HPL-2/HP1 and MET-2/SETDB1 bind distinct co-factors that promote heterochromatic foci, gene repression and organogenesis independently of H3K9 methylation

Histone post-translational modifications (PTMs) promote genome segregation into heterochromatin and euchromatin by recruiting reader proteins, including HP1 proteins that bind repressive H3 lysine 9 methylation (H3K9me). However, recent studies suggest that H3K9me readers and writers also have PTM-independent activities. Here we examine the PTM-independent roles of the key H3K9 methyltransferase, MET-2/SETDB1, and the C. elegans HP1 orthologue, HPL-2, in organogenesis. The two factors co-localize in sub-nuclear foci independently of each other, and their combined loss additively disrupts gene repression and vulval organogenesis. HPL-2 remains functional in the absence of H3K9me, and HPL-2 lacking its H3K9me-binding chromodomain still localizes to heterochromatin foci, restricting vulva formation and repressing developmental genes. While MET-2 requires the disordered protein LIN-65 to form foci and silence genes, HPL-2 functions together with LIN-13, which is bound through HPL-2’s chromoshadow domain. In conclusion, HPL-2 and MET-2 repress in parallel on largely H3K9me-independent pathways, requiring distinct cofactors; while H3K9me reinforces both.