Peptide Pull-Down (PPD) Assay for Identification and Characterization of Histone PTM Effectors

Introduction

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.

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Procedure

I. Design and synthesis of biotinylated “bait”-peptides: Biotinylated histone peptides with specific modifications can be chemically synthesized or obtained commercially. Peptides should be HPLC purified to >80% purity, aliquoted, lyophilized, and stored dry at -80°C. (See comment 1). When designing peptides consider the following:

Peptides should be approximately 15-20 amino acids in length with the modification of interest in the center of the sequence and at least 6-8 flanking residues on each side. (See note 1) Biotin should be conjugated to the N-termini of C-terminal histone peptides and the C-termini of N-terminal histone peptides For a negative control: corresponding unmodified peptides should be synthesized for each modified residue For a positive control: known histone PTM binding proteins can be used in conjunction with appropriately modified peptides (reviewed in Taverna et al. , 2007)

II. Preparation of peptide-bound resin: Biotinylated peptides are conjugated to avidin beads to generate the resin used for the peptide pulldown. All steps should be performed either on ice or at 4°C unless noted otherwise. The following is written for a single peptide and should be performed in parallel for all peptides of interest.

Resuspend 100 µg of biotinylated peptide in 400 µL PBS. (See note 2) Wash 400 µL avidin beads at least 3 times with 1 mL PBS + 0.1% Triton X-100, spin and remove supernatant. (See note 3) Apply the 400 µL re-suspended peptide to the 400 µL washed beads Incubate with rotation at room temperature for 3-5 hours to bind peptides to the beads. (See note 4) Wash bound beads at least 3 times with 1 mL PBS + 0.1% Triton X-100 to remove unbound peptide Resuspend peptide-conjugated beads in 400 µL PBS to yield a 50% slurry and store at 4°C. (See note 5) (See comment 2 and comment 3)

III. Peptide pull-down from a complex mixture of proteins: The peptide pull-down (PPD) is an unbiased assay to identify novel “reader” proteins that bind to specifically modified peptides from a complex protein mixture such as a nuclear extract.

General notes for this procedure are as follows:

At each step of the PPD assay ensure inclusion of protease inhibitors to minimize protein degradation and phosphatase inhibitors if working with phosphorylated proteins/peptides All steps should be performed on ice or at 4°C unless noted otherwise As a negative control perform the PPD with un-conjugated avidin beads in addition to any peptide-conjugated beads being tested Other buffers besides HEPES can be used in this assay: PBS or Tris buffers at various salt concentrations have not had obvious detrimental effects in our PPDs The following is written for a single peptide pull-down and should be performed in parallel for all peptides being tested

Prepare complex mixture of proteins: 1. Prepare fresh nuclear extract from 108 cells/pulldown using the cell type of choice and a standard high salt extraction protocol (Dignam et al. , 1983). (See note 6) 2. Adjust salt concentration of the extract to 150 mM and adjust the volume to 1 mL/108 cell equivalents (a total protein concentration of about 2-5 µg/µL) by either dialyzing against Buffer D(150 mM KCl) or diluting with Buffer D(no salt). (See note 7) 3. Add Triton X-100 to the extract to a final concentration of 0.1%. (See note 8) 4. Clear any precipitate from the extract by centrifuging in a microcentrifuge at max speed for 15-30 minutes and transferring the supernatant to a fresh tube. (See note 9) Pre-clear complex mixture: 5. Aliquot 80 µL of 50% un-conjugated avidin bead slurry (40 µL of beads) and remove supernatant. (See note 10) 6. Wash avidin beads at least 1X in 500 µL Buffer D(150 mM KCl). (See note 11) 7. Add the prepared nuclear extract to the washed avidin beads and incubate at 4°C with rotation for 30 minutes. (See note 12) 8. Pellet beads by centrifuging in a microcentrifuge at 500 RCF for 30 seconds and collect the supernatant in a fresh tube to use for pull-down. (See note 13) 9. Remove 15 uL of the cleared extract and save it as the PPD “input” fraction for later analysis. Prepare beads: 10. Aliquot 40 uL of 50% peptide-bound bead slurry (yields 20 uL of beads). (See note 14) 11. Pellet beads, remove all supernatant and ensure all aliquots have roughly equal amounts of beads. (See note 15) 12. Wash beads 1-3X in 500 uL Buffer D(150 mM KCl) and remove all supernatant. (See note 16) Peptide pull-down: 13. Add the pre-cleared extract to the 20 uL of washed peptide-bound beads. 14. Incubate with rotation for 2-16 hours at 4°C to allow binding to peptide-bound beads. (See note 17) 15. Pellet beads by centrifuging in a microcentrifuge at 500 RCF for 30 seconds. 16. Transfer all of the supernatant to a fresh tube and save as the PPD “flow-through” fraction for later analysis. 17. Wash beads at least 5X in 1 mL Buffer D(300 mM KCl) and then remove and discard all supernatant. (See note 18) 18. Wash beads one last time with 1 mL Low-HEPES Buffer and remove and discard all supernatant. (See note 19) Collect pull-down fractions: 19. Add 1-2 bead volumes (20-40 uL) of 100 mM glycine (pH 2.8) to the washed beads. (See note 20) 20. Incubate at room temperature for 10 minutes to acid-elute bound proteins. Flick tube gently during incubation to ensure efficient mixing of beads and elution buffer. (See note 21) 21. Transfer all of the eluate to a fresh tube with a capillary gel loading pipette tip. 22. Repeat steps 19-21 to perform a second acid-elution and pool both recovered eluates. (See note 22) 23. To neutralize the pH of the pooled eluates add 1/10 volume (4-8 uL) of 1 M Tris (pH 8.0). 24. Add the appropriate amount of 4X Laemmli sample buffer to the pooled eluates and save as the PPD “acid-elution” fraction for later analysis. 25. Resuspend acid-eluted beads in 1 bead volume (20uL) of 100 mM Tris (pH 8.0). 26. Add the appropriate amount of 4X Laemmli sample buffer and heat at 95°C for 5 minutes to denature any leftover bound protein. (See note 23) 27. Centrifuge in a microcentrifuge at max speed for 30 seconds, then vortex vigorously for 15 seconds and then centrifuge again. 28. Remove all supernatant as the PPD “denaturing-elution” fraction and save for analysis. Analyze PPD fractions and identify bound proteins: 29. Analyze “input”, “flow-through”, “acid-elution” and “denaturing-elution” fractions by SDS-PAGE followed by silver staining using a mass spectrometry compatible protocol. (See note 24) 30. Excise gel bands present in the modified peptide, but not unmodified peptide, pull-down lanes (“acid-elution” and/or “denaturing-elution”) and identify proteins by mass spectrometry. 31. Confirm the specific association of the identified protein with the modified peptide by analyzing the eluates by SDS-PAGE/immunoblotting with specific antibodies. Additionally, specific binding can be confirmed using recombinant protein in the PPD assay described below.