Identification of 'erasers' for lysine crotonylated histone marks using a chemical proteomics approach.

Xiucong Bao,Zheng Liu,Xiao-Meng Li,Chi Fat Wong,Xin Li,Xiang David Li,Jiangwen Zhang,Tangpo Yang,Quan Hao,Yi Wang

doi:10.7554/elife.02999

Abstract

Posttranslational modifications (PTMs) play a crucial role in a wide range of biological processes. Lysine crotonylation (Kcr) is a newly discovered histone PTM that is enriched at active gene promoters and potential enhancers in mammalian cell genomes. However, the cellular enzymes that regulate the addition and removal of Kcr are unknown, which has hindered further investigation of its cellular functions. Here we used a chemical proteomics approach to comprehensively profile 'eraser' enzymes that recognize a lysine-4 crotonylated histone H3 (H3K4Cr) mark. We found that Sirt1, Sirt2, and Sirt3 can catalyze the hydrolysis of lysine crotonylated histone peptides and proteins. More importantly, Sirt3 functions as a decrotonylase to regulate histone Kcr dynamics and gene transcription in living cells. This discovery not only opens opportunities for examining the physiological significance of histone Kcr, but also helps to unravel the unknown cellular mechanisms controlled by Sirt3, that have previously been considered solely as a deacetylase.

Highlights

Histone posttranslational modifications (PTMs) play a crucial role in regulating a wide range of biological processes, such as gene transcription, DNA replication, and chromosome segregation (Kouzarides, 2007)
To identify proteins that bind H3K4Cr with high selectivity and high affinity, we performed two types of culture (SILAC) based Protein Identification (CLASPI) experiments with cell lysates derived from HeLa S3 cells grown in medium containing either ‘heavy’ (13C, 15N-substitued arginine and lysine) or ‘light’ amino acids (Figure 1B)
In a ‘selectivity filter’ CLASPI experiment, the ‘heavy’ and ‘light’ cell lysates were photo-cross-linked with probe 1 and an unmodified H3 control probe, respectively, and pooled for the subsequent steps

Summary

Introduction

Histone posttranslational modifications (PTMs) play a crucial role in regulating a wide range of biological processes, such as gene transcription, DNA replication, and chromosome segregation (Kouzarides, 2007). The mechanistic insights into the regulation and functions of histone Kac remained challenging and elusive, until the identification and characterization of the enzymes responsible for the addition and removal of this PTM, which are known as histone acetyltransferases (Roth et al, 2001) and deacetylases (Sauve et al, 2006; Yang and Seto, 2008b; Haberland et al, 2009), respectively. Extensive studies have revealed that Kac plays an important role in controlling chromatin structure and gene transcription (Grunstein, 1997; Yang and Seto, 2008a).

Methods

Results

Conclusion