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.

The H3.3 K36M oncohistone disrupts the establishment of epigenetic memory through loss of DNA methylation

“…These cells also stably express the commonly used KRAB repressor domain from ZNF10 fused to the reverse tetracycline repressor (rTetR), allowing for targeted doxycycline (dox) dependent recruitment and subsequent long-term silencing of the reporter through deposition of H3K9me3 mediated heterochromatin. 28,45,49–52…”

“…These cells also stably express the commonly used KRAB repressor domain from ZNF10 fused to the reverse tetracycline repressor (rTetR), allowing for targeted doxycycline (dox) dependent recruitment and subsequent long-term silencing of the reporter through deposition of H3K9me3 mediated heterochromatin. 28,45,[49][50][51][52] To enable conditional expression of the oncohistone in the reporter cells, we stably expressed H3.3 K36M fused to an auxin inducible degron (AID, Fig. 1A right).…”

ProA and ProB repeat sequences shape genome organization, and enhancers open domains

“…We surmise that a large fraction of enhancers nonetheless perform a ProB function prior to induction. There are at least three possible explanations for why this is not generally apparent in the form of bound repressor TFs and repressive marks: (i) There is a conspicuous background of H3K9me3 signal across the whole genome, visible in both bulk ChIP and single-cell analyses, which would clearly mask a small peak; (ii) Highly transient binding of TFs such as Krüppel-associated box (KRAB) zinc-finger proteins (KZFPs) may evade detection by ChIP while being sufficient to maintain heterochromatin assembly over a region via discrete events of nucleation, due to memory effects within the heterochromatin system in a differentiated cell (111) (Box2). Consistent with this notion, even prototypical ProB TE most often do not appear as DHSs in spite of featuring both H3K9me3 and KZFP ChIP signals, indicative of transient TF binding.…”

“…meCpG renders the heterochromatin state of RepSeq more stable both within a cell cycle and through generations, by introducing some extra feed-forward and reciprocal loops in the heterochromatin regulatory network (21,248,249). CpG methylation therefore appears to guarantee the memory of a repressed state, which was formally demonstrated experimentally by artificially targeting DNMTs, or TET enzymes that revert meCpG, at reporter genes (111,177,178,241,250). Conversely, mutating DNMTs is embryonically lethal in mouse, due to an inability of embryo cells to securely silence prior or alternative cell identities (251).…”

Mapping the dynamics of epigenetic adaptation during heterochromatin misregulation

“…87 It remains to be seen whether other epigenetic regulators also exhibit hysteresis given the slow kinetics of establishing novel epigenetic states de novo. 88 In conclusion, our inducible system uniquely allowed us to capture the highly dynamic and unexpected changes in gene expression and chromatin states following acute heterochromatin misregulation in yeast populations, changes that would be otherwise obscured in conventional genetic assays. Using this approach, we could faithfully reconstruct the pathways that cells undertake prior to and during adaptation as well as investigate how the adapted state is memorized across multiple generations.…”