Abstract
ABSTRACT Ten-Eleven Translocation (TET) proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) leading to a dynamic epigenetic state of DNA that can influence transcription and chromatin organization. While TET proteins interact with complexes involved in transcriptional repression and activation, the overall understanding of the molecular mechanisms involved in TET-mediated regulation of gene expression still remains limited. Here, we show that TET proteins interact with the chromatin remodelling protein lymphoid-specific helicase (LSH/HELLS) in vivo and in vitro. In mouse embryonic fibroblasts (MEFs) and embryonic stem cells (ESCs) knock out of Lsh leads to a significant reduction of 5-hydroxymethylation amount in the DNA. Whole genome sequencing of 5hmC in wild-type versus Lsh knock-out MEFs and ESCs showed that in absence of Lsh, some regions of the genome gain 5hmC while others lose it, with mild correlation with gene expression changes. We further show that differentially hydroxymethylated regions did not completely overlap with differentially methylated regions indicating that changes in 5hmC distribution upon Lsh knock-out are not a direct consequence of 5mC decrease. Altogether, our results suggest that LSH, which interacts with TET proteins, contributes to the regulation of 5hmC levels and distribution in MEFs and ESCs.
Highlights
Ten-Eleven Translocation (TET) proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) leading to a dynamic epigenetic state of DNA that can influence transcription and chromatin organization
To explore the mechanisms of action of TET proteins, we used the already described HaloTag technology followed by mass-spectrometry analysis, in order to identify proteins co-immunoprecipitating with Halo-tagged-TET1, -TET2 and -TET3 protein expressed in human HEK293T cells [29]
We found by co-immunoprecipitation that endogenous LSH interacts with Flag-tagged catalytic domains (CD) of TET1, TET2 and TET3 but not with the Flag control (Figure 1a)
Summary
Ten-Eleven Translocation (TET) proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) leading to a dynamic epigenetic state of DNA that can influence transcription and chromatin organization. Our results suggest that LSH, which interacts with TET proteins, contributes to the regulation of 5hmC levels and distribution in MEFs and ESCs. 5-methylcytosine (or 5mC) is an essential DNA modification in mammals. Various TETs interacting partners have been described, shedding some light on how TET proteins could act on 5hmC levels and distribution Those include partners such as transcriptional factors/nuclear receptors (IDAX/CXXC4 [20], NANOG [21], PPARγ [22], SP1/PU.1 [23], EBF1 [24], PRDM14 [25], GADD45a [26], NF-κB [27] and ZSCAN4 [28], to name a few), chromatinassociated proteins involved in transcriptional activation (OGT and SET1/COMPASS complex [29]) or repression (SIN3A/HDACs, EZH2, NURD [29,30]) and the promyelocytic leukaemia (PML) protein [31]. Its deletion causes a lethal phenotype after birth with tissue-specific defects including skeletal defects, a smaller thymus and a barely detectable spleen [51,68,69]
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