Significance: Polycomb Repressive Complex 2 (PRC2) methylates histones to regulate multicellular development, maintenance of stem cell identity, X-chromosome inactivation, and other important processes. Given these essential roles, there is significant interest in identifying components that function with PRC2 to establish and maintain transcriptionally repressive heterochromatin. Here we document an unexpected new role for a well-studied and conserved chromatin remodeling factor, ISWI. We found that the Neurospora ISWI homolog is required for normal facultative heterochromatin structure and gene repression at PRC2 target regions, and we defined requirements for ATP-dependent catalytic activity and accessory regulatory proteins. These findings provide mechanistic insights into the formation and function of facultative heterochromatin in a model eukaryote.

Cellular differentiation is instructed by developmental regulators in coordination with chromatin remodeling complexes. Much information about their coordination comes from studies in the model ascomycetous yeasts. It is not clear, however, what kind of information that can be extrapolated to species of other phyla in Kingdom Fungi. In the basidiomycete Cryptococcus neoformans, the transcription factor Znf2 controls yeast-to-hypha differentiation. Through a forward genetic screen, we identified the basidiomycete-specific factor Brf1. We discovered Brf1 works together with Snf5 in the SWI/SNF chromatin remodeling complex in concert with existent Znf2 to execute cellular differentiation. We demonstrated that SWI/SNF assists Znf2 in opening the promoter regions of hyphal specific genes, including the ZNF2 gene itself. This complex also supports Znf2 to fully associate with its target regions. Importantly, our findings revealed key differences in composition and biological function of the SWI/SNF complex in the two major phyla of Kingdom Fungi.

Neurospora crassa contains a minimal Polycomb repression system, which provides rich opportunities to explore Polycomb-mediated repression across eukaryotes and enables genetic studies that can be difficult in plant and animal systems. Polycomb Repressive Complex 2 is a multi-subunit complex that deposits mono-, di-, and trimethyl groups on lysine 27 of histone H3, and trimethyl H3K27 is a molecular marker of transcriptionally repressed facultative heterochromatin. In mouse embryonic stem cells and multiple plant species, H2A.Z has been found to be colocalized with H3K27 methylation. H2A.Z is required for normal H3K27 methylation in these experimental systems, though the regulatory mechanisms are not well understood. We report here that Neurospora crassa mutants lacking H2A.Z or SWR-1, the ATP-dependent histone variant exchanger, exhibit a striking reduction in levels of H3K27 methylation. RNA-sequencing revealed downregulation of eed, encoding a subunit of PRC2, in an hH2Az mutant compared to wild type, and overexpression of EED in a ΔhH2Az;Δeed background restored most H3K27 methylation. Reduced eed expression leads to region-specific losses of H3K27 methylation, suggesting that differential dependence on EED concentration is critical for normal H3K27 methylation at certain regions in the genome.