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.

FIG. 1.

Domain organization of DIM-5 and other putative SET proteins of Neurospora crassa. Ten SET domain genes were identified in N. crassa: dim-5 (NCU04402.2); set-1 (NCU01206.2); set-2 (NCU00269.2); set-3 (NCU01932.2); set-4 (NCU04389.2); set-5 (NCU06119.2); set-6 (NCU09495.2); set-7 (NCU07496.2); set-8 (NCU01973.2); and set-9 (NCU08733.2) (4). Domains, identified using SMART (26, 41), are abbreviated as follows: AWS (Associated With SET, SM00570); SET (SM00317); pS (post-SET, SM00508); WW (SM00456), CC (coiled coil); PreSET (SM00468); AT-hook (SM00384); PHD zinc finger (SM00249); JmjC (SM0058); RRM 1 (SM00360). The set-2^RIP1 allele has predicted nonsense codons (indicated by asterisks) at positions 173, 178, 395, 410, 446, 485, 491, 553, 562, 595, 615, 617, 621, 639, 685, 723, and 787, and amino acid substitutions H235Y, S240L, H261Y, A365V, P378L, L393F, H416Y, R418W, L454F, R456C, T463I, H476Y, H479Y, L490F, T498I, T515I, H527Y, S532L, T582I, H593Y, S610F, H635Y, S636F, T645I, H675Y, H684Y, L692F, H742Y, A750V, S768L, R775C, P788S, T861I, and P862L. The sequence of set-2^RIP1 is available at GenBank (accession number AY971375).

FIG. 2.

SET-2 is essential for normal development in Neurospora crassa. (A) Poor conidiation in set-2^RIP1 and complementation of mutation. The wild type (1; N2952), a set-2^RIP1 strain (2; N2958), a set-2^RIP1 derivative in which the mutation was complemented by a C-terminal Flag-tagged version of set-2⁺ incorporated at the his-3 locus (3; N2962), and a set-2^RIP1 strain that has only a Flag tag at the his-3 locus (4; N2961) were grown at 32°C for 6 days on solid Vogel's medium N with 1.5% sucrose and supplemented with 0.2 mg/ml alanine and 0.05 mg/ml inositol. (B) The set-2^RIP1 mutant is female sterile. Reciprocal crosses between the wild type (N1) and a set-2^RIP1 strain (N2956) were accomplished using solid synthetic crossing medium with 0.1% sucrose as the carbon source and supplemented with 0.05 mg/ml inositol. The male parent was inoculated 4 days after the female parent. Photographs were taken after 18 and 58 days at 25°C. (C) Reduced and variable growth of set-2^RIP1 strains. Linear growth at 32°C of wild-type (N150, N623, N2952, N2953, N2954, and N2955) and set-2^RIP1 (N2949, N2956, N2957, N2958, N2959, and N2960) strains on the medium described in A were measured in race tubes (6, 38). Each value is an average of measurements from two tubes.

FIG. 3.

set-2^RIP1 mutant lacks SET-2 and H3 lysine 36 methylation. (A) Complementation of set-2^RIP1. Western blot analyses were performed using crude nuclear protein from the wild-type (N150), a set-2^RIP1 strain (N2956), and a set-2^RIP1 strain that has been complemented with a Flag-tagged set-2⁺ at the his-3 locus (N2962). (B) Wild-type N. crassa has mono-, di-, and trimethyl-lysine 36. Western analysis of wild-type (N150) and set-2^RIP1 (N2956) strains. (C) The dim-5 mutant has normal levels of lysine 36 methylation. Western analysis of nuclear proteins from wild-type (N150) and dim-5 (N2264) strains.

FIG. 4.

SET-2 preferentially methylates H3 in nucleosomes. (A) Lysates containing SET-2 from uninduced (U-SET-2) or IPTG-induced (I-SET-2) bacterial expression strains were used in HMT assays with chicken core histones or oligonucleosomes as substrates and ³H-labeled S-adenosyl-methionine (³H AdoMet). ³H incorporation was analyzed by the filter-binding assay (47). (B) Reaction products from HMT assays containing recombinant SET-2 from uninduced (U-SET-2) or IPTG-induced (I-SET-2) bacterial lysates, with chicken core histones or oligonucleosomes as substrates and ³H-labeled S-adenosyl-methionine were resolved on a sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis gel and examined by Coomassie staining (lower panel) and fluorography (upper panel). Asterisks indicate partial H3 breakdown product. (C) Reaction products from HMT assays containing recombinant SET-2 from uninduced (U-SET-2) or IPTG-induced (I-SET-2) bacterial lysates, with oligonucleosomes as substrates and AdoMet, were resolved on a sodium dodecyl sulfate-15% polyacrylamide gel electrophoresis gel and examined by Coomassie staining (lower panel) and Western blotting (upper panel).

FIG. 5.

hH3^K36L allele confers phenotypes similar to the set-2^RIP1 mutation. (A) Sequence of histone H3 amino-terminal tail. Lysines that are methylated are indicated in bold. (B) Strategy used for replacement of wild-type hH3 with hH3^K36L. (C) Southern analysis to confirm replacement. DNA isolated from wild-type hH3 (N2836) and hH3^K36L (N2970) was digested with HindIII. The blot was probed with a fragment indicated in B. The single ∼5.1-kb band indicates that the wild-type hH3 gene in N2836 was replaced by the mutated hH3^K36L::inl⁺ allele. (D and E) Poor growth and conidiation of the hH3^K36L strain. Wild-type (N150) and hH3^K36L (N2970) strains were grown as described for Fig. 2. Linear growth of the wild type (N150: square) and two hH3^K36L transformants (N2970 [triangles] and N2975 [circles]) were measured at 32°C in growth tubes.

FIG. 6.

H3 lysine 36 methylation is enriched in coding regions of actively transcribed genes. Chromatin immunoprecipitation analysis of regions across the mtr and hH3 genes. (A) Primers (Table 2) were designed to amplify 300-bp (a) or 400-bp (b, c, d, e, and f) fragments across a 2.4-kb region of the mtr gene. (B) PCRs were carried out to amplify region a with either b, c, d, e, or f. Samples are total input (1), no antibody (2), anti-H3K4me2 (3), and anti-H3K36me3 (4). (C) Relative enrichment of H3K4me2 and H3K36me3 at different regions across mtr. Relative enrichment values were calculated by dividing the ratio of band intensity for immunoprecipitated DNA/a region with ratio of intensities for total input/a region. Signal intensities are averages of PCRs from two independent chromatin immunoprecipitation experiments. All enrichments are standardized to that of region a. (D) Primers (Table 2) were designed to amplify 200-bp (a) or 300-bp (b and c) fragments across a 1.5-kb region of the hH3 gene. (E) PCRs were carried out to amplify region a with either b or c. (F) Relative enrichment of H3K4me2 and H3K36me3 at different regions across hH3.