UCSF

Female (XX) mouse embryonic stem cells (mESCs) differ from their male (XY) counterparts because they have lower levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). This difference in DNA modifications is a result of having two X chromosomes (Xs), both of which are active at this developmental stage. To test whether OGT is one of the X-linked proteins that regulate 5mC and 5hmC in mESCs, we manipulated OGT dose in XX and XY mESCs. We found that OGT abundance controls cytosine modifications, implicating OGT targets in 5mC and 5hmC regulation. Our quantitative comparison of the O-GlcNAcylated proteome in XX and XY mESCs revealed that O-GlcNAc modified TET3 peptides were more abundant in XX mESCs, which reflected an increase in TET3 amount in these cells. In addition to differing in abundance, TET3 and OGT distribution were also different in XX and XY mESCs. In XX cells, TET3 and OGT were enriched in the nucleus, while they were predominantly cytoplasmic in XY cells. TET3 and OGT occur in different high molecular weight complexes in XX and XY mESCs. When OGT is expressed from one X in XX mESCs, OGT and TET3 are predominantly cytoplasmic and 5mC/5hmC levels increase, indicating that OGT is one X-linked regulator of DNA cytosine modifications. To directly query whether TET3 is necessary for the female-specific 5mC and 5hmC in mESCs we generated homozygous TET3 mutant XX mESCs. In these cells, 5mC and 5hmC levels were decreased relative to wildtype XX mESCs, without dramatic gene expression changes. To investigate the developmental significance of TET3, examined the effects of this mutation on the ability of cells to undergo X chromosome inactivation (XCI) an epigenetic change that occurs when mESCs are differentiated into the next developmental stage, epiblast-like (mEpiL) cells. The establishment of XCI is characterized by the up-regulation of a non-coding RNA, Xist RNA, which remains in the nucleus and ‘coats’ the X concomitant with silencing. In TET3 mutant XX mEpiLCs Xist RNA exhibits abnormal distribution and silencing defects. These results link the activity of a dose-sensitive complex containing X and autosomal proteins to regulation of cytosine DNA modifications and XCI.