A dose-sensitive OGT-TET3 complex is necessary for normal Xist RNA distribution and function

Abstract

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. We identified an X-linked gene, Ogt, that controls levels of 5mC and 5hmC in mESCs. OGT is a post-translational modification enzyme and we identified the 5-methylcytosine dioxygenase TET3 as an OGT target that is differentially modified in XX and XY mESCs. In addition to influencing 5mC and 5hmC abundance, OGT dose also controls TET3 and OGT distribution. OGT and TET3 are predominantly nuclear in XX mESCs and cytoplasmic in XY mESCs. Furthermore, these proteins are present in different complexes in XX and XY mESCs. Mutational analysis revealed that TET3 determines the XX-specific abundance of 5mC and 5hmC in mESCs. While TET3 null XX mESCs exhibited modest changes in gene expression, there were substantial alterations upon differentiation into epiblast-like cells (mEpiLCs). In addition, these TET3 null XX mESCs did not undergo X-chromosome inactivation (XCI) when differentiated. These data suggest that an X-dose sensitive complex containing OGT and TET3 regulates cytosine modifications and XCI.