TET1 is controlled by pluripotency-associated factors in ESCs and downmodulated by PRC2 in differentiated cells and tissues

Abstract

Ten-eleven translocation (Tet) genes encode for a family of hydroxymethylase enzymes involved in regulating DNA methylation dynamics. Tet1 is highly expressed in mouse embryonic stem cells (ESCs) where it plays a critical role the pluripotency maintenance. Tet1 is also involved in cell reprogramming events and in cancer progression. Although the functional role of Tet1 has been largely studied, its regulation is poorly understood. Here we show that Tet1 gene is regulated, both in mouse and human ESCs, by the stemness specific factors Oct3/4, Nanog and by Myc. Thus Tet1 is integrated in the pluripotency transcriptional network of ESCs. We found that Tet1 is switched off by cell proliferation in adult cells and tissues with a consequent genome-wide reduction of 5hmC, which is more evident in hypermethylated regions and promoters. Tet1 downmodulation is mediated by the Polycomb repressive complex 2 (PRC2) through H3K27me3 histone mark deposition. This study expands the knowledge about Tet1 involvement in stemness circuits in ESCs and provides evidence for a transcriptional relationship between Tet1 and PRC2 in adult proliferating cells improving our understanding of the crosstalk between the epigenetic events mediated by these factors.

Figure 1.

Regulation of Tet1 in mouse ES cells. (A) Genomic view of RNA-seq and H3K4me3 in MEFs and mouse ESCs together with reconstructed Tet1 gene structure. (B) Isoform-specific RT-qPCR of Tet1 mRNA in the indicated samples. (C) qPCR of ChIP analysis for the indicated transcriptional factors on Tet1.1 promoter in mESCs. (D) Isoform-specific RT-qPCR of Tet1 mRNA in ESCs cultured without LIF for the indicated times. (E) Isoform-specific RT-qPCR of Tet1 mRNA in control and cMyc/Mycn, Nanog and Oct3/4 knockdowns ESCs. Error bars represent the standard deviation of three independent experiments. P-value was calculated by using t-test. (* = P-value < 0.01).

Figure 2.

Regulation of Tet1 in mouse proliferating cells. (A) Immunofluorescence analysis of Pcna and 5hmC in liver after hepatectomyzed the indicated time. DAPI is used for nuclei staining. (B) RT-qPCR of Tet1 and Pcna mRNA in liver after hepatectomy. (C) RT-qPCR of Tet1 mRNA in mouse embryo or in MEFs after several passages. (D) Dot-blot analysis of 5hmC and 5mC in mouse embryo or in MEFs after several passages. (E) Left panel: dot-blot analysis of 5hmC and 5mC in MEFs just extracted (p0) or maintained proliferating (p5) or blocked (by confluence or Mitomicyn C) from p0. Right panel: RT-qPCR of Tet1 mRNA in MEF in the same conditions. (F) Same experiment as in (E), but the blocking of proliferation was induced at p5 and maintained for other 5 days. Error bars represent the standard deviation of three independent experiments. P-value was calculated by using t-test.

Figure 3.

Tet1 regulates proliferation in primary cells. (A) Western blot analysis of Tet1 in control or Tet1 silenced MEF, after 24 h from silencing. Actb was used as loading control. (B) Dot-blot analysis of 5hmC and 5mC in Tet1 silenced MEFs. ssDNA was used as loading control. (C and D) FACS cell cycle and EdU incorporation analysis (at p2) in Tet1 silenced MEFs. (E) Cell growth assay in Tet1 silenced MEFs. (F) Dot-blot analysis of 5hmC and 5mC in control or Tet1 expressing MEFs at several passages. ssDNA was used as loading control. (G) FACS EdU incorporation analysis (at p2) in control or Tet1 expressing MEFs. Error bars represent the standard deviation of three independent experiments. (H) Cell growth assay in control or Tet1 expressing MEFs at several passages.

Figure 4.

Transcriptional regulation of Tet1.2 in proliferating cells. (A) RT-qPCR of Tet1 hnRNA in MEFs at the indicated passages. (B) Genomic view of the Tet1 promoters for the indicated ChIP-seq analysis in several mouse adult tissues. (C) qPCR of ChIP analysis for H3K4me3 and H3K27me3 in MEFs at the indicated passages. (D) qPCR of ChIP analysis for Ezh2 in MEFs at the indicated passages. (E) RT-qPCR of Suz12 and Tet1 mRNAs in control and Suz12 knockdown MEFs. (F) Western blot analysis of Tet1b in control or Suz12 silenced MEFs. Actb was used as loading control. (G) qPCR of ChIP analysis for H3K27me3 and Ezh2 in MEFs in the indicated knockdown. (H) Dot-blot analysis of 5hmC and 5mC in control or Suz12 silenced MEFs. ssDNA was used as loading control. Error bars represent the standard deviation of three independent experiments. P-value was calculated by using t-test. ** P-value < 0.001.

Figure 5.

Regulation of TET1 in human embryonic stem cells. (A) RT-qPCR of TET1 mRNA in the indicated samples. (B) Genomic view of RNA-seq and of the indicated ChIP-seq in human ESCs. (C) qPCR of ChIP analysis of the indicated transcriptional factors on the TET1 promoter in human ESCs. (D) RT-qPCR of TET1 mRNA in hESCs silenced for the indicated transcriptional factors. Error bars represent the standard deviation of three independent experiments. P-value was calculated by using t-test.

Figure 6.

Regulation of TET1 in human adult cells. (A) RT-qPCR of TET1 mRNA in HUVECs at the indicated passages (B) Dot-blot analysis of 5hmC and 5mC level in HUVECs at the indicated passages. ssDNA was used as a loading control. (C) Genomic view of the TET1 promoter for the indicated ChIP-seq analysis in hESCs and several human differentiated cells. (D) qPCR of ChIP analysis for H3K4me3 and H3K27me3 in HUVECs at the indicated passages. (E) qPCR of ChIP analysis for H3K4me3 and H3K27me3 in HUVECs at the indicated passages. Error bars represent the standard deviation of three independent experiments.