| Summary: | <p>During early development in female mammals one of the two X chromosomes is transcriptionally silenced, ensuring an equal dosage of X-linked genes relative to their XY male counterparts. This phenomenon is termed X chromosome inactivation (XCI). XCI is initiated by expression of the long noncoding RNA (lncRNA) Xist from the inactive X chromosome (Xi). Xist orchestrates molecular pathways in cis to establish transcriptional silencing and heterochromatinization of the Xi in a multi- stage process. A late stage of XCI is the methylation of CpG-rich regions associated with gene promoters, called CpG Islands (CGIs). Mammals express two highly similar de novo DNA methyl transferases, DNMT3A and DNMT3B. Intriguingly, only DNMT3B is crucial for Xi CGI methylation, and it is unclear what factor(s) imparts this specificity. </p>
<p>To address the mechanisms governing DNMT3B specificity, I have developed a sensitive experimental model system, utilising an XX mouse ES cell line with inducible Xist (iXist-ChrX) and quantitative MBD-seq, to conduct high-resolution, allele-specific analysis of XCI CGI methylation dynamics. This model was used to analyse Dnmt3b<sup>-/-</sup> cells and complementation lines expressing specific isoforms or mutants of DNMT3B. A detailed analysis of MBD-seq datasets unveiled that DNMT3B is dispensable for the methylation of a small group of X-linked CGIs and “CGI-like elements”, a novel group of CpG-rich regions reported in this work that possess enhancer-like chromatin features and gain methylation on the Xi. I demonstrate that the catalytically active isoform of DNMT3B, DNMT3B1, fully rescues CGI methylation in Dnmt3b<sup>-/-</sup> cells and that the widely expressed catalytically inactive isoform, DNMT3B3, does not contribute to Xi CGI methylation. I also explore the contribution of the less characterized DNMT3B N-terminal domain as a putative specificity imparting region, and report that this is indeed crucial for DNMT3B1 mediated CGI methylation on Xi and autosomes. Intriguingly, both complete or partial deletion of the N-terminal domain of DNMT3B1 abrogates the rescue of CGI methylation observed with full-length DNMT3B1. Furthermore, the N-terminal domain has a context-specific role in the genomic localization of DNMT3B1, being crucial for the localization/residence of DNMT3B1 at CGIs and the Xi.</p>
<p>By studying the effect of a broad range of DNMT3B manipulations on the methylation of X-linked CGIs and performing an exploratory proteomics analysis to identify proteins associated with DNMT3B before and after XCI, this work presents new insights into Xi CGI methylation and makes progress towards understanding the specificity of DNMT3B on the Xi.</p>
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