Requirements for Establishment and Epigenetic Inheritance of Mammalian Heterochromatin [CRISPR Screen]

Heterochromatic domains of DNA account for a large fraction of mammalian genomes and have critical roles in silencing of transposons and genes but the mechanisms that establish and maintain these domains are not fully understood. Here we use an inducible heterochromatin formation system combined with targeted CRISPR gene disruption to show that DNA sequence-independent and histone H3 lysine 9 methylation (H3K9me)-dependent heterochromatin can only be inherited during a limited number of cell divisions in mouse embryonic stem cells but becomes stable upon differentiation. We show that multiple H3K9 methyltransferases work together with DNA cytosine methyltransferases and other factors, including RNA processing proteins, to establish and maintain heterochromatin. In addition, we find that deletion of DHX9, an RNA/DNA helicase, results in defective heterochromatin maintenance associated with R-loop accumulation. Our findings suggest that components of H3K9 and DNA methylation pathways work in concert to ensure heterochromatin stability which additionally requires R-loop removal. CRISPR-Cas9 Screen to identify the molecular networks involved in establishment and maintenance of a silent state.