Membraneless pericentromeric heterochromatin (PCH) domains play vital roles in chromosome dynamics and genome stability. However, our current understanding of 3D genome organization does not include PCH domains because of technical challenges associated with repetitive sequences enriched in PCH genomic regions. We investigated the 3D architecture of Drosophila melanogaster PCH domains and their spatial associations with the euchromatic genome by developing a novel analysis method that incorporates genome-wide Hi-C reads originating from PCH DNA. Combined with cytogenetic analysis, we reveal a hierarchical organization of the PCH domains into distinct "territories." Strikingly, H3K9me2-enriched regions embedded in the euchromatic genome show prevalent 3D interactions with the PCH domain. These spatial contacts require H3K9me2 enrichment, are likely mediated by liquid-liquid phase separation, and may influence organismal fitness. Our findings have important implications for how PCH architecture influences the function and evolution of both repetitive heterochromatin and the gene-rich euchromatin.

Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context.Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.

Eukaryotic genomes are broadly divided between gene-rich euchromatin and the highly repetitive heterochromatin domain, which is enriched for proteins critical for genome stability and transcriptional silencing. This study shows that Drosophila KDM4A (dKDM4A), previously characterized as a euchromatic histone H3 K36 demethylase and transcriptional regulator, predominantly localizes to heterochromatin and regulates heterochromatin position-effect variegation (PEV), organization of repetitive DNAs, and DNA repair. We demonstrate that dKDM4A demethylase activity is dispensable for PEV. In contrast, dKDM4A enzymatic activity is required to relocate heterochromatic double-strand breaks outside the domain, as well as for organismal survival when DNA repair is compromised. Finally, DNA damage triggers dKDM4A-dependent changes in the levels of H3K56me3, suggesting that dKDM4A demethylates this heterochromatic mark to facilitate repair. We conclude that dKDM4A, in addition to its previously characterized role in euchromatin, utilizes both enzymatic and structural mechanisms to regulate heterochromatin organization and functions.

Repair of DNA double-strand breaks (DSBs) must be orchestrated properly within diverse chromatin domains in order to maintain genetic stability. Euchromatin and heterochromatin domains display major differences in histone modifications, biophysical properties, and spatiotemporal dynamics of DSB repair. However, it is unclear whether differential histone-modifying activities are required for DSB repair in these distinct domains. We showed previously that the Drosophila melanogaster KDM4A (dKDM4A) histone demethylase is required for heterochromatic DSB mobility. Here we used locus-specific DSB induction in Drosophila animal tissues and cultured cells to more deeply interrogate the impact of dKDM4A on chromatin changes, temporal progression, and pathway utilization during DSB repair. We found that dKDM4A promotes the demethylation of heterochromatin-associated histone marks at DSBs in heterochromatin but not euchromatin. Most importantly, we demonstrate that dKDM4A is required to complete DSB repair in a timely manner and regulate the relative utilization of homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways but exclusively for heterochromatic DSBs. We conclude that the temporal kinetics and pathway utilization during heterochromatic DSB repair depend on dKDM4A-dependent demethylation of heterochromatic histone marks. Thus, distinct pre-existing chromatin states require specialized epigenetic alterations to ensure proper DSB repair.

Double-strand breaks (DSBs) in heterochromatic repetitive DNAs pose significant threats to genome integrity, but information about how such lesions are processed and repaired is sparse. We observe dramatic expansion and dynamic protrusions of the heterochromatin domain in response to ionizing radiation (IR) in Drosophila cells. We also find that heterochromatic DSBs are repaired by homologous recombination (HR) but with striking differences from euchromatin. Proteins involved in early HR events (resection) are rapidly recruited to DSBs within heterochromatin. In contrast, Rad51, which mediates strand invasion, only associates with DSBs that relocalize outside of the domain. Heterochromatin expansion and relocalization of foci require checkpoint and resection proteins. Finally, the Smc5/6 complex is enriched in heterochromatin and is required to exclude Rad51 from the domain and prevent abnormal recombination. We propose that the spatial and temporal control of DSB repair in heterochromatin safeguards genome stability by preventing aberrant exchanges between repeats.

Chromatin environments differ greatly within a eukaryotic genome, depending on expression state, chromosomal location, and nuclear position. In genomic regions characterized by high repeat content and high gene density, chromatin structure must silence transposable elements but permit expression of embedded genes. We have investigated one such region, chromosome 4 of Drosophila melanogaster. Using chromatin-immunoprecipitation followed by microarray (ChIP-chip) analysis, we examined enrichment patterns of 20 histone modifications and 25 chromosomal proteins in S2 and BG3 cells, as well as the changes in several marks resulting from mutations in key proteins. Active genes on chromosome 4 are distinct from those in euchromatin or pericentric heterochromatin: while there is a depletion of silencing marks at the transcription start sites (TSSs), HP1a and H3K9me3, but not H3K9me2, are enriched strongly over gene bodies. Intriguingly, genes on chromosome 4 are less frequently associated with paused polymerase. However, when the chromatin is altered by depleting HP1a or POF, the RNA pol II enrichment patterns of many chromosome 4 genes shift, showing a significant decrease over gene bodies but not at TSSs, accompanied by lower expression of those genes. Chromosome 4 genes have a low incidence of TRL/GAGA factor binding sites and a low T(m) downstream of the TSS, characteristics that could contribute to a low incidence of RNA polymerase pausing. Our data also indicate that EGG and POF jointly regulate H3K9 methylation and promote HP1a binding over gene bodies, while HP1a targeting and H3K9 methylation are maintained at the repeats by an independent mechanism. The HP1a-enriched, POF-associated chromatin structure over the gene bodies may represent one type of adaptation for genes embedded in repetitive DNA.