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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 acetylation in sugarcane histone H4.(A) MS/MS spectrum of the doubly charged ion at m/z 768.9466 that corresponds to the histone H4 peptide (residues 4–17) prGKprGGKacGLGKprGGAKprR containing acetyl K8. (B) MS/MS spectrum of the [M+2H]2+ ion at m/z 761.9385 matching the H4 peptide prGKacGGKprGLGKacGGAKprR where K5 and K12 are acetylated. (C) MS/MS spectrum recorded on the [M+2H]2+ ion (m/z 754.9307) that corresponds to the peptide prGKacGGKacGLGKacGGAKprR where K5, K8 and K12 are acetylated. (D) The full acetylated peptide prGKacGGKacGLGKacGGAKacR at K5, K8, K12 and K16 was deduced from the MS/MS spectrum of the ion at m/z 747.9230. 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.g008: Lysine acetylation in sugarcane histone H4.(A) MS/MS spectrum of the doubly charged ion at m/z 768.9466 that corresponds to the histone H4 peptide (residues 4–17) prGKprGGKacGLGKprGGAKprR containing acetyl K8. (B) MS/MS spectrum of the [M+2H]2+ ion at m/z 761.9385 matching the H4 peptide prGKacGGKprGLGKacGGAKprR where K5 and K12 are acetylated. (C) MS/MS spectrum recorded on the [M+2H]2+ ion (m/z 754.9307) that corresponds to the peptide prGKacGGKacGLGKacGGAKprR where K5, K8 and K12 are acetylated. (D) The full acetylated peptide prGKacGGKacGLGKacGGAKacR at K5, K8, K12 and K16 was deduced from the MS/MS spectrum of the ion at m/z 747.9230. 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: A list of all modified peptides sequenced in the nanoLC-MS/MS analysis corresponding to histone Ss_H4.1 is shown in S4 Table. The modifications detected on these peptides include acetylation and methylation (Fig 7). From the modifications detected on histone Ss_H4.1, acetylation was found in all lysines present in the histone tail-domain (residues 5–20) and in a single threonine at position 30. Lysine acetylation is not only present in a single site but peptides in which two, three and even four lysines are acetylated could also be identified. In the peptide GKGGKGLGKGGAKR (residues 4–17) containing four out of the five lysines acetylated, we could detect combinations of two (i.e. K5-K12, K8-K16) and three (i.e. K5-K8-K12, K8-K12-K16) lysines and the fully acetylated peptides (S4 Table). Examples of the peptides sequenced containing one to four acetylated lysines are given in Fig 8A single residue H4K8 in the peptide prGKprGGKacGLGKprGGAKprR (residues 4–17) was determined to be acetylated from the MS/MS spectrum recorded from the doubly-charged ion at m/z 768.9466 (Fig 8A). Meanwhile, the MS/MS spectrum of the doubly-charged ion (m/z 761.9385) was shown to correspond to the peptide prGKacGGKprGLGKacGGAKprR where both K5 and K12 are acetylated (Fig 8B). The same peptide corresponding to residues 4–17 but containing three (K5ac, K8ac, K12ac) and four (K5ac, K8ac, K12ac, K16ac) acetyl groups were deduced from the MS/MS spectra recorded for the ions at m/z 754.9307 and m/z 747.9230, respectively (Fig 8C and 8D).


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 acetylation in sugarcane histone H4.(A) MS/MS spectrum of the doubly charged ion at m/z 768.9466 that corresponds to the histone H4 peptide (residues 4–17) prGKprGGKacGLGKprGGAKprR containing acetyl K8. (B) MS/MS spectrum of the [M+2H]2+ ion at m/z 761.9385 matching the H4 peptide prGKacGGKprGLGKacGGAKprR where K5 and K12 are acetylated. (C) MS/MS spectrum recorded on the [M+2H]2+ ion (m/z 754.9307) that corresponds to the peptide prGKacGGKacGLGKacGGAKprR where K5, K8 and K12 are acetylated. (D) The full acetylated peptide prGKacGGKacGLGKacGGAKacR at K5, K8, K12 and K16 was deduced from the MS/MS spectrum of the ion at m/z 747.9230. 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

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getmorefigures.php?uid=PMC4520453&req=5

pone.0134586.g008: Lysine acetylation in sugarcane histone H4.(A) MS/MS spectrum of the doubly charged ion at m/z 768.9466 that corresponds to the histone H4 peptide (residues 4–17) prGKprGGKacGLGKprGGAKprR containing acetyl K8. (B) MS/MS spectrum of the [M+2H]2+ ion at m/z 761.9385 matching the H4 peptide prGKacGGKprGLGKacGGAKprR where K5 and K12 are acetylated. (C) MS/MS spectrum recorded on the [M+2H]2+ ion (m/z 754.9307) that corresponds to the peptide prGKacGGKacGLGKacGGAKprR where K5, K8 and K12 are acetylated. (D) The full acetylated peptide prGKacGGKacGLGKacGGAKacR at K5, K8, K12 and K16 was deduced from the MS/MS spectrum of the ion at m/z 747.9230. 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: A list of all modified peptides sequenced in the nanoLC-MS/MS analysis corresponding to histone Ss_H4.1 is shown in S4 Table. The modifications detected on these peptides include acetylation and methylation (Fig 7). From the modifications detected on histone Ss_H4.1, acetylation was found in all lysines present in the histone tail-domain (residues 5–20) and in a single threonine at position 30. Lysine acetylation is not only present in a single site but peptides in which two, three and even four lysines are acetylated could also be identified. In the peptide GKGGKGLGKGGAKR (residues 4–17) containing four out of the five lysines acetylated, we could detect combinations of two (i.e. K5-K12, K8-K16) and three (i.e. K5-K8-K12, K8-K12-K16) lysines and the fully acetylated peptides (S4 Table). Examples of the peptides sequenced containing one to four acetylated lysines are given in Fig 8A single residue H4K8 in the peptide prGKprGGKacGLGKprGGAKprR (residues 4–17) was determined to be acetylated from the MS/MS spectrum recorded from the doubly-charged ion at m/z 768.9466 (Fig 8A). Meanwhile, the MS/MS spectrum of the doubly-charged ion (m/z 761.9385) was shown to correspond to the peptide prGKacGGKprGLGKacGGAKprR where both K5 and K12 are acetylated (Fig 8B). The same peptide corresponding to residues 4–17 but containing three (K5ac, K8ac, K12ac) and four (K5ac, K8ac, K12ac, K16ac) acetyl groups were deduced from the MS/MS spectra recorded for the ions at m/z 754.9307 and m/z 747.9230, respectively (Fig 8C and 8D).

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.