HP1-driven phase separation: liquid droplets or collapsed globules?

Faculty Development Competitive Research Grant Program (General) 2024-2026

Studies in Drosophila, human and mouse cells have shown that a significant fraction of the contact enrichments that emerge as B-type compartments result from HP1-driven phase separation. A key question, to be addressed here, is whether HP1-driven phase separation is the result of liquid-liquid phase separation (LLPS) forming “liquid droplets” consisting of a concentrated “solution” of HP1 proteins or is the result of a “collapsed globule” that results from polymer-polymer phase separation (PPPS) of HP1-containing heterochromatin-like domains/complexes (HLD/Cs). In this proposal our aim is to discriminate between these two mechanisms using 4C chromosome conformation capture to measure the (far) cis-contacts between the large (up to 4Mb) HP1-containing heterochromatin-like domains (HLDs) that constitute the B4 heterochromatic sub-compartment in Hi-C maps. To that end, we have generated a novel (unpublished) HP1 null cell line. The HP1 null cell line is a “tabula rasa” into which we can introduce constructs that express mutant forms of HP1 that have been implicated in LLPS or PPPS and thereby test whether these HP1 constructs can affect (far) cis-contacts between the KRAB-ZFP HLDs.