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The Fusarium graminearum histone H3 K27 methyltransferase KMT6 regulates development and expression of secondary metabolite gene clusters.

Connolly LR, Smith KM, Freitag M - PLoS Genet. (2013)

Bottom Line: By chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing.Di- or trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found in mutually exclusive regions of the genome.Taken together, we show that absence of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing, Oregon State University, Corvallis, Oregon, United States of America.

ABSTRACT
The cereal pathogen Fusarium graminearum produces secondary metabolites toxic to humans and animals, yet coordinated transcriptional regulation of gene clusters remains largely a mystery. By chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing. H3K27me3 was found predominantly in regions that lack synteny with other Fusarium species, generally subtelomeric regions. Di- or trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found in mutually exclusive regions of the genome. To find functions for H3K27me3, we deleted the gene for the putative H3K27 methyltransferase, KMT6, a homolog of Drosophila Enhancer of zeste, E(z). The kmt6 mutant lacks H3K27me3, as shown by western blot and ChIP-seq, displays growth defects, is sterile, and constitutively expresses genes for mycotoxins, pigments and other secondary metabolites. Transcriptome analyses showed that 75% of 4,449 silent genes are enriched for H3K27me3. A subset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in kmt6. A largely overlapping set of genes showed increased expression in kmt6. Almost 95% of the remaining 2,720 annotated silent genes showed no enrichment for either H3K27me3 or H3K4me2/3 in kmt6. In these cases mere absence of H3K27me3 was insufficient for expression, which suggests that additional changes are required to activate genes. Taken together, we show that absence of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity factors. Results from this study provide the framework for novel targeted strategies to control the "cryptic genome", specifically secondary metabolite expression.

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The kmt6 mutant lacks H3K27me3.A. Western analyses with acid-extracted histones from WT or kmt6, kmt1 and hpo mutants show absence of H3K27me3 in kmt6 but unchanged levels in kmt1, a mutant deficient in the H3K9 methyltransferase homologue of Su(var)3-9, Clr4 and DIM-5, and in hpo, a mutant in which the homologue of Heterochromatin Protein 1 (HP1) was deleted (L.R. Connolly and M. Freitag, in preparation). Two different antibodies (AM39535 and ab6147) were used. B. H3K27 trimethylation occurs in large blocks. H3K27me3 (orange) is mutually exclusive of H3K4me2 (green) and -me3 (blue), which are found in the same regions of chromosome 1 (see Fig. S3 for images of Chr. 2 to 4). Specific enrichment of H3K27me3 is lost in the kmt6 mutant but regained in a complemented strain (kmt6+).
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pgen-1003916-g002: The kmt6 mutant lacks H3K27me3.A. Western analyses with acid-extracted histones from WT or kmt6, kmt1 and hpo mutants show absence of H3K27me3 in kmt6 but unchanged levels in kmt1, a mutant deficient in the H3K9 methyltransferase homologue of Su(var)3-9, Clr4 and DIM-5, and in hpo, a mutant in which the homologue of Heterochromatin Protein 1 (HP1) was deleted (L.R. Connolly and M. Freitag, in preparation). Two different antibodies (AM39535 and ab6147) were used. B. H3K27 trimethylation occurs in large blocks. H3K27me3 (orange) is mutually exclusive of H3K4me2 (green) and -me3 (blue), which are found in the same regions of chromosome 1 (see Fig. S3 for images of Chr. 2 to 4). Specific enrichment of H3K27me3 is lost in the kmt6 mutant but regained in a complemented strain (kmt6+).

Mentions: To test if H3K27 methylation was altered in the kmt6 mutant, we purified histones and carried out western analyses with antibodies against methylated histones. H3K27me3 was present in WT but completely absent from the kmt6 mutant (Fig. 2A). We tested several different antibodies raised against H3K27me3 peptides (Fig. 2A, Fig. S2B). All showed absence of H3K27me3 in kmt6. Levels of H3K4me2 and another activating mark, H3K36me3, are equivalent in WT and kmt6 (Fig. S2B), suggesting that lack of H3K27me3 does not result in an overall increase in H3K4 or H3K36 methylation. H3K9me3, while present, proved difficult to detect in F. graminearum by western blot (Fig. S2B), matching our expectations from ChIP-seq (data not shown). Levels of H3K27me3 were not altered in strains in which H3K9me3 was abolished by deletion of the single Fusarium Su(var3-9) homologue (kmt1) or in which Heterochromatin Protein-1 (HP1) was deleted (hpo) (Fig. 2A). We conclude that KMT6 has specificity for H3K27, and that KMT6 is the sole or predominant H3K27 methyltransferase in F. graminearum.


The Fusarium graminearum histone H3 K27 methyltransferase KMT6 regulates development and expression of secondary metabolite gene clusters.

Connolly LR, Smith KM, Freitag M - PLoS Genet. (2013)

The kmt6 mutant lacks H3K27me3.A. Western analyses with acid-extracted histones from WT or kmt6, kmt1 and hpo mutants show absence of H3K27me3 in kmt6 but unchanged levels in kmt1, a mutant deficient in the H3K9 methyltransferase homologue of Su(var)3-9, Clr4 and DIM-5, and in hpo, a mutant in which the homologue of Heterochromatin Protein 1 (HP1) was deleted (L.R. Connolly and M. Freitag, in preparation). Two different antibodies (AM39535 and ab6147) were used. B. H3K27 trimethylation occurs in large blocks. H3K27me3 (orange) is mutually exclusive of H3K4me2 (green) and -me3 (blue), which are found in the same regions of chromosome 1 (see Fig. S3 for images of Chr. 2 to 4). Specific enrichment of H3K27me3 is lost in the kmt6 mutant but regained in a complemented strain (kmt6+).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814326&req=5

pgen-1003916-g002: The kmt6 mutant lacks H3K27me3.A. Western analyses with acid-extracted histones from WT or kmt6, kmt1 and hpo mutants show absence of H3K27me3 in kmt6 but unchanged levels in kmt1, a mutant deficient in the H3K9 methyltransferase homologue of Su(var)3-9, Clr4 and DIM-5, and in hpo, a mutant in which the homologue of Heterochromatin Protein 1 (HP1) was deleted (L.R. Connolly and M. Freitag, in preparation). Two different antibodies (AM39535 and ab6147) were used. B. H3K27 trimethylation occurs in large blocks. H3K27me3 (orange) is mutually exclusive of H3K4me2 (green) and -me3 (blue), which are found in the same regions of chromosome 1 (see Fig. S3 for images of Chr. 2 to 4). Specific enrichment of H3K27me3 is lost in the kmt6 mutant but regained in a complemented strain (kmt6+).
Mentions: To test if H3K27 methylation was altered in the kmt6 mutant, we purified histones and carried out western analyses with antibodies against methylated histones. H3K27me3 was present in WT but completely absent from the kmt6 mutant (Fig. 2A). We tested several different antibodies raised against H3K27me3 peptides (Fig. 2A, Fig. S2B). All showed absence of H3K27me3 in kmt6. Levels of H3K4me2 and another activating mark, H3K36me3, are equivalent in WT and kmt6 (Fig. S2B), suggesting that lack of H3K27me3 does not result in an overall increase in H3K4 or H3K36 methylation. H3K9me3, while present, proved difficult to detect in F. graminearum by western blot (Fig. S2B), matching our expectations from ChIP-seq (data not shown). Levels of H3K27me3 were not altered in strains in which H3K9me3 was abolished by deletion of the single Fusarium Su(var3-9) homologue (kmt1) or in which Heterochromatin Protein-1 (HP1) was deleted (hpo) (Fig. 2A). We conclude that KMT6 has specificity for H3K27, and that KMT6 is the sole or predominant H3K27 methyltransferase in F. graminearum.

Bottom Line: By chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing.Di- or trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found in mutually exclusive regions of the genome.Taken together, we show that absence of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Center for Genome Research and Biocomputing, Oregon State University, Corvallis, Oregon, United States of America.

ABSTRACT
The cereal pathogen Fusarium graminearum produces secondary metabolites toxic to humans and animals, yet coordinated transcriptional regulation of gene clusters remains largely a mystery. By chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq) we found that regions with secondary metabolite clusters are enriched for trimethylated histone H3 lysine 27 (H3K27me3), a histone modification associated with gene silencing. H3K27me3 was found predominantly in regions that lack synteny with other Fusarium species, generally subtelomeric regions. Di- or trimethylated H3K4 (H3K4me2/3), two modifications associated with gene activity, and H3K27me3 are predominantly found in mutually exclusive regions of the genome. To find functions for H3K27me3, we deleted the gene for the putative H3K27 methyltransferase, KMT6, a homolog of Drosophila Enhancer of zeste, E(z). The kmt6 mutant lacks H3K27me3, as shown by western blot and ChIP-seq, displays growth defects, is sterile, and constitutively expresses genes for mycotoxins, pigments and other secondary metabolites. Transcriptome analyses showed that 75% of 4,449 silent genes are enriched for H3K27me3. A subset of genes that were enriched for H3K27me3 in WT gained H3K4me2/3 in kmt6. A largely overlapping set of genes showed increased expression in kmt6. Almost 95% of the remaining 2,720 annotated silent genes showed no enrichment for either H3K27me3 or H3K4me2/3 in kmt6. In these cases mere absence of H3K27me3 was insufficient for expression, which suggests that additional changes are required to activate genes. Taken together, we show that absence of H3K27me3 allowed expression of an additional 14% of the genome, resulting in derepression of genes predominantly involved in secondary metabolite pathways and other species-specific functions, including putative secreted pathogenicity factors. Results from this study provide the framework for novel targeted strategies to control the "cryptic genome", specifically secondary metabolite expression.

Show MeSH
Related in: MedlinePlus