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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE212152 | GEO | 2025/09/17

REPOSITORIES: GEO

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Project description:Heterochromatic DNA domains play important roles in the regulation of gene expression and maintenance of genome stability by silencing repetitive DNA elements and transposons. In organisms ranging from fission yeast to mammals, heterochromatin assembly at DNA repeats involves the activity of small noncoding RNAs (sRNAs) associated with the RNA interference (RNAi) pathway. Typically, sRNAs, originating from long noncoding RNAs transcribed from DNA repeats, guide Argonaute-containing effector complexes to complementary nascent RNAs to initiate histone H3 lysine 9 di- and tri-methylation (H3K9me2 and H3K9me3, respectively) and heterochromatin formation. H3K9me is in turn required for recruitment of RNAi to chromatin and promotes sRNA generation. However, how heterochromatin formation, which silences transcription, can proceed by a co-transcriptional mechanism that also promotes sRNA generation remains paradoxical. Here, using Clr4, the fission yeast homolog of mammalian SUV39H H3K9 methyltransferases, we designed active site mutations, which allow H3K9me2 catalysis but block the transition to H3K9me3. We show that H3K9me2 defines a functionally distinct heterochromatin state that is sufficient for RNAi-dependent co-transcriptional gene silencing (CTGS). Unlike H3K9me3 domains, which are transcriptionally silent, H3K9me2 domains are transcriptionally active, contain modifications associated with euchromatic transcription, and couple RNAi-mediated transcript degradation to the establishment of H3K9me domains. The two H3K9me states recruit reader proteins with different efficiencies, explaining their different downstream silencing functions. Furthermore, transition from H3K9me2 to H3K9me3 is required for RNAi-independent epigenetic inheritance of H3K9me domains. Our findings demonstrate that H3K9me2 and H3K9me3 define functionally distinct heterochromatin states and uncover a mechanism for formation of transcriptionally permissive heterochromatin that is compatible with its broadly conserved role in RNAi-mediated genome defense.

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

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