Unlike the binding of effector proteins and antibodies, which is primarily measured through indirect fluorescence hybridization, the activity of histone-modifying enzymes such as kinases and methyltransferases can be measured directly using radioisotopes
Unlike the binding of effector proteins and antibodies, which is primarily measured through indirect fluorescence hybridization, the activity of histone-modifying enzymes such as kinases and methyltransferases can be measured directly using radioisotopes. link between histone posttranslational modifications (PTMs) and transcriptional regulation (Brownell et al., 1996; Taunton, Hassig, KX2-391 2HCl & Schreiber, 1996). Since then, a significant effort has been placed on the identification and biological characterization of histone PTMs, which function in DNA-templated processes such as transcription, recombination, and DNA repair (Kouzarides, 2007). The N- and C-terminal tails of the four core histone proteins are rich in amino acids that are known sites of PTM (Fig. 6.1). These PTMs include, but are not limited to, methylation, acetylation, and ubiquitination of lysine, and phosphorylation of serine and threonine. How the myriad of known histone PTMs function has remained a fundamental question in modern biology. Open in a separate window Physique 6.1 A representation of select posttranslational Mouse monoclonal to EphA5 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). It is thought that histone PTMs function to regulate the diverse activities associated with chromatin (Gardner, Allis, & Strahl, 2011; Kornberg & Lorch, 1999; Kouzarides, 2007). The histone code hypothesis, formally introduced just over a decade ago, suggests that histone PTMs function in a combinatorial fashion to KX2-391 2HCl regulate chromatin structure and function (Jenuwein & Allis, 2001; Strahl & Allis, 2000). We now know that histone PTMs such as lysine acetylation can directly alter the physical structure of chromatin by charge neutralization (Shogren-Knaak et al., 2006), and PTMs can also serve as binding sites for effector proteins that contain one or more well-characterized protein folds (Taverna, Li, Ruthenburg, Allis, & Patel, 2007). For example, methyllysine can be recognized by motifs like chromodomains and herb homeodomains (PHD), while acetyllysine can be recognized by bromodomains. Increasing evidence is emerging to support the histone code hypothesis, in the context of both effector protein binding and antibody recognition (Bock et KX2-391 2HCl al., 2011; Fuchs & Strahl, 2011; Garske et al., 2010), and more recently around the multivalent engagement of effector proteins with chromatin through linked recognition domains (Allis & Muir, 2011; Ruthenburg, Li, Patel, & Allis, 2007). A notable example of the latter is the recent finding that the paired bromodomain and PHD finger of the BPTF subunit of the NURF chromatin-remodeling complex simultaneously engage nucleosomes bearing H4 lysine 16 acetylation (H4K16ac) and H3 lysine 4 trimethylation (H3K4me3), respectively (Ruthenburg et al., 2011). In the past few years, a number of successful strategies have been used to discover and characterize proteins (and their domains) that recognize histones and histone PTMs. These range from high-throughput batch screening of purified proteins using large peptide libraries on beads (Garske et al., 2010), discovery-based approaches utilizing stable isotope labeling with amino acids in culture (SILAC) combined with peptide and/or nucleosomal pull-downs (Vermeulen et al., 2011), and a number of peptide microarray-based approaches utilizing purified recombinant proteins (Bock et al., 2011; Bua et al., 2009; Nady, Min, Kareta, Chedin, & Arrowsmith, 2008). Bead-based approaches have significant advantages in the large library size that can be created but require labor-intensive screening to identify hits. SILAC-based approaches offer tremendous potential to identify novel histone-interacting proteins and protein complexes. However, they do not report around the domain name, or protein from within a complex, that binds to the peptide or nucleosome used in the pull-down. Peptide microarrays provide an extremely rapid and robust method for measuring binding to a large number of peptide substrates, although the development of the peptide library itself may be time intensive. While each KX2-391 2HCl of the different approaches to identify and characterize histone-interacting proteins have their advantages and disadvantages, it is clear that these approaches are valuable ways to characterize histone interactions and are fundamental to advancing the chromatin field. Here, we describe a microarray approach using high-purity biotinylated peptides spotted onto streptavidin-coated glass slides. We describe the peptide synthesis and the microarray fabrication as well as the methodology for using these microarrays to probe the binding of not only histone-binding proteins but also PTM-specific antibodies and.