Identifying novel proteins recognizing histone modifications using peptide pull-down assay

Abstract

Post-translational modifications of histones have been correlated with virtually all chromatin-templated processes, including gene expression regulation, DNA replication, mitosis and meiosis, and DNA repair. In order to better understand the mechanistic basis by which histone modifications participate in the control of cellular processes, it is essential to identify and characterize downstream effector proteins, or "readers", that are responsible for recognizing different marks and translating them into specific biological outcomes. Ideally, identification of potential histone-binding effectors should occur in an unbiased fashion. Although in the recent years much progress has been made in identifying readers of histone modifications, in particular methylation, recognition of the majority of known histone marks is still poorly understood. Here I describe a simple and unbiased biochemical pull-down assay that allows for the identification of novel histone effector proteins and utilizes biotinylated histone peptides modified at various residues. I provide detailed protocols and suggestions for troubleshooting.

Figure

Schematics of the peptide pull-down assay. Biotinylated histone peptides either unmodified, or modified at specific residues are immobilized on avidin beads and incubated with extract. To illustrate the principle of the assay, H3 peptide, either unmodified, or trimethylated at lysine 4 (K4), or trimethylated at lysine 9 (K9) is shown. Specific effector proteins bind to histone peptides in a modification dependent manner. This is illustrated by specific recognition of the H3 K4me3 peptide by BPTF (shown in blue), and of the H3 K9me3 peptide by HP1 (shown in red). Bound proteins are then eluted from the avidin beads, and resolved by SDS PAGE. Proteins present in the modified, but not unmodified peptide pull-down lane represent candidate readers of a specific histone modification. For example, BPTF is specifically present in the H3 K4me3 pull-down, whereas HP1 is recovered in the H3 K9me3 pull-down.