Top-down and Middle-down Protein Analysis Reveals that Intact and Clipped Human Histones Differ in Post-translational Modification Patterns*

Andrey Tvardovskiy,Krzysztof Wrzesinski,Simone Sidoli,Stephenj Fey,Adelina Rogowska-Wrzesinska,Olen Jensen

doi:10.1074/mcp.m115.048975

Andrey Tvardovskiy, Krzysztof Wrzesinski + Show 4 more

Open Access

https://doi.org/10.1074/mcp.m115.048975

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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.

Highlights

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
By using antibodies (Abs) specific for the C-terminal part of histone H3 we reproducibly observed two faster migrating H3 bands in whole-cell extracts (WCEs) isolated from 3D culture, but not in C3A or HeLa grown in conventional 2D culture (Fig. 1A)
To confirm the obtained results we analyzed histone fraction isolated from 3D culture using SDS-PAGE and liquid chromatographytandem mass spectrometry (LC-MS/MS) after limited in-gel trypsin digestion

Summary

Introduction

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. Evidence is emerging that ”classical” histone PTMs, such as methylation, acetylation, and phosphorylation at distinct residues, and proteolytic processing of nucleosome proteins, known as “histone clipping,” can be involved in regulation of key cellular processes, such as transcriptional regulation, cell differentiation, and senescence [1,2,3,4,5,6,7]. Santos-Rosa et al demonstrated a serine protease activity in S. cerevisiae that cleaves histone H3 after residue Ala (A21) during sporulation and stationary phase [1]. Duncan et al demonstrated that histone H3 is proteolytically cleaved by the enzyme Cathepsin L1 (CTSL1) at several sites between residues A21 and S28 during mouse ESC differentiation [5]. The “in vitro” proteolytic activity of CTSL1 was found to be dependent on the H3 N-tail PTM status. H3 clipping was demonstrated in human ESCs [6]

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