Active and repressive chromatin are interspersed without spreading in an imprinted gene cluster in the mammalian genome

The Igf2r imprinted cluster is an epigenetic silencing model in which expression of a ncRNA silences multiple genes in cis. Here, we map a 250 kb region in mouse embryonic fibroblast cells to show that histone modifications associated with expressed and silent genes are mutually exclusive and localized to discrete regions. Expressed genes were modified at promoter regions by H3K4me3 + H3K4me2 + H3K9Ac and on putative regulatory elements flanking active promoters by H3K4me2 + H3K9Ac. Silent genes showed two types of nonoverlapping profile. One type spread over large domains of tissue-specific silent genes and contained H3K27me3 alone. A second type formed localized foci on silent imprinted gene promoters and a nonexpressed pseudogene and contained H3K9me3 + H4K20me3 +/- HP1. Thus, mammalian chromosome arms contain active chromatin interspersed with repressive chromatin resembling the type of heterochromatin previously considered a feature of centromeres, telomeres, and the inactive X chromosome.     

Effects of tethering HP1 to euchromatic regions of the Drosophila genome

Heterochromatin protein 1 (HP1) is a conserved non-histone chromosomal protein enriched in heterochromatin. On Drosophila polytene chromosomes, HP1 localizes to centric and telomeric regions, along the fourth chromosome, and to specific sites within euchromatin. HP1 associates with centric regions through an interaction with methylated lysine nine of histone H3, a modification generated by the histone methyltransferase SU(VAR)3-9. This association correlates with a closed chromatin configuration and silencing of euchromatic genes positioned near heterochromatin. To determine whether HP1 is sufficient to nucleate the formation of silent chromatin at non-centric locations, HP1 was tethered to sites within euchromatic regions of Drosophila chromosomes. At 25 out of 26 sites tested, tethered HP1 caused silencing of a nearby reporter gene. The site that did not support silencing was upstream of an active gene, suggesting that the local chromatin environment did not support the formation of silent chromatin. Silencing correlated with the formation of ectopic fibers between the site of tethered HP1 and other chromosomal sites, some containing HP1. The ability of HP1 to bring distant chromosomal sites into proximity with each other suggests a mechanism for chromatin packaging. Silencing was not dependent on SU(VAR)3-9 dosage, suggesting a bypass of the requirement for histone methylation.     

HP1a Recruitment to Promoters Is Independent of H3K9 Methylation in Drosophila melanogaster

Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila melanogaster three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1, and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, we have examined correlations between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. We show here that Su(var)3-9 controls H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. Our results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.