The establishment of HP1-independent heterochromatin reveals an essential role for HP1 proteins in maintaining epigenetic memory.
H3K9 methylation (H3K9me) marks transcriptionally silent genomic regions called heterochromatin. A conserved class of HP1 proteins are critically required to establish and  maintain heterochromatin. HP1 proteins bind to H3K9me, recruit factors that promote heterochromatin formation, and oligomerize to form phase-separated condensates. We do not  understand how HP1 protein binding to heterochromatin establishes and maintains transcriptional silencing. Here, we demonstrate that the S.pombe HP1 homolog, Swi6, can be  completely bypassed to establish silencing at ectopic and endogenous loci when an H3K4 methyltransferase, Set1 and an H3K14 acetyltransferase, Mst2 are deleted. Deleting Set1 and  Mst2 enhances Clr4 enzymatic activity, leading to higher H3K9me levels and increased spreading. In contrast, Swi6 and its capacity to oligomerize were indispensable during epigenetic maintenance. Our results demonstrate the role of HP1 proteins in regulating histone modification crosstalk during establishment and identifies a genetically separable function in  maintaining epigenetic memory. Overall design: Comparative gene expression profiling analysis of RNA-seq data, performed in triplicate, under several genotypic backgrounds. Strains contained a tethering site to nucleate heterochromatin at an ectopic locus with a TetR-Clr4-I fusion (-Tet). To release the fusion protein, strains were treated with Tetracycline (+Tet).