| INVESTIGATION Normal Patterns of Histone H3K27 Methylation Require the Histone Variant H2A.Z in Neurospora crassa Abigail J. Courtney,* Masayuki Kamei,* Aileen R. Ferraro,* Kexin Gai,† Qun He,† Shinji Honda,‡ and Zachary A. Lewis*,1 *Department of Microbiology, University of Georgia, Athens, Georgia 30602, †State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China, and ‡Division of Chromosome Biology, Faculty of Medical Sciences, University of Fukui, 910-1193, Japan ORCID IDs: 0000-0001-5319-2630 (A.J.C.); 0000-0003-0918-4486 (A.R.F.); 0000-0002-1735-8266 (Z.A.L.) ABSTRACT 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 DhH2Az;Deed 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. KEYWORDS H2A.Z; EED; PRC2; H3K27 methylation N eukaryotes, DNA-dependent processes in the nucleus are H3K27isamolecularmarkerof transcriptionally re- Iregulated by chromatin-based mechanisms (Luger 2003). pressed facultative heterochromatin (Cao et al. 2002; One heavily studied group of proteins that are particularly Czermin et al. 2002; Kuzmichev et al. 2002; Müller et al. important for maintaining stable gene repression are the 2002). PcG proteins are absent from the model yeasts, polycomb group (PcG) proteins. In plants and animal cells, PcG Saccharomyces cerevisiae and Schizosaccharomyces pombe, proteins assemble into polycomb repressive complexes 1 and but core PRC2 components have been identified and char- 2 (PRC1 and PRC2), which play key roles in repression of acterized in several fungi, including Neurospora crassa, developmental genes (as reviewed in Müller (1995), Hennig Fusarium graminearum, Cryptococcus neoformans, Epichloë and Derkacheva (2009), Simon and Kingston (2009), festucae,andFusarium fujikuroi (Veerappan et al. 2008; Schuettengruber et al. (2017), Kuroda et al. (2020)). PRC2 Aramayo and Selker 2013; Connolly et al. 2013; Jamieson is a multi-subunit complex that deposits mono-, di-, and tri- et al. 2013; Chujo and Scott 2014; Schotanus et al. 2015; methyl groups on lysine 27 of histone H3, and trimethyl Studt et al. 2016). In these fungi, PRC2 is required for re- Copyright © 2020 by the Genetics Society of America pression of key fungal genes, suggesting that this enzyme doi: https://doi.org/10.1534/genetics.120.303442 complex is functionally conserved between fungi, plants, Manuscript received April 9, 2020; accepted for publication July 5, 2020; published Early Online July 10, 2020. and animals (Connolly et al. 2013; Jamieson et al. 2013; Supplemental material available at figshare: https://doi.org/10.25386/genetics. Dumesic et al. 2015). 12486482. 1Correspondingauthor:UniversityofGeorgia,DavisonLifeSciences,120EGreenSt., In N. crassa, the catalytic subunit of PRC2 is SET-7, a pro- B402, Athens, GA 30602. E-mail: [email protected] tein with homology to EZH1/EZH2 in humans and curly leaf Genetics, Vol. 216, 51–66 September 2020 51 (CLF), medea (MEA), or swinger (SWN) in Arabidopsis (Wang et al. 2018). Colocalization of SUZ12, a subunit of (Jones and Gelbart 1993; Abel et al. 1996; Chen et al. PRC2, and H2A.Z has been found in mESCs at developmen- 1996; Goodrich et al. 1997; Grossniklaus et al. 1998; tally important genes, such as HOX clusters (Creyghton Czermin et al. 2002; Kuzmichev et al. 2002; Chanvivattana et al. 2008). In addition, H2A.Z is differentially modified et al. 2004; Schwartz and Pirrotta 2007). Neurospora EED is at its N- and C- terminal tails at bivalent domains that are essential for catalysis and is a homolog of mammalian en- “poised” for activation or repression upon differentiation hanced ectoderm development (EED), Drosophila extra sex (Ku et al. 2012; Surface et al. 2016). N-terminal acetylation combs (Esc) and Arabidopsis fertilization independent endo- (acH2A.Z) or C-terminal ubiquitylation (H2A.Zub) repress sperm (FIE) (Schumacher et al. 1998; Ng et al. 2000; or stimulate the action of PRC2 through interactions with Chanvivattana et al. 2004). SUZ-12 is the third essential com- the transcriptional activator BRD2 or the PcG protein com- ponent of PRC2 in Neurospora and is named SUZ12 in hu- plex PRC1 (Surface et al. 2016). It is important to note that mans, su(z)12 in Drosophila and embryonic flower 2 (EMF2), functional studies of H2A.Z are challenging because this vernalization 2 (VER2), or fertilization-independent seed 2 histone variant is essential for viability in most organisms, (FIS2) in Arabidopsis (Birve et al. 2001; Chanvivattana et al. including Drosophila, Tetrahymena,mouse,andXenopus 2004). N. crassa CAC-3/NPF is an accessory subunit homol- (van Daal and Elgin 1992; Iouzalen et al. 1996; Liu et al. ogous to mammalian retinoblastoma binding protein 46/48 1996; Clarkson et al. 1999; Faast et al. 2001; Ridgway et al. (RBAP46/68) in humans, and multicopy suppressor of 2004). IRA1-5 (MSI1-5) in Arabidopsis (Huang et al. 1991; Qian In Arabidopsis thaliana, a genetic interaction between et al. 1993; Derkacheva et al. 2013). In contrast to PRC2, PICKLE (PKL), a chromatin remodeler that promotes PRC1 components appear to be absent from the fungal king- H3K27me3, and PIE-1 (homolog to SWR-1), the remodeler dom (Jamieson et al. 2013; Lewis 2017). which deposits H2A.Z, was recently reported (Carter et al. The presence of a minimal polycomb repressive system in 2018). PKL has been found by ChIP-seq at loci enriched for well studied fungi such as N. crassa provides an opportunity H3K27me3, and is proposed to determine levels of H3K27me3 to explore the diversity of polycomb-mediated repression at repressed genes in Arabidopsis (Zhang et al. 2012). In rice across eukaryotes and enables genetic studies that can be callus and seedlings, H2A.Z is found at the 59 and 39 ends of difficult in plant and animal systems. Indeed, genetic studies genes that are highly expressed. In repressed genes, H2A.Z is have provided insights into PRC2 control in Neurospora.De- found along the gene body, and this pattern closely mimics letion of cac-3/npf causes region-specific losses of H3K27me3 the presence of H3K27me3 (Zhang et al. 2017). This is a at telomere-proximal domains, and telomere repeat sequences notable difference between plants and other eukaryotes. are sufficient to nucleate a new domain of H3K27me3-enriched We investigated the relationship between H2A.Z and PRC2 chromatin (Jamieson et al. 2013, 2018). In constitutive het- in the filamentous ascomycete Neurospora crassa, and report erochromatin domains, heterochromatin protein-1 (HP1) that H2A.Z is required for normal enrichment of H3K27me2/ prevents accumulation of H3K27me3 (Basenko et al. 2015; 3 across the genome. Our findings show that loss of H2A.Z Jamieson et al. 2016). Thus, regulation of H3K27 methyl- leads to region-specific losses of H3K27me2/3 in N. crassa. ation occurs at multiple levels. Despite recent advances, the Expression levels of eed, encoding a PRC2 subunit, are re- mechanisms that regulate PRC2 in fungal systems and eu- duced in the absence of H2A.Z and ectopic expression of karyotes in general is poorly understood. eed can restore H3K27me2/3 in an H2A.Z-deficient strain. In addition to the core histones (H2A, H2B, H3, and H4), Together, these data suggest that H2A.Z regulates facultative eukaryotes also encode nonallelic histone variants. One of the heterochromatin through transcriptional regulation of the most conserved and extensively studied histone variants is PRC2 component EED and points to differential requirements H2A.Z, which is enriched proximal to transcription start sites for EED at discrete PRC2-target domains. (TSS) and in vertebrate enhancers (Guillemette et al. 2005; Barski et al. 2007; Creyghton et al. 2008; Bargaje et al. 2012; Weber et al. 2014; Latorre et al. 2015; Dai et al. 2017; Gómez- Materials and Methods Zambrano et al. 2019). Functional studies of H2A.Z have Strains and growth media linked presence of this variant in nucleosomes to gene acti- vation, gene repression, maintaining chromatin accessibility, Strains used in this study are listed in Supplemental Material, and a multitude of other functions (Adam et al. 2001; Table S1. Strains were grown at 32° in Vogel’s minimal me- Meneghini et al. 2003; Rangasamy et al. 2004; Bruce et al. dium (VMM) with 1.5% sucrose or glucose for DNA-based 2005; Guillemette et al. 2005; Dhillon et al. 2006; Xu et al. protocols and RNA-based protocols, respectively (Davis and 2012; Hu et al. 2013; Neves et al. 2017). Notably, H2A.Z has de Serres 1970). Liquid cultures were shaken at 180 rpm. been implicated in the direct regulation of H3K27 methyl- Crosses were performed on synthetic crossing (SC) medium ation in mouse embryonic stem cells (mESCs) and in plants in the dark at room temperature (Davis and de Serres 1970). (Surface et al. 2016; Zhang et al. 2017; Carter et al. 2018).