Understand How X-chromosome Inactivation is Maintained in Mouse Cells

Silent chromatin states

How are silent chromatin states such as inactivated Xi inherited during mitotic cell division?

In an effort to understand how heterochromatin structure is maintained in mammalian cells, we are studying how the inactivated X-chromosome is maintained in female mouse cells because inactivated X-chromosome utilizes distinct known silencing pathways to initiate and maintain silencing. These include methylation of H3 lysine 27 (H3K27me3), methylation of lysine 9 of H3 (H3K9me3), non-coding RNA(Xist) and DNA methylation. Therefore, understanding on how inactivated X-chromosome is inactivated will shed light on the interplay of these diverse silencing mechanisms. Towards this end, we have developed a mouse cell line in which the inactivated X-chromosome is marked by green fluorescent protein. We used this cell line to perform a genome wide shRNA screen to identify genes that, when depleted, resulted in expression of the GFP transgene. Using this approach, we identified over 32 candidates involved in the maintenance of X-chromosome inactivation. Most of these gene products have not been implicated in silencing of the X-chromosome or heterochromatin silencing in general. We are selecting a few genes for further study to understand how they function in transcriptional silencing in mammalian cells. We hope that these studies will not only increase our understanding of how silencing of the inactivated X-chromosome and heterochromatin in general is maintained, but also shed light on how silencing of tumor suppressor genes is maintained in cancer cells.