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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.


Comparative analysis of post-translational modifications in the canonical Ss_H3.1 and Ss_H3.3 variant.Relative amounts of the different modifications were calculated for the peptides corresponding to residues 27–40 of H3.1 (KSAPATGGVKKPHR) and H3.3 (KSAPTTGGVKKPHR). Only the most abundant isoforms are shown.
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pone.0134586.g006: Comparative analysis of post-translational modifications in the canonical Ss_H3.1 and Ss_H3.3 variant.Relative amounts of the different modifications were calculated for the peptides corresponding to residues 27–40 of H3.1 (KSAPATGGVKKPHR) and H3.3 (KSAPTTGGVKKPHR). Only the most abundant isoforms are shown.

Mentions: We identified an H3.3 variant in sugarcane that, like sequences of all the plant H3.3 identified so far, was different from the H3.1 in only four positions. Due to this sequence similarity, we could not differentiate between both variants for most of the length of the proteins, except for two peptides corresponding to residues 27–40 and 41–49 [17]. In this region we found acetylation and methylation at lysine K27 and K36. Relative quantification of the H3.1 peptides indicates that while K27 is highly methylated at this position (32% me1, 14% me2 and 4% me3), the levels of K27ac were extremely low (0.01%) (Fig 6). The position K36 was highly monomethylated (37%) whereas the di- and trimethyl forms were extremely low, > 0.01% and 3% respectively. Fragments methylated at both positions, i.e. 3% K27me1^K36me1, 0.8% K27me2^K36me1, 1% K27me1^K36me2 (Fig 6), were also detected. In the peptides corresponding to H3.3, the methylation levels of K27 were consistently lower than those for H3.1 (30% me1, 10% me2 and 2% me3) especially K27me3 (Fig 6). Acetylation of K27 was also found close to 1%. Compared to H3.1, the levels of H3K36me1 were lower in H3.3 (9%), yet fairly high levels of di- and trimethylation of H3K36 were found in this variant (respectively, 4% and 9%). The levels of the peptides modified at both K27 and K36 were also higher in H3.3, i.e. 10% K27me1^K36me1, 4.7% K27me2^K36me1, 3% K27me1^K36me2 (Fig 6).


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)

Comparative analysis of post-translational modifications in the canonical Ss_H3.1 and Ss_H3.3 variant.Relative amounts of the different modifications were calculated for the peptides corresponding to residues 27–40 of H3.1 (KSAPATGGVKKPHR) and H3.3 (KSAPTTGGVKKPHR). Only the most abundant isoforms are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134586.g006: Comparative analysis of post-translational modifications in the canonical Ss_H3.1 and Ss_H3.3 variant.Relative amounts of the different modifications were calculated for the peptides corresponding to residues 27–40 of H3.1 (KSAPATGGVKKPHR) and H3.3 (KSAPTTGGVKKPHR). Only the most abundant isoforms are shown.
Mentions: We identified an H3.3 variant in sugarcane that, like sequences of all the plant H3.3 identified so far, was different from the H3.1 in only four positions. Due to this sequence similarity, we could not differentiate between both variants for most of the length of the proteins, except for two peptides corresponding to residues 27–40 and 41–49 [17]. In this region we found acetylation and methylation at lysine K27 and K36. Relative quantification of the H3.1 peptides indicates that while K27 is highly methylated at this position (32% me1, 14% me2 and 4% me3), the levels of K27ac were extremely low (0.01%) (Fig 6). The position K36 was highly monomethylated (37%) whereas the di- and trimethyl forms were extremely low, > 0.01% and 3% respectively. Fragments methylated at both positions, i.e. 3% K27me1^K36me1, 0.8% K27me2^K36me1, 1% K27me1^K36me2 (Fig 6), were also detected. In the peptides corresponding to H3.3, the methylation levels of K27 were consistently lower than those for H3.1 (30% me1, 10% me2 and 2% me3) especially K27me3 (Fig 6). Acetylation of K27 was also found close to 1%. Compared to H3.1, the levels of H3K36me1 were lower in H3.3 (9%), yet fairly high levels of di- and trimethylation of H3K36 were found in this variant (respectively, 4% and 9%). The levels of the peptides modified at both K27 and K36 were also higher in H3.3, i.e. 10% K27me1^K36me1, 4.7% K27me2^K36me1, 3% K27me1^K36me2 (Fig 6).

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.