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. 2014 Jan 28;111(4):1361-6.
doi: 10.1073/pnas.1322921111. Epub 2014 Jan 13.

Distinct roles of the methylcytosine oxidases Tet1 and Tet2 in mouse embryonic stem cells

Yun Huang  1 , Lukas Chavez, Xing Chang, Xue Wang, William A Pastor, Jinsuk Kang, Jorge A Zepeda-Martínez, Utz J Pape, Steven E Jacobsen, Bjoern Peters, Anjana Rao
Affiliations

Distinct roles of the methylcytosine oxidases Tet1 and Tet2 in mouse embryonic stem cells

Yun Huang et al. Proc Natl Acad Sci U S A. .

Abstract

Dioxygenases of the Ten-Eleven Translocation (TET) family are 5-methylcytosine oxidases that convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in DNA. We show that Tet1 and Tet2 have distinct roles in regulating 5hmC in mouse embryonic stem cells (mESC). Tet1 depletion diminishes 5hmC levels at transcription start sites (TSS), whereas Tet2 depletion is predominantly associated with decreased 5hmC in gene bodies. Enrichment of 5hmC is observed at the boundaries of exons that are highly expressed, and Tet2 depletion results in substantial loss of 5hmC at these boundaries. In contrast, at promoter/TSS regions, Tet2 depletion results in increased 5hmC, potentially because of the redundant activity of Tet1. Together, the data point to a complex interplay between Tet1 and Tet2 in mESC, and to distinct roles for these two proteins in regulating promoter, exon, and polyadenylation site usage in cells.

Keywords: DNA demethylation; DNA hydroxymethylation; DNA methylation; epigenetics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Distinct 5hmC distribution in Tet1 kd or Tet2 kd mESC. (A) Western blot showing that v6.5 mESC with stable shRNA-mediated depletion of Tet1 and Tet2 show ∼90% depletion of Tet1 and Tet2 protein levels, with no or little change in protein levels of the other Tet protein. (B) Levels of 5hmC in parental v6.5 ESC and ESC stably depleted of Tet1 or Tet2, quantified by dot blot using anti-CMS antiserum. (Lower) A dot-blot with twofold dilutions of genomic DNA is shown. (Upper) Quantification based on a standard curve of twofold dilutions of a 5hmC-containing oligonucleotide. (C) Immunocytochemical staining of 5hmC in parental, Tet1 or Tet2 deleted mESC. Left, DAPI; Right, 5hmC. Consistent with the dot-blot assay, 5hmC staining is weaker in Tet2 KO mESC than that in Tet1 KO mESC. (D) Distribution of averaged 5hmC enrichment at all genes in parental, Tet1 kd, or Tet2 kd mESC. (E) Distribution of averaged 5hmC enrichment in parental mESC ranked by gene expression. (F) Distribution of average 5hmC enrichment at high, intermediate, low and non-expressed genes in parental (black), Tet1 kd (blue) and Tet2 kd (red) mESCs. For a heatmap representation of the data in Fig. 1 E and F, see SI Appendix, Fig. S2.
Fig. 2.
Fig. 2.
Distribution of DHMRs in Tet1 kd and Tet2 kd mESC. (A) Venn diagram showing the overlap of DHMRs in Tet1 kd compared with Tet2 kd mESC. (B) Genomic distribution of DHMRs in Tet1 kd or Tet2 kd mESC. DHMRs with gain of 5hmC in Tet2 kd specifically show enrichment at promoter-TSS regions. An alternative representation is shown in SI Appendix, Fig. S5A. (C and D) Heat map of histone modification and transcription factor enrichment at all DHMRs with gain of 5hmC (C), or DHMRs with gain of 5hmC at promoter regions (D), in Tet2kd mESC. (E) Relation of differentially methylated (y axis) and hydroxymethylated (x axis) 300-bp windows (assessed by MeDIP and CMS-IP, respectively) in Tet2 kd mESC. Only 300-bp windows with significant changes in both 5hmC and 5mC are shown. In all cases (Left, all windows; Center, windows overlapping with promoters; Right, windows overlapping with enhancers defined by the presence of H3K4me1 but the absence of H3K4me3), the majority of windows show loss of both 5hmC and 5mC.
Fig. 3.
Fig. 3.
Differentially expressed genes and 5hmC distribution in Tet1 kd or Tet2 kd mESC compared with parental mESC. (A) Genes differentially expressed in Tet1 kd and Tet2 kd compared with parental mESC were identified using DESeq. The numbers of genes significantly up- or down-regulated in Tet1 and Tet2 kd mESC are indicated. Three genes (Uty, Ddx3y, Eif2s3y) located on the Y chromosome were highly down-regulated in both Tet1 and Tet2 kd mESC. (B) Venn diagram showing the overlap of differentially expressed genes in Tet1 kd and Tet2 kd mESC (also see SI Appendix, Fig. S7). (C) Correlation of significant change of gene expression (y axis) with promoter (−1 kb to +0.5 kb relative to the TSS; Upper) and gene body (Lower) 5hmC (x axis) in Tet1 kd (blue) and Tet2 kd (red) mESC. The figure shows all genes for which RNA-seq and promoter or gene body CMS-IP data are available. (D) Graphical representation of C.
Fig. 4.
Fig. 4.
Differential exon use and 5hmC distribution at exons in parental, Tet1 kd, and Tet2 kd mESC. (A) Enrichment of 5hmC at exon start and exon end sites for exons with high (top 10%, black), intermediate (10–90%, green), low (bottom 10%, purple), or no (blue) expression in parental mESC. (B) Numbers of differentially expressed exons in Tet1 kd and Tet2 kd. All affected exons, exons located within differentially expressed genes; selectively affected exons, exons that are differentially expressed relative to the entire transcript. (C) Venn diagram showing the overlap of selectively affected exons in Tet1kd or Tet2kd mESC. (D) Differentially expressed exons were analyzed by DEseq. Exons located within the three most significantly down-regulated genes on the Y chromosome (Uty, Ddx3y, and Eif2s3y) are highlighted. (E) Distribution of averaged 5hmC enrichment at exon start (Left) and end (Right) sites for the top 10% of high-expressed exons in parental, Tet1 kd and Tet2 kd mESC.
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