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Dynamics of de novo heterochromatin assembly and disassembly at replication forks ensures fork stability

ABSTRACT: Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here, we discover a checkpoint regulated cascade of chromatin signaling that activates 5 the histone methyltransferase EHMT2/G9a to catalyze heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fiber approaches, we show that G9a together with SUV39h1 induces chromatin compaction by accumulating the repressive modifications, H3K9me1/me2/me3, in the vicinity of stressed replication forks. This closed conformation is also favored by the G9a-dependent exclusion of the H3K9-demethylase 10 JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering ssDNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help explaining chemotherapy resistance and poor prognosis observed in cancer patients displaying elevated level of G9a/H3K9me3.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

TISSUE(S): Fibroblast, Embryonic Stem Cell

SUBMITTER: Jeroen Demmers

LAB HEAD: Jeroen Demmers

PROVIDER: PXD041742 | Pride | 2023-05-12

REPOSITORIES: Pride

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			Action	DRS
	2315_E_CalvinLo_15juni21_Silac_HU_L_HU_G9al_H_CV_50.raw	Raw
	2315_E_CalvinLo_15juni21_Silac_HU_L_HU_G9al_H_CV_60.raw	Raw
	2315_E_CalvinLo_15juni21_Silac_HU_L_HU_G9al_H_CV_75.raw	Raw
	2315_E_CalvinLo_15juni21_Silac_UT_L_UT_G9al_H_CV_60.raw	Raw
	2315_E_CalvinLo_15juni21_Silac_UT_L_UT_G9al_H_CV_75.raw	Raw

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Dynamics of de novo heterochromatin assembly and disassembly at replication forks ensures fork stability

Project description:Chromatin is dynamically reorganized when DNA replication forks are challenged. However, the process of epigenetic reorganization and its implication for fork stability is poorly understood. Here, we discover a checkpoint regulated cascade of chromatin signaling that activates the histone methyltransferase EHMT2/G9a to catalyze heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fiber approaches, we show that G9a together with SUV39h1 induces chromatin condensation by accumulating the silent mark, H3K9me1/2/3, in the vicinity of stressed replication forks. This closed conformation is also favored by the G9a-dependent exclusion of the H3K9-demethylase JMJD1A/KDM3A, which facilitates heterochromatin disassembly upon fork restart. Untimely heterochromatin disassembly from stressed forks by KDM3A enables PRIMPOL access, triggering ssDNA gaps formation and sensitizing cells towards chemotherapeutic drugs. These findings may help explaining chemotherapy resistance and poor prognosis observed in cancer patients displaying elevated level of G9a/H3K9me3.modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.

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