- Histones, as part of nucleosomes, are responsible for DNA packaging in chromosomes. They also affect DNA expression by a multitude of post-translational modifications, especially prevalent on the amino terminus of histones. Co-activator protein complexes “write”, “read”, or “erase” histone modifications, and in balance determine which sections of DNA are free of histones and thus can be transcribed or “expressed”. The effects of histone H3 on gene silencing, DNA methylation, and centromere formation and maintenance has been studied in the model fungus, Neurospora crassa. Certain point mutations in the amino terminal tail of H3 can abrogate DNA methylation. We extended previous work by systematically mutating amino acids along the entire H3 protein, replacing the normal gene with mutated copies in both N. crassa and Fusarium graminearum. Point mutations were introduced into plasmids by a modified “QuickChange” method and transformed into each fungus by homologous recombination. Transformants were screened for proper integration, and strains were crossed to reporter strains to yield haploid progeny with mutated hH3 genes in combination with cytological markers. Cytology was combined with molecular and morphological assays to determine how exactly point mutations affect centromere function and DNA methylation in Neurospora, and gene silencing by H3K27 methylation in Fusarium.
- Key Words: Histone H3, gene silencing, centromeres, DNA methylation, Fusarium, Neurospora, chromatin