25 Neurospora crassa contains a minimal Polycomb repression system, which provides rich 26 opportunities to explore Polycomb-mediated repression across eukaryotes and enables genetic 27 studies that can be difficult in plant and animal systems. Polycomb Repressive Complex 2 is a 28 multi-subunit complex that deposits mono-, di-, and tri-methyl groups on lysine 27 of histone 29 H3, and tri-methyl H3K27 is a molecular marker of transcriptionally repressed facultative 30 heterochromatin. In mouse embryonic stem cells and multiple plant species, H2A.Z has been 31 found to be co-localized with H3K27 methylation. H2A.Z is required for normal H3K27 32 methylation in these experimental systems, though the regulatory mechanisms are not well 33 understood. We report here that Neurospora crassa mutants lacking H2A.Z or SWR-1, the ATP-34 dependent histone variant exchanger, exhibit a striking reduction in levels of H3K27 35 methylation. RNA-sequencing revealed downregulation of eed, encoding a subunit of PRC2, in 36 an hH2Az mutant compared to wild type and overexpression of EED in a ∆hH2Az;∆eed 37 background restored most H3K27 methylation. Reduced eed expression leads to region-specific 38 losses of H3K27 methylation suggesting that EED-dependent mechanisms are critical for normal 39 H3K27 methylation at certain regions in the genome. 40 41 42 43 44 45 46 In eukaryotes, DNA-dependent processes in the nucleus are regulated by chromatin-67 based mechanisms (1). One heavily studied group of proteins that are particularly important for 68 maintaining stable gene repression are the Polycomb Group (PcG) proteins. In plants and animal 69 cells, PcG proteins assemble into Polycomb Repressive Complexes 1 and 2 (PRC1 and PRC2), 70 which play key roles in repression of developmental genes, as reviewed in (2-6). PRC2 is a 71 multi-subunit complex that deposits mono-, di-, and tri-methyl groups on lysine 27 of histone 72 H3, and tri-methyl H3K27 is a molecular marker of transcriptionally repressed facultative 73 heterochromatin (7-10). PcG proteins are absent from the model yeasts, Saccharomyces 74 cerevisiae and Schizosaccharomyces pombe, but core PRC2 components have been identified 75 and characterized in several fungi, including Neurospora crassa, Fusarium graminearum, 76 Cryptococcus neoformans, Epichloë festucae, and Fusarium fujikoroi (11)(12)(13)(14)(15)(16)(17). In these fungi, 77 PRC2 is required for repression of key fungal genes suggesting that this enzyme complex is 78 functionally conserved between fungi, plants, and animals (13, 14, 18).
79In N. crassa, the catalytic subunit of PRC2 is SET-7, a protein with homology to 80 EZH1/EZH2 in humans and curly leaf (CLF), medea (MEA), or swinger (SWN) in Arabidopsis 81 (9, 10,(19)(20)(21)(22)(23)(24)(25). N. crassa EED and SUZ12 are respectively homologous to Drosophila Esc and 82 su(z)12, human EED and SUZ12, and Arabidopsis fertilization independent endosperm (FIE; a 83 homolog of EED), and SUZ12 homologs embryonic flower 2 (EMF2), vernalization 2 (VER2), 84 or fertilization in...