Biochemical Analysis of Histone Succinylation.

Atsushi Yokoyama,Akira Sugawara,Shogo Katsura

doi:10.1155/2017/8529404

Abstract

Posttranslational modification (PTM) of proteins is used to regulate protein activity and stability. Histone PTMs are regarded as some of the most important, as they can directly regulate gene expression through chromatin reorganization. Recently, histone proteins were found to undergo succinylation, adding to other well-known PTMs such as acetylation, methylation, and phosphorylation. However, there is little information regarding the enzyme which catalyzes histone lysine succinylation. In fact, it is unclear whether this reaction is enzymatic. In this study, we tested histone succinylation activity in vitro using cell nuclear extracts of HepG2 cells. Although whole nuclear extracts did not show histone succinylation activity, we found that an SP 1.0 M KCl fraction of nuclear extracts indeed had such activity. These data offer the first direct evidence that histone succinylation is an enzymatic PTM as are other histone codes in the nucleus.

Highlights

Since the first identification of ε-acetylation of lysine in histone proteins [1, 2], several hundred protein posttranslational modifications (PTMs) have been identified in both histone and nonhistone proteins
Histone proteins are dynamically modified at specific amino acid residues by a variety of histone-modifying enzymes such as histone acetyl transferases (HATs) and histone methyl transferases (HMTs)
Whole nuclear extracts did not show histone succinylation activity, we found that the strong cation exchange column-binding fraction (HiTrap SP column, 1.0 M KCl-eluted fraction) of nuclear extracts possessed succinylation activity

Summary

Introduction

Since the first identification of ε-acetylation of lysine in histone proteins [1, 2], several hundred protein posttranslational modifications (PTMs) have been identified in both histone and nonhistone proteins. We tested histone succinylation activity in vitro using nuclear extracts. Total reaction mixtures (250 μL) contained the following: 30 mM Tris, pH 7.5, 50 nCi 14C-labeled acyl-coenzyme A, 1 mM 2-mercaptoethanol, and 300 μg of a protein extract with or without 2 μg of calf thymus histone.

Results

Conclusion