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Analysis of Histones H3 and H4 Reveals Novel and Conserved Post-Translational Modifications in Sugarcane.

Moraes I, Yuan ZF, Liu S, Souza GM, Garcia BA, Casas-Mollano JA - PLoS ONE (2015)

Bottom Line: As a result, modifications, alone or in combination, are important determinants of chromatin states.Several modifications conserved in other plants, and also novel modified residues, were identified.Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

ABSTRACT
Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is "written" in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate. Thus, identifying and characterizing these modifications and the proteins related to them is the initial step to understanding the mechanisms of gene regulation and in the future may even provide tools for breeding programs. Several studies over the past years have contributed to increase our knowledge of epigenetic gene regulation in model organisms like Arabidopsis, yet this field remains relatively unexplored in crops. In this study we identified and initially characterized histones H3 and H4 in the monocot crop sugarcane. We discovered a number of histone genes by searching the sugarcane ESTs database. The proteins encoded correspond to canonical histones, and their variants. We also purified bulk histones and used them to map post-translational modifications in the histones H3 and H4 using mass spectrometry. Several modifications conserved in other plants, and also novel modified residues, were identified. In particular, we report O-acetylation of serine, threonine and tyrosine, a recently identified modification conserved in several eukaryotes. Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species. To our knowledge, this is the first report of histones H3 and H4 as well as their post-translational modifications in sugarcane, and will provide a starting point for the study of chromatin regulation in this crop.

No MeSH data available.


Distribution patterns of histone post-translational modifications in sugarcane.(A) Immunoblot analysis of global histone H3 modifications in sugarcane tissues. (B) Sub-nuclear localization of H3K4me1, H3K4me3, H3K9me2, H3K27me3 and H3K9ac. (C) Chromatin distribution of sugarcane and Arabidopsis; white arrows show DAPI densely stained regions in sugarcane, representing heterochromatic blocks. In Arabidopsis, the chromocenters are well defined regions of heterochromatin (yellow arrows). (D) H3T3ph (red signals) does not co-localize with actively transcribed regions rich in RNA Polymerase II (green signals). Instead, it appears to be associated with silent chromatin; DAPI densely stained regions (grey nucleus, blue arrows) coincide with H3T3ph brighter foci (red nucleus, blue arrows), whereas weaker/absent H3T3ph regions (red nucleus, orange arrows) coincide with the less condensed chromatin poorly stained with DAPI (grey nucleus, orange arrows). Bars = 5 μm.
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pone.0134586.g010: Distribution patterns of histone post-translational modifications in sugarcane.(A) Immunoblot analysis of global histone H3 modifications in sugarcane tissues. (B) Sub-nuclear localization of H3K4me1, H3K4me3, H3K9me2, H3K27me3 and H3K9ac. (C) Chromatin distribution of sugarcane and Arabidopsis; white arrows show DAPI densely stained regions in sugarcane, representing heterochromatic blocks. In Arabidopsis, the chromocenters are well defined regions of heterochromatin (yellow arrows). (D) H3T3ph (red signals) does not co-localize with actively transcribed regions rich in RNA Polymerase II (green signals). Instead, it appears to be associated with silent chromatin; DAPI densely stained regions (grey nucleus, blue arrows) coincide with H3T3ph brighter foci (red nucleus, blue arrows), whereas weaker/absent H3T3ph regions (red nucleus, orange arrows) coincide with the less condensed chromatin poorly stained with DAPI (grey nucleus, orange arrows). Bars = 5 μm.

Mentions: To investigate the conservation of nuclear distribution of histone modifications between sugarcane and other plant species, we applied immunostaining using antibodies against H3K4me1, H3K4me3, H3K9me2, H3K27me3, H3K9ac and H3T3ph in nuclei isolated from root cells of sugarcane. Prior to immunostaining, we confirmed the presence of these modifications and the specificity of the antibodies on purified histones from sugarcane leaves and stems. Immunoblot analysis indicates that H3K4me1, H3K4me3, H3K9me2, H3K27me3 and H3K9ac, modifications previously identified by nanoLC-MS/MS, are indeed present in sugarcane histones from leaves and stems. Although we could not confidently detect histone phosphorylation in our analysis we decided to use a site specific antibody to analyze this modification. In plants, phosphorylation at serine residues is associated with the pericentromeric region, while threonine phosphorylation has been linked to chromosome condensation. Because these roles are conserved among all the plants studied so far, we decided to use an antibody against H3T3ph to investigate its localization in sugarcane nuclei (Fig 10).


Analysis of Histones H3 and H4 Reveals Novel and Conserved Post-Translational Modifications in Sugarcane.

Moraes I, Yuan ZF, Liu S, Souza GM, Garcia BA, Casas-Mollano JA - PLoS ONE (2015)

Distribution patterns of histone post-translational modifications in sugarcane.(A) Immunoblot analysis of global histone H3 modifications in sugarcane tissues. (B) Sub-nuclear localization of H3K4me1, H3K4me3, H3K9me2, H3K27me3 and H3K9ac. (C) Chromatin distribution of sugarcane and Arabidopsis; white arrows show DAPI densely stained regions in sugarcane, representing heterochromatic blocks. In Arabidopsis, the chromocenters are well defined regions of heterochromatin (yellow arrows). (D) H3T3ph (red signals) does not co-localize with actively transcribed regions rich in RNA Polymerase II (green signals). Instead, it appears to be associated with silent chromatin; DAPI densely stained regions (grey nucleus, blue arrows) coincide with H3T3ph brighter foci (red nucleus, blue arrows), whereas weaker/absent H3T3ph regions (red nucleus, orange arrows) coincide with the less condensed chromatin poorly stained with DAPI (grey nucleus, orange arrows). Bars = 5 μm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4520453&req=5

pone.0134586.g010: Distribution patterns of histone post-translational modifications in sugarcane.(A) Immunoblot analysis of global histone H3 modifications in sugarcane tissues. (B) Sub-nuclear localization of H3K4me1, H3K4me3, H3K9me2, H3K27me3 and H3K9ac. (C) Chromatin distribution of sugarcane and Arabidopsis; white arrows show DAPI densely stained regions in sugarcane, representing heterochromatic blocks. In Arabidopsis, the chromocenters are well defined regions of heterochromatin (yellow arrows). (D) H3T3ph (red signals) does not co-localize with actively transcribed regions rich in RNA Polymerase II (green signals). Instead, it appears to be associated with silent chromatin; DAPI densely stained regions (grey nucleus, blue arrows) coincide with H3T3ph brighter foci (red nucleus, blue arrows), whereas weaker/absent H3T3ph regions (red nucleus, orange arrows) coincide with the less condensed chromatin poorly stained with DAPI (grey nucleus, orange arrows). Bars = 5 μm.
Mentions: To investigate the conservation of nuclear distribution of histone modifications between sugarcane and other plant species, we applied immunostaining using antibodies against H3K4me1, H3K4me3, H3K9me2, H3K27me3, H3K9ac and H3T3ph in nuclei isolated from root cells of sugarcane. Prior to immunostaining, we confirmed the presence of these modifications and the specificity of the antibodies on purified histones from sugarcane leaves and stems. Immunoblot analysis indicates that H3K4me1, H3K4me3, H3K9me2, H3K27me3 and H3K9ac, modifications previously identified by nanoLC-MS/MS, are indeed present in sugarcane histones from leaves and stems. Although we could not confidently detect histone phosphorylation in our analysis we decided to use a site specific antibody to analyze this modification. In plants, phosphorylation at serine residues is associated with the pericentromeric region, while threonine phosphorylation has been linked to chromosome condensation. Because these roles are conserved among all the plants studied so far, we decided to use an antibody against H3T3ph to investigate its localization in sugarcane nuclei (Fig 10).

Bottom Line: As a result, modifications, alone or in combination, are important determinants of chromatin states.Several modifications conserved in other plants, and also novel modified residues, were identified.Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil.

ABSTRACT
Histones are the main structural components of the nucleosome, hence targets of many regulatory proteins that mediate processes involving changes in chromatin. The functional outcome of many pathways is "written" in the histones in the form of post-translational modifications that determine the final gene expression readout. As a result, modifications, alone or in combination, are important determinants of chromatin states. Histone modifications are accomplished by the addition of different chemical groups such as methyl, acetyl and phosphate. Thus, identifying and characterizing these modifications and the proteins related to them is the initial step to understanding the mechanisms of gene regulation and in the future may even provide tools for breeding programs. Several studies over the past years have contributed to increase our knowledge of epigenetic gene regulation in model organisms like Arabidopsis, yet this field remains relatively unexplored in crops. In this study we identified and initially characterized histones H3 and H4 in the monocot crop sugarcane. We discovered a number of histone genes by searching the sugarcane ESTs database. The proteins encoded correspond to canonical histones, and their variants. We also purified bulk histones and used them to map post-translational modifications in the histones H3 and H4 using mass spectrometry. Several modifications conserved in other plants, and also novel modified residues, were identified. In particular, we report O-acetylation of serine, threonine and tyrosine, a recently identified modification conserved in several eukaryotes. Additionally, the sub-nuclear localization of some well-studied modifications (i.e., H3K4me3, H3K9me2, H3K27me3, H3K9ac, H3T3ph) is described and compared to other plant species. To our knowledge, this is the first report of histones H3 and H4 as well as their post-translational modifications in sugarcane, and will provide a starting point for the study of chromatin regulation in this crop.

No MeSH data available.