Limits...
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.


Relative abundance of histone H3 (residues 9–26) acetylation and methylation in sugarcane.(A) Percent relative amounts of peptide isoforms containing residues 9–17 of histone H3. * Peptide isoforms containing a single acetylation at K9 or K14 could not be separated by nanoLC. ▼ Peptide isoforms containing a single acetylation on S10 or T11 could not be separated by nanoLC. (B) Relative amounts of peptide isoforms containing residues 18–26 of histone H3. Only the most abundant isoforms are shown.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520453&req=5

pone.0134586.g005: Relative abundance of histone H3 (residues 9–26) acetylation and methylation in sugarcane.(A) Percent relative amounts of peptide isoforms containing residues 9–17 of histone H3. * Peptide isoforms containing a single acetylation at K9 or K14 could not be separated by nanoLC. ▼ Peptide isoforms containing a single acetylation on S10 or T11 could not be separated by nanoLC. (B) Relative amounts of peptide isoforms containing residues 18–26 of histone H3. Only the most abundant isoforms are shown.

Mentions: Propionylation of histones blocks lysine residues allowing trypsin to cleave only C-terminal to arginine and generating defined peptides. We took advantage of this property and used the ion currents of each peptide isoforms to calculate the relative abundance of their associated modifications [17]. In the histone H3 tail we calculated the relative abundance of modifications present in the peptides corresponding to residues 9–17, 18–26 and 27–40. The predominant modifications on the H3 peptide at residues 9–17 was H3K9 methylation. The highest levels correspond to K9me1 (22%) and K9me2 (38%) whereas K9me3 was lower than 2% (Fig 5A). The levels of acetylation were lower than those observed in Arabidopsis, but still more than 10% of the lysines are acetylated at either K9 or K14 (Fig 5A). Since K9 and K14 acetylated peptides co-elute in the nanoLC, we used the MS/MS spectra to calculate the abundance of each modification. We found the levels of K9 acetylation to be 50 times lower than those of K14. Similarly, the levels of the doubly acetylated peptide (at K9 and K14) turned out to be very low compared to K14 acetylation and only 2% of the peptides contain the double modification, methyl K9 and acetyl K14 (Fig 5A). These observations indicate that in sugarcane K14 is the predominant acetylation mark and not K9. Peptides corresponding to residues 18–26 of histone H3 were found mostly unmodified (78%) or acetylated at K18 (4%), at K23 (13%) or at both residues (3%) (Fig 5B). Methylation of K18 and K23 on the contrary were shown to be present at very low levels (~0.1%) (Fig 5B). The relative abundance of serine and threonine (S/T) O-acetylation was very low (1% or less) in all the organisms examined which is in stark contrast to the much higher levels of lysine acetylation [22]. In sugarcane, we observed a similar disparity between the levels of S/T and lysine acetylation (Fig 5A and 5B). The relative level of H3T22ac is less than 0.5% whereas H3S10ac was at 2.5%. Although sugarcane H3S10ac, combined with H3T11ac, level is slightly higher than the 1% observed for other organisms, it is still very low compared to the relative levels of lysine acetylation.


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)

Relative abundance of histone H3 (residues 9–26) acetylation and methylation in sugarcane.(A) Percent relative amounts of peptide isoforms containing residues 9–17 of histone H3. * Peptide isoforms containing a single acetylation at K9 or K14 could not be separated by nanoLC. ▼ Peptide isoforms containing a single acetylation on S10 or T11 could not be separated by nanoLC. (B) Relative amounts of peptide isoforms containing residues 18–26 of histone H3. 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.g005: Relative abundance of histone H3 (residues 9–26) acetylation and methylation in sugarcane.(A) Percent relative amounts of peptide isoforms containing residues 9–17 of histone H3. * Peptide isoforms containing a single acetylation at K9 or K14 could not be separated by nanoLC. ▼ Peptide isoforms containing a single acetylation on S10 or T11 could not be separated by nanoLC. (B) Relative amounts of peptide isoforms containing residues 18–26 of histone H3. Only the most abundant isoforms are shown.
Mentions: Propionylation of histones blocks lysine residues allowing trypsin to cleave only C-terminal to arginine and generating defined peptides. We took advantage of this property and used the ion currents of each peptide isoforms to calculate the relative abundance of their associated modifications [17]. In the histone H3 tail we calculated the relative abundance of modifications present in the peptides corresponding to residues 9–17, 18–26 and 27–40. The predominant modifications on the H3 peptide at residues 9–17 was H3K9 methylation. The highest levels correspond to K9me1 (22%) and K9me2 (38%) whereas K9me3 was lower than 2% (Fig 5A). The levels of acetylation were lower than those observed in Arabidopsis, but still more than 10% of the lysines are acetylated at either K9 or K14 (Fig 5A). Since K9 and K14 acetylated peptides co-elute in the nanoLC, we used the MS/MS spectra to calculate the abundance of each modification. We found the levels of K9 acetylation to be 50 times lower than those of K14. Similarly, the levels of the doubly acetylated peptide (at K9 and K14) turned out to be very low compared to K14 acetylation and only 2% of the peptides contain the double modification, methyl K9 and acetyl K14 (Fig 5A). These observations indicate that in sugarcane K14 is the predominant acetylation mark and not K9. Peptides corresponding to residues 18–26 of histone H3 were found mostly unmodified (78%) or acetylated at K18 (4%), at K23 (13%) or at both residues (3%) (Fig 5B). Methylation of K18 and K23 on the contrary were shown to be present at very low levels (~0.1%) (Fig 5B). The relative abundance of serine and threonine (S/T) O-acetylation was very low (1% or less) in all the organisms examined which is in stark contrast to the much higher levels of lysine acetylation [22]. In sugarcane, we observed a similar disparity between the levels of S/T and lysine acetylation (Fig 5A and 5B). The relative level of H3T22ac is less than 0.5% whereas H3S10ac was at 2.5%. Although sugarcane H3S10ac, combined with H3T11ac, level is slightly higher than the 1% observed for other organisms, it is still very low compared to the relative levels of lysine acetylation.

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.