Peptide microarrays to interrogate the "histone code"

Abstract

Histone posttranslational modifications (PTMs) play a pivotal role in regulating the dynamics and function of chromatin. Supported by an increasing body of literature, histone PTMs such as methylation and acetylation function together in the context of a "histone code," which is read, or interpreted, by effector proteins that then drive a functional output in chromatin (e.g., gene transcription). A growing number of domains that interact with histones and/or their PTMs have been identified. While significant advances have been made in our understanding of how these domains interact with histones, a wide number of putative histone-binding motifs have yet to be characterized, and undoubtedly, novel domains will continue to be discovered. In this chapter, we provide a detailed method for the construction of combinatorially modified histone peptides, microarray fabrication using these peptides, and methods to characterize the interaction of effector proteins, antibodies, and the substrate specificity of histone-modifying enzymes. We discuss these methods in the context of other available technologies and provide a user-friendly approach to enable the exploration of histone-protein-enzyme interactions and function.

Figure 6.1

A representation of select posttranslational modifications (PTMs) on human histones. Depicted are the PTMs that are most amenable for peptide synthesis, such as acetylation (ac), methylation (me), and phosphorylation (P). *Histone lysine methylation occurs in three forms (mono-, di-, and trimethylation), as does arginine methylation (monomethylation, symmetric, and asymmetric dimethylation).

Figure 6.2

Chemical derivatives used for the synthesis of modified histone tails suitable for arraying. (A) N- and C-terminal biotin tags with PEG linkers. (B) Derivatives used for PTM incorporation.

Figure 6.3

Slide design and workflow for peptide arraying. (A) A suggested layout of peptides for printing, as detailed in Chapter 3. (B) Depiction of the stepwise procedure for peptide arraying of an effector protein from peptide immobilization to antibody hybridization and visualization. Positive interactions are visualized as red fluorescence. All printed spots show green fluorescence from the biotinylated fluorescein printing control.

Figure 6.4

Typical data presentation for peptide array results. (A) Scatter plot showing the reproducibility of two arrays probed with an H3K4me3 antibody (Millipore #07-473, Lot #DAM1623866). Analysis of H3K4 methylation states shows no binding to H3K4me1-containing peptides (green), weak affinity for most H3K4me2-containing peptides (blue), and strong affinity for most H3K4me3-containing peptides (red) with the exception of several combinatorial PTMs that perturb binding to H3K4me3. All other peptides, including H3 peptides with no H3K4 methylation, are shown in black. (B) Array analysis of the PHD finger of the Rag2 V(D)J recombination factor identifies specificity for H3K4me3 as previously described (Ramon-Maiques et al., 2007). Peptide interactions were normalized to the average intensity of H3K4me3. Intensities were compared by two-way analysis of variance with 99% confidence intervals (**). (C) Heatmap depicting the effects of combinatorial PTMs on the binding of the Rag2 PHD finger to H3K4me3-containing peptides. Binding intensities are represented relative to H3K4me3 (0, white). Enhanced (1, red) and occluded (−1, blue) interactions are depicted. The heatmap was generated using JavaTreeView (Saldanha, 2004).