Peptide microarrays to interrogate the "histone code".
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.
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Scott B
SB
Rothbart
Krzysztof
K
Krajewski
Brian D
BD
Strahl
Stephen M
SM
Fuchs
D000107:Acetylation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D057075:Enzyme Assays; D004798:Enzymes / Q000737:chemistry; D044127:Epigenesis, Genetic; D005449:Fluorenes / Q000737:chemistry; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry*; D019151:Peptide Library; D040081:Protein Array Analysis; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D000596:Amino Acids; D004798:Enzymes; D005449:Fluorenes; D006657:Histones; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010446:Peptide Fragments; D019151:Peptide Library