Requirements for establishment and epigenetic stability of mammalian heterochromatin

Abstract Heterochromatic domains of DNA account for a large fraction of mammalian genomes and play critical roles in silencing 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 a CRISPR-based genetic screen to investigate the requirements for the establishment and maintenance of heterochromatin in mouse embryonic stem cells (mESCs). We show that DNA sequence-independent and histone H3 lysine 9 methylation (H3K9me)-dependent heterochromatin can be inherited for a limited number of cell divisions in mESCs but becomes stable upon differentiation. We provide evidence that the increased stability of heterochromatin in differentiated cells results from the downregulation of one or more enzymes that erase H3K9me and DNA methylation. Moreover, we show that in addition to components of the H3K9 and DNA methylation pathways, heterochromatin maintenance requires DHX9 and other RNA processing proteins. DHX9 is an RNA/DNA helicase with previously described roles in preventing genomic instability resulting from transcription-associated replication stress. We found that deletion of DHX9 results in defective heterochromatin inheritance and is associated with increased transcription of major satellite repeats, accumulation of R-loops, and loss of H3K9me. Our findings define the requirements for the establishment and epigenetic inheritance of mammalian heterochromatin and suggest that R-loops and replication stress lead to epigenetic instability.