The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components

Monica Soldi,Tiziana Bonaldi

doi:10.1074/mcp.m112.024307

Abstract

Chromatin is a highly dynamic, well-structured nucleoprotein complex of DNA and proteins that controls virtually all DNA transactions. Chromatin dynamicity is regulated at specific loci by the presence of various associated proteins, histones, post-translational modifications, histone variants, and DNA methylation. Until now the characterization of the proteomic component of chromatin domains has been held back by the challenge of enriching distinguishable, homogeneous regions for subsequent mass spectrometry analysis. Here we describe a modified protocol for chromatin immunoprecipitation combined with quantitative proteomics based on stable isotope labeling by amino acids in cell culture to identify known and novel histone modifications, variants, and complexes that specifically associate with silent and active chromatin domains. Our chromatin proteomics strategy revealed unique functional interactions among various chromatin modifiers, suggesting new regulatory pathways, such as a heterochromatin-specific modulation of DNA damage response involving H2A.X and WICH, both enriched in silent domains. Chromatin proteomics expands the arsenal of tools for deciphering how all the distinct protein components act together to enforce a given region-specific chromatin status.

Highlights

In eukaryotes, genetic information is stored in chromatin, a highly structured nucleoprotein complex that mediates the coordinated regulation of gene expression
Affinity-interaction assays using histone-peptide baits in conjunction with proteomics based on stable isotope labeling by amino acids in cell culture (SILAC) were recently developed to pull down and identify proteins interacting with distinct H3 and H4 trimethyl lysines [20, 21]
The approach has since been modified into the SILAC nucleosome affinity purification method, whereby in vitro reconstituted nucleosomes are used as bait in affinity purification assays, which are followed by mass spectrometry (MS) in order to obtain a “modification binding profile” of proteins whose interactions are regulated by the cooperative contribution of both DNA methylation and histone post-translational modifications (hPTMs) [22]

Summary

Introduction

Genetic information is stored in chromatin, a highly structured nucleoprotein complex that mediates the coordinated regulation of gene expression. In addition to hPTMs, chromatin is modulated by the local enrichment of a distinct set of histone variants, binding proteins, including various ATP-dependent remodeling activities, DNA methylation, and differential nucleosome density.

Results

Conclusion