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.

No abstract

Histone H3 lysine 9 methylation (H3K9me) epigenetically silences gene expression by forming heterochromatin. Proteins called HP1, which contain specialized reader domains, bind to H3K9me and recruit factors that regulate epigenetic silencing. Though these interactions have been identifiedin vitro, we do not understand how HP1 proteins specifically and selectively bind to heterochromatin-associated factors within the nucleus. Using fission yeast as a model system, we measured the single-molecule dynamics associated with two archetypal HP1 paralogs, Swi6 and Chp2, and inferred how they form complexes with their interacting partners: Epe1, a putative H3K9 demethylase; Clr3, a histone deacetylase; and Mit1, a chromatin remodeler. Through a series of genetic perturbations that affect H3K9 methylation and HP1-mediated recruitment, we were able to track altered diffusive properties associated with each HP1 protein and its binding partner. Our findings show that the HP1-interacting proteins we investigated only co-localize with Swi6 and Chp2 at sites of H3K9me. When H3K9me is absent, Epe1 and Swi6 exhibit diffusive states consistent with off-chromatin interactions. Our results suggest that histone modifications like H3K9 methylation are not simply inert binding platforms but rather, they can shift the balance of HP1 complex assembly toward a predominantly chromatin-bound state. By inferring protein-protein interactions based on the altered mobilities of proteins in living cells, we propose that H3K9 methylation can stimulate the assembly of diverse HP1-associated complexes on chromatin.

A classical and well-established mechanism that enables cells to adapt to new and adverse conditions is the acquisition of beneficial genetic mutations. Much less is known about epigenetic mechanisms that allow cells to develop novel and adaptive phenotypes without altering their genetic blueprint. It has been recently proposed that histone modifications, such as heterochromatin-defining H3K9 methylation (H3K9me), normally reserved to maintain genome integrity, can be redistributed across the genome to establish new and potentially adaptive phenotypes. To uncover the dynamics of this process, we developed a precision engineered genetic approach to trigger H3K9me redistribution on demand in fission yeast. This enabled us to trace genome-scale RNA and chromatin changes over time prior to and during adaptation in long-term continuous cultures. Establishing adaptive H3K9me occurs over remarkably slow time-scales relative to the initiating stress. During this time, we captured dynamic H3K9me redistribution events ultimately leading to cells converging on an optimal adaptive solution. Upon removal of stress, cells relax to new transcriptional and chromatin states rather than revert to their initial (ground) state, establishing a tunable memory for a future adaptive epigenetic response. Collectively, our tools uncover the slow kinetics of epigenetic adaptation that allow cells to search for and heritably encode adaptive solutions, with implications for drug resistance and response to infection.