Role of hp1 proteins in murine 3d genomic architecture

Abstract

Proper spatial organization of the eukaryotic genome is critical for cellular function, yet the precise biophysical mechanisms governing higher-order chromatin folding remain incompletely understood. Recent studies have indicated that highly-conserved HP1 proteins are good candidates for regulating folding of the Genome into the interphase nucleus, but rigorous functional analyses in mammalian cells is currently lacking. Here, we show that the mammalian HP1 paralog family (HP1α, HP1β, and HP1γ) are dynamic thermodynamic drivers of murine 3D genome architecture, mediating polymer-polymer micro-phase separation of H3K9me3-marked Heterochromatin-Like Domains/Complexes (HLD/Cs). By integrating high-resolution Hi-C contact mapping with targeted genetic ablations in murine embryonic stem cells (mESCs), we show key functions of HP1 proteins in folding the mammalian genome into the interphase nucleus. First, targeted depletion of the HP1 network indicates that HP1 proteins mediate the contact enrichments that emerge as B-type compartmental domains in Hi-C maps, while leaving cohesin-driven Topologically Associating Domains (TADs) intact. Second, systematic paralog dissection demonstrates that HP1 isoforms play non-redundant roles. HP1 proteins operate within a tightly calibrated equilibrium where HP1α acts as a regulator of spatial clustering of HLD/Cs, and must be present in the proper stoichiometric ratio with HP1β and HP1γ. Third, the contact enrichments that result from interactions of HLD/Cs are size-dependent. Ablation of the HP1 network causes the dis-assembly of large megabase-scale domains and a significant shift from cis to trans interactions, while smaller domains hyper-cluster. Finally, analysis of the constitutive 10-5 triple-knockout (TKO) mutant reveals an architectural plasticity. In the permanent absence of HP1, the genome appears to activate a latent rescue network, where compartmentalization is partially rescued. In light of recent studies, we suggest that this partial rescue is a consequence of substituting the H3K9me3/HP1 axis with a Polycomb-mediated pathway (H3K27me3 and uH2A).