Novel regulators of the HUSH epigenetic repressor complex (360G-Wellcome-109074_Z_15_A)

Epigenetic regulation of chromatin structure is fundamental to eukaryotic gene expression. The critical role of histone modifications in regulating chromatin has been established in genetic screens in Drosophila. Less information is available in more diverse organisms due, at least in part, to their genetic intractability. Using a gene-trap mutagenesis screen, the Lehner group recently discovered a novel gene silencing complex, the Human Silencing Hub (HUSH), which acts as a repressive H3K9me3 reader-writer complex through recruitment of the SETDB1 methyltransferase. However, regulation of the HUSH complex remains unexplored, and no associated demethylase has been identified. Here, I propose two complementary approaches to identifying novel regulatory components of the HUSH pathway: (A) A forward genetic approach, which will use a genome-wide CRISPR activation library. In a HUSH responsive GFPdim reporter cell line, activation of a HUSH regulatory gene will reverse reporter gene silencing. Following enrichment for rare cells reverting to a GFPbright reporter phenotype, candidate regulatory genes can be identified by next-generation sequencing. (B) A proteomic approach will use proximity-dependent biotinylation to mark HUSH interaction partners for identification by mass spectrometry. In both approaches, candidate genes will be validated and characterised biochemically to elucidate their mechanistic role in the regulation of heterochromatin.