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Polycomb repressive complex 2 (PRC2) is responsible for the trimethylation of lysine 27 of histone H3 (H3K27me3)-mediated transcriptional silencing. At present, its biological roles in the devastating rice pathogenic fungus Ustilaginoidea virens remain unclear. In this study, we analyzed the function of a putative PRC2 catalytic subunit UvKmt6. The results showed that disruption of UvKMT6 resulted in reduced growth, conidiation and pathogenicity in U. virens. Furthermore, UvKmt6 is essential for establishment of H3K27me3 modification, which covers 321 genes in the genome. Deletion of UvKMT6 led to transcriptional derepression of 629 genes, 140 of which were occupied with H3K27me3 modification. Consistent with RNA-seq and ChIP-seq analysis, UvKmt6 was further confirmed to participate in the transcriptional repression of genes encoding effectors and genes associated with secondary metabolites production, such as PKSs, NRPSs and Cytochrome P450s. Notably, we found that UvKmt6 is involved in transcriptional repression of oxidative, osmotic, cell wall and nutrient starvation stresses response-related genes. From the perspective of gene expression and phenotype, in addition to the relatively conservative role in fungal development, virulence and production of secondary metabolites, we further reported that UvKmt6-mdediated H3K27me3 plays a critical role in the response to various stresses in U. virens.

EZH2 inhibition can decrease global histone H3 lysine 27 trimethylation (H3K27me3) and thereby reactivates silenced tumor suppressor genes. Inhibition of EZH2 is regarded as an option for therapeutic cancer intervention. To identify novel small-molecule (SMOL) inhibitors of EZH2 in drug discovery, trustworthy cellular assays amenable for phenotypic high-throughput screening (HTS) are crucial. We describe a reliable approach that quantifies changes in global levels of histone modification marks using high-content analysis (HCA). The approach was validated in different cell lines by using small interfering RNA and SMOL inhibitors. By automation and miniaturization from a 384-well to 1536-well plate, we demonstrated its utility in conducting phenotypic HTS campaigns and assessing structure-activity relationships (SAR). This assay enables screening of SMOL EZH2 inhibitors and can advance the mechanistic understanding of H3K27me3 suppression, which is crucial with regard to epigenetic therapy. We observed that a decrease in global H3K27me3, induced by EZH2 inhibition, comprises two distinct mechanisms: (1) inhibition of de novo DNA methylation and (II) inhibition of dynamic, replication-independent H3K27me3 turnover. This report describes an HCA assay for primary HTS to identify, profile, and optimize cellular active SMOL inhibitors targeting histone methyltransferases, which could benefit epigenetic drug discovery. [ABSTRACT FROM PUBLISHER]

Polycomb repressive complex 2 (PRC2) is responsible for the trimethylation of lysine 27 of histone H3 (H3K27me3)-mediated transcriptional silencing. At present, its biological roles in the devastating rice pathogenic fungus Ustilaginoidea virens remain unclear. In this study, we analyzed the function of a putative PRC2 catalytic subunit UvKmt6. The results showed that disruption of UvKMT6 resulted in reduced growth, conidiation and pathogenicity in U. virens . Furthermore, UvKmt6 is essential for establishment of H3K27me3 modification, which covers 321 genes in the genome. Deletion of UvKMT6 led to transcriptional derepression of 629 genes, 140 of which were occupied with H3K27me3 modification. Consistent with RNA-seq and ChIP-seq analysis, UvKmt6 was further confirmed to participate in the transcriptional repression of genes encoding effectors and genes associated with secondary metabolites production, such as PKS s, NRPS s and Cytochrome P450s . Notably, we found that UvKmt6 is involved in transcriptional repression of oxidative, osmotic, cell wall and nutrient starvation stresses response-related genes. From the perspective of gene expression and phenotype, in addition to the relatively conservative role in fungal development, virulence and production of secondary metabolites, we further reported that UvKmt6-mdediated H3K27me3 plays a critical role in the response to various stresses in U. virens .