Research Activity
Regulation of Heterochromatin Spreading
Maintenance of open and repressed chromatin states is crucial for genome stability and regulation of gene expression. To study the genes involved in maintaining chromatin states, we generated a random mutant library in Schizosaccharomyces pombe and monitored silencing of reporter genes inserted into the euchromatic region, adjacent to the heterochromatic mating type locus. This screen identified several genes, including a novel gene, named epe1 (Enhancement of Position Effect), that encodes a protein with a JmjC domain. Mutational inactivation of epe1 promotes propagation of heterochromatin across boundary elements and stabilization of methylated H3K9. This observation is consistent with the proposition that Epe1, like other JmjC proteins, is a putative histone demethylase. However, no demethylase activity has been shown to be associated with Epe1. Additional possible mechanisms for Epe1 anti-silencing activity involve destabilization of heterochromatin by up-regulation of histone turnover. Since its identification, Epe1 was shown to be recruited to heterochromatin domains by Swi6 (HP1) and to be eliminated from internal parts of these domains by the conserved Cul4-Ddb1Cdt2 ubiquitin ligase. In addition to its activity at boundary elements, Epe1 functions as an anti-silencer at constitutive, as well as facultative heterochromatin domains. Remarkably, inactivation of Epe1 allows inheritance of methylated H3K9 trans-generationally through many mitotic divisions.
A similar genetic screen for genes that regulate heterochromatin spreading revealed members of the RNA polymerase II-associated factor 1 complex (PafC). We showed that like Epe1, members of PafC (Paf1 and Leo1) are required to prevent spreading of heterochromatin into euchromatin. Employing a novel recombination-induced tag exchange (RITE) assay, we showed that loss of Leo1–Paf1 increases heterochromatin stability at several facultative heterochromatin loci in an RNAi-independent manner. Instead, deletion of Leo1 decreases nucleosome turnover, leading to heterochromatin stabilization. Our data reveal that Leo1–Paf1, like Epe1, promotes chromatin state fluctuations by enhancing histone turnover.
