GSE38568
Distinct roles of Tet1 and Tet2 in mouse embryonic stem cells (MeDIP-seq)
The TET proteins TET1, TET2 and TET3 constitute a new family of dioxygenases that utilize molecular oxygen and the cofactors Fe(II) and 2-oxoglutarate to convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in DNA. Tet1 and Tet2 are expressed at high levels in mouse embryonic stem cells, and together are responsible for essentially all the oxidized methylcytosine present in these cells. Here we show that Tet1 and Tet2 have independent as well as overlapping roles in embryonic stem cells. Genes with Tet1-regulated 5hmC at transcription start sites (TSS) were expressed at lower levels. These TSS were occupied by polycomb repressor complexes (PRC), and were enriched for “bivalent” H3K27me3 and H3K4me3 marks; in contrast, genes with Tet2-regulated 5hmC at their TSS were expressed at higher levels and these TSS showed no enrichment for bivalent marks and PRC. When 5hmC levels were monitored by CMS-IP, the major effects of Tet1 and Tet2 were to diminish 5hmC levels at transcription start sites (TSS) and intron-exon boundaries, respectively. By RNA sequencing, Tet1 depletion altered the expression of entire genes, whereas Tet2 depletion selectively altered the expression of individual exons. Since loss-of-function mutations in TET2 and in splicing factors are both associated with haematopoietic malignancies in humans, we investigated the possible relationship between Tet2 and pre-mRNA splicing. 5hmC levels were selectively depleted in exons positively regulated by Tet2 (i.e. whose expression was downregulated upon Tet2 depletion), and CTCF binding to sites in the vicinity of downregulated exons was diminished by Tet2 depletion. Together, these data indicate that Tet1 and Tet2 play distinct roles in regulating 5hmC distribution and pre-mRNA splicing in embryonic stem cells, and suggest that one mechanism by which Tet2 controls the inclusion of individual exons is by facilitating CTCF binding in the vicinity. Overall design: Mapping of 5-hydroxymethylcytosine in ES, Tet1 knockdown and Tet2 knockdown cells by anti-CMS methodologies, and in input cells respetively.