X-inactivation (XCI) is an epigenetic phenomenon resulting in transcriptional silencing to attain gene dosage parity between XX females and XY males. XCI involves coating of the non-coding Xist RNA followed by depletion of euchromatic marks and shortly after accumulation of heterochromatic marks. The chromatin modifying activities that are involved in these steps and their functional interplay remain poorly understood however. We will specifically assess how euchromatic marks, such as H3K4 methylation and H3 and H4 acetylation, are lost shortly after Xist RNA coating and the importance of this loss in gene silencing and heterochromatin formation. First, we will perform functional studies in differentiating XX ES cells, including knockdown of chromatin modifying candidates, such as H3K4 demethylases and histone deacetylases. Second, we will investigate how specific X-linked loci behave during XCI, using a Lac or Tet operator tagging system. This will allow us to (a) visualise a locus in living cells and assess the exact timing of recruitment of proteins of interest (fused to a fluorescent protein), in control and knockdown situations; and (b) target specific chromatin factors (using the Lac or Tet repressor fusion) to a locus in order to assess the impact of chromatin changes on XCI. In summary, the two complementary strategies outlined here should provide us with new insights into the roles that various chromatin changes associated with XCI actually play in this process. This should also give new insights into the process of gene regulation in other systems.
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