ABSTRACT
Both RNAi-dependent and -independent mechanisms have been implicated in the establishment of heterochromatin domains, which may be stabilized by feedback loops involving chromatin proteins and modifications of histones and DNA. Neurospora crassa sports features of heterochromatin found in higher eukaryotes, namely cytosine methylation (5mC), methylation of histone H3 lysine 9 (H3K9me), and heterochromatin protein 1 (HP1), and is a model to investigate heterochromatin establishment and maintenance. We mapped the distribution of HP1, 5mC, H3K9me3, and H3K4me2 at 100 bp resolution and explored their interplay. HP1, H3K9me3, and 5mC were extensively co-localized and defined 44 heterochromatic domains on linkage group VII, all relics of repeat-induced point mutation. Interestingly, the centromere was found in an approximately 350 kb heterochromatic domain with no detectable H3K4me2. 5mC was not found in genes, in contrast to the situation in plants and animals. H3K9me3 is required for HP1 localization and DNA methylation in N. crassa. In contrast, we found that localization of H3K9me3 was independent of 5mC or HP1 at virtually all heterochromatin regions. In addition, we observed complete restoration of DNA methylation patterns after depletion and reintroduction of the H3K9 methylation machinery. These data show that A:T-rich RIP'd DNA efficiently directs methylation of H3K9, which in turn, directs methylation of associated cytosines.
Subject(s)
Heterochromatin/metabolism , Neurospora crassa/genetics , Point Mutation , Repetitive Sequences, Nucleic Acid/physiology , Chromosome Mapping , Chromosomes, Fungal/genetics , DNA Methylation/physiology , Eukaryotic Initiation Factors/genetics , Evolution, Molecular , Heterochromatin/genetics , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Mutagenesis/genetics , Mutagenesis/physiology , Organisms, Genetically Modified , Point Mutation/genetics , Repetitive Sequences, Nucleic Acid/geneticsABSTRACT
BACKGROUND: Silencing of genes inserted near telomeres provides a model to investigate the function of heterochromatin. We initiated a study of telomeric silencing in Neurospora crassa, a fungus that sports DNA methylation, unlike most other organisms in which telomeric silencing has been characterized. RESULTS: The selectable marker, hph, was inserted at the subtelomere of Linkage Group VR in an nst-1 (neurospora sir two-1) mutant and was silenced when nst-1 function was restored. We show that NST-1 is an H4-specific histone deacetylase. A second marker, bar, tested at two other subtelomeres, was similarly sensitive to nst-1 function. Mutation of three additional SIR2 homologues, nst-2, nst-3 and nst-5, partially relieved silencing. Two genes showed stronger effects: dim-5, which encodes a histone H3 K9 methyltransferase and hpo, which encodes heterochromatin protein-1. Subtelomeres showed variable, but generally low, levels of DNA methylation. Elimination of DNA methylation caused partial derepression of one telomeric marker. Characterization of histone modifications at subtelomeric regions revealed H3 trimethyl-K9, H3 trimethyl-K27, and H4 trimethyl-K20 enrichment. These modifications were slightly reduced when telomeric silencing was compromised. In contrast, acetylation of histones H3 and H4 increased. CONCLUSION: We demonstrate the presence of telomeric silencing in Neurospora and show a dependence on histone deacetylases and methylation of histone H3 lysine 9. Our studies also reveal silencing functions for DIM-5 and HP1 that appear independent of their role in de novo DNA methylation.
ABSTRACT
Methylation of cytosines silences transposable elements and selected cellular genes in mammals, plants, and some fungi. Recent findings have revealed mechanistic connections between DNA methylation and features of specialized condensed chromatin, "heterochromatin." In Neurospora crassa, DNA methylation depends on trimethylation of Lys9 in histone H3 by DIM-5. Heterochromatin protein HP1 binds methylated Lys9 in vitro. We therefore investigated the possibility that a Neurospora HP1 homolog reads the methyl-Lys9 mark to signal DNA methylation. We identified an HP1 homolog and showed that it is essential for DNA methylation, is localized to heterochromatic foci, and that this localization is dependent on the catalytic activity of DIM-5. We conclude that HP1 serves as an adaptor between methylated H3 Lys9 and the DNA methylation machinery. Unlike mutants that lack DNA methyltransferase, mutants with defects in the HP1 gene hpo exhibit severe growth defects, suggesting that HP1 is required for processes besides DNA methylation.
