphosphate (dATP), deoxyuridine triphosphate (dUTP), deoxyguanosine triphosphate (dGTP) and deoxycytidine triphosphate (dCTP) were purchased from New England Biolabs (Ipswich, MA, USA). .. SYBR Gold was purchased from Life Technologies (Carlsbad, CA, USA).

    Purification:

    Article Title: RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation
    Article Snippet: .. 50 or 500 ng of purified DNA (total plasmid, or alkaline lysis, or total genomic preparation) were incubated in 20 μl of 1× Thermopol buffer (NEB) containing either no enzyme or 1.5-2.5 units of RNase HII (NEB). .. DNA was incubated with 2.5 units of RNase HI (Ambion or ThermoScientific) in 20 μl of 1× RNase HI buffer (ThermoScientific): 20 mM Tris HCl (pH 7.8), 40 mM KCl, 8 mM MgCl2 , 1 mM DTT.

    Incubation:

    Article Title: Large-Scale Photolithographic Synthesis of Chimeric DNA/RNA Hairpin Microarrays To Explore Sequence Specificity Landscapes of RNase HII Cleavage
    Article Snippet: .. RNase HII Assays and Data Analysis After the deprotection procedure, the hairpins folded and the slides were incubated with a buffered solution of E. coli recombinant RNase HII (5 units, New England Biolabs M0288S) at 37 °C [10 mM KCl, 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 The recorded cleavage efficiency is an average from five independent measurements (±standard deviation). .. The 20 best cleaved hairpin sequences in each series (top 2% of 1024 combinations) were used for motif searching, which was rendered as a sequence logo using Weblogo 3.6 ( http://weblogo.threeplusone.com ).

    Article Title: RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation
    Article Snippet: .. 50 or 500 ng of purified DNA (total plasmid, or alkaline lysis, or total genomic preparation) were incubated in 20 μl of 1× Thermopol buffer (NEB) containing either no enzyme or 1.5-2.5 units of RNase HII (NEB). .. DNA was incubated with 2.5 units of RNase HI (Ambion or ThermoScientific) in 20 μl of 1× RNase HI buffer (ThermoScientific): 20 mM Tris HCl (pH 7.8), 40 mM KCl, 8 mM MgCl2 , 1 mM DTT.

    Alkaline Lysis:

    Article Title: RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation
    Article Snippet: .. 50 or 500 ng of purified DNA (total plasmid, or alkaline lysis, or total genomic preparation) were incubated in 20 μl of 1× Thermopol buffer (NEB) containing either no enzyme or 1.5-2.5 units of RNase HII (NEB). .. DNA was incubated with 2.5 units of RNase HI (Ambion or ThermoScientific) in 20 μl of 1× RNase HI buffer (ThermoScientific): 20 mM Tris HCl (pH 7.8), 40 mM KCl, 8 mM MgCl2 , 1 mM DTT.

    Standard Deviation:

    Article Title: Large-Scale Photolithographic Synthesis of Chimeric DNA/RNA Hairpin Microarrays To Explore Sequence Specificity Landscapes of RNase HII Cleavage
    Article Snippet: .. RNase HII Assays and Data Analysis After the deprotection procedure, the hairpins folded and the slides were incubated with a buffered solution of E. coli recombinant RNase HII (5 units, New England Biolabs M0288S) at 37 °C [10 mM KCl, 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 The recorded cleavage efficiency is an average from five independent measurements (±standard deviation). .. The 20 best cleaved hairpin sequences in each series (top 2% of 1024 combinations) were used for motif searching, which was rendered as a sequence logo using Weblogo 3.6 ( http://weblogo.threeplusone.com ).

    Recombinant:

    Article Title: Large-Scale Photolithographic Synthesis of Chimeric DNA/RNA Hairpin Microarrays To Explore Sequence Specificity Landscapes of RNase HII Cleavage
    Article Snippet: .. RNase HII Assays and Data Analysis After the deprotection procedure, the hairpins folded and the slides were incubated with a buffered solution of E. coli recombinant RNase HII (5 units, New England Biolabs M0288S) at 37 °C [10 mM KCl, 20 mM Tris-HCl, 10 mM (NH4 )2 SO4 , 2 mM MgSO4 The recorded cleavage efficiency is an average from five independent measurements (±standard deviation). .. The 20 best cleaved hairpin sequences in each series (top 2% of 1024 combinations) were used for motif searching, which was rendered as a sequence logo using Weblogo 3.6 ( http://weblogo.threeplusone.com ).

    Plasmid Preparation:

    Article Title: RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation
    Article Snippet: .. 50 or 500 ng of purified DNA (total plasmid, or alkaline lysis, or total genomic preparation) were incubated in 20 μl of 1× Thermopol buffer (NEB) containing either no enzyme or 1.5-2.5 units of RNase HII (NEB). .. DNA was incubated with 2.5 units of RNase HI (Ambion or ThermoScientific) in 20 μl of 1× RNase HI buffer (ThermoScientific): 20 mM Tris HCl (pH 7.8), 40 mM KCl, 8 mM MgCl2 , 1 mM DTT.

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    New England Biolabs adomet
    Human METTL12 is a protein-specific <t>MTase.</t> A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 <t>H]AdoMet</t> and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.
    Adomet, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    New England Biolabs non radioactive adomet
    Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 <t>H]-AdoMet</t> and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading
    Non Radioactive Adomet, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/non radioactive adomet/product/New England Biolabs
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    Human METTL12 is a protein-specific MTase. A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.

    Journal: The Journal of Biological Chemistry

    Article Title: Uncovering human METTL12 as a mitochondrial methyltransferase that modulates citrate synthase activity through metabolite-sensitive lysine methylation

    doi: 10.1074/jbc.M117.808451

    Figure Lengend Snippet: Human METTL12 is a protein-specific MTase. A , targeting of METTL12 gene in human HAP1 WT cells by CRISPR/Cas9 generated METTL12 KO cells containing a 1 base pair insertion in the METTL12 gene, located upstream of motif Post I, resulting in generation of truncated METTL12 protein. The dashed lines interrupting the open reading frames correspond to 177 nucleotides, i.e. 59 amino acids. B , METTL12-dependent protein methylation in cell extracts. Mitochondrial extracts from HAP1 WT or METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12. Methylation reactions were separated by SDS-PAGE and transferred to a membrane. Methylation was visualized by fluorography ( top ) of the Ponceau S-stained membrane ( bottom ). Arrows indicate the positions of the ∼48 kDa substrate and METTL12. C , D107A mutation abrogates enzymatic activity of METTL12. Mitochondrial extracts from METTL12 KO cells were incubated with [ 3 H]AdoMet and recombinant human METTL12, either WT or D107A-mutated. Methylation was analyzed as in B . Note: in panels B and C different levels of background (non-METTL12-dependent) methylation are observed; this is likely due to differences in the purity of the mitochondrial extracts.

    Article Snippet: For scintillation counting and titration experiments, MTase reactions contained [3 H]AdoMet, which was diluted with nonradioactive AdoMet (New England BioLabs).

    Techniques: CRISPR, Generated, Methylation, Incubation, Recombinant, SDS Page, Staining, Mutagenesis, Activity Assay

    Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN 13 ), N7- or 2′O-methylated capped RNA substrates ( 7Me GpppAN 13 or GpppA 2′OMe N 13 ), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.

    Journal: Nucleic Acids Research

    Article Title: mRNA maturation in giant viruses: variation on a theme

    doi: 10.1093/nar/gkv224

    Figure Lengend Snippet: Mg18 2′O MTase activity. AdoMet-dependent MTase assays were performed on equimolar amounts of short capped RNAs substrates (GpppAN 13 ), N7- or 2′O-methylated capped RNA substrates ( 7Me GpppAN 13 or GpppA 2′OMe N 13 ), A. castellanii mRNAs or homopolymeric poly (U), (C), (G) and (A). Human N7 MTase and Vaccinia virus VP39 were use as controls.

    Article Snippet: MTase activity assays were performed at 30°C in 40 mM Tris-HCl pH 7.5, 1 mM DTT, 1 mM MgCl2 , 0.7 μM RNA substrates, 10 μM AdoMet (NEB), and 0.03 mCi/ml 3 H-AdoMet (GE Healthcare).

    Techniques: Activity Assay, Methylation

    Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Journal: Journal of Bacteriology

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿

    doi: 10.1128/JB.00108-07

    Figure Lengend Snippet: Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Article Snippet: To investigate methylation and protection by the recombinant protein, we incubated 1 μg of a PCR fragment containing one GANTC site (778 bp, amplified with 1351GANTCF and 1351GANTCR) (Table ) with NEB2 buffer, S -adenosylmethionine (New England BioLabs), and different M.HpyAIV concentrations (0, 200, 400, 800, and 1,200 nM) and performed incubation for 1 h at room temperature followed by protein inactivation at 95°C for 10 min.

    Techniques: Purification, Incubation, Polymerase Chain Reaction, Concentration Assay, In Vitro

    Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 H]-AdoMet and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading

    Journal: Nature Communications

    Article Title: The dual methyltransferase METTL13 targets N terminus and Lys55 of eEF1A and modulates codon-specific translation rates

    doi: 10.1038/s41467-018-05646-y

    Figure Lengend Snippet: Identification of METTL13 as an eEF1A-specific methyltransferase. a Workflow of mass spectrometry-based quantitative peptide pull-down screen. Synthetic peptides corresponding N-terminally trimethylated (Nt-Me3) and unmethylated (Nt-Me0) eEF1A were used as baits to enrich proteins from HAP-1 cell extracts. b . c Domain organization of METTL13. The boundaries for used constructs encompassing the N-terminal (MT13-N) and the C-terminal (MT13-C) methyltransferase domains are indicated. d , e Evaluation of METTL13 constructs for eEF1A-specific methyltransferase activity. MT13-N ( d ) and MT13-C ( e ) were incubated with [ 3 H]-AdoMet and eEF1A1 carrying an N-terminal or C-terminal His-tag in the absence of cofactors and in the presence of either GDP or GTP. Methylation was visualized by fluorography (top panels) and the membranes were stained with Ponceau S (bottom panels) to assess protein loading

    Article Snippet: For quantitative MTase assays, [3 H]-AdoMet was diluted with non-radioactive AdoMet (New England Biolabs) ([AdoMet]total = 32.6 μM) .

    Techniques: Mass Spectrometry, Construct, Activity Assay, Incubation, Methylation, Staining