title: Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns.
abstract: Post-translational modifications (PTMs) of histone proteins play a fundamental role in regulation of DNA-templated processes. There is also growing evidence that proteolytic cleavage of histone N-terminal tails, known as histone clipping, influences nucleosome dynamics and functional properties. Using top-down and middle-down protein analysis by mass spectrometry, we report histone H2B and H3 N-terminal tail clipping in human hepatocytes and demonstrate a relationship between clipping and co-existing PTMs of histone H3. Histones H2B and H3 undergo proteolytic processing in primary human hepatocytes and the hepatocellular carcinoma cell line HepG2/C3A when grown in spheroid (3D) culture, but not in a flat (2D) culture. Using tandem mass spectrometry we localized four different clipping sites in H3 and one clipping site in H2B. We show that in spheroid culture clipped H3 proteoforms are mainly represented by canonical histone H3, whereas in primary hepatocytes over 90% of clipped H3 correspond to the histone variant H3.3. Comprehensive analysis of histone H3 modifications revealed a series of PTMs, including K14me1, K27me2/K27me3, and K36me1/me2, which are differentially abundant in clipped and intact H3. Analysis of co-existing PTMs revealed negative crosstalk between H3K36 methylation and H3K23 acetylation in clipped H3. Our data provide the first evidence of histone clipping in human hepatocytes and demonstrate that clipped H3 carry distinct co-existing PTMs different from those in intact H3.
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology and §Center for Epigenetics, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK - 5230 Odense M, Denmark.
authors.forename: Andrey
authors.initials: A
authors.lastname: Tvardovskiy
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology and.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Wrzesinski
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology and §Center for Epigenetics, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK - 5230 Odense M, Denmark.
authors.forename: Simone
authors.initials: S
authors.lastname: Sidoli
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology and.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Fey
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology and §Center for Epigenetics, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK - 5230 Odense M, Denmark.
authors.forename: Adelina
authors.initials: A
authors.lastname: Rogowska-Wrzesinska
authors.affiliation: From the ‡Department of Biochemistry and Molecular Biology and §Center for Epigenetics, VILLUM Center for Bioanalytical Sciences, University of Southern Denmark, Campusvej 55, DK - 5230 Odense M, Denmark jenseno@bmb.sdu.dk.
authors.forename: Ole N
authors.initials: ON
authors.lastname: Jensen
mesh_terms: D000107:Acetylation; D018929:Cell Culture Techniques; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006657:Histones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D006657:Histones