SUMMARY CTCF/cohesin play a central role in insulator function and higher-order chromatin organization of mammalian genomes. Recent studies identified a correlation between the orientation of CTCF-binding sites (CBSs) and chromatin loops. To test the functional significance of this observation, we combined CRISPR/Cas9-based genomic-DNA-fragment editing with chromosome-conformation-capture experiments to show that the location and relative orientations of CBSs determine the specificity of long-range chromatin looping in mammalian genomes, using protocadherin (Pcdh) and β-globin as model genes. Inversion of CBS elements within the Pcdh enhancer reconfigures the topology of chromatin loops between the distal enhancer and target promoters, and alters gene-expression patterns. Thus, although enhancers can function in an orientation-independent manner in reporter assays, in the native chromosome context the orientation of at least some enhancers carrying CBSs can determine both the architecture of topological chromatin domains and enhancer/promoter specificity. The findings reveal how 3D chromosome architecture can be encoded by genome sequence.

Summary HP1 proteins are central to the assembly and spread of heterochromatin containing histone H3K9 methylation. The chromodomain (CD) of HP1 proteins specifically recognizes the methyl mark on H3 peptides, but the same extent of specificity is not observed within chromatin. The chromoshadow domain of HP1 proteins promotes homodimerization, but this alone cannot explain heterochromatin spread. Using the S. pombe HP1 protein, Swi6, we show that recognition of H3K9 methylated chromatin in vitro relies on a newly identified interface between two CDs. This interaction causes Swi6 to tetramerize on a nucleosome, generating two vacant CD sticky ends. On nucleosomal arrays, methyl-mark recognition is highly sensitive to inter-nucleosomal distance, suggesting that the CD sticky ends bridge nearby methylated nucleosomes. Strengthening the CD-CD interaction enhances silencing and heterochromatin spread in vivo. Our findings suggest that recognition of methylated nucleosomes and HP1 spread on chromatin are structurally coupled, and imply that methylation and nucleosome arrangement synergistically regulate HP1 function.

Gene silencing is essential for regulating cell fate in eukaryotes. Altered chromatin architectures contribute to maintaining the silenced state in a variety of species. The silent information regulator (SIR) proteins regulate mating type in S. cerevisiae. One of these proteins, Sir3, interacts directly with the nucleosome to help generate silenced domains. We determined the crystal structure of a complex of the yeast Sir3 BAH domain and the nucleosome core particle at 3.0 Å resolution. We see multiple molecular interactions between the protein surfaces of the nucleosome and the BAH domain that explain numerous genetic mutations. These interactions are accompanied by structural rearrangements in both the nucleosome and the BAH domain. The structure explains how covalent modifications on H4 K16 and H3 K79 regulate formation of a silencing complex that contains the nucleosome as a central component.

Heterochromatin protein 1 (HP1) proteins were originally identified as critical components in heterochromatin mediated gene silencing and are now recognized to play essential roles in several other processes including gene activation. Several eukaryotes possess more than one HP1 paralog. Despite high sequence conservation, the HP1 paralogs achieve diverse functions. Further, in many cases, the same HP1 paralog is implicated in multiple functions. Recent biochemical studies have revealed interesting paralog-specific biophysical differences and unanticipated conformational versatility in HP1 proteins that may account for this functional promiscuity. Here, we review these findings and describe a molecular framework that aims to link the conformational flexibility of HP1 proteins observed in vitro with their functional promiscuity observed in vivo.

A hallmark of histone H3 lysine 9 (H3K9) methylated heterochromatin, conserved from fission yeast,Schizosaccharomyces pombe (S. pombe), to humans, is its ability to spread to adjacent genomic regions1–6. Central to heterochromatin spread is the heterochromatin protein 1 (HP1), which recognizes H3K9 methylated chromatin, oligomerizes, and forms a versatile platform that participates in diverse nuclear functions, ranging from gene silencing to chromosome segregation1–6. How HP1 proteins assemble on methylated nucleosomal templates and how the HP1-nucleosome complex achieves functional versatility remain poorly understood. Here, we show that binding of the major S. pombe HP1 protein, Swi6, to methylated nucleosomes drives a switch from an auto-inhibited state to a spreading competent state. In the auto-inhibited state, a histone mimic sequence in one Swi6 monomer blocks methyl mark recognition by the chromodomain of another monomer. Auto-inhibition is relieved by recognition of two template features, the H3K9 methyl mark and nucleosomal DNA. Cryo-Electron Microscopy (EM) based reconstruction of the Swi6-nucleosome complex provides the overall architecture of the spreading-competent state in which two unbound chromodomain sticky ends appear exposed. Disruption of the switch between the auto-inhibited and spreading competent state disrupts heterochromatin assembly and gene silencing in vivo. These findings are reminiscent of other conditionally activated polymerization processes, such as actin nucleation, and open up a new class of regulatory mechanisms that operate on chromatin in vivo.