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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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Heatmaps for individual genes in primary (A) and secondary (B) metabolism (left panels) and clusters of genes around eight centers (right panels).Few primary metabolic genes are affected by kmt6 mutation compared to the majority of secondary metabolic genes that have increased expression in kmt6. Values for the log2 of the ratio of RPKMs for kmt6/WT or high/low nitrogen conditions for each strain are color-coded and the scale shown on the right. For example, in the left image of (A) red represents log2 (kmt6/WT) of 10, which means the kmt6 RPKM is 1024-fold higher than the WT RPKM. Primary metabolic genes in clusters 2 and 5, upregulated in kmt6, and cluster 7, downregulated in high nitrogen, are listed in Table S2 with their putative functions. Secondary metabolic genes in clusters 1, 4, and 7, upregulated in kmt6, and cluster 8, downregulated in high nitrogen, are listed in Table S2 with their putative functions.
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pgen-1003916-g006: Heatmaps for individual genes in primary (A) and secondary (B) metabolism (left panels) and clusters of genes around eight centers (right panels).Few primary metabolic genes are affected by kmt6 mutation compared to the majority of secondary metabolic genes that have increased expression in kmt6. Values for the log2 of the ratio of RPKMs for kmt6/WT or high/low nitrogen conditions for each strain are color-coded and the scale shown on the right. For example, in the left image of (A) red represents log2 (kmt6/WT) of 10, which means the kmt6 RPKM is 1024-fold higher than the WT RPKM. Primary metabolic genes in clusters 2 and 5, upregulated in kmt6, and cluster 7, downregulated in high nitrogen, are listed in Table S2 with their putative functions. Secondary metabolic genes in clusters 1, 4, and 7, upregulated in kmt6, and cluster 8, downregulated in high nitrogen, are listed in Table S2 with their putative functions.

Mentions: We immediately realized that regions of KMT6-dependent repression are home to secondary metabolite (SM) gene clusters, and thus generated heatmaps of expression changes for all primary and secondary metabolite genes to visualize the effect of high compared to low nitrogen and kmt6 mutation on expression of these genes. Growth in low and high nitrogen was compared because nitrogen is a known regulator of many SM gene clusters [37]. Primary metabolite (PM) genes were largely unaffected by either mutation of kmt6 or growth in high nitrogen (Fig. 6A). Specific sets of genes, summarized in the clustered heatmap with 8 k-means (Fig. 6A, right panel) stand out as being repressed in high nitrogen (245 genes in cluster 7), or induced by kmt6 mutation (41 genes in cluster 5 and 49 genes in cluster 2). The genes repressed in high nitrogen include six out of 17 genes in the gluconeogenesis I pathway, and several genes for carbohydrate metabolism including glycolytic enzymes (Table S2). The 90 genes induced in kmt6 are enriched for genes involved in carbohydrate binding and degradation, peptidases, and cell signaling components. Five of the 90 genes, although classified as primary metabolic genes, are part of SM clusters (carB, fus1, tri5, a kinase belonging to the zon pathway, and FGSG_10615).


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)

Heatmaps for individual genes in primary (A) and secondary (B) metabolism (left panels) and clusters of genes around eight centers (right panels).Few primary metabolic genes are affected by kmt6 mutation compared to the majority of secondary metabolic genes that have increased expression in kmt6. Values for the log2 of the ratio of RPKMs for kmt6/WT or high/low nitrogen conditions for each strain are color-coded and the scale shown on the right. For example, in the left image of (A) red represents log2 (kmt6/WT) of 10, which means the kmt6 RPKM is 1024-fold higher than the WT RPKM. Primary metabolic genes in clusters 2 and 5, upregulated in kmt6, and cluster 7, downregulated in high nitrogen, are listed in Table S2 with their putative functions. Secondary metabolic genes in clusters 1, 4, and 7, upregulated in kmt6, and cluster 8, downregulated in high nitrogen, are listed in Table S2 with their putative functions.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003916-g006: Heatmaps for individual genes in primary (A) and secondary (B) metabolism (left panels) and clusters of genes around eight centers (right panels).Few primary metabolic genes are affected by kmt6 mutation compared to the majority of secondary metabolic genes that have increased expression in kmt6. Values for the log2 of the ratio of RPKMs for kmt6/WT or high/low nitrogen conditions for each strain are color-coded and the scale shown on the right. For example, in the left image of (A) red represents log2 (kmt6/WT) of 10, which means the kmt6 RPKM is 1024-fold higher than the WT RPKM. Primary metabolic genes in clusters 2 and 5, upregulated in kmt6, and cluster 7, downregulated in high nitrogen, are listed in Table S2 with their putative functions. Secondary metabolic genes in clusters 1, 4, and 7, upregulated in kmt6, and cluster 8, downregulated in high nitrogen, are listed in Table S2 with their putative functions.
Mentions: We immediately realized that regions of KMT6-dependent repression are home to secondary metabolite (SM) gene clusters, and thus generated heatmaps of expression changes for all primary and secondary metabolite genes to visualize the effect of high compared to low nitrogen and kmt6 mutation on expression of these genes. Growth in low and high nitrogen was compared because nitrogen is a known regulator of many SM gene clusters [37]. Primary metabolite (PM) genes were largely unaffected by either mutation of kmt6 or growth in high nitrogen (Fig. 6A). Specific sets of genes, summarized in the clustered heatmap with 8 k-means (Fig. 6A, right panel) stand out as being repressed in high nitrogen (245 genes in cluster 7), or induced by kmt6 mutation (41 genes in cluster 5 and 49 genes in cluster 2). The genes repressed in high nitrogen include six out of 17 genes in the gluconeogenesis I pathway, and several genes for carbohydrate metabolism including glycolytic enzymes (Table S2). The 90 genes induced in kmt6 are enriched for genes involved in carbohydrate binding and degradation, peptidases, and cell signaling components. Five of the 90 genes, although classified as primary metabolic genes, are part of SM clusters (carB, fus1, tri5, a kinase belonging to the zon pathway, and FGSG_10615).

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