Application of Celluspots peptide arrays for the analysis of the binding specificity of epigenetic reading domains to modified histone tails

Abstract

BackgroundEpigenetic reading domains are involved in the regulation of gene expression and chromatin state by interacting with histones in a post-translational modification specific manner. A detailed knowledge of the target modifications of reading domains, including enhancing and inhibiting secondary modifications, will lead to a better understanding of the biological signaling processes mediated by reading domains.ResultsWe describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. To validate the method, we tested the binding specificities of seven known epigenetic reading domains on Celluspots peptide arrays, viz. the HP1ß and MPP8 Chromo domains, JMJD2A and 53BP1 Tudor domains, Dnmt3a PWWP domain, Rag2 PHD domain and BRD2 Bromo domain. In general, the binding results agreed with literature data with respect to the primary specificity of the reading domains, but in almost all cases we obtained additional new information concerning the influence of secondary modifications surrounding the target modification.ConclusionsWe conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides.

Lamin B Receptor Recognizes Specific Modifications of Histone H4 in Heterochromatin Formation

“…Another possible determinant of specificity might be modification patterns surrounding H4K20. Here we have presented evidence that LBR recognizes H4K20me2 in combination with certain surrounding modified residues, and this is distinct from the binding specificity of 53BP1 (31). Although the roles of H4R19 and H4R23 methylation have not been identified, the combination appears to be important for LBR functions; that is, for chromatin compaction and transcriptional repression.…”

“…1, B and C). First, we used a Celluspots peptide array comprising 384 histone tail peptides with various combinations of modifications; this array is a powerful tool for determining the specificity of histone modification-recognition domains (31). The array was incubated with the GST-fused wild-type NP domain (NP WT ) and then probed with anti-GST antibody.…”

Analyzing N-terminal Arginylation through the Use of Peptide Arrays and Degradation Assays

“…This technology makes it possible to produce a number of virtually identical copies of a peptide array from a single set of peptide-synthesizing reactions (102). CelluSpots arrays have been successfully used to examine the specificity of histone-modifying enzymes for amino acid sequences in histones; to analyze, in vitro, the binding specificity of antibodies; to characterize specific binding of SH2 (Src homology 2 domain) protein regions to phosphotyrosine-containing peptides; and to survey the specificities of phosphokinases and kinase inhibitors (103)(104)(105).…”

Application of histone modification-specific interaction domains as an alternative to antibodies

“…On the peptide array, MPHOSPH8 Chromo bound most prominently to H3K9me3 peptides and to a lesser extent to H3K9me2 and H3K27me3 peptides ( Fig. 2A; Bock et al 2011b). Akin to the Chromo domain of CBX1 (Supplemental Fig.…”