Facultative heterochromatin in Fusarium: Control of gene regulation by Polycomb Repressive Complex 2

Polycomb Group (PcG) proteins generate facultative heterochromatin by trimethylating histone H3 lysine 27 (H3K27me3). Members of the conserved Polycomb Repressive Complex 2 (PRC2) include the H3K27 methyltransferase, KMT6, and its binding partners SUZ12, EED, and –at least sometimes– MSL1. In humans, mutation of PRC2 components result in developmental defects, inherited diseases and sporadic cancers. Deletion of kmt6, eed, or suz12 in Fusarium graminearum leads to complete loss of H3K27me3, which is accompanied by pleiotropic developmental defects, but deletion of msl1 has no such drastic effects. Close to a quarter of all genes are increased in expression by more than fourfold in kmt6, eed, or suz12 mutants, and many are involved in development, secondary metabolism and pathogenicity. We will report on the in vivo and in vitro effects of PRC2 subunit mutations. Minor changes in the primary sequence of KMT6 resulted in complete or intermediate loss of function. Cytology and ChIP-seq showed partial mislocalization of KMT6-GFP in some of these strains. Several mutations affected the allosteric regulation of KMT6 by the EED or SUZ12 subunits. To uncover suppressors of H3K27me3 silencing, and to identify functional equivalents of PRC1 (an animal complex that binds H3K27me3 yet does not exist in fungi), we developed a forward genetics approach, relying on de-repression of a neo reporter gene in a reliably silenced region. Dozens of primary mutants, called defective in silencing (dis) or drug response attenuated (dat), were classified by distinct growth phenotypes and global gene expression patterns evidenced by transcriptome sequencing. Mutations in dis and dat genes were identified by bulk segregant analyses followed by sequencing. The first mutant, dis1, carries a truncated eed gene, and completely phenocopies the deletion mutant we constructed. We will report on additional components of the PcG silencing system.