cpg methyltransferase m sssi  (New England Biolabs)


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    Name:
    CpG Methyltransferase M SssI
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    CpG Methyltransferase M SssI 500 units
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    m0226l
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    Category:
    DNA Methylases
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    New England Biolabs cpg methyltransferase m sssi
    CpG Methyltransferase M SssI
    CpG Methyltransferase M SssI 500 units
    https://www.bioz.com/result/cpg methyltransferase m sssi/product/New England Biolabs
    Average 99 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    cpg methyltransferase m sssi - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma"

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2010.01124.x

    Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P
    Figure Legend Snippet: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P

    Techniques Used: Methylation, Binding Assay, Activity Assay, In Vitro, Electrophoretic Mobility Shift Assay, Mutagenesis, DNA Methylation Assay, Transfection, Construct, Luciferase

    2) Product Images from "Nucleofection of Expression Vectors Induces a Robust Interferon Response and Inhibition of Cell Proliferation"

    Article Title: Nucleofection of Expression Vectors Induces a Robust Interferon Response and Inhibition of Cell Proliferation

    Journal: DNA and Cell Biology

    doi: 10.1089/dna.2012.1950

    Production of IFNβ by NIH3T3 cells after nucleofection with methylated or unmethylated phGfΔG plasmid DNA. (A) The phGfΔG plasmid was treated with the methyltransferase M. SssI to methylate the CpG motifs. Prevention of the digestion by Hpa II indicated the methylation state of the plasmid. Untreated plasmid was used as a positive control of Hpa II digestion. (B) Cells were transfected either with unmethylated or methylated plasmid, and the levels of IFNβ production were assayed by ELISA. The data represent the values of two independent experiments.
    Figure Legend Snippet: Production of IFNβ by NIH3T3 cells after nucleofection with methylated or unmethylated phGfΔG plasmid DNA. (A) The phGfΔG plasmid was treated with the methyltransferase M. SssI to methylate the CpG motifs. Prevention of the digestion by Hpa II indicated the methylation state of the plasmid. Untreated plasmid was used as a positive control of Hpa II digestion. (B) Cells were transfected either with unmethylated or methylated plasmid, and the levels of IFNβ production were assayed by ELISA. The data represent the values of two independent experiments.

    Techniques Used: Methylation, Plasmid Preparation, Positive Control, Transfection, Enzyme-linked Immunosorbent Assay

    3) Product Images from "Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing"

    Article Title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3073

    MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P
    Figure Legend Snippet: MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P

    Techniques Used: Activity Assay, Sequencing, Lambda DNA Preparation, Methylation Sequencing, Immunoprecipitation, DNA Immunoprecipitation Sequencing, Modification

    4) Product Images from "Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells"

    Article Title: Epigenetic Regulation and Functional Characterization of MicroRNA-142 in Mesenchymal Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0079231

    Analyses of methylation status of CGIs associated mir-142 and in vitro methylation of its upstream regulatory region. ( A ) Methylation-specific PCR (MSP) analyses of the CGIs, before and after treatment with 5-Aza-2′-deoxycytidine (5-Aza). U and M, unmethylated and methylated products. Both standard gel images and 3D densitograms of the signal intensities are shown. ( B ) Schematic representation of the mir-142 locus. Locations of MSP amplicon #1 and #2 are indicated by arrows. 18 CpGs (region #1) and 14 CpGs (region #2) were subjected to bisulfite sequencing. CpG-containing transcription factor binding motifs are enclosed by boxes (see Figure 3B for more information). E-box, enhancer box. Numbers indicate the position relative to pre-miR-142 (+1 to +87). ( C ) Bisulfite sequencing of DNA from MG-63 cells before and after treatment with 5-Aza for 72 hours, and untreated IOR/OS14 cells. The Wilcoxon signed rank test was used to test for statistical differences between treated and untreated MG-63 cells, as described in more details in Materials and Methods . A P value ≤0.05 was considered as significant. ( D ) Bisulfite sequencing of immortalized bone marrow-derived stromal cells (iMSC#3) and primary osteoblasts. ( E ) Bisulfite sequencing of K562 leukemia- and peripheral blood progenitor cells. Black and white circles represent methylated and unmethylated CpGs, respectively, and each row represents a single clone. Grey circles, not determined. Ten clones were sequenced (n = 10), with the exception of MG-63 cells (region #1, n = 13; region #2, n = 12). ( F ) The 2,031 bp upstream region of pre-mir-142 was cloned into the promoter-less luciferase reporter construct pCpGL-basic and in vitro methylated with M.SssI (pCpGL/2031_M.SssI) or mock-methylated (pCpGL/2031_mock). All three constructs were individually transfected into U-2 OS cells along with a Renilla reporter construct. The luciferase activity was measured after 48 hours and calculated relative to that of pCpGL-basic (set to 1). Each histogram shows the average relative luciferase activity, and the error bars show the standard deviation of biological experiments (n≥5). The Wilcoxon signed rank test was used to test for statistical differences and the P values are shown above the histograms. A P value ≤0.05 was considered as significant.
    Figure Legend Snippet: Analyses of methylation status of CGIs associated mir-142 and in vitro methylation of its upstream regulatory region. ( A ) Methylation-specific PCR (MSP) analyses of the CGIs, before and after treatment with 5-Aza-2′-deoxycytidine (5-Aza). U and M, unmethylated and methylated products. Both standard gel images and 3D densitograms of the signal intensities are shown. ( B ) Schematic representation of the mir-142 locus. Locations of MSP amplicon #1 and #2 are indicated by arrows. 18 CpGs (region #1) and 14 CpGs (region #2) were subjected to bisulfite sequencing. CpG-containing transcription factor binding motifs are enclosed by boxes (see Figure 3B for more information). E-box, enhancer box. Numbers indicate the position relative to pre-miR-142 (+1 to +87). ( C ) Bisulfite sequencing of DNA from MG-63 cells before and after treatment with 5-Aza for 72 hours, and untreated IOR/OS14 cells. The Wilcoxon signed rank test was used to test for statistical differences between treated and untreated MG-63 cells, as described in more details in Materials and Methods . A P value ≤0.05 was considered as significant. ( D ) Bisulfite sequencing of immortalized bone marrow-derived stromal cells (iMSC#3) and primary osteoblasts. ( E ) Bisulfite sequencing of K562 leukemia- and peripheral blood progenitor cells. Black and white circles represent methylated and unmethylated CpGs, respectively, and each row represents a single clone. Grey circles, not determined. Ten clones were sequenced (n = 10), with the exception of MG-63 cells (region #1, n = 13; region #2, n = 12). ( F ) The 2,031 bp upstream region of pre-mir-142 was cloned into the promoter-less luciferase reporter construct pCpGL-basic and in vitro methylated with M.SssI (pCpGL/2031_M.SssI) or mock-methylated (pCpGL/2031_mock). All three constructs were individually transfected into U-2 OS cells along with a Renilla reporter construct. The luciferase activity was measured after 48 hours and calculated relative to that of pCpGL-basic (set to 1). Each histogram shows the average relative luciferase activity, and the error bars show the standard deviation of biological experiments (n≥5). The Wilcoxon signed rank test was used to test for statistical differences and the P values are shown above the histograms. A P value ≤0.05 was considered as significant.

    Techniques Used: Methylation, In Vitro, Polymerase Chain Reaction, Amplification, Methylation Sequencing, Binding Assay, Derivative Assay, Clone Assay, Luciferase, Construct, Transfection, Activity Assay, Standard Deviation

    5) Product Images from "Epigenetic Control of Viral Life-Cycle by a DNA-Methylation Dependent Transcription Factor"

    Article Title: Epigenetic Control of Viral Life-Cycle by a DNA-Methylation Dependent Transcription Factor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025922

    Evaluation of predicted ZREs in three EBV promoters. A. Summary of the known (filled box) and predicted (open box) core ZRE sequences within the proximal 500 nucleotides of the indicated BZLF1 , BRLF1 and BMRF1 promoters. The arrows represent the transcription start sites. Stars represent CpG ZREs. B. Double strand oligonucleotides were generated with the core ZRE sequence and at least 10 nucleotides of cognate sequence on either side. Following radio labeling, these were incubated with in vitro translated Zta and subject to EMSA. The reactions contained no protein, 0, control lysate, C or Zta, Z. The DNA probes are indicated above with their originating promoters. C. Double strand oligonucleotides were generated with the core ZRE sequence and 10 nucleotides of cognate sequence on either side. Following radio labeling, these were subject to in vitro methylation with SssI methyl transferase (+), or a mock reaction (−). Subsequently, they were incubated with in vitro translated Zta and subject to EMSA. The reactions contained control lysate, C; or Zta, Z.
    Figure Legend Snippet: Evaluation of predicted ZREs in three EBV promoters. A. Summary of the known (filled box) and predicted (open box) core ZRE sequences within the proximal 500 nucleotides of the indicated BZLF1 , BRLF1 and BMRF1 promoters. The arrows represent the transcription start sites. Stars represent CpG ZREs. B. Double strand oligonucleotides were generated with the core ZRE sequence and at least 10 nucleotides of cognate sequence on either side. Following radio labeling, these were incubated with in vitro translated Zta and subject to EMSA. The reactions contained no protein, 0, control lysate, C or Zta, Z. The DNA probes are indicated above with their originating promoters. C. Double strand oligonucleotides were generated with the core ZRE sequence and 10 nucleotides of cognate sequence on either side. Following radio labeling, these were subject to in vitro methylation with SssI methyl transferase (+), or a mock reaction (−). Subsequently, they were incubated with in vitro translated Zta and subject to EMSA. The reactions contained control lysate, C; or Zta, Z.

    Techniques Used: Generated, Sequencing, Labeling, Incubation, In Vitro, Methylation

    Zta recognition and methylation dependence of PFM CpG5 predicted CpG containing ZREs. A. Flow diagram illustrating the information flow from the PFM to the predictions of novel ZREs in the EBV genome and their subsequent evaluation. B. Core heptamer sequences, in both forward and reverse complement, of PFM CpG5 predicted CpG containing ZREs within the EBV genome. C. PFM CpG5 was used to predict the potential for further ZREs in the EBV genome. Double strand oligonucleotides were generated. Following radio labeling, these were subject to in vitro methylation with SssI methyl transferase (+), or a mock reaction (−). Subsequently, they were incubated with in vitro translated Zta and subject to EMSA. The reactions contained control lysate, C; or Zta, Z.
    Figure Legend Snippet: Zta recognition and methylation dependence of PFM CpG5 predicted CpG containing ZREs. A. Flow diagram illustrating the information flow from the PFM to the predictions of novel ZREs in the EBV genome and their subsequent evaluation. B. Core heptamer sequences, in both forward and reverse complement, of PFM CpG5 predicted CpG containing ZREs within the EBV genome. C. PFM CpG5 was used to predict the potential for further ZREs in the EBV genome. Double strand oligonucleotides were generated. Following radio labeling, these were subject to in vitro methylation with SssI methyl transferase (+), or a mock reaction (−). Subsequently, they were incubated with in vitro translated Zta and subject to EMSA. The reactions contained control lysate, C; or Zta, Z.

    Techniques Used: Methylation, Flow Cytometry, Generated, Labeling, In Vitro, Incubation

    6) Product Images from "Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells"

    Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0096512

    Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p
    Figure Legend Snippet: Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p

    Techniques Used: Methylation, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Binding Assay, SDS Page, Western Blot, Generated, Staining, Labeling, Cell Culture, Real-time Polymerase Chain Reaction, Incubation

    7) Product Images from "Hydrophobicity of Methylated DNA as a Possible Mechanism for Gene Silencing"

    Article Title: Hydrophobicity of Methylated DNA as a Possible Mechanism for Gene Silencing

    Journal: Physical biology

    doi: 10.1088/1478-3975/9/6/065001

    The 2905 bp DNA template (A) showing Ava1 restriction sites. The sequence contains 951 cytosines (32.2% C content) and 259 occurrences of the 5’CG3’ motif (the target for CpG Methyltransferase) on one strand. (B) Ava1 digests of DNA incubated
    Figure Legend Snippet: The 2905 bp DNA template (A) showing Ava1 restriction sites. The sequence contains 951 cytosines (32.2% C content) and 259 occurrences of the 5’CG3’ motif (the target for CpG Methyltransferase) on one strand. (B) Ava1 digests of DNA incubated

    Techniques Used: Sequencing, Incubation

    8) Product Images from "Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing"

    Article Title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3073

    MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P
    Figure Legend Snippet: MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P

    Techniques Used: Activity Assay, Sequencing, Lambda DNA Preparation, Methylation Sequencing, Immunoprecipitation, DNA Immunoprecipitation Sequencing, Modification

    9) Product Images from "High throughput screening identifies SOX2 as a Super Pioneer Factor that inhibits DNA methylation maintenance at its binding sites"

    Article Title: High throughput screening identifies SOX2 as a Super Pioneer Factor that inhibits DNA methylation maintenance at its binding sites

    Journal: bioRxiv

    doi: 10.1101/2020.02.10.941682

    Validation of the experimental approach (Hi-TransMet) to test for TF effect on DNA methylation. ( a ) Schematic representation of Hi-TransMet. PF motifs flanked by unique barcode sequences were individually cloned at the center of an intermediate-CpG content bacterial DNA fragment (FR1) within an RMCE donor plasmid. Motif-containing plasmid libraries were transfected into mESCs following in vitro methylation via M.SssI enzyme (+SssI) or without further treatment (–SssI). Insertion of the –SssI library results in de novo methylation. Methylation levels in cells that underwent successful recombination were analyzed using universal bisulfite PCR primers designed around the inserted motifs (blue and green arrows). ( b ) Results allow to classify the screened PFs essentially in three classes: I) PFs that cannot bind methylated DNA or induce changes in DNA methylation; II) PFs that are able to bind unmethylated DNA and protect it from methylation; III) PFs that are able to bind methylated DNA and induce DNA demethylation. ( c ) Validation of the experimental approach by Bisulfite Sanger Sequencing in cell lines containing FR1 with CTCF-motifs only. Upon insertion, in the –SssI condition, FR1 undergoes de novo methylation. Lower methylation is observed in the presence of the CTCF WT but not Sc motif. In the +SssI condition, high levels of DNA methylation were retained by FR1 in the absence of the motif and in the presence of the CTCF Sc motif. In the presence of the CTCF WT motif, a reduction of DNA methylation levels is observed. Vertical bars correspond to CpG positions, and the color code corresponds to the percentage of methylation calculated for each CpG with a minimum coverage of 10 bisulfite reads. ( d ) CTCF binding in the FR1 was verified by ChIP.
    Figure Legend Snippet: Validation of the experimental approach (Hi-TransMet) to test for TF effect on DNA methylation. ( a ) Schematic representation of Hi-TransMet. PF motifs flanked by unique barcode sequences were individually cloned at the center of an intermediate-CpG content bacterial DNA fragment (FR1) within an RMCE donor plasmid. Motif-containing plasmid libraries were transfected into mESCs following in vitro methylation via M.SssI enzyme (+SssI) or without further treatment (–SssI). Insertion of the –SssI library results in de novo methylation. Methylation levels in cells that underwent successful recombination were analyzed using universal bisulfite PCR primers designed around the inserted motifs (blue and green arrows). ( b ) Results allow to classify the screened PFs essentially in three classes: I) PFs that cannot bind methylated DNA or induce changes in DNA methylation; II) PFs that are able to bind unmethylated DNA and protect it from methylation; III) PFs that are able to bind methylated DNA and induce DNA demethylation. ( c ) Validation of the experimental approach by Bisulfite Sanger Sequencing in cell lines containing FR1 with CTCF-motifs only. Upon insertion, in the –SssI condition, FR1 undergoes de novo methylation. Lower methylation is observed in the presence of the CTCF WT but not Sc motif. In the +SssI condition, high levels of DNA methylation were retained by FR1 in the absence of the motif and in the presence of the CTCF Sc motif. In the presence of the CTCF WT motif, a reduction of DNA methylation levels is observed. Vertical bars correspond to CpG positions, and the color code corresponds to the percentage of methylation calculated for each CpG with a minimum coverage of 10 bisulfite reads. ( d ) CTCF binding in the FR1 was verified by ChIP.

    Techniques Used: DNA Methylation Assay, Clone Assay, Plasmid Preparation, Transfection, In Vitro, Methylation, Polymerase Chain Reaction, Sequencing, Binding Assay, Chromatin Immunoprecipitation

    10) Product Images from "Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma"

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/j.1582-4934.2010.01124.x

    Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P
    Figure Legend Snippet: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P

    Techniques Used: Methylation, Binding Assay, Activity Assay, In Vitro, Electrophoretic Mobility Shift Assay, Mutagenesis, DNA Methylation Assay, Transfection, Construct, Luciferase

    11) Product Images from "Hypomethylation at non-CpG/CpG sites in the promoter of HIF-1α gene combined with enhanced H3K9Ac modification contribute to maintain higher HIF-1α expression in breast cancer"

    Article Title: Hypomethylation at non-CpG/CpG sites in the promoter of HIF-1α gene combined with enhanced H3K9Ac modification contribute to maintain higher HIF-1α expression in breast cancer

    Journal: Oncogenesis

    doi: 10.1038/s41389-019-0135-1

    Non-CpHs methylation can silence HIF-1α gene transcription. a Analyzing in vitro DNA-methyltransferase, MSP I and MSss I recognition sites within HIF-1α gene promoter. b The methylation at CpC and CpG sites could cause transcriptional repression of HIF-1α gene by using luciferase activity assay. Data are presented as the means ± SD of three independent experiments. * p
    Figure Legend Snippet: Non-CpHs methylation can silence HIF-1α gene transcription. a Analyzing in vitro DNA-methyltransferase, MSP I and MSss I recognition sites within HIF-1α gene promoter. b The methylation at CpC and CpG sites could cause transcriptional repression of HIF-1α gene by using luciferase activity assay. Data are presented as the means ± SD of three independent experiments. * p

    Techniques Used: Methylation, In Vitro, Luciferase, Activity Assay

    12) Product Images from "Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource"

    Article Title: Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx900

    Determination of de novo and maintenance CpG methylation in Tnp1 and Pfn3 minigenes with distinct splicing capacities. ( A ) Schematic representation of regions interrogated by bisulfite-pyrosequencing (BS-pyroseq) in the Tnp1 and Pfn3 minigenes. ( B ) Per-base heatmap for percent CpG methylation at the 7 interrogated sites within Tnp1 exon 2. Values represent averages from duplicate BS-pyroseq performed on biological replicates of Dox-induced Tnp1 host cells transfected with vector encoding DNMT3A, DNMT3B or control (left). Average methylation across the seven sites in DNMT3-transfected and control cells (right), mean ± SEM. ( C ) BS-pyroseq of the 18 CpGs overlapping the Pfn3 ORF, as in (A). P -values = two-tailed Student's t -test comparing average methylation DNMT3-transfected cells versus control, as indicated. ( D ) CpG-free Tnp1 expression vector was methylated in vitro using M.SssI and stably transfected into 293T cells for random integration. ( E ) MSRE-qPCR for relative Tnp1 DNA methylation from all stable gDNA and ΔSS minigene clones at 21, 49 and 77 days post-transfection summarized in boxplot; white diamonds indicate average Tnp1 methylation from seven and six independent Tnp1 gDNA and ΔSS clones, respectively. Student's t -test comparing mean methylation between indicated pairs yielded no significant differences (NS).
    Figure Legend Snippet: Determination of de novo and maintenance CpG methylation in Tnp1 and Pfn3 minigenes with distinct splicing capacities. ( A ) Schematic representation of regions interrogated by bisulfite-pyrosequencing (BS-pyroseq) in the Tnp1 and Pfn3 minigenes. ( B ) Per-base heatmap for percent CpG methylation at the 7 interrogated sites within Tnp1 exon 2. Values represent averages from duplicate BS-pyroseq performed on biological replicates of Dox-induced Tnp1 host cells transfected with vector encoding DNMT3A, DNMT3B or control (left). Average methylation across the seven sites in DNMT3-transfected and control cells (right), mean ± SEM. ( C ) BS-pyroseq of the 18 CpGs overlapping the Pfn3 ORF, as in (A). P -values = two-tailed Student's t -test comparing average methylation DNMT3-transfected cells versus control, as indicated. ( D ) CpG-free Tnp1 expression vector was methylated in vitro using M.SssI and stably transfected into 293T cells for random integration. ( E ) MSRE-qPCR for relative Tnp1 DNA methylation from all stable gDNA and ΔSS minigene clones at 21, 49 and 77 days post-transfection summarized in boxplot; white diamonds indicate average Tnp1 methylation from seven and six independent Tnp1 gDNA and ΔSS clones, respectively. Student's t -test comparing mean methylation between indicated pairs yielded no significant differences (NS).

    Techniques Used: CpG Methylation Assay, Transfection, Plasmid Preparation, Methylation, Two Tailed Test, Expressing, In Vitro, Stable Transfection, Real-time Polymerase Chain Reaction, DNA Methylation Assay, Clone Assay

    13) Product Images from "Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat"

    Article Title: Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat

    Journal: Journal of Neuroendocrinology

    doi: 10.1111/jne.12371

    Methylation of CpG (cytosine‐phosphate‐guanine) sites on the Avp promoter in vitro . The substitution (C‐A) at CpG sites in the Avp promoter by overlap extension polymerase chain reaction was used to prevent methylation at specific CpG sites. The mutation sites are shown in ( a ). The mutated plasmids were subsequently methylated by methyltransferase enzyme. ( b ) Successful methylation was determined using methylation sensitive restriction enzyme Pml I. (c, d ) Luciferase assays were performed by co‐transfection of plasmid expressing Creb3l1 and 350 bp Avp promoter contructs with ( c ) unmethylated and ( d ) methylated plasmid. Error bars indicate the mean ± SEM (n = 4 per group). *P
    Figure Legend Snippet: Methylation of CpG (cytosine‐phosphate‐guanine) sites on the Avp promoter in vitro . The substitution (C‐A) at CpG sites in the Avp promoter by overlap extension polymerase chain reaction was used to prevent methylation at specific CpG sites. The mutation sites are shown in ( a ). The mutated plasmids were subsequently methylated by methyltransferase enzyme. ( b ) Successful methylation was determined using methylation sensitive restriction enzyme Pml I. (c, d ) Luciferase assays were performed by co‐transfection of plasmid expressing Creb3l1 and 350 bp Avp promoter contructs with ( c ) unmethylated and ( d ) methylated plasmid. Error bars indicate the mean ± SEM (n = 4 per group). *P

    Techniques Used: Methylation, In Vitro, Overlap Extension Polymerase Chain Reaction, Mutagenesis, Luciferase, Cotransfection, Plasmid Preparation, Expressing

    Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P
    Figure Legend Snippet: Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P

    Techniques Used: Methylation, Clone Assay

    14) Product Images from "Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation"

    Article Title: Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029939

    Methylation of CpG sites in the vav 1 promoter impairs expression of the reporter gene in various cell lines. (A) Le2 plasmid, either un-treated or methylated by CpG methyltransferase (M.SssI), was incubated with HpaII and analyzed on a gel. The plasmid treated with M.SssI was not digested by HpaII, indicating that methylation was successful. (B) Unmethylated or methylated Le2 was transfected into Jurkat T cells, U937 myeloid cells and H441 lung cancer cells. The luciferase activity of these plasmids was measured 24 hr after transfection. Fold induction of luciferase activity was calculated relative to the activity in cells transfected with unmethylated Le2. Each point is the mean of three experiments. (***) indicates p
    Figure Legend Snippet: Methylation of CpG sites in the vav 1 promoter impairs expression of the reporter gene in various cell lines. (A) Le2 plasmid, either un-treated or methylated by CpG methyltransferase (M.SssI), was incubated with HpaII and analyzed on a gel. The plasmid treated with M.SssI was not digested by HpaII, indicating that methylation was successful. (B) Unmethylated or methylated Le2 was transfected into Jurkat T cells, U937 myeloid cells and H441 lung cancer cells. The luciferase activity of these plasmids was measured 24 hr after transfection. Fold induction of luciferase activity was calculated relative to the activity in cells transfected with unmethylated Le2. Each point is the mean of three experiments. (***) indicates p

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

    15) Product Images from "The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes"

    Article Title: The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000935

    Experimental design of MeDIP analysis. A : Schematic representation of the experimental setup for the analysis of CpG methylation patterns. The KSHV episome in infected cells is expected to be partially methylated, as indicated by black and white circles which symbolize methylated or unmethylated CpG dinucleotides, respectively. Genomic DNA was isolated from such cells and the samples were subjected to immunoprecipitation using a methylcytidine specific antibody (MeDIP procedure), followed by hybridization of the precipitated samples versus the input on tiling microarrays. For each probe, an enrichment score E S was calculated, which represents the ratio of MeDIP over input fluorescence signals. The efficiency of the immunoprecipitation depends on the total number of methylated CpG motifs in a given fragment and E S is thus a function of the extend of methylation as well as local CpG frequencies. Therefore, to obtain reference values which signify maximum methylation for each probe, we generated a positive control by subjecting KSHV bacmids to CpG methylation in vitro . The bacmid was mixed with cellular DNA to simulate the host background and subjected to the same MeDIP procedure as samples from infected cells. Similarly, a negative control of unmethylated bacmid was prepared to control for cross-hybridization of unspecific background. After normalization of the array data using a spike-in control (see Material Methods for details), background-corrected methylation values M S and M P were calculated for each probe by subtraction of the corresponding negative control value. B : Confirmation of successful in vitro methylation of KSHV bacmids used as a positive control. A bacmid carrying the complete KSHV genome (BAC36 [36] ) was methylated using M.SssI, a methyltransferase specific for CpG dinucleotides. Methylated or unmethylated bacmids were subjected to restriction digestion using the methylation sensitive enzyme HpaII and its isoschizomer MspI, which cuts regardless of methylation. Methylated bacmids were resistant to HpaII digestion, signifying complete methylation.
    Figure Legend Snippet: Experimental design of MeDIP analysis. A : Schematic representation of the experimental setup for the analysis of CpG methylation patterns. The KSHV episome in infected cells is expected to be partially methylated, as indicated by black and white circles which symbolize methylated or unmethylated CpG dinucleotides, respectively. Genomic DNA was isolated from such cells and the samples were subjected to immunoprecipitation using a methylcytidine specific antibody (MeDIP procedure), followed by hybridization of the precipitated samples versus the input on tiling microarrays. For each probe, an enrichment score E S was calculated, which represents the ratio of MeDIP over input fluorescence signals. The efficiency of the immunoprecipitation depends on the total number of methylated CpG motifs in a given fragment and E S is thus a function of the extend of methylation as well as local CpG frequencies. Therefore, to obtain reference values which signify maximum methylation for each probe, we generated a positive control by subjecting KSHV bacmids to CpG methylation in vitro . The bacmid was mixed with cellular DNA to simulate the host background and subjected to the same MeDIP procedure as samples from infected cells. Similarly, a negative control of unmethylated bacmid was prepared to control for cross-hybridization of unspecific background. After normalization of the array data using a spike-in control (see Material Methods for details), background-corrected methylation values M S and M P were calculated for each probe by subtraction of the corresponding negative control value. B : Confirmation of successful in vitro methylation of KSHV bacmids used as a positive control. A bacmid carrying the complete KSHV genome (BAC36 [36] ) was methylated using M.SssI, a methyltransferase specific for CpG dinucleotides. Methylated or unmethylated bacmids were subjected to restriction digestion using the methylation sensitive enzyme HpaII and its isoschizomer MspI, which cuts regardless of methylation. Methylated bacmids were resistant to HpaII digestion, signifying complete methylation.

    Techniques Used: Methylated DNA Immunoprecipitation, CpG Methylation Assay, Infection, Methylation, Isolation, Immunoprecipitation, Hybridization, Fluorescence, Generated, Positive Control, In Vitro, Negative Control

    Related Articles

    In Vitro:

    Article Title: DNA Methylation Signatures Triggered by Prenatal Maternal Stress Exposure to a Natural Disaster: Project Ice Storm
    Article Snippet: .. In vitro pCpGL-SCG5 promoter methylation Plasmid constructs were methylated in vitro using CpG methyltransferase (M.Sss I) (New England Biolabs). .. Cell line and transfection Human embryonic kidney 293 cells (HEK293) (ATCC) were transiently transfected using calcium phosphate method based on Rouleau et al .

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice
    Article Snippet: .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. .. Methylated DNA was then purified using the QIAquick gel extraction kit (Qiagen) and quantified by NanoDrop (Thermo Scientific NanoDrop products, Wilmington, DE).

    Methylation:

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma
    Article Snippet: .. The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned. .. The DNA binding reaction was carried out in a 20 μl reaction mixture containing Gel Shift Binding Buffer (Promega), 10 μg nuclear extract and the biotin-labelled probe or methylated biotin-labelled probe, with or without 100-fold molar excess unlabelled competitor.

    Article Title: DNA Methylation Signatures Triggered by Prenatal Maternal Stress Exposure to a Natural Disaster: Project Ice Storm
    Article Snippet: .. In vitro pCpGL-SCG5 promoter methylation Plasmid constructs were methylated in vitro using CpG methyltransferase (M.Sss I) (New England Biolabs). .. Cell line and transfection Human embryonic kidney 293 cells (HEK293) (ATCC) were transiently transfected using calcium phosphate method based on Rouleau et al .

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice
    Article Snippet: .. M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions. .. Methylated DNA was then purified using the QIAquick gel extraction kit (Qiagen) and quantified by NanoDrop (Thermo Scientific NanoDrop products, Wilmington, DE).

    Article Title: Investigation of the Role of DNA Methylation in the Expression of ERBB2 in Human Myocardium
    Article Snippet: .. The −499 ERBB2 -pCpGL reporter plasmid was methylated using the CpG methyltransferases M.SssI or HpaII (New England Biolabs, Massachusetts, USA). .. The extent of methylation was determined by bisulfite sequencing PCR.

    Construct:

    Article Title: DNA Methylation Signatures Triggered by Prenatal Maternal Stress Exposure to a Natural Disaster: Project Ice Storm
    Article Snippet: .. In vitro pCpGL-SCG5 promoter methylation Plasmid constructs were methylated in vitro using CpG methyltransferase (M.Sss I) (New England Biolabs). .. Cell line and transfection Human embryonic kidney 293 cells (HEK293) (ATCC) were transiently transfected using calcium phosphate method based on Rouleau et al .

    Purification:

    Article Title: Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation
    Article Snippet: .. The specific activity of the purified M.Sss I enzyme was determined using CpG146 DNA as substrate, in comparison with the activity unit of the NEB M.Sss I enzyme (cat. M0226M), by quantifying the amounts of the Eco72 I‐digested DNA fragments. .. Large‐scale methylation of CpG146 DNA and its verification For the preparation of CpG‐methylated nucleosomal DNA, 50 μg·mL−1 of the CpG146 DNA was incubated at 37 °C for 16 h with M.Sss I (6 units·μg−1 DNA), in 10 mm Tris‐HCl buffer (pH 8.0), containing 50 mm NaCl, 2.5 mm EDTA, and 640 μm SAM.

    Incubation:

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma
    Article Snippet: .. The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned. .. The DNA binding reaction was carried out in a 20 μl reaction mixture containing Gel Shift Binding Buffer (Promega), 10 μg nuclear extract and the biotin-labelled probe or methylated biotin-labelled probe, with or without 100-fold molar excess unlabelled competitor.

    Activity Assay:

    Article Title: Latency of Viral Expression In Vivo Is Not Related to CpG Methylation in the U3 Region and Part of the R Region of the Long Terminal Repeat of Bovine Leukemia Virus
    Article Snippet: .. Nonetheless, we also showed that the transactivation activity of LTR was reduced after treatment with the CpG methyltransferase Sss I. .. These results raise the possibility that CpG methylation is required for silencing induction but not for maintenance of BLV silencing.

    Article Title: Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation
    Article Snippet: .. The specific activity of the purified M.Sss I enzyme was determined using CpG146 DNA as substrate, in comparison with the activity unit of the NEB M.Sss I enzyme (cat. M0226M), by quantifying the amounts of the Eco72 I‐digested DNA fragments. .. Large‐scale methylation of CpG146 DNA and its verification For the preparation of CpG‐methylated nucleosomal DNA, 50 μg·mL−1 of the CpG146 DNA was incubated at 37 °C for 16 h with M.Sss I (6 units·μg−1 DNA), in 10 mm Tris‐HCl buffer (pH 8.0), containing 50 mm NaCl, 2.5 mm EDTA, and 640 μm SAM.

    Plasmid Preparation:

    Article Title: DNA Methylation Signatures Triggered by Prenatal Maternal Stress Exposure to a Natural Disaster: Project Ice Storm
    Article Snippet: .. In vitro pCpGL-SCG5 promoter methylation Plasmid constructs were methylated in vitro using CpG methyltransferase (M.Sss I) (New England Biolabs). .. Cell line and transfection Human embryonic kidney 293 cells (HEK293) (ATCC) were transiently transfected using calcium phosphate method based on Rouleau et al .

    Article Title: Investigation of the Role of DNA Methylation in the Expression of ERBB2 in Human Myocardium
    Article Snippet: .. The −499 ERBB2 -pCpGL reporter plasmid was methylated using the CpG methyltransferases M.SssI or HpaII (New England Biolabs, Massachusetts, USA). .. The extent of methylation was determined by bisulfite sequencing PCR.

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    New England Biolabs m sssi cpg methyltransferase
    DNA methylation of Gpx1 decreases gene expression. In vitro methylation of Gpx1 target region inhibited transcriptional activity, as measured by a luciferase reporter assay. (A) Schematic representation of the plasmid construction containing the Gpx1 <t>CpG</t> island region. (B) Luciferase activity ratio of methylated <t>(M.SssI</t> treated) to unmethylated control (CTL) plasmids containing a CpG-free promoter or the Gpx1 CpG island region. The assay was repeated 4 times and data are mean ± SEM. *: p
    M Sssi Cpg Methyltransferase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    DNA methylation of Gpx1 decreases gene expression. In vitro methylation of Gpx1 target region inhibited transcriptional activity, as measured by a luciferase reporter assay. (A) Schematic representation of the plasmid construction containing the Gpx1 CpG island region. (B) Luciferase activity ratio of methylated (M.SssI treated) to unmethylated control (CTL) plasmids containing a CpG-free promoter or the Gpx1 CpG island region. The assay was repeated 4 times and data are mean ± SEM. *: p

    Journal: PLoS ONE

    Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice

    doi: 10.1371/journal.pone.0151526

    Figure Lengend Snippet: DNA methylation of Gpx1 decreases gene expression. In vitro methylation of Gpx1 target region inhibited transcriptional activity, as measured by a luciferase reporter assay. (A) Schematic representation of the plasmid construction containing the Gpx1 CpG island region. (B) Luciferase activity ratio of methylated (M.SssI treated) to unmethylated control (CTL) plasmids containing a CpG-free promoter or the Gpx1 CpG island region. The assay was repeated 4 times and data are mean ± SEM. *: p

    Article Snippet: M. SssI CpG methyltransferase (New England Biolabs, Frankfurt, Germany) was used for in vitro methylation according to manufacturer’s instructions.

    Techniques: DNA Methylation Assay, Expressing, In Vitro, Methylation, Activity Assay, Luciferase, Reporter Assay, Plasmid Preparation, CTL Assay

    Biochemical methylation of CpG dinucleotide‐containing nucleosomal DNA (CpG146). Lanes are as follows: M, 10‐bp DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA; cat. 10821‐015); (+), presence; and (−), absence of the respective enzyme shown on the left. (A) Comparison of the CpG methyltransferase M.Sss I enzymatic activities. CpG146 DNA was methylated with M.Sss I enzymes as follows: lanes 3–5, M.Sss I purchased from New England Biolabs (NEB, cat. M0226M); and lanes 6–11, the M.Sss I protein purified in this study. In lanes 1 and 2, CpG146 DNAs were incubated in the methylation reaction buffer, in the absence of M.Sss I. Each DNA sample was methylated with an increasing amount of M.Sss I, and the DNA from each reaction was purified, digested with the methyl‐sensitive restriction endonuclease Eco72 I, and electrophoresed. The specific units of the NEB M.Sss I enzyme used in each reaction are as follows: lane 3, 2.0 units·μg −1 DNA; lane 4, 3.0 units·μg −1 DNA; and lane 5, 4.0 units·μg −1 DNA. The amount of the purified M.Sss I protein used in each reaction is as follows: lane 6, 0.032 μg·μg −1 DNA; lane 7, 0.063 μg·μg −1 DNA; lane 8, 0.13 μg·μg −1 DNA; lane 9, 0.25 μg·μg −1 DNA; lane 10, 0.50 μg·μg −1 DNA; and lane 11, 1.0 μg·μg −1 DNA. The positions of the 146‐bp CpG146 nucleosomal DNA, the half unit of CpG146, and the digested DNAs are shown by a red arrow, a black arrow, and a gray square bracket, respectively. (B) Confirmation of the CpG‐methylated CpG146 DNA by Eco72 I digestion. The digestion was performed with 20 units·μg −1 DNA (1.8 units·pmol −1 DNA). Lanes 1 and 3, unmodified CpG146 DNA; lanes 2 and 4, M.Sss I‐treated CpG146 DNA. In lanes 3 and 4, the nucleosomal DNA was subsequently incubated with the methylation‐sensitive restriction enzyme Eco72 I. (C) Confirmation of the CpG‐methylated CpG146 DNA by Msp JI digestion. The digestion was performed with 5 units·μg −1 DNA (0.45 units·pmol −1 DNA). Lanes 1 and 3, unmodified CpG146 DNA; lanes 2 and 4, M.Sss I‐treated CpG146 DNA. In lanes 3 and 4, the nucleosomal DNA was subsequently incubated with the methylation‐dependent restriction enzyme Msp JI.

    Journal: FEBS Open Bio

    Article Title: Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation

    doi: 10.1002/2211-5463.12064

    Figure Lengend Snippet: Biochemical methylation of CpG dinucleotide‐containing nucleosomal DNA (CpG146). Lanes are as follows: M, 10‐bp DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA; cat. 10821‐015); (+), presence; and (−), absence of the respective enzyme shown on the left. (A) Comparison of the CpG methyltransferase M.Sss I enzymatic activities. CpG146 DNA was methylated with M.Sss I enzymes as follows: lanes 3–5, M.Sss I purchased from New England Biolabs (NEB, cat. M0226M); and lanes 6–11, the M.Sss I protein purified in this study. In lanes 1 and 2, CpG146 DNAs were incubated in the methylation reaction buffer, in the absence of M.Sss I. Each DNA sample was methylated with an increasing amount of M.Sss I, and the DNA from each reaction was purified, digested with the methyl‐sensitive restriction endonuclease Eco72 I, and electrophoresed. The specific units of the NEB M.Sss I enzyme used in each reaction are as follows: lane 3, 2.0 units·μg −1 DNA; lane 4, 3.0 units·μg −1 DNA; and lane 5, 4.0 units·μg −1 DNA. The amount of the purified M.Sss I protein used in each reaction is as follows: lane 6, 0.032 μg·μg −1 DNA; lane 7, 0.063 μg·μg −1 DNA; lane 8, 0.13 μg·μg −1 DNA; lane 9, 0.25 μg·μg −1 DNA; lane 10, 0.50 μg·μg −1 DNA; and lane 11, 1.0 μg·μg −1 DNA. The positions of the 146‐bp CpG146 nucleosomal DNA, the half unit of CpG146, and the digested DNAs are shown by a red arrow, a black arrow, and a gray square bracket, respectively. (B) Confirmation of the CpG‐methylated CpG146 DNA by Eco72 I digestion. The digestion was performed with 20 units·μg −1 DNA (1.8 units·pmol −1 DNA). Lanes 1 and 3, unmodified CpG146 DNA; lanes 2 and 4, M.Sss I‐treated CpG146 DNA. In lanes 3 and 4, the nucleosomal DNA was subsequently incubated with the methylation‐sensitive restriction enzyme Eco72 I. (C) Confirmation of the CpG‐methylated CpG146 DNA by Msp JI digestion. The digestion was performed with 5 units·μg −1 DNA (0.45 units·pmol −1 DNA). Lanes 1 and 3, unmodified CpG146 DNA; lanes 2 and 4, M.Sss I‐treated CpG146 DNA. In lanes 3 and 4, the nucleosomal DNA was subsequently incubated with the methylation‐dependent restriction enzyme Msp JI.

    Article Snippet: The specific activity of the purified M.Sss I enzyme was determined using CpG146 DNA as substrate, in comparison with the activity unit of the NEB M.Sss I enzyme (cat. M0226M), by quantifying the amounts of the Eco72 I‐digested DNA fragments.

    Techniques: Methylation, Purification, Incubation

    Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma

    doi: 10.1111/j.1582-4934.2010.01124.x

    Figure Lengend Snippet: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P

    Article Snippet: The methylation of the CD147 promoter probe was obtained through incubation with CpG methyltransferase M.SssI (New England BioLabs) as above-mentioned.

    Techniques: Methylation, Binding Assay, Activity Assay, In Vitro, Electrophoretic Mobility Shift Assay, Mutagenesis, DNA Methylation Assay, Transfection, Construct, Luciferase

    Inhibition of the activity of BLV LTR by CpG methyltransferase Sss I. Human embryonic kidney 293T cells were transfected with pME18Neo that encoded the wild-type Tax protein or with the control plasmid pME18Neo, pGV-BLTR that had been treated ( Sss I) or not treated (-) with Sss I, and the reference plasmid pRL-SV40. At 60 h after transfection, cells were recovered and the activities of firefly luciferase and Renilla luciferase were measured in cell lysates. For each sample, relative raw light units were calculated by dividing the activity of firefly luciferase by that of Renilla luciferase.

    Journal: Journal of Virology

    Article Title: Latency of Viral Expression In Vivo Is Not Related to CpG Methylation in the U3 Region and Part of the R Region of the Long Terminal Repeat of Bovine Leukemia Virus

    doi: 10.1128/JVI.77.7.4423-4430.2003

    Figure Lengend Snippet: Inhibition of the activity of BLV LTR by CpG methyltransferase Sss I. Human embryonic kidney 293T cells were transfected with pME18Neo that encoded the wild-type Tax protein or with the control plasmid pME18Neo, pGV-BLTR that had been treated ( Sss I) or not treated (-) with Sss I, and the reference plasmid pRL-SV40. At 60 h after transfection, cells were recovered and the activities of firefly luciferase and Renilla luciferase were measured in cell lysates. For each sample, relative raw light units were calculated by dividing the activity of firefly luciferase by that of Renilla luciferase.

    Article Snippet: Nonetheless, we also showed that the transactivation activity of LTR was reduced after treatment with the CpG methyltransferase Sss I.

    Techniques: Inhibition, Activity Assay, Transfection, Plasmid Preparation, Luciferase