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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.


Lysine methylation and threonine acetylation in sugarcane histone H4.(A) MS/MS spectrum of the doubly charged ion at m/z 768.9646 indicating H4K5me3 in the H4 peptide prGKme3GGKprGLGKprGGAKprR (residues 4–17). (B) MS/MS spectrum [M+2H]2+ ion at m/z 747.4198 corresponding to the peptide prDNIQGITacKme1PAIR (residues 24–35) containing H4T30ac and H4K31me1. Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.
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pone.0134586.g009: Lysine methylation and threonine acetylation in sugarcane histone H4.(A) MS/MS spectrum of the doubly charged ion at m/z 768.9646 indicating H4K5me3 in the H4 peptide prGKme3GGKprGLGKprGGAKprR (residues 4–17). (B) MS/MS spectrum [M+2H]2+ ion at m/z 747.4198 corresponding to the peptide prDNIQGITacKme1PAIR (residues 24–35) containing H4T30ac and H4K31me1. Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.

Mentions: In the same way as acetylation, methylation was detected in all lysines corresponding to the tail domain in the histone H4 (Fig 7). For instance, H4K5me3 in the peptide prGKme3GGKprGLGKprGGAKprR (residues 4–17) was deduced from the MS/MS spectrum of the doubly-charged ion at m/z 768.9646 (Fig 9A). In addition to modifications in the tail domain, a lysine present in the globular domain, H4K31, was also found methylated. Methylation of H4K31 was identified from the MS/MS spectrum recorded from the [M+2H]2+ ion at m/z 747.4198 that matched the H4 peptide prDNIQGITacKme1PAIR (residues 24–35) were H4K31 is monomethylated (Fig 9B). H4T30 was also found acetylated in this same peptide. Thus, in contrast to lysine acetylation, methylation occurs not only in the histone tail, but also in the globular domain.


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)

Lysine methylation and threonine acetylation in sugarcane histone H4.(A) MS/MS spectrum of the doubly charged ion at m/z 768.9646 indicating H4K5me3 in the H4 peptide prGKme3GGKprGLGKprGGAKprR (residues 4–17). (B) MS/MS spectrum [M+2H]2+ ion at m/z 747.4198 corresponding to the peptide prDNIQGITacKme1PAIR (residues 24–35) containing H4T30ac and H4K31me1. Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134586.g009: Lysine methylation and threonine acetylation in sugarcane histone H4.(A) MS/MS spectrum of the doubly charged ion at m/z 768.9646 indicating H4K5me3 in the H4 peptide prGKme3GGKprGLGKprGGAKprR (residues 4–17). (B) MS/MS spectrum [M+2H]2+ ion at m/z 747.4198 corresponding to the peptide prDNIQGITacKme1PAIR (residues 24–35) containing H4T30ac and H4K31me1. Sequence of the modified peptide and the measured mass of the precursor ion are shown in the figure inset. N-terminal and lysine propionylation, products of the chemical derivatization, are indicated by pr.
Mentions: In the same way as acetylation, methylation was detected in all lysines corresponding to the tail domain in the histone H4 (Fig 7). For instance, H4K5me3 in the peptide prGKme3GGKprGLGKprGGAKprR (residues 4–17) was deduced from the MS/MS spectrum of the doubly-charged ion at m/z 768.9646 (Fig 9A). In addition to modifications in the tail domain, a lysine present in the globular domain, H4K31, was also found methylated. Methylation of H4K31 was identified from the MS/MS spectrum recorded from the [M+2H]2+ ion at m/z 747.4198 that matched the H4 peptide prDNIQGITacKme1PAIR (residues 24–35) were H4K31 is monomethylated (Fig 9B). H4T30 was also found acetylated in this same peptide. Thus, in contrast to lysine acetylation, methylation occurs not only in the histone tail, but also in the globular domain.

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.