| Histone modification and DNA methylation have been the subject of intense study in recent years, and interesting connections between these two epigenetic silencing mechanisms have been discovered. In the filamentous fungus Neurospora crassa, treatment with the histone deacetylase inhibitor Trichostatin A (TSA) results in sequence-specific loss of DNA methylation. I was interested in investigating this novel phenomenon. No genes encoding proteins responsible for histone modifications had been isolated from Neurospora when I began this project.; I isolated and characterized four histone deacetylase (HDAC) genes from Neurospora crassa (hda-1–hda-4). These genes comprise the entire ‘classical’ RPD3-type HDAC family from this fungus. I created null mutants of hda-1, hda-2, and hda-4, and two partially functional mutants of the hda-3 gene. I found evidence that HDA-3 is essential for viability, and production of asexual spores. Mutation of either hda-1 or hda-3 affected DNA methylation at specific chromosomal loci, much like treatment of hda+ strains with TSA. Western blot analysis revealed differential histone acetylation in HDAC mutants. Results suggest that HDA-1 or HDA-3 is the major target of TSA treatment. Changes in both methylation and phosphorylation of histone H3 were also observed in these mutants. Mutation of HDA-2 and HDA-4 did not affect DNA methylation at any loci tested. Both of these mutant strains exhibited invasive growth on solid medium.; I isolated and mutated Neurospora genes encoding three histone acetyltransferases (HATs), and one co-repressor and investigated their effects on DNA methylation. The GNAT (Gcn5-similar N-acetyltransferase) family HATs ngf-1 and elp-3, were isolated, and found to have no effect on DNA methylation. Strains mutated in ngf-1 had a slow-growth phenotype, as well as a lack of aerial hyphae. Mutant elp-3 strains grew slowly, but showed no other phenotypes. A null mutation was created in the MYST (MOZ, Ybf2/Sas3, Sas2, and Tip60) family HAT gene hbo-1 . The mutant strain grew slowly and colonially, but no effect on DNA methylation either with or without TSA-treatment was observed. Mutations in the Neurospora homologue of the co-repressor sin-3 were also obtained. This gene is likely essential for viability, and no effects on DNA methylation were observed in the mutant isolated. |
