Genome-wide chromatin conformation capture (Hi-C) is used to investigate three-dimensional genome organization in Schizosaccharomyces pombe; small domains of chromatin interact locally on chromosome arms to form globules, which depend on cohesin but not heterochromatin for formation, and heterochromatin at centromeres and telomeres provides crucial structural constraints to shape genome architecture. Mammalian and Drosophila genomes are organized into megabase-sized topological associated domains (TADs), the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF). Here, Shiv Grewal and colleagues have performed Hi-C analysis to investigate three-dimensional genome organization in the fission yeast Schizosaccharomyces pombe, which has conserved features and mechanisms present in higher eukaryotes. The authors describe TAD-like structures in yeast for the first time. They also find smaller domains of local chromatin interactions on chromosome arms which they term 'globules'. The formation of globules requires cohesin but not heterochromatin, whereas heterochromatin seems to have a complementary role in distinct aspects of genome architecture such as at centromeres and telomeres. Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions1,2,3,4,5,6,7. How local chromatin interactions govern higher-order folding of chromatin fibres and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis8 to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes9. Our analyses of wild-type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form ‘globules’. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains9,10,11, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fibre compaction at centromeres and promotes prominent inter-arm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions.

Heterochromatin assembly requires methylation of histone H3 lysine 9 (H3K9me) and serves as a paradigm for understanding the importance of histone modifications in epigenetic genome control. Heterochromatin is nucleated at specific genomic sites and spreads across extended chromosomal domains to promote gene silencing. Moreover, heterochromatic structures can be epigenetically inherited in a self-templating manner, which is critical for stable gene repression. The spreading and inheritance of heterochromatin are believed to be dependent on preexisting H3K9 tri-methylation (H3K9me3), which is recognized by the histone methyltransferase Clr4/Suv39h via its chromodomain, to promote further deposition of H3K9me. However, the process involving the coupling of the “read” and “write” capabilities of histone methyltransferases is poorly understood. From an unbiased genetic screen, we characterize a dominant-negative mutation in histone H3 (H3^G13D) that impairs the propagation of endogenous and ectopic heterochromatin domains in the fission yeast genome. H3^G13D blocks methylation of H3K9 by the Clr4/Suv39h methyltransferase and acts in a dosage-dependent manner to interfere with the spreading and maintenance of heterochromatin. Our analyses show that the incorporation of unmethylatable histone H3^G13D into chromatin decreases H3K9me3 density and thereby compromises the read-write capability of Clr4/Suv39h. Consistently, enhancing the affinity of Clr4/Suv39h for methylated H3K9 is sufficient to overcome the defects in heterochromatin assembly caused by H3^G13D. Our work directly implicates methylated histones in the transmission of epigenetic memory and shows that a critical density threshold of H3K9me3 is required to promote epigenetic inheritance of heterochromatin through the read-write mechanism. Significance In multicellular organisms, a single genome gives rise to a multitude of cell types by enforcing appropriate gene expression patterns. Epigenetic mechanisms involving modification of histones, including tri-methylation of histone H3 lysine 9 (H3K9me3), assemble and propagate repressive heterochromatin to prevent untimely gene expression. Dysregulation of epigenetic gene-silencing mechanisms is a hallmark of a variety of diseases including cancer. However, the requirements for epigenetic transmission of heterochromatin are not well understood. This study reveals the mechanism by which methylated histones provide an epigenetic template for heterochromatin propagation. We discover that a critical threshold of H3K9me3 is required for effective chromatin-association of the histone methyltransferase, which binds to and catalyzes additional H3K9me to propagate heterochromatin and enforce stable gene silencing.

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 signalling that activates the histone methyltransferase EHMT2/G9a to catalyse heterochromatin assembly at stressed replication forks. Using biochemical and single molecule chromatin fibre 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 favoured 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 single-stranded DNA gap formation and sensitizing cells towards chemotherapeutic drugs. These findings may help in explaining chemotherapy resistance and poor prognosis observed in patients with cancer displaying elevated levels of G9a/H3K9me3.

The interplay of SMARCAD1-mediated active fork and BRCA1-mediated stalled fork stability pathways maintains genome integrity.