Systematic Identification of Methyllysine-Driven Interactions for Histone and Nonhistone Targets

Abstract

An important issue in epigenetic research is to understand how the numerous methylation marks associated with histone and certain nonhistone proteins are recognized and interpreted by the hundreds of chromatin-binding modules (CBMs) in a cell to control chromatin state, gene expression, and other cellular functions. We have assembled a peptide chip that represents known and putative lysine methylation marks on histones and p53 and probed the chip for binding to a group of CBMs to obtain a comprehensive interaction network mediated by lysine methylation. Interactions revealed by the peptide array screening were validated by in-solution binding assays. This study not only recapitulated known interactions but also uncovered new ones. A novel heterochromatin protein 1 beta (HP1β) chromodomain-binding site on histone H3, H3K23me, was discovered from the peptide array screen and subsequently verified by mass spectrometry. Data from peptide pull-down and colocalization in cells suggest that, besides the H3K9me mark, H3K23me may play a role in facilitating the recruitment of HP1β to the heterochromatin. Extending the peptide array and mass spectrometric approach presented here to more histone marks and CBMs would eventually afford a comprehensive specificity and interaction map to aid epigenetic studies.

Loss of the Methyl Lysine Effector Protein PHF20 Impacts the Expression of Genes Regulated by the Lysine Acetyltransferase MOF

“…1B). By reversing this experiment and using recombinant effector molecules to probe a library of arrayed peptides representing histone modifications, we consistently observe a striking overlap in the binding profile between the Tudor domains of DNA damage repair protein 53BP1 and that of PHF20 (12). Therefore, we initially predicted that PHF20 may also have a role in DNA damage response and repair.…”

A Comprehensive Immunoreceptor Phosphotyrosine-based Signaling Network Revealed by Reciprocal Protein–Peptide Array Screening

“…Peptide Array Preparation and Probing-Peptides were printed on glass slides as neutravidin complexes (20). Specifically, the biotinpeptide was incubated with neutravidin in 3:1 ratio in PBS, pH 7.5, to form a complex.…”

“…1). Biotin-tagged peptides were printed on glass slides in quadruplets as neutravidin conjugates (20). The peptide array was subsequently probed for binding to SH2 domains (supplemental Fig.…”

Histone Recognition by Tandem Modules and Modulation by Multiple PTMs

“…In addition to the abovementioned cell-based in vivo or semi in vivo characterization of histone PTM patterns and their functional readout, in vitro peptide or reader library-based technologies have been developed for systematic analysis of the recognition between reader modules and multiply modified histone peptides (Bua et al 2009;Garske et al 2010;Kim et al 2006;Liu et al 2010). These technologies allow biophysical probing and profiling of "mark-reader" pairs and their potential for multivalent engagement assuming that the combinatorial readout of modifications would render the strongest binding.…”