title: Identification of novel post-translational modifications in linker histones from chicken erythrocytes.
abstract: Chicken erythrocyte nuclei were digested with micrococcal nuclease and fractionated by centrifugation in low-salt buffer into soluble and insoluble fractions. Post-translational modifications of the purified linker histones of both fractions were analyzed by LC-ESI-MS/MS. All six histone H1 subtypes (H1.01, H1.02, H1.03, H1.10, H1.1L and H1.1R) and histone H5 were identified. Mass spectrometry analysis enabled the identification of a wide range of PTMs, including N(α)-terminal acetylation, acetylation, formylation, phosphorylation and oxidation. A total of nine new modifications in chicken linker histones were mapped, most of them located in the N-terminal and globular domains. Relative quantification of the modified peptides showed that linker histone PTMs were differentially distributed among both chromatin fractions, suggesting their relevance in the regulation of chromatin structure. The analysis of our results combined with previously reported data for chicken and some mammalian species showed that most of the modified positions were conserved throughout evolution, highlighting their importance in specific linker histone functions and epigenetics.
  
  
  CONCLUSIONS
  Post-translational modifications of linker histones could have a role in the regulation of gene expression through the modulation of chromatin higher-order structure and chromatin remodeling. Finding new PTMs in linker histones is the first step to elucidate their role in the histone code. In this manuscript we report nine new post-translational modifications of the linker histones from chicken erythrocytes, one in H5 and eight in the H1 subtypes. Chromatin fractionated by centrifugation in low-salt buffer resulted in two fractions with different contents and compositions of linker histones and enriched in specific core histone PTMs. Of particular interest is the fact that linker histone PTMs were differentially distributed in both chromatin fractions, suggesting specific functions. Future studies are needed to establish the interplay between PTMs of linker and core histones in order to fully understand chromatin regulation. A protein sequence alignment summarizing the PTMs found to date in chicken, mouse, rat and humans showed that, while many of the modified positions were conserved between these species, the type of modification often varied depending on the species or the cellular type. This finding suggests an important role for the PTMs in the regulation of linker histone functions.
authors.affiliation: Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Bettina
authors.initials: B
authors.lastname: Sarg
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Biociencias, Universidad Autónoma de Barcelona, Bellaterra, Barcelona, Spain.
authors.forename: Rita
authors.initials: R
authors.lastname: Lopez
authors.affiliation: Division of Clinical Biochemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Herbert
authors.initials: H
authors.lastname: Lindner
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Biociencias, Universidad Autónoma de Barcelona, Bellaterra, Barcelona, Spain.
authors.forename: Inma
authors.initials: I
authors.lastname: Ponte
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Biociencias, Universidad Autónoma de Barcelona, Bellaterra, Barcelona, Spain.
authors.forename: Pedro
authors.initials: P
authors.lastname: Suau
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Biociencias, Universidad Autónoma de Barcelona, Bellaterra, Barcelona, Spain. Electronic address: alicia.roque@uab.es.
authors.forename: Alicia
authors.initials: A
authors.lastname: Roque
mesh_terms: D000818:Animals; D030161:Avian Proteins / Q000378:metabolism*; D002645:Chickens; D002843:Chromatin / Q000378:metabolism*; D004900:Erythroblasts / Q000378:metabolism*; D006657:Histones / Q000378:metabolism*; D051379:Mice; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017434:Protein Structure, Tertiary; D051381:Rats
chemical_list: D030161:Avian Proteins; D002843:Chromatin; D006657:Histones
keywords: Chicken erythrocyte; Chromatin fractionation; Epigenetics; Linker histone PTMs; Mass spectrometry