Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component

Post-translational modification of histone tails is thought to modulate higher-order chromatin structure(1-3). Combinations of modifications including acetylation, phosphorylation and methylation have been proposed to provide marks recognized by specific proteins(4). This is exemplified, in both mammalian cells and fission yeast, by transcriptionally silent constitutive pericentric heterochromatin. Such heterochromatin contains histones that are generally hypoacetylated(5) and methylated by Suv39h methyltransferases at lysine 9 of histone H3 (H3- K9)(6,7). Each of these modification states has been implicated in the maintenance of HP1 protein-binding at pericentric heterochromatin, in transcriptional silencing and in centromere function(7-12). In particular, H3-K9 methylation is thought to provide a marking system for the establishment and maintenance of stably repressed regions and heterochromatin subdomains(3,13). To address the question of how these two types of modifications, as well as other unidentified parameters, function to maintain pericentric heterochromatin, we used a combination of histone deacetylase inhibitors, RNAse treatments and an antibody raised against methylated branched H3- K9 peptides. Our results show that both H3- K9 acetylation and methylation can occur on independent sets of H3 molecules in pericentric heterochromatin. In addition, we identify an RNA-and histone modification-dependent structure that brings methylated H3- K9 tails together in a specific configuration required for the accumulation of HP1 proteins in these domains.