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Regulation of metabolic gene clusters in Arabidopsis thaliana.

Nützmann HW, Osbourn A - New Phytol. (2014)

Bottom Line: We show that the transcript levels of genes within two metabolic clusters are coordinately reduced in an arp6 and h2a.z background.We further show that nucleosome stability within the cluster regions is higher in the arp6 background compared with the wild-type.These results implicate ARP6 and H2A.Z in the regulation of metabolic clusters in Arabidopsis thaliana through localized chromatin modifications that enable the coordinate expression of groups of contiguous genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

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Analysis of nucleosome occupancy within the thalianol cluster. (a) Comparison of nucleosome positioning at the transcriptional start site of the THAS gene in roots of Arabidopsis thaliana Col-0 wild-type (solid line) and arp6 mutant (dashed line) lines (left); Col-0 roots (solid line) and leaves (dashed line) (right).The −1 and +1 indicate the first positioned nucleosome upstream and downstream of the putative transcriptional start site. (b) Relative abundance of stably positioned nucleosomes within the thalianol cluster and at the At5g47970 flanking gene in roots and leaves of seedlings of Col-0 wild-type and arp6 mutant lines. The position of each analysed nucleosome is indicated in the map of the cluster region. The −1 and +1 indicate the first positioned nucleosome upstream and downstream of the putative transcriptional start site of the respective gene. Nucleosome abundance was measured by qRT-PCR relative to the stably positioned −1 nucleosome at the At4g07700 reference locus. Results for representative experiments are shown (error bars indicate standard deviation of two biological replicates). Significant differences between wild-type and mutants lines (five experiments, 10 biological replicates) (t-test): *P < 0.05; **P < 0.01; ***P < 0.001.
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fig04: Analysis of nucleosome occupancy within the thalianol cluster. (a) Comparison of nucleosome positioning at the transcriptional start site of the THAS gene in roots of Arabidopsis thaliana Col-0 wild-type (solid line) and arp6 mutant (dashed line) lines (left); Col-0 roots (solid line) and leaves (dashed line) (right).The −1 and +1 indicate the first positioned nucleosome upstream and downstream of the putative transcriptional start site. (b) Relative abundance of stably positioned nucleosomes within the thalianol cluster and at the At5g47970 flanking gene in roots and leaves of seedlings of Col-0 wild-type and arp6 mutant lines. The position of each analysed nucleosome is indicated in the map of the cluster region. The −1 and +1 indicate the first positioned nucleosome upstream and downstream of the putative transcriptional start site of the respective gene. Nucleosome abundance was measured by qRT-PCR relative to the stably positioned −1 nucleosome at the At4g07700 reference locus. Results for representative experiments are shown (error bars indicate standard deviation of two biological replicates). Significant differences between wild-type and mutants lines (five experiments, 10 biological replicates) (t-test): *P < 0.05; **P < 0.01; ***P < 0.001.

Mentions: We next analysed the local genome accessibility at the thalianol cluster using micrococcal nuclease accessibility assays. Addition of micrococcal nuclease to nuclear chromatin leads to digestion of nucleosome-free DNA, while nucleosome-bound DNA is protected. Subsequent quantification of undigested DNA enables the identification of nucleosome positions and their stability at these sites. Initially, we compared the nucleosome abundance at the transcriptional start-site of the THAS gene within the thalianol gene cluster. As expected we found two well-positioned nucleosomes, the −1 nucleosome, just upstream of the transcriptional start site and the +1 nucleosome, just downstream (Fig.4a). In preliminary experiments we found that the −1 and +1 nucleosomes were less abundant in chromatin preparations from wild-type roots compared with the transcriptionally less active arp6 roots, or to wild-type leaves (where the cluster is inactive) (Fig.4a). We then broadened our analysis to the whole cluster (Fig.4b). We detected well-positioned nucleosomes around the putative transcriptional start sites of thalianol cluster genes and elsewhere within the gene cluster, and measured the abundance of these nucleosomes in chromatin isolated from the roots and leaves of wild-type and arp6 mutant Arabidopsis thaliana lines. In seven out of the eight positioned nucleosomes analysed we found a significant increase in nucleosome occupancy in the arp6 mutant roots compared with the wild-type (Fig.4b). Surprisingly, we found higher nucleosome occupancy not only at the transcriptional start sites of cluster genes but also at intergenic regions within the cluster, in line with the cluster-wide enrichment of H2A.Z.


Regulation of metabolic gene clusters in Arabidopsis thaliana.

Nützmann HW, Osbourn A - New Phytol. (2014)

Analysis of nucleosome occupancy within the thalianol cluster. (a) Comparison of nucleosome positioning at the transcriptional start site of the THAS gene in roots of Arabidopsis thaliana Col-0 wild-type (solid line) and arp6 mutant (dashed line) lines (left); Col-0 roots (solid line) and leaves (dashed line) (right).The −1 and +1 indicate the first positioned nucleosome upstream and downstream of the putative transcriptional start site. (b) Relative abundance of stably positioned nucleosomes within the thalianol cluster and at the At5g47970 flanking gene in roots and leaves of seedlings of Col-0 wild-type and arp6 mutant lines. The position of each analysed nucleosome is indicated in the map of the cluster region. The −1 and +1 indicate the first positioned nucleosome upstream and downstream of the putative transcriptional start site of the respective gene. Nucleosome abundance was measured by qRT-PCR relative to the stably positioned −1 nucleosome at the At4g07700 reference locus. Results for representative experiments are shown (error bars indicate standard deviation of two biological replicates). Significant differences between wild-type and mutants lines (five experiments, 10 biological replicates) (t-test): *P < 0.05; **P < 0.01; ***P < 0.001.
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Related In: Results  -  Collection

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fig04: Analysis of nucleosome occupancy within the thalianol cluster. (a) Comparison of nucleosome positioning at the transcriptional start site of the THAS gene in roots of Arabidopsis thaliana Col-0 wild-type (solid line) and arp6 mutant (dashed line) lines (left); Col-0 roots (solid line) and leaves (dashed line) (right).The −1 and +1 indicate the first positioned nucleosome upstream and downstream of the putative transcriptional start site. (b) Relative abundance of stably positioned nucleosomes within the thalianol cluster and at the At5g47970 flanking gene in roots and leaves of seedlings of Col-0 wild-type and arp6 mutant lines. The position of each analysed nucleosome is indicated in the map of the cluster region. The −1 and +1 indicate the first positioned nucleosome upstream and downstream of the putative transcriptional start site of the respective gene. Nucleosome abundance was measured by qRT-PCR relative to the stably positioned −1 nucleosome at the At4g07700 reference locus. Results for representative experiments are shown (error bars indicate standard deviation of two biological replicates). Significant differences between wild-type and mutants lines (five experiments, 10 biological replicates) (t-test): *P < 0.05; **P < 0.01; ***P < 0.001.
Mentions: We next analysed the local genome accessibility at the thalianol cluster using micrococcal nuclease accessibility assays. Addition of micrococcal nuclease to nuclear chromatin leads to digestion of nucleosome-free DNA, while nucleosome-bound DNA is protected. Subsequent quantification of undigested DNA enables the identification of nucleosome positions and their stability at these sites. Initially, we compared the nucleosome abundance at the transcriptional start-site of the THAS gene within the thalianol gene cluster. As expected we found two well-positioned nucleosomes, the −1 nucleosome, just upstream of the transcriptional start site and the +1 nucleosome, just downstream (Fig.4a). In preliminary experiments we found that the −1 and +1 nucleosomes were less abundant in chromatin preparations from wild-type roots compared with the transcriptionally less active arp6 roots, or to wild-type leaves (where the cluster is inactive) (Fig.4a). We then broadened our analysis to the whole cluster (Fig.4b). We detected well-positioned nucleosomes around the putative transcriptional start sites of thalianol cluster genes and elsewhere within the gene cluster, and measured the abundance of these nucleosomes in chromatin isolated from the roots and leaves of wild-type and arp6 mutant Arabidopsis thaliana lines. In seven out of the eight positioned nucleosomes analysed we found a significant increase in nucleosome occupancy in the arp6 mutant roots compared with the wild-type (Fig.4b). Surprisingly, we found higher nucleosome occupancy not only at the transcriptional start sites of cluster genes but also at intergenic regions within the cluster, in line with the cluster-wide enrichment of H2A.Z.

Bottom Line: We show that the transcript levels of genes within two metabolic clusters are coordinately reduced in an arp6 and h2a.z background.We further show that nucleosome stability within the cluster regions is higher in the arp6 background compared with the wild-type.These results implicate ARP6 and H2A.Z in the regulation of metabolic clusters in Arabidopsis thaliana through localized chromatin modifications that enable the coordinate expression of groups of contiguous genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Metabolic Biology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.

Show MeSH
Related in: MedlinePlus