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TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells.

Jin C, Lu Y, Jelinek J, Liang S, Estecio MR, Barton MC, Issa JP - Nucleic Acids Res. (2014)

Bottom Line: TET1-FL specifically accumulates 5-hydroxymethylcytosine at the edges of hypomethylated CGIs, while knockdown of endogenous TET1 induces methylation spreading from methylated edges into hypomethylated CGIs.We also found that gene expression changes after TET1-FL overexpression are relatively small and independent of its dioxygenase function.Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically prevents aberrant methylation spreading into CGIs in differentiated cells.

View Article: PubMed Central - PubMed

Affiliation: The Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA Department of Biochemistry and Molecular Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, PA 19140, USA.

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TET1-FL induces specific accumulation of 5hmC at the edges of hypomethylated CGIs. (A) Examples of hMeDIP-Seq profiles in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Gene distribution (exons) and CGIs are indicated below the graph. (B) The genomic distributions of 5hmC peaks detected in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Upstream: −5 to −1 kb relative to TSS; promoter: −1 to 0.5 kb relative to TSS; downstream: −0.5 to 1 kb relative to TES; intergenic: 1 kb from TES to −5 kb of downstream gene. (C) Distributions of 5hmC tag across gene bodies in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Each gene body was normalized to 0–100%. (D and E) Distributions of 5hmC tag around TSSs that overlap CGIs (D) or non-CGIs (E). (F) Distributions of 5hmC tag across CGIs which locates in gene promoters. Total 13 913 out of 27 639 CGIs reside in promoter in human genome. (G and H) Distributions of 5hmC tag across the non-promoter CGIs which were either hypomethylated (methylation < 10%, G) or hypermethylated (methylation > 50%, H) based on DREAM data. Total 739 hypomethylated and 1059 hypermethylated non-promoter CGIs were covered by DREAM results and analyzed for 5hmC distribution. The cells overexpressing (m)TET1-CD or (m)TET1-FL were collected by FACS 3 or 7 days after transfection, respectively. Each CGI in (F–H) was normalized to 0–100%. The key to (C)–(H) is at the bottom.
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Figure 3: TET1-FL induces specific accumulation of 5hmC at the edges of hypomethylated CGIs. (A) Examples of hMeDIP-Seq profiles in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Gene distribution (exons) and CGIs are indicated below the graph. (B) The genomic distributions of 5hmC peaks detected in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Upstream: −5 to −1 kb relative to TSS; promoter: −1 to 0.5 kb relative to TSS; downstream: −0.5 to 1 kb relative to TES; intergenic: 1 kb from TES to −5 kb of downstream gene. (C) Distributions of 5hmC tag across gene bodies in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Each gene body was normalized to 0–100%. (D and E) Distributions of 5hmC tag around TSSs that overlap CGIs (D) or non-CGIs (E). (F) Distributions of 5hmC tag across CGIs which locates in gene promoters. Total 13 913 out of 27 639 CGIs reside in promoter in human genome. (G and H) Distributions of 5hmC tag across the non-promoter CGIs which were either hypomethylated (methylation < 10%, G) or hypermethylated (methylation > 50%, H) based on DREAM data. Total 739 hypomethylated and 1059 hypermethylated non-promoter CGIs were covered by DREAM results and analyzed for 5hmC distribution. The cells overexpressing (m)TET1-CD or (m)TET1-FL were collected by FACS 3 or 7 days after transfection, respectively. Each CGI in (F–H) was normalized to 0–100%. The key to (C)–(H) is at the bottom.

Mentions: As the primary product of TET-catalyzed 5mC oxidation reaction, 5hmC also serves as a critical intermediate for TET-induced DNA demethylation (13,15,17,18). We therefore asked whether the differences in demethylation induction between TET1-CD and TET1-FL were due to their different regulation of 5hmC distribution. We performed genome-wide mapping of 5hmC in HEK293T cells with 5hmC antibody-based hMeDIP-seq (Figure 3A and Supplementary Figure S3). Once again, we used as controls mTET1-CD and mTET1-FL, which cannot catalyze 5mC oxidation. In these two control transfections, a similar number of 5hmC peaks and an almost identical distribution pattern were detected (Figure 3B) with 5hmC enrichment around promoters but even distribution in exons and introns at a relatively low level (Figure 3C and Supplementary Figure S4A and B). The enrichment of 5hmC around TSSs is similar to what was previously observed in mESCs (25,42). Interestingly, 5hmC density showed a dip in CGI-overlapped TSSs but peaked at non-CGI-overlapped TSSs (Figure 3D and E).


TET1 is a maintenance DNA demethylase that prevents methylation spreading in differentiated cells.

Jin C, Lu Y, Jelinek J, Liang S, Estecio MR, Barton MC, Issa JP - Nucleic Acids Res. (2014)

TET1-FL induces specific accumulation of 5hmC at the edges of hypomethylated CGIs. (A) Examples of hMeDIP-Seq profiles in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Gene distribution (exons) and CGIs are indicated below the graph. (B) The genomic distributions of 5hmC peaks detected in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Upstream: −5 to −1 kb relative to TSS; promoter: −1 to 0.5 kb relative to TSS; downstream: −0.5 to 1 kb relative to TES; intergenic: 1 kb from TES to −5 kb of downstream gene. (C) Distributions of 5hmC tag across gene bodies in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Each gene body was normalized to 0–100%. (D and E) Distributions of 5hmC tag around TSSs that overlap CGIs (D) or non-CGIs (E). (F) Distributions of 5hmC tag across CGIs which locates in gene promoters. Total 13 913 out of 27 639 CGIs reside in promoter in human genome. (G and H) Distributions of 5hmC tag across the non-promoter CGIs which were either hypomethylated (methylation < 10%, G) or hypermethylated (methylation > 50%, H) based on DREAM data. Total 739 hypomethylated and 1059 hypermethylated non-promoter CGIs were covered by DREAM results and analyzed for 5hmC distribution. The cells overexpressing (m)TET1-CD or (m)TET1-FL were collected by FACS 3 or 7 days after transfection, respectively. Each CGI in (F–H) was normalized to 0–100%. The key to (C)–(H) is at the bottom.
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Figure 3: TET1-FL induces specific accumulation of 5hmC at the edges of hypomethylated CGIs. (A) Examples of hMeDIP-Seq profiles in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Gene distribution (exons) and CGIs are indicated below the graph. (B) The genomic distributions of 5hmC peaks detected in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Upstream: −5 to −1 kb relative to TSS; promoter: −1 to 0.5 kb relative to TSS; downstream: −0.5 to 1 kb relative to TES; intergenic: 1 kb from TES to −5 kb of downstream gene. (C) Distributions of 5hmC tag across gene bodies in HEK293T cells overexpressing (m)TET1-CD or (m)TET1-FL. Each gene body was normalized to 0–100%. (D and E) Distributions of 5hmC tag around TSSs that overlap CGIs (D) or non-CGIs (E). (F) Distributions of 5hmC tag across CGIs which locates in gene promoters. Total 13 913 out of 27 639 CGIs reside in promoter in human genome. (G and H) Distributions of 5hmC tag across the non-promoter CGIs which were either hypomethylated (methylation < 10%, G) or hypermethylated (methylation > 50%, H) based on DREAM data. Total 739 hypomethylated and 1059 hypermethylated non-promoter CGIs were covered by DREAM results and analyzed for 5hmC distribution. The cells overexpressing (m)TET1-CD or (m)TET1-FL were collected by FACS 3 or 7 days after transfection, respectively. Each CGI in (F–H) was normalized to 0–100%. The key to (C)–(H) is at the bottom.
Mentions: As the primary product of TET-catalyzed 5mC oxidation reaction, 5hmC also serves as a critical intermediate for TET-induced DNA demethylation (13,15,17,18). We therefore asked whether the differences in demethylation induction between TET1-CD and TET1-FL were due to their different regulation of 5hmC distribution. We performed genome-wide mapping of 5hmC in HEK293T cells with 5hmC antibody-based hMeDIP-seq (Figure 3A and Supplementary Figure S3). Once again, we used as controls mTET1-CD and mTET1-FL, which cannot catalyze 5mC oxidation. In these two control transfections, a similar number of 5hmC peaks and an almost identical distribution pattern were detected (Figure 3B) with 5hmC enrichment around promoters but even distribution in exons and introns at a relatively low level (Figure 3C and Supplementary Figure S4A and B). The enrichment of 5hmC around TSSs is similar to what was previously observed in mESCs (25,42). Interestingly, 5hmC density showed a dip in CGI-overlapped TSSs but peaked at non-CGI-overlapped TSSs (Figure 3D and E).

Bottom Line: TET1-FL specifically accumulates 5-hydroxymethylcytosine at the edges of hypomethylated CGIs, while knockdown of endogenous TET1 induces methylation spreading from methylated edges into hypomethylated CGIs.We also found that gene expression changes after TET1-FL overexpression are relatively small and independent of its dioxygenase function.Thus, our results identify TET1 as a maintenance DNA demethylase that does not purposely decrease methylation levels, but specifically prevents aberrant methylation spreading into CGIs in differentiated cells.

View Article: PubMed Central - PubMed

Affiliation: The Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, TX 77030, USA Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA Department of Biochemistry and Molecular Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030, USA Fels Institute for Cancer Research and Molecular Biology, Temple University, Philadelphia, PA 19140, USA.

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