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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 regulates gene transcription independent of its demethylating activity. (A and B) Effect of TET1 knockdown on the expressions of its target genes with (A) or without (B) increased DNA methylation spreading into their CGI promotes. Data represent mean ± SD (n = 3). *P < 0.05, **P < 0.01 by Student's t-test compared to shControl cells. (C) RNA-seq transcriptional landscapes of the TET1 gene in the vector control and (m)TET1-FL- or (m)TET1-CD-overexpressing HEK293T cells. The two introduced substitution mutations in mTET1-FL and mTET1-CD were also validated by RNA-seq. (D and E) Scatter plots comparing the RNA-seq-derived gene expression profiles among the vector control, TET1-FL and mTET1-FL overexpressions (D), and among the vector control, TET1-CD and mTET1-CD overexpressions (E). Pearson correlation coefficients, r, are listed in each graph. (F) Venn diagram showing the overlap of differentially expressed genes (>2-fold change, FDR < 0.05) of TET1-FL, mTET1-FL, TET1-CD and mTET1-CD overexpressions. (G) Heatmap showing hierarchical clustering of differentially expressed genes of TET1-FL, mTET1-FL, TET1-CD and mTET1-CD overexpressions. All cells were collected by FACS 3 days after transfection.
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Figure 7: TET1 regulates gene transcription independent of its demethylating activity. (A and B) Effect of TET1 knockdown on the expressions of its target genes with (A) or without (B) increased DNA methylation spreading into their CGI promotes. Data represent mean ± SD (n = 3). *P < 0.05, **P < 0.01 by Student's t-test compared to shControl cells. (C) RNA-seq transcriptional landscapes of the TET1 gene in the vector control and (m)TET1-FL- or (m)TET1-CD-overexpressing HEK293T cells. The two introduced substitution mutations in mTET1-FL and mTET1-CD were also validated by RNA-seq. (D and E) Scatter plots comparing the RNA-seq-derived gene expression profiles among the vector control, TET1-FL and mTET1-FL overexpressions (D), and among the vector control, TET1-CD and mTET1-CD overexpressions (E). Pearson correlation coefficients, r, are listed in each graph. (F) Venn diagram showing the overlap of differentially expressed genes (>2-fold change, FDR < 0.05) of TET1-FL, mTET1-FL, TET1-CD and mTET1-CD overexpressions. (G) Heatmap showing hierarchical clustering of differentially expressed genes of TET1-FL, mTET1-FL, TET1-CD and mTET1-CD overexpressions. All cells were collected by FACS 3 days after transfection.

Mentions: DNA methylation of CGI promoters is associated with repressed gene transcription (2,46), and TET1 has previously been reported to affect gene expression (21,22,25,28). We therefore asked whether TET1 is required for the active transcription of target genes by preventing de novo DNA methylation spreading into the CGI promoters. We first analyzed the effect of TET1 knockdown on the expression of target genes previously analyzed. TET1 knockdown reduced the expression of only three out of five genes for which we confirmed DNA methylation spreading into their promoter CGIs (Figure 7A), and no or inconsistent expression changes of other three genes that had unchanged DNA methylation at their promoter CGIs (Figure 7B). We next used cDNA microarrays to analyze whole genome gene expression changes after TET1 knockdown. Using a criterion of 1.5-fold expression change, 89 upregulated and 97 downregulated genes were identified in both TET1 knockdown cell clones (Supplementary Figure S12A), and some of them were further validated by RT-qPCR (Supplementary Figure S12B and C). Three of these validated downregulated genes were further proved to be TET1 target genes but did not consistently gain DNA methylation spreading in their CGI promoters after TET1 knockdown (Supplementary Figure S12D and E). Thus, consistent with previous results indicating that depletion of Tet1 induces similar gene expression changes in wild type and Dnmt TKO mESCs (25), our data suggest relatively minor effects of TET1 on gene transcription in HEK293T cells that are both DNA methylation-dependent and -independent.


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 regulates gene transcription independent of its demethylating activity. (A and B) Effect of TET1 knockdown on the expressions of its target genes with (A) or without (B) increased DNA methylation spreading into their CGI promotes. Data represent mean ± SD (n = 3). *P < 0.05, **P < 0.01 by Student's t-test compared to shControl cells. (C) RNA-seq transcriptional landscapes of the TET1 gene in the vector control and (m)TET1-FL- or (m)TET1-CD-overexpressing HEK293T cells. The two introduced substitution mutations in mTET1-FL and mTET1-CD were also validated by RNA-seq. (D and E) Scatter plots comparing the RNA-seq-derived gene expression profiles among the vector control, TET1-FL and mTET1-FL overexpressions (D), and among the vector control, TET1-CD and mTET1-CD overexpressions (E). Pearson correlation coefficients, r, are listed in each graph. (F) Venn diagram showing the overlap of differentially expressed genes (>2-fold change, FDR < 0.05) of TET1-FL, mTET1-FL, TET1-CD and mTET1-CD overexpressions. (G) Heatmap showing hierarchical clustering of differentially expressed genes of TET1-FL, mTET1-FL, TET1-CD and mTET1-CD overexpressions. All cells were collected by FACS 3 days after transfection.
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Figure 7: TET1 regulates gene transcription independent of its demethylating activity. (A and B) Effect of TET1 knockdown on the expressions of its target genes with (A) or without (B) increased DNA methylation spreading into their CGI promotes. Data represent mean ± SD (n = 3). *P < 0.05, **P < 0.01 by Student's t-test compared to shControl cells. (C) RNA-seq transcriptional landscapes of the TET1 gene in the vector control and (m)TET1-FL- or (m)TET1-CD-overexpressing HEK293T cells. The two introduced substitution mutations in mTET1-FL and mTET1-CD were also validated by RNA-seq. (D and E) Scatter plots comparing the RNA-seq-derived gene expression profiles among the vector control, TET1-FL and mTET1-FL overexpressions (D), and among the vector control, TET1-CD and mTET1-CD overexpressions (E). Pearson correlation coefficients, r, are listed in each graph. (F) Venn diagram showing the overlap of differentially expressed genes (>2-fold change, FDR < 0.05) of TET1-FL, mTET1-FL, TET1-CD and mTET1-CD overexpressions. (G) Heatmap showing hierarchical clustering of differentially expressed genes of TET1-FL, mTET1-FL, TET1-CD and mTET1-CD overexpressions. All cells were collected by FACS 3 days after transfection.
Mentions: DNA methylation of CGI promoters is associated with repressed gene transcription (2,46), and TET1 has previously been reported to affect gene expression (21,22,25,28). We therefore asked whether TET1 is required for the active transcription of target genes by preventing de novo DNA methylation spreading into the CGI promoters. We first analyzed the effect of TET1 knockdown on the expression of target genes previously analyzed. TET1 knockdown reduced the expression of only three out of five genes for which we confirmed DNA methylation spreading into their promoter CGIs (Figure 7A), and no or inconsistent expression changes of other three genes that had unchanged DNA methylation at their promoter CGIs (Figure 7B). We next used cDNA microarrays to analyze whole genome gene expression changes after TET1 knockdown. Using a criterion of 1.5-fold expression change, 89 upregulated and 97 downregulated genes were identified in both TET1 knockdown cell clones (Supplementary Figure S12A), and some of them were further validated by RT-qPCR (Supplementary Figure S12B and C). Three of these validated downregulated genes were further proved to be TET1 target genes but did not consistently gain DNA methylation spreading in their CGI promoters after TET1 knockdown (Supplementary Figure S12D and E). Thus, consistent with previous results indicating that depletion of Tet1 induces similar gene expression changes in wild type and Dnmt TKO mESCs (25), our data suggest relatively minor effects of TET1 on gene transcription in HEK293T cells that are both DNA methylation-dependent and -independent.

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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Related in: MedlinePlus