Periodic Reporting for period 4 - XPRESS (Exploring mechanisms of gene repression and escape during X-chromosome inactivation)
Periodo di rendicontazione: 2020-05-01 al 2021-04-30
In the XPRESS ERC grant we have provided key insights into the molecular mechanisms that ensure the variable silencing of genes during XCI. The use of an embryonic stem cell (ESC) line with highly polymorphic X chromosomes, in which Xist RNA could be induced on one X chromosome upon Doxycycline treatment, provided a powerful system in which to decipher the molecular basis of XCI. Different levels of epigenetic control were explored by studying chromosome wide changes at the nucleotide level using genomics approaches, and investigating the factors involved in these changes using both biochemical and imaging approaches. We also used genetic engineering and fluorescently labelled cells to screen for new factors involved in XCI and to test the functions of cis and trans regulatory candidates that regulate Xist-mediated gene silencing. The results obtained from this work have major implications on our understanding of how chromatin changes are functionally linked to gene expression changes in the context of mammalian development.
Another major contribution of this work was to decipher the roles of some of the key factors involved in the initiation of XCI. One such factor we explored was the SPEN protein, a previously identified partner of Xist RNA. We found that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and in embryonic stem cells. SPEN was found to be dispensable for maintenance of XCI in neural progenitors, but its depletion nevertheless led to increased expression of genes that escape XCI. We also elucidated SPEN’s mode of action and the other factors that it interacts with. We found that SPEN is recruited to the X chromosome by Xist RNA, but specifically targets only the enhancers and promoters of transcriptionally active genes, rapidly disengaging from chromatin as soon as gene silencing is achieved. This suggests that active transcription is required to tether SPEN to chromatin. We also defined the SPOC domain as the major effector of the gene-silencing function of SPEN. Indeed, artificial tethering SPOC to Xist RNA was shown to be sufficient to mediate silencing of many genes along the X when endogenous SPEN was depleted. We also identified the protein partners of SPOC, which include NCoR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in the regulation of transcription initiation and elongation. Based on our findings, we have proposed that SPEN acts as a molecular integrator for the initiation of XCI, bridging Xist RNA with the transcription machinery, as well as with nucleosome remodellers and histone deacetylases at active enhancers and promoters.
Overall, the results from the XPRESS ERC have provided new insights into mechanisms of gene regulation in general and how they relate to non-coding RNAs, chromosome architecture and chromatin states. Our findings are relevant not only to the silencing of the X chromosome in mammals, but more generally for our understanding of how and why genes become silenced and others remain expressed in diseases such as cancer, where epigenetic mechanisms become disrupted. Indeed some of the key factors involved in XCI that we are studying are also implicated in cancers (eg SPEN/SHARP, RNA binding proteins, HDACs, Polycomb group factors), such as leukemia. Indeed, we hope that our work will lead to new hypotheses that can be tested using animal models and human cell lines, exploring the roles that SPEN and other Xist RNA associated factors have in cancer. Such work should open up potential avenues of research with prognostic and therapeutic value.