HP1 dimerization but not LLPS drives the condensation and segregation of H3K9me-marked chromatin

Heterochromatin protein 1 (HP1) is a conserved chromatin-associated factor implicated in the establishment and maintenance of H3K9me-marked heterochromatin, potentially through phase separation-mediated condensation. Whether HP1 in molecular terms works primarily via dimerization or liquid-liquid phase separation (LLPS) is unresolved. Using the C. elegans HP1 orthologue HPL-2 and a combined in vitro-in vivo approach, we systematically dissected the molecular determinants of HPL-2 function in heterochromatin condensation. Through specific mutants, we demonstrate that HPL-2 dimerization, but not LLPS, is essential for condensing H3K9me chromatin arrays in vitro and for maintaining H3K9me heterochromatin foci in C. elegans embryos. We further show that HPL-2 dimerization is sufficient to mediate segregation of H3K9me from unmodified chromatin arrays in vitro, generating biphasic condensates reminiscent of cellular heterochromatin domains. Surprisingly, HPL-2 mutants defective in condensation cause only minor transcriptional changes at canonical heterochromatin loci, implying that HP1-dependent heterochromatin foci and gene silencing are not tightly coupled in vivo. Nonetheless, these mutant C. elegans exhibit profound physiological and developmental defects. Our findings establish dimerization as the principal molecular mechanism of HP1-driven H3K9me-chromatin condensation, elucidate the auxiliary role of LLPS, and reveal the uncoupling between HP1-dependent heterochromatin and transcriptional regulation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/687974v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@ec0e06org.highwire.dtl.DTLVardef@18cbdd3org.highwire.dtl.DTLVardef@1028422org.highwire.dtl.DTLVardef@31123f_HPS_FORMAT_FIGEXP M_FIG C_FIG