H3K9 methylation-independent activity for HPL-2/HP1 in heterochromatin foci, gene repression, and organogenesis

Differentiated cells rely on segregation of the genome into heterochromatin and euchromatin, but the mechanisms that establish these domains in vivo are incompletely understood. The current models suggest that heterochromatin is marked by histone H3 lysine 9 methylation (H3K9me), which recruits heterochromatin protein 1 (HP1), to compact chromatin and repress transcription. In C. elegans, the SETDB1 homolog MET-2 is essential for H3K9me-mediated gene silencing; however, MET-2 also localizes to heterochromatic foci independently of H3K9me and exhibits non-catalytic functions that support both germline and somatic development. Here we extend these findings by examining the classical H3K9me reader HP1. We show that HPL-2/HP1 deficient in methyl-lysine binding or in the absence of H3K9me can still repress transcription, promote organogenesis and localize to heterochromatin foci. Whereas activity remains in the absence of the HPL-2:H3K9me interaction, complete loss of met-2 and hpl-2 shows synergistic phenotypes for organogenesis and gene de-repression, underscoring the existence of additional, H3K9me-independent functions. HPL-2 and MET-2 require distinct binding partners for their localization and activity: MET-2 associates with the disordered protein LIN-65/ATF7IP, while HPL-2 depends on the multi-zinc finger protein LIN-13, which interacts with the HPL-2 chromoshadow domain. Our findings suggest that HPL-2 and MET-2 operate in parallel pathways, localizing to heterochromatic foci and promoting gene silencing and organogenesis, with H3K9me acting as a reinforcing, but non-essential, element in these processes. Overall design: RNA was extracted from early wild-type (N2 Bristol), hpl-2 (hpl-2(tm1489)), hpl-2-ffaa (hpl-2-FFAA(px112)), and hpl-2 met-2 (met-2(n4256) hpl-2(tm1489) III/hT2 [bli-4(e937) let-?(q782) qIs48] (I;III)) C. elegans embryos from a synchronized culture grown at 20°C, using Trizol. Embryos were freeze cracked five times, then RNA was extracted with choloroform followed by isopropanol precipitation. Further purification was performed with the RNA Clean and Concentrator kit (Zymo). Libraries were produced using the Stranded Total RNA Prep Ligation with Ribo-Zero Plus Kit (Illumina). rRNA was depleted using the Ribo-Zero Plus kit supplemented with custom made C. elegans specific rDNA oligos (IDT). Libraries were profiled in a DNA 1000 Chip on a 2100 Bioanalyser (Agilent technologies) and quantified using the Qubit 1x dsDNA HS Assay Kit, in a Qubit 4.0 Fluorometer (Life technologies). Equimolar amounts of indexed libraries were pooled and sequenced on a NextSeq 2000 (Illumina).