Methylation of Histone H3 Lysine 36 Is Required for Normal Development in Neurospora crassa

Abstract

The SET domain is an evolutionarily conserved domain found predominantly in histone methyltransferases (HMTs). The Neurospora crassa genome includes nine SET domain genes (set-1 through set-9) in addition to dim-5, which encodes a histone H3 lysine 9 HMT required for DNA methylation. We demonstrate that Neurospora set-2 encodes a histone H3 lysine 36 (K36) methyltransferase and that it is essential for normal growth and development. We used repeat induced point mutation to make a set-2 mutant (set-2 RIP1 ) with multiple nonsense mutations. Western analyses revealed that the mutant lacks SET-2 protein and K36 methylation. An amino-terminal fragment that includes the AWS, SET, and post-SET domains of SET-2 proved sufficient for K36 HMT activity in vitro. Nucleosomes were better substrates than free histones. The set-2 RIP1 mutant grows slowly, conidiates poorly, and is female sterile. Introducing the wild-type gene into the mutant complemented the defects, confirming that they resulted from loss of set-2 function. We replaced the wild-type histone H3 gene (hH3) with an allele producing a Lys to Leu substitution at position 36 and found that this hH3 K36L mutant phenocopied the set-2 RIP1 mutant, confirming that the observed defects in growth and development result from inability to methylate K36 of H3. Finally, we used chromatin immunoprecipitation to demonstrate that actively transcribed genes in Neurospora crassa are enriched for H3 methylated at lysines 4 and 36. Taken together, our results suggest that methylation of K36 in Neurospora crassa is essential for normal growth and development.In eukaryotes DNA is wrapped around histones, which are subject to a variety of covalent modifications (e.g., methylation, acetylation, phosphorylation, and ubiquitylation) (51, 54). These modifications are believed to alter chromatin structure by influencing histone-DNA, histone-histone, and histone-nonhistone protein interactions (16, 55). Permutations of histone modifications constitute a "histone code" that can impact chromatin-templated processes, including DNA methylation, heterochromatin formation, transcription, and DNA replication and repair (18,46). The SET [Su(var)3-9, Enhancer-of-zeste, Trithorax] domain histone methyltransferases (HMTs) constitute one group of evolutionarily conserved chromatin-modifying proteins (10,12,19,23,24,40,45,60). A well-characterized example is the Neurospora crassa DIM-5 protein, which trimethylates lysine 9 of histone H3 and directs DNA methylation (48,49,58,59). In addition to DIM-5, the N. crassa genome encodes nine other putative SET domain proteins (Fig.

Suppression of WHITE COLLAR-independent frequency Transcription by Histone H3 Lysine 36 Methyltransferase SET-2 Is Necessary for Clock Function in Neurospora

“…Obviously, frq is not the only gene that SET-2 directly regulated. In 2005, Adhvaryu et al have found that SET-2 is required for Neurospora growth and development (26), indicating that SET-2 regulate genes in these processes. Furthermore, previous study in Saccharomyces cerevisiae has shown that ϳ25% of the total genome displayed a significant increase of H4ac following deletion of Set-2.…”

“…After electrophoresis, proteins were transferred onto PVDF membrane, and Western blot analysis was performed. RNA was extracted as described previously and then analyzed by Northern blotting (26). For Northern blot analysis, equal amounts of total RNA (20 g) were loaded onto 1.3% agarose gels for electrophoresis, the RNAs were transferred to nylon membrane.…”

“…SET-2 is the histone H3K36 methyltransferase in Neurospora, which is required for growth and development (26). The homolog of SET-2 in yeast Set2, it directly regulates gene transcription through interacting with phosphorylated RNA po-lymerase II CTD.…”

Genome-wide redistribution of H3K27me3 is linked to genotoxic stress and defective growth

“…We performed ChIP-seq for H3K27me3 in set-1 and set-2 mutants, which are deficient for H3K4 and H3K36 methylation, respectively (Fig. S2) (70). We observed changes in the relative level of H3K27me3 enrichment in the set-2 strain at some Pc-target domains, but the global pattern of H3K27me3 was similar to wild type in both strains.…”

Structural dynamics of protein lysine methylation and demethylation

“…Both Arabidopsis KYP and SUVH6 have a tyrosine at the switch position and are primarily monoMTases [57]. Neurospora crassa SET2 includes F296 at the switch position and the ezyme can add up to three methyl groups on histone H3 Lys-36 [58]. Chlamydomonas Set1 contains Y1768 at the switch position which would predict to generate mono-methylated Lys-4 on histone H3, an epigenetic mark for repressed euchromatin in Chlamydomonas [59].…”