The RPD3L deacetylation complex is required for facultative heterochromatin repression in Neurospora crassa [ChIP-seq]
Repression of facultative heterochromatin is essential for developmental processes in numerous organisms.  Methylation of histone H3 lysine 27 (H3K27) by Polycomb repressive complex 2 is a prominent feature of facultative heterochromatin in both fungi and higher eukaryotes.  Although this methylation is frequently crucial for silencing, the detailed mechanism of repression remains poorly understood.  We utilized a forward genetics approach to identify three previously uncharacterized genes required to maintain silencing at facultative heterochromatin genes in Neurospora crassa: sds3 (NCU01599), rlp1 (RPD3L protein 1; NCU09007) and rlp2 (RPD3L protein 2; NCU02898).  We found that SDS3, RLP1, and RLP2 associate with N. crassa homologs of the Saccharomyces cerevisiae RPD3L complex and are required for repression of a subset of H3K27-methylated genes.  Loss of these genes does not lead to loss of H3K27 methylation but increases acetylation of histone H3 lysine 14 at upregulated genes, and SDS3 binding levels are highest at genes derepressed upon loss of sds3, rlp1, and rlp2, implying that RPD3L-driven deacetylation is a previously undescribed factor required for silencing of facultative heterochromatin in N. crassa. Overall design: To analyze levels of histone modifications in RPD3L and RPD3S mutants, we performed ChIP-seq in ?sds3, ?rlp1, ?rlp2, ?eaf-3, and strain N6279 treated with TSA with antibodies against H3K27me2me3, H3K9ac, and H3K14ac.