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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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Specific gene clusters are upregulated in kmt6.A. All genes in the fusarin C cluster are upregulated (A, FGSG_07797; B, FGSG_07798, fus1/pks10; C, FGSG_13222, fus2; D, FGSG_13223, fus3; E, FGSG_07800, fus4; F, FGSG_07801, fus5; G, FGSG_07802, fus6; H, FGSG_07803, fus7; I, FGSG_07804, fus8 cytochrome P450; J, FGSG_07805). B. The carotenoid cluster is derepressed in kmt6. A. Histone modification state and expression of carotenoid biosynthetic genes (A, FGSG_03063, ammonium permease; B, FGSG_03064, rhodopsin; C, FGSG_03065, al-1/carB phytoene dehydrogenase; D, FGSG_03066, al-2/carRA phytoene synthase; E, FGSG_03067, cao-1/carX carotenoid dioxygenase; F, FGSG_03068, carR transcriptional regulator; G, FGSG_03069, dihydrodipicolinate synthetase; H, FGSG_12520, RING finger protein; I, FGSG_03070; J, FGSG_03071, FAD-dependent oxidoreductase). Genes al-1, al-2 and FGSG_03069 are most strongly upregulated, and the transcription factor carR is 3-fold induced in low nitrogen (log2 = 1.63), and 1.5-fold induced in high nitrogen (log2 = 0.6). In A and B, the log2-fold change in expression has a solid line for the x-axis, and dashed lines at 2 (4-fold increase) and 6 (64-fold increase). Only statistically significant values are shown (p<0.001). C. Lack of KMT6 induces production of carotenoids and accumulation in the culture liquid at low but especially high nitrogen levels. WT and kmt6 were incubated for 7 days at 28C in liquid ICI with 6 mM or 60 mM NH4NO3.
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pgen-1003916-g008: Specific gene clusters are upregulated in kmt6.A. All genes in the fusarin C cluster are upregulated (A, FGSG_07797; B, FGSG_07798, fus1/pks10; C, FGSG_13222, fus2; D, FGSG_13223, fus3; E, FGSG_07800, fus4; F, FGSG_07801, fus5; G, FGSG_07802, fus6; H, FGSG_07803, fus7; I, FGSG_07804, fus8 cytochrome P450; J, FGSG_07805). B. The carotenoid cluster is derepressed in kmt6. A. Histone modification state and expression of carotenoid biosynthetic genes (A, FGSG_03063, ammonium permease; B, FGSG_03064, rhodopsin; C, FGSG_03065, al-1/carB phytoene dehydrogenase; D, FGSG_03066, al-2/carRA phytoene synthase; E, FGSG_03067, cao-1/carX carotenoid dioxygenase; F, FGSG_03068, carR transcriptional regulator; G, FGSG_03069, dihydrodipicolinate synthetase; H, FGSG_12520, RING finger protein; I, FGSG_03070; J, FGSG_03071, FAD-dependent oxidoreductase). Genes al-1, al-2 and FGSG_03069 are most strongly upregulated, and the transcription factor carR is 3-fold induced in low nitrogen (log2 = 1.63), and 1.5-fold induced in high nitrogen (log2 = 0.6). In A and B, the log2-fold change in expression has a solid line for the x-axis, and dashed lines at 2 (4-fold increase) and 6 (64-fold increase). Only statistically significant values are shown (p<0.001). C. Lack of KMT6 induces production of carotenoids and accumulation in the culture liquid at low but especially high nitrogen levels. WT and kmt6 were incubated for 7 days at 28C in liquid ICI with 6 mM or 60 mM NH4NO3.

Mentions: A. NRPS (blue, top), PKS (gray, center) and Cytochrome P450 (light blue, bottom) genes were mapped to the four chromosomes, in relation to H3K27me3 (low nitrogen conditions). Almost all genes are in regions covered by H3K27me3. . Changes in transcription for low nitrogen conditions are shown in purple as log2 value of the RPKM ratio of kmt6 vs. WT. B. Heatmaps showing expression changes and H3K27me3 enrichment of three classes of SM genes, cytochrome P450s, PKSs, and NRPSs. Legends to heatmaps as described in Fig. 8. H3K27me3 enrichment is shown as log2 value of the NLCS.


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)

Specific gene clusters are upregulated in kmt6.A. All genes in the fusarin C cluster are upregulated (A, FGSG_07797; B, FGSG_07798, fus1/pks10; C, FGSG_13222, fus2; D, FGSG_13223, fus3; E, FGSG_07800, fus4; F, FGSG_07801, fus5; G, FGSG_07802, fus6; H, FGSG_07803, fus7; I, FGSG_07804, fus8 cytochrome P450; J, FGSG_07805). B. The carotenoid cluster is derepressed in kmt6. A. Histone modification state and expression of carotenoid biosynthetic genes (A, FGSG_03063, ammonium permease; B, FGSG_03064, rhodopsin; C, FGSG_03065, al-1/carB phytoene dehydrogenase; D, FGSG_03066, al-2/carRA phytoene synthase; E, FGSG_03067, cao-1/carX carotenoid dioxygenase; F, FGSG_03068, carR transcriptional regulator; G, FGSG_03069, dihydrodipicolinate synthetase; H, FGSG_12520, RING finger protein; I, FGSG_03070; J, FGSG_03071, FAD-dependent oxidoreductase). Genes al-1, al-2 and FGSG_03069 are most strongly upregulated, and the transcription factor carR is 3-fold induced in low nitrogen (log2 = 1.63), and 1.5-fold induced in high nitrogen (log2 = 0.6). In A and B, the log2-fold change in expression has a solid line for the x-axis, and dashed lines at 2 (4-fold increase) and 6 (64-fold increase). Only statistically significant values are shown (p<0.001). C. Lack of KMT6 induces production of carotenoids and accumulation in the culture liquid at low but especially high nitrogen levels. WT and kmt6 were incubated for 7 days at 28C in liquid ICI with 6 mM or 60 mM NH4NO3.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003916-g008: Specific gene clusters are upregulated in kmt6.A. All genes in the fusarin C cluster are upregulated (A, FGSG_07797; B, FGSG_07798, fus1/pks10; C, FGSG_13222, fus2; D, FGSG_13223, fus3; E, FGSG_07800, fus4; F, FGSG_07801, fus5; G, FGSG_07802, fus6; H, FGSG_07803, fus7; I, FGSG_07804, fus8 cytochrome P450; J, FGSG_07805). B. The carotenoid cluster is derepressed in kmt6. A. Histone modification state and expression of carotenoid biosynthetic genes (A, FGSG_03063, ammonium permease; B, FGSG_03064, rhodopsin; C, FGSG_03065, al-1/carB phytoene dehydrogenase; D, FGSG_03066, al-2/carRA phytoene synthase; E, FGSG_03067, cao-1/carX carotenoid dioxygenase; F, FGSG_03068, carR transcriptional regulator; G, FGSG_03069, dihydrodipicolinate synthetase; H, FGSG_12520, RING finger protein; I, FGSG_03070; J, FGSG_03071, FAD-dependent oxidoreductase). Genes al-1, al-2 and FGSG_03069 are most strongly upregulated, and the transcription factor carR is 3-fold induced in low nitrogen (log2 = 1.63), and 1.5-fold induced in high nitrogen (log2 = 0.6). In A and B, the log2-fold change in expression has a solid line for the x-axis, and dashed lines at 2 (4-fold increase) and 6 (64-fold increase). Only statistically significant values are shown (p<0.001). C. Lack of KMT6 induces production of carotenoids and accumulation in the culture liquid at low but especially high nitrogen levels. WT and kmt6 were incubated for 7 days at 28C in liquid ICI with 6 mM or 60 mM NH4NO3.
Mentions: A. NRPS (blue, top), PKS (gray, center) and Cytochrome P450 (light blue, bottom) genes were mapped to the four chromosomes, in relation to H3K27me3 (low nitrogen conditions). Almost all genes are in regions covered by H3K27me3. . Changes in transcription for low nitrogen conditions are shown in purple as log2 value of the RPKM ratio of kmt6 vs. WT. B. Heatmaps showing expression changes and H3K27me3 enrichment of three classes of SM genes, cytochrome P450s, PKSs, and NRPSs. Legends to heatmaps as described in Fig. 8. H3K27me3 enrichment is shown as log2 value of the NLCS.

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