HP6/Umbrea, a rapidly evolving Drosophila HP1-family paralog, is a candidate HP1a-recruited plasticizer of heterochromatin

Isotype specific loss of HP1α but not of HP1β uncovers genomic regions that behave as HP1α-dependent common fragile sites

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.

The novel HP1a interactor Clump/CG30403 safeguards transposon silencing and reproductive resilience in the Drosophila ovary

Heterochromatin and its core effectors from the Heterochromatin Protein 1 (HP1) family are essential for epigenetic silencing of repetitive regions and genome integrity from yeast to humans. However, how HP1s and associated factors regulate heterochromatin properties in vivo to sustain silencing across development, aging, and environmental factors, remains incompletely understood. Here, we identify Clump/CG30403 — a previously uncharacterized MADF-BESS domain protein — as a novel heterochromatin factor required for robust transposon silencing during Drosophila oogenesis and sustained fertility with age and temperature stress. Clump/CG30403 interacts with the main HP1-family silencing effector Su(var)205/HP1a through a noncanonical binding motif within its large intrinsically disordered region. Notably, in the absence of Clump/CG30403, HP1a mobility and silencing capacity are compromised despite largely unperturbed genomic distribution, showing that HP1a presence alone is insufficient for repression. We also show that Clump/CG30403 uniquely accumulates at its own promoter to self-repress and prevent ectopic aggregation, revealing a feedback mechanism to constrain protein dosage and phase behavior. Overall, we propose that Clump/CG30403 is a HP1 corepressor that acts as a tightly calibrated safeguard of the heterochromatin environment properties to ensure stable silencing, genome integrity, and persistent reproductive function.

ORC binding to Heterochromatin Protein 1 through intrinsically disordered regions is required for heterochromatin structure and function

Outside-in progression of heterochromatin replication and exclusion of CDC45 from the PCH domain in Drosophila

Heterochromatin replication isn’t random or uniform, but occurs in a characteristic spatial and temporal pattern. Previous studies produced conflicting models for pericentric heterochromatin (PCH) replication, suggesting either that heterochromatic sequences translocate to the domain surface for replication, or that replication can also occur internally through localized decondensation. To distinguish true overlap from peripheral enrichment around an irregular PCH domain, we developed FOC-Map, a colocalization analysis approach that combines segmentation of one channel with binning of the other. Applying FOC-Map to three-dimensional Airyscan imaging of cultured Drosophila cells, we find that replication foci at the onset of late S-phase are confined to the outer boundary of the PCH domain, forming a shell-like pattern with little overlap into the HP1a-rich interior. As late S-phase progresses, replication foci are observed within the domain, localizing to low-HP1a regions interspersed between more condensed regions. We then assessed the distribution of CDC45, a rate-limiting replication initiation factor, and found that CDC45 foci are depleted from the PCH domain throughout the cell cycle. We propose that low levels of CDC45 within HP1a-rich PCH limit replication initiation to the domain periphery, giving rise to the shell-like pattern of replication foci that progressively works inward until PCH replication is complete.

Proximity labeling at H3K9me3 reveals VRK-1 regulate global chromatin distribution in C. elegans

Heterochromatin marked by histone H3 lysine 9 di- or trimethylation (H3K9me2/3) underpins transcriptional silencing and nuclear organization, yet its full complement of associated proteins remains incompletely defined. Here, we apply ChromID proximity labelling with the mouse HP1β chromodomains to map the H3K9me3-proximal proteome in Caenorhabditis elegans, recovering known heterochromatin factors alongside previously uncharacterized candidates. We pursued one such candidate, the vaccinia-related kinase VRK-1, because of its established but poorly understood links to chromatin organization. Intriguingly, VRK-1 dynamically relocates from a broad nuclear distribution to the nuclear periphery upon azide or heat stress. Following these stresses, bulk chromatin exhibits similarly increased peripheral enrichment and apparent compaction, as assessed by radial fluorescence profiles. Although VRK-1 is not necessary for stress-induced chromatin reorganization, decompaction and repositioning of chromatin away from the nuclear envelope during recovery requires VRK-1. VRK-1 depletion leads to persistent perinuclear chromatin retention and compromises post-stress survival. Furthermore, loss of VRK-1 catalytic activity results in over-retention of chromatin at the nuclear periphery under normal growth conditions; this phenotype can be reversed by depletion of a key VRK-1 substrate at the nuclear envelope BAF-1. Our findings identify VRK-1 as a key regulator that controls the interaction of chromatin with the nuclear lamina through regulation of BAF-1.

Plant LHP1 as a Context-Dependent Regulator of Polycomb-Associated Chromatin.

Like heterochromatin protein 1 (LHP1) is a conserved HP1-like chromatin protein in land plants and a major Polycomb-associated factor that recognizes H3K27me3 enriched euchromatic domains. Recent biochemical, genomic, and structural studies show that LHP1 acts as a modular chromatin scaffold whose regulatory output depends on local histone modifications, RNA interactions, transcriptional factors, and PRC1/PRC2-associated partners. In this review, we outline emerging roles of LHP1 in Polycomb-mediated repression, chromatin-state maintenance, transcriptional responsiveness, hormone signaling, stress responses, and TE-proximal gene regulation. We also examine the structural features and evolutionary diversification of LHP1 homologs across land plants and assess how duplicated LHP1 copies may contribute to species-specific developmental and stress-adaptive traits. Rather than presenting LHP1 as a universal master regulator, we emphasize its context-dependent functions and distinguish experimentally supported mechanisms from correlative or still unresolved models. Finally, we highlight future directions involving genome editing, epigenomic profiling, chromatin conformation analysis, and targeted epigenome engineering to clarify how LHP1 associated modules may be exploited for crop improvement and postharvest quality management.