Maintenance of ovarian cell identity is required throughout life to prevent the activation of the testicular program, but the epigenetic mechanisms underlying this process remain poorly understood. Although TRIM28 is required to prevent granulosa-to-Sertoli transdifferentiation, it can act both as a regulator of H3K9me3-dependent heterochromatin and as a transcriptional activator through its E3 SUMOligase activity. Here, we combined CUT&RUN, ATAC-seq and RNA-seq to define the respective contributions of these activities to maintain ovarian cell identity. Strikingly, only a small fraction of TRIM28-bound regions was associated with H3K9me3. Although Trim28 deletion induced focal H3K9me3 loss, it had limited transcriptional consequences and primarily affected repetitive elements rather than regions controlling testis-determining genes. In contrast, Trim28 loss led to reductions in chromatin accessibility and H3K27ac at regions enriched for ovarian transcription factor (TF) motifs FOXL2, NR5A2, ESR2 and RUNX1. Moreover, TRIM28 was frequently co-localized with these TFs on chromatin, and the accessibility and the SUMOylation at these co-bound regions were reduced by Trim28 deletion. Together, our findings identify TRIM28 as a central organizer of ovarian TF hubs whose predominant function is to preserve granulosa cell identity through stabilization of lineage-specific TFs rather than H3K9me3-dependent heterochromatin. GRAPHICAL ABSTRACT
Laura Sitkiewicz, Florian Chaleil, Gaby Granès et al.· bioRxiv· 0 citations
HP1 proteins are highly evolutionarily conserved chromatin-associated factors known to play essential roles in genome stability and nuclear organization. In mammals, three HP1 isoforms, HP1α, HP1β and HP1γ, have been described, but their individual functions remain incompletely characterized. Here, we inactivated HP1α or HP1β in different cell lines and quantified chromosomal breaks on metaphase spreads in the presence or absence of aphidicolin-induced replication stress. Loss of HP1α, but not of HP1β, led to a significant increase of chromosomal breaks on chromosome arms and within pericentromeric heterochromatin under these conditions. Mechanistically, loss of HP1α was associated with a reduction in replication fork velocity, suggesting that HP1α deficiency induces a replication stress that sensitizes specific genomic loci to replication perturbation. Consistent with this, HP1α loss was associated with a moderate but consistent increase in γH2AX and 53BP1 foci, an increased occurrence of DNA synthesis during mitosis, and enhanced recruitment of FANCD2, all recognized as hallmarks of common fragile site (CFS) expression. In addition, rescue experiments using a chromodomain mutant HP1α (V22M) unable to bind H3K9me3 indicated that HP1α protective function over these specific foci did not require its interaction with this histone mark. Altogether, these data indicate that, independently of its binding to H3K9me3, HP1α stabilizes specific genomic regions that behave as HP1α-dependent fragile sites, at least in part by regulating replication fork progression, limiting mitotic DNA synthesis possibly by competing with FANCD2 for chromatin access at these regions.
Katherine Yaacoub, Thanh Nhan Nguyen, Eric Julien et al.· bioRxiv· 0 citations
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