Post-translational modifications (PTMs) of histones specify regulatory functions on chromatin through the recruitment of downstream effectors or “readers”, that can specifically recognize different PTMs and translate epigenetic marks into a functionally relevant outcome (reviewed in Taverna et al., 2007). To comprehend the complexity of epigenetic regulation, it is essential to not only catalogue histone PTMs and their patterns, but also to understand roles that histone PTMs and their effectors play in biological processes. An important component of this understanding will come through identification of histone PTM binding proteins. To this end, the peptide pull-down (PPD) assay provides a simple and effective tool to identify and characterize such reader proteins.
The general principle of the PPD is as follows. Biotinylated histone tail peptides containing a specific histone PTM and corresponding control unmodified peptides, are immobilized onto avidin-conjugated beads. The beads are incubated with a sample of interest, such as nuclear extract or purified recombinant protein, and washed to remove unbound proteins. Bound proteins can then be eluted and analyzed by SDS/PAGE and visualized by protein staining. By comparing proteins bound to modified versus unmodified peptides it is possible to identify candidate “reader” proteins for specific histone PTMs.
The PPD assay relies on two critical assumptions: 1) the “bait” peptides structurally mimic the histone region of interest, and 2) the length of the “bait” peptide is sufficient for recognition by the candidate reader protein(s). Histones are most heavily modified at their N- and C-terminal tails, which are relatively unstructured and jut away from the nucleosome/DNA core; thus short peptides are probably adequate structural-mimics. Also, structural studies of known histone PTM readers have revealed that generally fewer than ten residues of histone sequence are required for recognition (reviewed in Taverna et al., 2007). However, there are still considerable limitations to the PPD assay. First, the recognition of some histone modifications (e.g., those within globular domains) may require structures that cannot be modeled by short, synthetic peptides. Second, the affinity of the reader of interest for its bait-peptide must be sufficiently high (low micromolar range) such that the interaction can withstand the washing conditions necessary to clear away non-specific binders. Third, the PPD assay is not quantitative.
Despite these limitations, the PPD assay has considerable advantages and applications. Namely, it enables an unbiased approach to identifying new epigenetic readers from a complex mixture of proteins. Second, it allows for the determination of substrate preference, specificity and recognition domains of candidate readers. Finally, it is a simple assay to perform, requires few reagents, and is relatively inexpensive.
1 Department of Biochemistry
2 Departments of Chemical and Systems Biology
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