Method Article

Assays for Validating Histone Acetyltransferase Inhibitors

DOI:

10.3791/61289

⸱

August 6th, 2020

 , 
1Department of Anatomy and Cell Biology, and UF Health Cancer Center, University of Florida College of Medicine

In This Article

Summary

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Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and ChIP-qPCR.

Abstract

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Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene expression. KATs, such as CBP/p300, are under intense investigation as therapeutic targets due to their critical role in tumorigenesis of diverse cancers. The development of novel small molecule inhibitors targeting the histone acetyltransferase (HAT) function of KATs is challenging and requires robust assays that can validate the specificity and potency of potential inhibitors.

This article outlines a pipeline of three methods that provide rigorous in vitro validation for novel HAT inhibitors (HATi). These methods include a test tube HAT assay, Chromatin Hyperacetylation Inhibition (ChHAI) assay, and Chromatin Immunoprecipitation-quantitative PCR (ChIP-qPCR). In the HAT assay, recombinant HATs are incubated with histones in a test tube reaction, allowing for acetylation of specific lysine residues on the histone tails. This reaction can be blocked by a HATi and the relative levels of site-specific histone acetylation can be measured via immunoblotting. Inhibitors identified in the HAT assay need to be confirmed in the cellular environment.

The ChHAI assay uses immunoblotting to screen for novel HATi that attenuate the robust hyperacetylation of histones induced by a histone deacetylase inhibitor (HDACi). The addition of an HDACi is helpful because basal levels of histone acetylation can be difficult to detect via immunoblotting.

The HAT and ChHAI assays measure global changes in histone acetylation, but do not provide information regarding acetylation at specific genomic regions. Therefore, ChIP-qPCR is used to investigate the effects of HATi on histone acetylation levels at gene regulatory elements. This is accomplished through selective immunoprecipitation of histone-DNA complexes and analysis of the purified DNA through qPCR. Together, these three assays allow for the careful validation of the specificity, potency, and mechanism of action of novel HATi.

Introduction

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Lysine acetyltransferases (KATs) catalyze the acetylation of lysine residues on both histone and non-histone proteins1,2,3,4. Recent research reveals that KATs and their acetyltransferase function can promote solid tumor growth4,5,6,7,8,9. For example, CREB-binding protein (CBP)/p300 are two paralogous KATs that regulate numerous signaling pathways in cancer2,3. CBP/p300 have a well characterized histone acetyltransferase (HAT) function and catalyze Histone 3 Lysine 27 acetylation (H3K27ac)2,4,5,10,11, an important marker for active enhancers, promoter regions and active gene transcription12,13,14. CBP/p300 serve as critical co-activators for pro-growth signaling pathways in solid tumors by activating transcription of oncogenes through acetylation of histones and other transcription factors4,9,15,16,17,18. Due to their role in tumor progression, CBP/p300 and other KATs are under investigation for the development of novel inhibitors that block their oncogenic function4,5,6,7,8,9,18,19,20. A-485 and GNE-049 represent two successful attempts to develop potent and specific inhibitors for CBP/p3004,9. Additional inhibitors are currently under investigation for CBP/p300 and other KATs.

The quality of previously described KAT inhibitors (KATi) is being called into question, with many inhibitors showing off target effects and poor characterization21. Therefore, rigorous characterization and validation of novel drug candidates is essential for the development of high-quality chemical probes. Outlined here are three protocols that form a pipeline for screening and rigorously validating the potency and specificity of novel KATi, with a specific focus on inhibiting the HAT function (HATi) of KATs. CBP/p300 and their inhibitors are used as examples, but these protocols can be adapted for other KATs that have a HAT function7.

The first protocol is an in vitro histone acetyltransferase (HAT) assay that utilizes purified recombinant p300 and histones in a controlled test tube reaction. This assay is simple to perform, is cost-effective, can be used to screen compounds in a low throughput setting, and does not require radioactive materials. In this protocol, recombinant p300 catalyzes lysine acetylation on histone tails during a brief incubation period and the levels of histone acetylation are measured using standard immunoblotting procedures. The enzymatic reaction can be performed in the presence or absence of CBP/p300 inhibitors to screen for compounds that reduce histone acetylation. Additionally, the HAT assay can be used to verify whether novel compounds are selective for CBP/p300 by assessing their activity against other purified KATs, such as PCAF. The HAT assay is an excellent starting point for investigating novel inhibitors due to its simplicity, low cost, and the ability to determine the potency/selectivity of an inhibitor. Indeed, this protocol is often used in the literature as an in vitro screen5,10. However, inhibitors identified in the HAT assay are not always effective in cell culture because a test tube reaction is much simpler than a living cell system. Therefore, it is essential to further characterize inhibitors in cell culture experiments22,23.

The second protocol in the pipeline is the Chromatin Hyperacetylation Inhibition (ChHAI) assay. This cell based assay utilizes histone deacetylase inhibitors (HDACi) as a tool to hyperacetylate histones in chromatin before co-incubation with a HATi24. Basal histone acetylation can be low in cell culture, making it difficult to probe for via immunoblotting without the addition of an HDACi to increase acetylation. The purpose of the ChHAI assay is to identify novel HATi that can attenuate the increase in histone acetylation caused by HDAC inhibition. The advantages of this assay include its low cost, relative ease to perform, and the use of cells in culture, which provides more physiological relevance than the test tube HAT assay. Similar to the HAT assay, this protocol uses standard immunoblotting for data collection.

The HAT and ChHAI assays provide data about the potency of novel compounds for inhibiting global histone acetylation, but do not provide insight into how these compounds affect modifications at specific genomic regions. Therefore, the final protocol, Chromatin Immunoprecipitation-quantitative Polymerase Chain Reaction (ChIP-qPCR) is a cell culture experiment that investigates DNA-protein interactions at specific regions of the genome. In the ChIP protocol, chromatin is crosslinked to preserve DNA-protein interactions. The chromatin is then extracted from cells and the DNA-protein complex undergoes selective immunoprecipitation for the protein of interest (e.g., using an antibody specific for H3K27ac). The DNA is then purified and analyzed using qPCR. For example, ChIP-qPCR can be used to determine if a novel HATi downregulates histone acetylation at individual oncogenes, such as Cyclin D125. While ChIP-qPCR is a common technique used in the field, it can be difficult to optimize4,10,26. This protocol provides tips for avoiding potential pitfalls that can occur while performing the ChIP-qPCR procedure and includes quality control checks that should be performed on the data.

When used together, these three protocols allow for the rigorous characterization and validation of novel HATi. Additionally, these methods offer many advantages because they are easy to perform, relatively cheap and provide data on global as well as regional histone acetylation.

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Protocol

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1. In vitro HAT assay

  1. Buffer preparation
    NOTE: See Table 1 for buffer recipes.
    1. Prepare 5x assay buffer and 6x Sodium Dodecyl Sulfate (SDS) and store at -20 °C. Aliquot SDS in 1 mL aliquots.
    2. Prepare 10x SDS gel running buffer and 10x TBST and store at room temperature.
    3. Prepare 1x transfer buffer and store at 4 °C.
      CAUTION: Check safety data sheet for all chemicals used in this protocol. SDS, DTT, and bromophenol blue are toxic and should not be ingested, inhaled, or exposed to the skin or eyes. Please see safety data sheet for proper handling procedures. Please use a chemical fume hood for handling dangerous chemicals.
  2. HAT reaction
    NOTE: Anacardic acid is a known p300 inhibitor3 and is used as an example to demonstrate how the HAT assay can identify novel p300 inhibitors. See Supplementary Protocol (Schematic 1) for a schematic of step 1.2.1.
    1. Prepare the following enzymatic reaction in a 0.2 mL PCR tube: 2 μL of 5x assay buffer, 1 μL of purified p300 (0.19 μg/μL ), 1 μL of anacardic acid (HATi) or DMSO control diluted in 1x assay buffer and 2 μL of autoclaved ddH2O. Pre-incubate this mixture for 10 min at room temperature. Then add 3 μL of 100 μM Acetyl-CoA and 1 μL of purified H3.1 (0.2 μg/μL) to the reaction.
    2. Incubate the complete reaction mixture at 30 °C for 1 h in a PCR thermal cycler.
    3. Add 2-mercaptoethanol at a 1:10 ratio to the 6x SDS sample buffer.
    4. Remove samples from the PCR thermal cycler and add 2 µL of 6x SDS (with 2-mercaptoethanol added) to the reaction mix.
      CAUTION: 2-mercaptoethanol is toxic and should be used inside a chemical fume hood. Please see safety data sheet for proper handling.
    5. Heat samples at 95 °C for 5 min on a heat block and cool on ice. Store the samples at -20 or -80 °C or perform gel electrophoresis and immunoblotting as detailed below.
  3. Gel electrophoresis and immunoblotting
    NOTE: If unfamiliar with gel electrophoresis and immunoblotting, see this standard procedure27 for additional details on how to perform steps 1.3.1-1.3.17. Additional information can be found here28,29,30,31,32,33.
    1. Pipette 10 μL of samples (from step 1.2.5.) into the wells of a 4-20% gradient polyacrylamide gel. Pipette 5 μL of protein ladder into one of the wells as a molecular weight reference. Run the gel at 120 V for 90 min using a gel tank.
    2. Transfer the gel to a polyvinylidene difluoride (PVDF) membrane at 100 V for 70 min using a transfer tank.
    3. Remove the membrane from the transfer apparatus and place it in a plastic container. Block the membrane by adding 1x TBST (containing 5% milk) to the container and gently shake for 1 h at room temperature.
    4. Remove the 1x TBST from step 1.3.3. Incubate the membrane overnight with selected site-specific acetyl antibodies (e.g., H3K18ac or H3K27ac primary antibodies at a 1:5,000 dilution in 1x TBST containing 5% milk) at 4 °C with gentle shaking.
    5. Remove the primary antibody solution. Wash the membrane 2x with 1x TBST (no milk) at room temperature with gentle shaking for 15 min each wash.
    6. Dilute the secondary antibody at 1:20,000 in 1x TBST (containing 5% milk) and incubate the membrane for 1 h at room temperature with gentle shaking.
    7. Remove the secondary antibody solution. Wash the membrane 2x with 1x TBST (no milk) at room temperature with gentle shaking for 15 min each wash.
    8. Drain the 1x TBST from the membrane. Mix HRP substrate peroxide solution and HRP substrate luminol solution in a 1:1 ratio (1 mL of each) and pipette 2 mL of the combined solution to the membrane surface.
    9. Incubate the solution with the membrane for 5 min at room temperature.
    10. Drain excess chemiluminescent substrate from the membrane onto a paper towel and place the membrane in plastic wrap inside an x-ray cassette holder.
    11. Move to a dark room dedicated to x-ray film processing. Expose the membrane to an x-ray film by placing the film on top of the membrane and closing the cassette for 30 s.
      NOTE: Time of contact between the film and membrane must be determined experimentally. Strong signals will need short exposures (seconds) and weaker signals may need longer exposures.
    12. Remove the x-ray film from the cassette and process the film by running it through an x-ray film processor. See the manufacturer manuals for specific instructions on how to process the x-ray film.
    13. Remove the membrane from the plastic wrap and wash it with ddH2O for 5 min at room temperature with gentle shaking.
    14. Incubate the membrane with 0.2 M NaOH for 5 min at room temperature with gentle shaking.
    15. Wash the membrane with ddH2O for 5 min at room temperature with gentle shaking.
    16. Block the membrane by adding 1x TBST (containing 5% milk) to the container and gently shake for 1 h at room temperature.
    17. Add the next primary antibody dilution (e.g., probe for H3K27ac if first antibody used was H3K18ac) and shake overnight at 4 °C. Repeat steps 1.3.4-1.3.17 until all antibody probes are completed.

2. ChHAI assay

  1. In vitro drug treatments and analysis of acetylated histones
    NOTE: A-485 is a potent and well characterized p300 HATi2,4 . This inhibitor will be utilized in the remaining assays due to its efficacy and specificity in cell culture. MS-275 (Entinostat)24 is an HDACi that markedly increases histone acetylation levels and is used to facilitate easier detection of acetylation probes with standard immunoblotting. See Supplementary Protocol (Schematic 2) for a schematic of the drug dilutions used in step 2.1.
    1. Seed 100,000 MCF-7 cells in a 12 well plate and allow cells to grow to 80-90% confluency in 1 mL of cell culture medium. Mark the wells for the following experimental design: well 1: DMSO control (reference point); well 2: A-485 (3 μM); well 3: A-485 (10 μM); well 4: MS-275 (3 μM); well 5: MS-275 (3 μM) + A-485 (3 μM); well 6: MS-275 (3 μM) + A-485 (10 μM).
      NOTE: For culturing MCF-7 cells, use complete DMEM media and allow cells to grow at 37 °C with 5% CO2. See Table 1 for complete DMEM recipe.
    2. At 24 h after seeding, pipette 4 mL of complete DMEM media to a sterile 15 mL conical tube. Pipette 2 μL of MS-275 (6 mM in DMSO) to the 4 mL of medium for a final concentration of 3 μM MS-275.
    3. Pipette 2 μL of DMSO to 4 mL of medium in a separate sterile 15 mL conical tube.
      CAUTION: Please check safety data sheet for proper handling of DMSO. Some glove types are not rated for handling DMSO.
    4. Aspirate the cell culture medium from wells 4-6 and pipette 1 mL of 3 μM MS-275 in medium (step 2.1.2) to each well. Discard unused diluted MS-275.
    5. Aspirate the cell culture medium from wells 1-3 and pipette 1 mL of diluted DMSO (step 2.1.3) to each well. Discard unused diluted DMSO.
    6. Return the cells to the incubator and incubate for 4 h to allow accumulation of acetylated histones in cells exposed to MS-275 (wells 4-6).
      NOTE: MS-275 is an HDACi and will cause histone hyperacetylation24. This 4 h pre-incubation is necessary to allow MS-275 to induce hyperacetylation before the addition of A-485, which reduces histone acetylation2,4.
    7. After 4 h of incubation with MS-275, prepare the following dilutions in separate sterile 1.5 mL tubes by pipetting: 1.0 μL of DMSO to 1 mL of DMEM media; 0.5 μL of DMSO and 0.5 μL A-485 (6 mM) to 1 mL of DMEM media; 0.5 μL of DMSO and 0.5 μL of A-485 (20 mM) to 1 mL of DMEM media; 0.5 μL of DMSO and 0.5 μL of MS-275 (6 mM) to 1 mL of DMEM media; 0.5 μL of A-485 (6 mM) and 0.5 μL of MS-275 (6 mM) to 1 mL of DMEM media; 0.5 μL of A-485 (20 mM) and 0.5 μL of MS-275 (6 mM) to 1 mL of DMEM media.
    8. Aspirate the cell culture medium from wells 1-6 and pipette 1 mL of dilution 1 to well 1, dilution 2 to well 2, dilution 3 to well 3, dilution 4 to well 4, dilution 5 to well 5, and dilution 6 to well 6.
      NOTE: A general rule is to balance DMSO (solvent) content between experimental groups and not to exceed 0.1% DMSO content in cell culture to avoid cellular toxicity and changes in proliferation.
    9. Return the cells to the incubator and culture for 20 h.
    10. After 20 h, aspirate the cell culture medium from wells 1-6.
    11. Wash the cells by pipetting 1 mL of PBS to wells 1-6. Aspirate the PBS.
    12. Add 100 μL of 1x passive lysis buffer (see Table 1) to wells 1-6. Store cell culture plate (with samples in passive lysis buffer) at −80 °C overnight for freeze-thaw and lysis of cells.
      CAUTION: Please check safety data sheet for all chemicals before making buffers. CDTA can cause serious eye damage and irritation.
    13. Thaw samples at room temperature with gentle shaking for 10 min. Transfer samples to separate 1.5 mL tubes and immediately place on ice.
    14. Measure protein concentration of each sample. Protein concentration can be determined using several well-established protocols34.
    15. Equilibrate protein concentration between samples 1-6 (in an equal volume) using 1x passive lysis buffer to dilute, as necessary.
    16. Add 2-mercaptoethanol at a 1:10 ratio to 6x SDS sample buffer.
    17. Add 6x SDS sample buffer with 2-mercaptoethanol to samples 1-6 to a final concentration of 1x SDS sample buffer.
    18. Heat samples at 95 °C for 5 min on a heat block and cool on ice. Samples can be stored at -20 °C or -80 °C until step 2.1.19.
    19. Pipette a volume containing 30 μg of protein for samples 1-6 to the wells in a 4-20% gradient polyacrylamide gel. Perform immunoblotting procedure according to protocol described in Protocol 1.

3. ChIP-qPCR

NOTE: The protocol below is described for inhibitors of p300 as an example.

  1. Buffer preparation
    NOTE: See Table 1 for buffer recipes. The general steps of the ChIP protocol (e.g. buffer recipes, wash times and centrifugation times) below are modified and adapted from the manufacturer’s recommendations of a commercially available kit (see Table of Materials) and from the literature35,36.
    1. Prepare ChIP dilution buffer, nuclei swelling buffer, low salt wash buffer, high salt wash buffer, LiCl wash buffer and TE buffer. Store at 4 °C.
    2. Prepare SDS lysis buffer, 10x glycine buffer and ChIP elution buffer. Store at room temperature.
      CAUTION: Please check safety data sheet for all chemicals before making buffers to ensure proper handling.
  2. Drug treatment
    NOTE: See Supplementary Protocol (Schematic 3) for a schematic of the drug dilutions used in step 3.2.
    1. Seed MCF-7 cells in two 15 cm culture dishes and grow cells to 90% confluency in 12 mL of the complete DMEM medium. Mark the dishes for the following experimental design: Dish 1: DMSO control (reference point); Dish 2: A-485 (3 μM).
      NOTE: For culturing MCF-7 cells, use complete DMEM media and grow at 37 °C with 5% CO2. See Table 1 for complete DMEM recipe.
    2. In a sterile 15 mL conical tube, pipette 12 mL of DMEM media and pipette 6 μL of DMSO. Mix well.
    3. In a separate sterile 15 mL conical tube, pipette 12 mL of DMEM media and pipette 6 μL of A-485 (6 mM in DMSO) to get a final concentration of 3 µM A-485. Mix well.
    4. Aspirate the media from Dish 1 and 2.
    5. Add the 12 mL of diluted DMSO in DMEM (step 3.2.2.) to Dish 1.
    6. Add the 12 mL of 3 µM A-485 in DMEM (step 3.2.3.) to Dish 2.
    7. Return the cell culture dishes to the incubator and incubate for 24 h.
  3. Cell fixation
    1. Pipette 330 μL (27.5 µL per mL) of 37% formaldehyde to the complete media and gently swirl the plate to mix.
      CAUTION: Formaldehyde is toxic. Please see safety data sheet for proper handling procedures.
    2. Incubate for 10 min at room temperature.
    3. Pipette 2 mL of 10x glycine to the plate and swirl to mix.
    4. Incubate for 5 min at room temperature.
    5. After incubation, place dishes on ice and thaw an aliquot of protease inhibitor cocktail.
    6. Prepare the following solutions for both the DMSO and A-485 samples using the buffers prepared in step 3.1.
      1. 2 mL of PBS with a 1:1,000 dilution of the protease inhibitor cocktail
      2. 1 mL of nuclei swelling buffer with a 1:1,000 dilution of the protease inhibitor cocktail
      3. 0.5 mL of SDS lysis buffer with a 1:1,000 dilution of the protease inhibitor cocktail
    7. Aspirate the media from the cell culture dishes and wash the cells twice with 15 mL of cold PBS.
    8. Pipette 2 mL of PBS with the protease inhibitor cocktail (step 3.3.6.1) to the cell culture dishes. Lift the cells into solution using a cell scraper.
    9. Transfer the cell suspension to a microcentrifuge tube. Collect remaining cells with additional PBS if necessary.
    10. Spin tubes at 800 x g at 4 °C for 5 min to pellet the cells.
    11. Aspirate the supernatant and pipette 1 mL of nuclei swelling buffer with the protease inhibitor cocktail (step 3.3.6.2) to the pellet. Resuspend the pellet and incubate on ice for 10 min.
    12. Centrifuge the tubes at 2,700 x g at 4 °C for 5 min to pellet nuclei.
    13. Aspirate the supernatant and pipette 0.5 mL of SDS lysis buffer with the protease inhibitor cocktail (step 3.3.6.3) to the pellet. Resuspend the pellet and incubate on ice for 10 min.
    14. Store the chromatin samples at -80 °C until step 3.4.1 or proceed immediately to step 3.4.
  4. DNA sonication
    1. Transfer 130 µL of chromatin from DMSO sample (step 3.3.14) to two DNA sonication tubes using a pipette (130 µL each).
    2. Transfer 130 µL of chromatin from A-485 sample (step 3.3.14) to two DNA sonication tubes using a pipette (130 µL each).
    3. Sonicate the DNA to roughly 150-200 base pair fragments using the following sonicator settings: Peak Incident Power (W) of 175, Duty Factor of 10%, 200 Cycles per Burst and 430 s treatment time.
      NOTE: Sonication settings may differ between models and settings may need to be adjusted to achieve appropriate fragment size for different cell lines.
    4. Keep samples on ice after sonication.
    5. Transfer the sonicated chromatin to a 1.5 mL tube using a pipette and centrifuge at 10,000 x g at 4 °C for 10 min to pellet debris.
    6. Pipette the supernatant (contains sonicated chromatin) to a new tube and discard the debris. Sonicated chromatin can be stored at -80 °C.
  5. Chromatin immunoprecipitation (ChIP)
    NOTE: See Supplementary Protocol (Schematic 3) for a schematic of the IP groups in Step 3.5.
    1. Measure the protein content of the sonicated chromatin for the DMSO and A-485 samples from Step 3.4.6. Protein content can be measured using well-established protocols34.
      NOTE: For simplicity, this protocol will assume protein content is equal and 100 µL of sonicated chromatin will be used. Otherwise, protein content will need to be equilibrated between all samples in an equal volume.
    2. Pipette 100 µL of DMSO sonicated chromatin to two 1.5 mL tubes (100 µL each). Pipette 400 µL of ChIP dilution buffer (containing a 1:1,000 dilution of the protease inhibitor cocktail) to each tube to bring total volume up to 500 µL. Remove 5 µL of the solution from one of the tubes and store at -20 °C as DMSO Input.
    3. Pipette 100 µL of A-485 sonicated chromatin to two 1.5 mL tubes (100 µL each). Pipette 400 µL of ChIP dilution buffer (containing a 1:1000 dilution of the protease inhibitor cocktail) to each tube to bring total volume up to 500 µL. Remove 5 µL of the solution from one of the tubes and store at -20 °C as A-485 Input.
    4. Using a pipette, add the immunoprecipitation (IP) antibody (e.g. non-specific IgG control or H3K27ac specific antibody) to the corresponding tubes for the DMSO and A-485 samples: IP #1 DMSO chromatin with IgG antibody (5-10 µg); IP #2 DMSO chromatin with H3K27ac antibody (5-10 µg); IP #3 A-485 chromatin with IgG antibody (5-10 µg); IP #4 A-485 chromatin with H3K27ac antibody (5-10 µg).
    5. Add 20 µL of protein A magnetic beads to each tube. Make sure the beads are well resuspended.
    6. Rotate the samples overnight at 4 °C.
    7. Pellet the protein A magnetic beads using a magnetic separator and remove the supernatant. Do not disturb the beads.
    8. Wash the beads with 500 µL to 1 mL of the low salt wash buffer and rotate for 5 min at 4 °C. Perform a quick spin down, pellet the beads using a magnetic separator, and remove the supernatant.
    9. Wash the beads with 500 µL to 1 mL of the high salt wash buffer and rotate for 5 min at 4 °C. Perform a quick spin down, pellet the beads using a magnetic separator, and remove the supernatant.
    10. Wash the beads with 500 µL to 1 mL of the LiCl wash buffer and rotate for 5 min at 4 °C. Perform a quick spin down, pellet the beads using a magnetic separator, and remove the supernatant.
    11. Wash the beads with 500 µL to 1 mL of TE buffer and rotate for 5 min at 4 °C. Perform a quick spin down. Keep the beads in the TE buffer until step 3.5.14.
    12. Remove Input samples (from step 3.5.2 and 3.5.3) from freezer and keep on ice.
    13. Thaw an aliquot of Proteinase K.
    14. Pellet the beads using a magnetic separator and remove the TE buffer from the beads (from step 3.5.11).
    15. Add 100 µL ChIP elution buffer + 1 µL of Proteinase K to every sample, including the Input samples. Incubate samples with shaking at 62 °C for 2 h using a thermocycler.
    16. After 2 h, heat samples to 95 °C for 10 min using a thermocycler.
    17. Cool the samples to room temperature.
    18. Pellet magnetic beads using a magnetic separator and transfer supernatant (contains the DNA of interest) to a new 1.5 mL tube.
    19. Purify the DNA using a standard PCR cleanup kit.
    20. The purified DNA can be stored at -20 °C and can be used as templates in standard qPCR protocols. Follow manufacturer protocols for running qPCR.
  6. ChIP-qPCR data analysis
    NOTE: Two common methods to analyze ChIP-qPCR results are fold enrichment over the IgG antibody and the 1% Input method. An excellent template for both analysis methods is provided by a commercial source can be used to quickly calculate fold enrichment for each IP antibody/target of interest37.
    1. To calculate fold enrichment and % Input, copy and paste the ΔCt values from the qPCR data obtained for each antibody (non-specific IgG, the IP H3K27ac antibody, and the 1% Input) into the corresponding region in the analysis template and the Fold Enrichment and Yield % Input will automatically populate.

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Results

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The in vitro histone acetyltransferase (HAT) assay can be used to probe for compounds that inhibit p300 HAT activity towards a histone substrate. Figure 1A provides an experimental schematic for the HAT assay. Anacardic acid, a known HATi3,38, was utilized in this assay in a concentration range from 12.5-100 µM. At 100 µM, anacardic acid downregulates p300 catalyzed histone acetylation at Histone 3, Lysines 9 and 18 versus the contro...

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Discussion

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Lysine acetyltransferases (KATs) acetylate several lysine residues on histone tails and transcription factors to regulate gene transcription2,3. Work in the last two decades has revealed that KATs, such as CBP/p300, PCAF and GCN5, interact with oncogenic transcription factors and help drive tumor growth in several solid tumor types4,5,9,15

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Disclosures

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The authors have no conflicts of interest or disclosures to make.

Acknowledgements

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This work was supported by grants from James and Esther King Biomedical Research Program (6JK03 and 20K07), and Bankhead-Coley Cancer Research Program (4BF02 and 6BC03), Florida Department of Health, Florida Breast Cancer Foundation, and UF Health Cancer Center. Additionally, we would like to thank Dr. Zachary Osking and Dr. Andrea Lin for their support during the publication process.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1.5 ml tubeFisher Scientific05-408-129For all methods
10 cm dishSarstedt AG & Co.83.3902For cell culture of MCF-7 cells
10 ul tipsFisher Scientific02-707-454For all Methods
1000 ul tipsCorning4846For all Methods
10X Glycine bufferFor Method 3. See Table 1 for recipe.
10X Running BufferFor Methods 1 and 2. See Table 1 for recipe.
10X TBSTFor Methods 1 and 2. See Table 1 for recipe.
12 well plateCorning3513For Method 2
15 cm dishSarstedt AG & Co.83.3903For Method 3
15 ml conical tubeSanta Cruz Biotechnologysc-200249For Methods 2 and 3
1X TBST with 5% milk and 0.02% Sodium AzideFor Methods 1 and 2. Can be used to dilute primary antibodies that will be used more than once. Allows for short-term storage of primary antibody dilutions. Do not use for secondary antibody diluton. CAUTION: Sodium Azide is toxic.
1X TBST with 5% milkFor Methods 1 and 2. Used to block PVDF membrane and for antibody diltions. See Table 1 for recipe.
200 ul tipsCorning4844For all Methods
2-mercaptoethanolSigma-AldrichM3148for SDS sample buffer preparation
4-20% polyacrylamide gelThermo Fisher: InvitrogenXP04205BOXFor Methods 1 and 2
5X Assay bufferFor Method 1. See Table 1 for recipe.
5X Passive lysis bufferFor Method 2. See Table 1 for recipe.
6X Sodium Dodecyl Sulfate (SDS)For Methods 1 and 2. See Table 1 for recipe.
A-485MedChemExpressHY-107455CBP/p300 Inhbitor for use in Methods 2 and 3. Dissolved in DMSO.
Acetyl-CBP(K1535)/p300(K1499) antibodyCell Signaling Technology4771For Method 1
Acetyl-CoASigma-AldrichA2056for use in Method 1
Acetyl-Histone H3 (Lys 27) antibody (H3K27ac)Cell Signaling TechnologyCST 8173antoibodies for H3K27ac for immunoblots and ChIP
Acetyl-Histone H3 (Lys18) antibody (H3K18ac)Cell Signaling TechnologyCST 9675antoibodies for H3K18ac for immunoblots and ChIP
alpha tubulin antibodyMillipore SigmaT5168For Method 2. Dilute 1:20,000
Anacardic acidCayman Chemical13144For Method 1
anti-mouse IgG HRP linked secondary antibodyCell Signaling Technology7076For Methods 1 and 2. Dilute 1:10,000
anti-rabbit IgG secondary antibodyJackson ImmunoResearch711-035-152For Methods 1 and 2. Dilute 1:10,000 to 1:20,000
Autoradiography filmMIDSCIBX810For Methods 1 and 2
Belly Dancer Rotating PlatformStovall Life Science Incorporatednot availableFor Methods 1 and 2
Bovine Calf Serum (BCS)HyCloneSH30072.03cell culture media
Bovine Serum Albumin (BSA)Sigma-AldrichA2153for buffer preparation
Bromophenol BlueSigma-AldrichB0126for SDS sample buffer preparation
CDTASpectrum Chemical125572-95-4For buffer preparation
cell scraperMillipore SigmaCLS3010For Method 3
ChIP dilution bufferFor Method 3. See Table 1 for recipe.
ChIP Elution BufferFor Method 3. See Table 1 for recipe.
Complete DMEM for MCF-7 CellsFor Methods 2 and 3. See Table 1 for recipe.
Covaris 130 µl microTUBECovaris520045Sonication tube for use with Covaris S220 in Method 3
Covaris S220 Focused-ultrasonicatorCovarisS220DNA sonicator for use in Method 3
Dimethyl sulfoxide (DMSO)Sigma-Aldrich41639for drug dilution and vehicle control treatment
DL-Dithiothreitol (DTT)Sigma-Aldrich43815for SDS sample buffer preparation
DMEMCorning10-013-CVcell culture media
EDTAFisher ScientificBP120-1for buffer preparation
Example transfer tank and transfer apparatusBio-rad1704070For Methods 1 and 2
EZ-Magna ChIP A/G Chromatin Immunoprecipitation KitMillipore Sigma17-10086For Method 3
FK228 (Romidepsin)Cayman Chemical128517-07-7HDAC Inhibitor for use in Method 2
Formaldehyde solutionSigma-AldrichF8775for cell fixation
glycerolFisher ScientificBP229-1For buffer preparation
glycineSigma-AldrichG7126for buffer preparation
HEPESSigma-Aldrich54457for buffer preparation
High salt wash bufferFor Method 3
IGEPAL (NP-40)Sigma-AldrichI3021for buffer preparation
Immobilon Chemiluminescent HRP SubstrateMillipore SigmaWBKLS0500For Methods 1 and 2
KClFisher ScientificBP366-500for buffer preparation
LiClSigma-AldrichL9650For buffer preparation
LiCl wash bufferFor Method 3. See Table 1 for recipe.
Low salt wash bufferFor Method 3. See Table 1 for recipe.
Magnetic SeparatorPromegaZ5341For use in Method 3
MethanolSigma-Aldrich494437For buffer preparation
Mini gel tankInvitrogenA25977For Methods 1 and 2
MS-275 (Entinostat)Cayman Chemical209783-80-2HDAC Inhibitor for use in Method 2. Dissolved in DMSO.
NaClFisher Scientific7647-14-5for buffer preparation
NaOHFisher ScientificS318-100for buffer preparation in Methods 1 and 2
Normal Rabbit IgGBethyl LaboratoriesP120-101Control rabbit antibody for use in Method 3
Nuclei swelling bufferFor Method 3. See Table 1 for recipe.
PCR Cleanup KitQiagen28104For use in Method 3
Penicillin/Streptomycin 100XCorning30-002-CIcell culture media
Phosphate-buffered saline (PBS)Corning21-040-CVFor Methods 2 and 3
PIPESSigma-Aldrich80635for buffer preparation
powdered milkNestle CarnationFor Methods 1 and 2
Power Pac 200 for western blot transferBio-radFor Methods 1 and 2
Power Pac 3000 for SDS gel runningBio-radFor Methods 1 and 2
Prestained Protein LadderThermo Fisher26616For Methods 1 and 2
Protease Inhibitor CocktailSigma-AldrichPI8340for use in Method 3
Protein A Magentic BeadsNew England BioLabsS1425SFor use in Method 3
Proteinase KNew England BioLabsP8107SFor use in Method 3
PTC-100 Programmable Thermal ControllerMJ Research Inc.PTC-100For Method 1
PVDF Transfer MembraneMillipore SigmaIEVH00005For Methods 1 and 2
Recombinant H3.1New England BioLabsM2503Sfor use in Method 1
Recombinant p300ENZO Life SciencesBML-SE451-0100for use in Method 1
SAHA (Vorinostat)Cayman Chemical149647-78-9HDAC Inhibitor for use in Method 2
SDS lysis bufferFor Method 3. See Table 1 for recipe.
Sodium AzideFisher Scientific26628-22-8For Methods 1 and 2. CAUTION: Sodium Azide is toxic. See SDS for proper handling.
Sodium BicarbonateFisher ScientificS233-500for buffer preparation
Sodium deoxycholateSigma-AldrichD6750for buffer preparation
Sodium dodecyl sulfate (SDS)Sigma-Aldrich71725for SDS sample buffer preparation
Standard HeatblockVWR Scientific ProductsMPN: 949030For Methods 1 and 2
Table top centrifugeEppendorf5417RFor all methods
TE bufferFor Method 3. See Table 1 for recipe.
Transfer bufferFor Methods 1 and 2. See Table 1 for recipe.
Trichostatin ACayman Chemical58880-19-6HDAC Inhibitor for use in Method 2
TrisFisher ScientificBP152-5for buffer preparation
Triton X-100Sigma-AldrichT8787for buffer preparation
Tween 20Sigma-Aldrich9005-64-5for buffer preparation in Methods 1 and 2
X-ray film processorKonica Minolta Medical & Graphic, Inc.SRX-101AFor Methods 1 and 2

References

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  1. Simon, R. P., Robaa, D., Alhalabi, Z., Sippl, W., Jung, M. KATching-Up on Small Molecule Modulators of Lysine Acetyltransferases. Journal of Medicinal Chemistry. 59 (4), 1249-1270 (2016).
  2. Weinert, B. T., et al. Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome. Cell. 174 (1), 231-244 (2018).
  3. Dancy, B. M., Cole, P. A. Protein lysine acetylation by p300/CBP. Chemical Reviews. 115 (6), 2419-2452 (2015).
  4. Lasko, L. M., et al. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours. Nature. 550 (7674), 128-132 (2017).
  5. Yang, H., et al. Small-molecule inhibitors of acetyltransferase p300 identified by high-throughput screening are potent anticancer agents. Molecular Cancer Therapeutics. 12 (5), 610-620 (2013).
  6. Baell, J. B., et al. Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth. Nature. 560 (7717), 253-257 (2018).
  7. Coffey, K., et al. Characterisation of a Tip60 specific inhibitor, NU9056, in prostate cancer. Plos One. 7 (10), 45539(2012).
  8. Majaz, S., et al. Histone acetyl transferase GCN5 promotes human hepatocellular carcinoma progression by enhancing AIB1 expression. Cell & Bioscience. 6, 47(2016).
  9. Jin, L., et al. Therapeutic Targeting of the CBP/p300 Bromodomain Blocks the Growth of Castration-Resistant Prostate Cancer. Cancer Research. 77 (20), 5564-5575 (2017).
  10. Raisner, R., et al. Enhancer Activity Requires CBP/P300 Bromodomain-Dependent Histone H3K27 Acetylation. Cell Reports. 24 (7), 1722-1729 (2018).
  11. Jin, Q., et al. Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation. The EMBO Journal. 30 (2), 249-262 (2011).
  12. Pradeepa, M. M. Causal role of histone acetylations in enhancer function. Transcription. 8 (1), 40-47 (2017).
  13. Creyghton, M. P., et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proceedings of the National Academy of Sciences of the United States of America. 107 (50), 21931-21936 (2010).
  14. Wang, Z., et al. Combinatorial patterns of histone acetylations and methylations in the human genome. Nature Genetics. 40 (7), 897-903 (2008).
  15. Ianculescu, I., Wu, D. Y., Siegmund, K. D., Stallcup, M. R. Selective roles for cAMP response element-binding protein binding protein and p300 protein as coregulators for androgen-regulated gene expression in advanced prostate cancer cells. The Journal of Biological Chemistry. 287 (6), 4000-4013 (2012).
  16. Zhong, J., et al. p300 acetyltransferase regulates androgen receptor degradation and PTEN-deficient prostate tumorigenesis. Cancer Research. 74 (6), 1870-1880 (2014).
  17. Fu, M., et al. p300 and p300/cAMP-response element-binding protein-associated factor acetylate the androgen receptor at sites governing hormone-dependent transactivation. The Journal of Biological Chemistry. 275 (27), 20853-20860 (2000).
  18. Emami, K. H., et al. A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected]. Proceedings of the National Academy of Sciences of the United States of America. 101 (34), 12682-12687 (2004).
  19. Stimson, L., et al. Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity. Molecular Cancer Therapeutics. 4 (10), 1521-1532 (2005).
  20. Modak, R., et al. Probing p300/CBP associated factor (PCAF)-dependent pathways with a small molecule inhibitor. ACS Chemical Biology. 8 (6), 1311-1323 (2013).
  21. Dahlin, J. L., et al. Assay interference and off-target liabilities of reported histone acetyltransferase inhibitors. Nature Communications. 8 (1), 1527(2017).
  22. Liao, D. Identification and characterization of small-molecule inhibitors of lysine acetyltransferases. Methods in Molecular Biology. 1238, 539-548 (2015).
  23. Gardberg, A. S., et al. Make the right measurement: Discovery of an allosteric inhibition site for p300-HAT. Structural dynamics. 6 (5), Melville, N.Y. 054702(2019).
  24. Ceccacci, E., Minucci, S. Inhibition of histone deacetylases in cancer therapy: lessons from leukaemia. British Journal of Cancer. 114 (6), 605-611 (2016).
  25. Tashiro, E., Tsuchiya, A., Imoto, M. Functions of cyclin D1 as an oncogene and regulation of cyclin D1 expression. Cancer Science. 98 (5), 629-635 (2007).
  26. Collas, P. The current state of chromatin immunoprecipitation. Molecular Biotechnology. 45 (1), 87-100 (2010).
  27. JoVE. JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. The Western Blot. JoVE. , Cambridge, MA. (2020).
  28. Gooderham, K. Transfer techniques in protein blotting. Methods in Molecular Biology. 1, 165-178 (1984).
  29. Westermeier, R. Electrophoresis in practice: A guide to methods and applications of DNA and protein separations. , Wiley. (2004).
  30. Towbin, H., Staehelin, T., Gordon, J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America. 76 (9), 4350-4354 (1979).
  31. Kurien, B. T., Scofield, R. H. Nonelectrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens to obtain 12 immunoblots. Methods in Molecular Biology. 536, 55-65 (2009).
  32. Kyhse-Andersen, J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. Journal of Biochemical and Biophysical Methods. 10 (3-4), 203-209 (1984).
  33. Tovey, E. R., Baldo, B. A. Comparison of semi-dry and conventional tank-buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes. Electrophoresis. 8 (9), 384-387 (1987).
  34. Greenfield, E. A. Protein Quantitation. Cold Spring Harbor Protocols. 2018 (6), (2018).
  35. Barrow, J. J., Masannat, J., Bungert, J. Neutralizing the function of a β-globin-associated cis-regulatory DNA element using an artificial zinc finger DNA-binding domain. Proceedings of the National Academy of Sciences of the United States of America. 109 (44), 17948-17953 (2012).
  36. Leach, K. M., et al. Characterization of the human beta-globin downstream promoter region. Nucleic Acids Research. 31 (4), 1292-1301 (2003).
  37. ChIP-qPCR and Data Analysis. Sigma-Aldrich. , Available from: https://www.sigmaaldrich.com/technical-documents/articles/biology/chip-qpcr-data-analysis.html (2020).
  38. Balasubramanyam, K., Swaminathan, V., Ranganathan, A., Kundu, T. K. Small molecule modulators of histone acetyltransferase p300. The Journal of Biological Chemistry. 278 (21), 19134-19140 (2003).
  39. Using ImageJ to quantify blots. Diamantina Institute - University of Queensland. , Available from: https://di.uq.edu.au/community-and-alumni/sparq-ed-services/using-imagej-quantify-blots (2020).
  40. Kalkhoven, E., et al. Loss of CBP acetyltransferase activity by PHD finger mutations in Rubinstein-Taybi syndrome. Human Molecular Genetics. 12 (4), 441-450 (2003).
  41. Ortega, E., et al. Transcription factor dimerization activates the p300 acetyltransferase. Nature. 562 (7728), 538-544 (2018).
  42. Koutelou, E., Hirsch, C. L., Dent, S. Y. R. Multiple faces of the SAGA complex. Current Opinion in Cell Biology. 22 (3), 374-382 (2010).
  43. Wang, Y., et al. Identification of histone deacetylase inhibitors with benzoylhydrazide scaffold that selectively inhibit class I histone deacetylases. Chemistry & Biology. 22 (2), 273-284 (2015).
  44. Hu, E., et al. Identification of novel isoform-selective inhibitors within class I histone deacetylases. The Journal of Pharmacology and Experimental Therapeutics. 307 (2), 720-728 (2003).
  45. Lee, B. I., et al. MS-275, a histone deacetylase inhibitor, selectively induces transforming growth factor beta type II receptor expression in human breast cancer cells. Cancer Research. 61 (3), 931-934 (2001).
  46. Leus, N. G. J., et al. HDAC1-3 inhibitor MS-275 enhances IL10 expression in RAW264.7 macrophages and reduces cigarette smoke-induced airway inflammation in mice. Scientific Reports. 7, 45047(2017).
  47. Rossi, L., et al. HDAC1 inhibition by MS-275 in mesothelial cells limits cellular invasion and promotes MMT reversal. Scientific Reports. 8 (1), 8492(2018).

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Histone Acetyltransferase InhibitorsHAT AssayChHAI AssayChIP qPCRHistone AcetylationImmunoblottingProteinase KMagnetic BeadsqPCR AnalysisEpigenetic Regulation

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