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.

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

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</s...

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.

<table><tbody><tr><td>1.5 ml tube</td><td>Fisher Scientific</td><td>05-408-129</td><td>For all methods</td></tr><tr><td>10 cm dish</td><td>Sarstedt AG &amp;amp; Co.</td><td>83.3902</td><td>For cell culture of MCF-7 cells</td></tr><tr><td>10 ul tips</td><td>Fisher Scientific</td><td>02-707-454</td><td>For all Methods</td></tr><tr><td>1000 ul tips</td><td>Corning</td><td>4846</td><td>For all Methods</td></tr><tr><td>10X Glycine buffer</td><td></td><td></td><td>For Method 3. See Table 1 for recipe.</td></tr><tr><td>10X Running Buffer</td><td></td><td></td><td>For Methods 1 and 2. See Table 1 for recipe.</td></tr><tr><td>10X TBST</td><td></td><td></td><td>For Methods 1 and 2. See Table 1 for recipe.</td></tr><tr><td>12 well plate</td><td>Corning</td><td>3513</td><td>For Method 2</td></tr><tr><td>15 cm dish</td><td>Sarstedt AG &amp;amp; Co.</td><td>83.3903</td><td>For Method 3</td></tr><tr><td>15 ml conical tube</td><td>Santa Cruz Biotechnology</td><td>sc-200249</td><td>For Methods 2 and 3</td></tr><tr><td>1X TBST with 5% milk and 0.02% Sodium Azide</td><td></td><td></td><td>For 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.</td></tr><tr><td>1X TBST with 5% milk</td><td></td><td></td><td>For Methods 1 and 2. Used to block PVDF membrane and for antibody diltions. See Table 1 for recipe.</td></tr><tr><td>200 ul tips</td><td>Corning</td><td>4844</td><td>For all Methods</td></tr><tr><td>2-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td>for SDS sample buffer preparation</td></tr><tr><td>4-20% polyacrylamide gel</td><td>Thermo Fisher: Invitrogen</td><td>XP04205BOX</td><td>For Methods 1 and 2</td></tr><tr><td>5X Assay buffer</td><td></td><td></td><td>For Method 1. See Table 1 for recipe.</td></tr><tr><td>5X Passive lysis buffer</td><td></td><td></td><td>For Method 2. See Table 1 for recipe.</td></tr><tr><td>6X Sodium Dodecyl Sulfate (SDS)</td><td></td><td></td><td>For Methods 1 and 2. See Table 1 for recipe.</td></tr><tr><td>A-485</td><td>MedChemExpress</td><td>HY-107455</td><td>CBP/p300 Inhbitor for use in Methods 2 and 3. Dissolved in DMSO.</td></tr><tr><td>Acetyl-CBP(K1535)/p300(K1499) antibody</td><td>Cell Signaling Technology</td><td>4771</td><td>For Method 1</td></tr><tr><td>Acetyl-CoA</td><td>Sigma-Aldrich</td><td>A2056</td><td>for use in Method 1</td></tr><tr><td>Acetyl-Histone H3 (Lys 27) antibody (H3K27ac)</td><td>Cell Signaling Technology</td><td>CST 8173</td><td>antoibodies for H3K27ac for immunoblots and ChIP</td></tr><tr><td>Acetyl-Histone H3 (Lys18) antibody (H3K18ac)</td><td>Cell Signaling Technology</td><td>CST 9675</td><td>antoibodies for H3K18ac for immunoblots and ChIP</td></tr><tr><td>alpha tubulin antibody</td><td>Millipore Sigma</td><td>T5168</td><td>For Method 2. Dilute 1:20,000</td></tr><tr><td>Anacardic acid</td><td>Cayman Chemical</td><td>13144</td><td>For Method 1</td></tr><tr><td>anti-mouse IgG HRP linked secondary antibody</td><td>Cell Signaling Technology</td><td>7076</td><td>For Methods 1 and 2. Dilute 1:10,000</td></tr><tr><td>anti-rabbit IgG secondary antibody</td><td>Jackson ImmunoResearch</td><td>711-035-152</td><td>For Methods 1 and 2. Dilute 1:10,000 to 1:20,000</td></tr><tr><td>Autoradiography film</td><td>MIDSCI</td><td>BX810</td><td>For Methods 1 and 2</td></tr><tr><td>Belly Dancer Rotating Platform</td><td>Stovall Life Science Incorporated</td><td>not available</td><td>For Methods 1 and 2</td></tr><tr><td>Bovine Calf Serum (BCS)</td><td>HyClone</td><td>SH30072.03</td><td>cell culture media</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A2153</td><td>for buffer preparation</td></tr><tr><td>Bromophenol Blue</td><td>Sigma-Aldrich</td><td>B0126</td><td>for SDS sample buffer preparation</td></tr><tr><td>CDTA</td><td>Spectrum Chemical</td><td>125572-95-4</td><td>For buffer preparation</td></tr><tr><td>cell scraper</td><td>Millipore Sigma</td><td>CLS3010</td><td>For Method 3</td></tr><tr><td>ChIP dilution buffer</td><td></td><td></td><td>For Method 3. See Table 1 for recipe.</td></tr><tr><td>ChIP Elution Buffer</td><td></td><td></td><td>For Method 3. See Table 1 for recipe.</td></tr><tr><td>Complete DMEM for MCF-7 Cells</td><td></td><td></td><td>For Methods 2 and 3. See Table 1 for recipe.</td></tr><tr><td>Covaris 130 &amp;micro;l microTUBE</td><td>Covaris</td><td>520045</td><td>Sonication tube for use with Covaris S220 in Method 3</td></tr><tr><td>Covaris S220 Focused-ultrasonicator</td><td>Covaris</td><td>S220</td><td>DNA sonicator for use in Method 3</td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>41639</td><td>for drug dilution and vehicle control treatment</td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>43815</td><td>for SDS sample buffer preparation</td></tr><tr><td>DMEM</td><td>Corning</td><td>10-013-CV</td><td>cell culture media</td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP120-1</td><td>for buffer preparation</td></tr><tr><td>Example transfer tank and transfer apparatus</td><td>Bio-rad</td><td>1704070</td><td>For Methods 1 and 2</td></tr><tr><td>EZ-Magna ChIP A/G Chromatin Immunoprecipitation Kit</td><td>Millipore Sigma</td><td>17-10086</td><td>For Method 3</td></tr><tr><td>FK228 (Romidepsin)</td><td>Cayman Chemical</td><td>128517-07-7</td><td>HDAC Inhibitor for use in Method 2</td></tr><tr><td>Formaldehyde solution</td><td>Sigma-Aldrich</td><td>F8775</td><td>for cell fixation</td></tr><tr><td>glycerol</td><td>Fisher Scientific</td><td>BP229-1</td><td>For buffer preparation</td></tr><tr><td>glycine</td><td>Sigma-Aldrich</td><td>G7126</td><td>for buffer preparation</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>54457</td><td>for buffer preparation</td></tr><tr><td>High salt wash buffer</td><td></td><td></td><td>For Method 3</td></tr><tr><td>IGEPAL (NP-40)</td><td>Sigma-Aldrich</td><td>I3021</td><td>for buffer preparation</td></tr><tr><td>Immobilon Chemiluminescent HRP Substrate</td><td>Millipore Sigma</td><td>WBKLS0500</td><td>For Methods 1 and 2</td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>BP366-500</td><td>for buffer preparation</td></tr><tr><td>LiCl</td><td>Sigma-Aldrich</td><td>L9650</td><td>For buffer preparation</td></tr><tr><td>LiCl wash buffer</td><td></td><td></td><td>For Method 3. See Table 1 for recipe.</td></tr><tr><td>Low salt wash buffer</td><td></td><td></td><td>For Method 3. See Table 1 for recipe.</td></tr><tr><td>Magnetic Separator</td><td>Promega</td><td>Z5341</td><td>For use in Method 3</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>494437</td><td>For buffer preparation</td></tr><tr><td>Mini gel tank</td><td>Invitrogen</td><td>A25977</td><td>For Methods 1 and 2</td></tr><tr><td>MS-275 (Entinostat)</td><td>Cayman Chemical</td><td>209783-80-2</td><td>HDAC Inhibitor for use in Method 2. Dissolved in DMSO.</td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>7647-14-5</td><td>for buffer preparation</td></tr><tr><td>NaOH</td><td>Fisher Scientific</td><td>S318-100</td><td>for buffer preparation in Methods 1 and 2</td></tr><tr><td>Normal Rabbit IgG</td><td>Bethyl Laboratories</td><td>P120-101</td><td>Control rabbit antibody for use in Method 3</td></tr><tr><td>Nuclei swelling buffer</td><td></td><td></td><td>For Method 3. See Table 1 for recipe.</td></tr><tr><td>PCR Cleanup Kit</td><td>Qiagen</td><td>28104</td><td>For use in Method 3</td></tr><tr><td>Penicillin/Streptomycin 100X</td><td>Corning</td><td>30-002-CI</td><td>cell culture media</td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Corning</td><td>21-040-CV</td><td>For Methods 2 and 3</td></tr><tr><td>PIPES</td><td>Sigma-Aldrich</td><td>80635</td><td>for buffer preparation</td></tr><tr><td>powdered milk</td><td>Nestle Carnation</td><td></td><td>For Methods 1 and 2</td></tr><tr><td>Power Pac 200 for western blot transfer</td><td>Bio-rad</td><td></td><td>For Methods 1 and 2</td></tr><tr><td>Power Pac 3000 for SDS gel running</td><td>Bio-rad</td><td></td><td>For Methods 1 and 2</td></tr><tr><td>Prestained Protein Ladder</td><td>Thermo Fisher</td><td>26616</td><td>For Methods 1 and 2</td></tr><tr><td>Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>PI8340</td><td>for use in Method 3</td></tr><tr><td>Protein A Magentic Beads</td><td>New England BioLabs</td><td>S1425S</td><td>For use in Method 3</td></tr><tr><td>Proteinase K</td><td>New England BioLabs</td><td>P8107S</td><td>For use in Method 3</td></tr><tr><td>PTC-100 Programmable Thermal Controller</td><td>MJ Research Inc.</td><td>PTC-100</td><td>For Method 1</td></tr><tr><td>PVDF Transfer Membrane</td><td>Millipore Sigma</td><td>IEVH00005</td><td>For Methods 1 and 2</td></tr><tr><td>Recombinant H3.1</td><td>New England BioLabs</td><td>M2503S</td><td>for use in Method 1</td></tr><tr><td>Recombinant p300</td><td>ENZO Life Sciences</td><td>BML-SE451-0100</td><td>for use in Method 1</td></tr><tr><td>SAHA (Vorinostat)</td><td>Cayman Chemical</td><td>149647-78-9</td><td>HDAC Inhibitor for use in Method 2</td></tr><tr><td>SDS lysis buffer</td><td></td><td></td><td>For Method 3. See Table 1 for recipe.</td></tr><tr><td>Sodium Azide</td><td>Fisher Scientific</td><td>26628-22-8</td><td>For Methods 1 and 2. CAUTION: Sodium Azide is toxic. See SDS for proper handling.</td></tr><tr><td>Sodium Bicarbonate</td><td>Fisher Scientific</td><td>S233-500</td><td>for buffer preparation</td></tr><tr><td>Sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td>for buffer preparation</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma-Aldrich</td><td>71725</td><td>for SDS sample buffer preparation</td></tr><tr><td>Standard Heatblock</td><td>VWR Scientific Products</td><td>MPN: 949030</td><td>For Methods 1 and 2</td></tr><tr><td>Table top centrifuge</td><td>Eppendorf</td><td>5417R</td><td>For all methods</td></tr><tr><td>TE buffer</td><td></td><td></td><td>For Method 3. See Table 1 for recipe.</td></tr><tr><td>Transfer buffer</td><td></td><td></td><td>For Methods 1 and 2. See Table 1 for recipe.</td></tr><tr><td>Trichostatin A</td><td>Cayman Chemical</td><td>58880-19-6</td><td>HDAC Inhibitor for use in Method 2</td></tr><tr><td>Tris</td><td>Fisher Scientific</td><td>BP152-5</td><td>for buffer preparation</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T8787</td><td>for buffer preparation</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>9005-64-5</td><td>for buffer preparation in Methods 1 and 2</td></tr><tr><td>X-ray film processor</td><td>Konica Minolta Medical &amp;amp; Graphic, Inc.</td><td>SRX-101A</td><td>For Methods 1 and 2</td></tr></tbody></table>

assays for validating histone acetyltransferase inhibitors

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.

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 &#181;M. At 100 &#181;M, anacardic acid downregulates p300 catalyzed histone acetylation at Histone 3, Lysines 9 and 18 versus ...

Watch this Scientific Journal Video about Assays for Validating Histone Acetyltransferase Inhibitors at JoVE.com

Assays for Validating Histone Acetyltransferase Inhibitors

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.

Lysine acetyltransferases (KATs) catalyze acetylation of lysine residues on histones and other proteins to regulate chromatin dynamics and gene ...

assays-for-validating-histone-acetyltransferase-inhibitors

Research

JoVE Journal

Cancer Research

6.5K Views.  University of Florida College of Medicine. 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.

Video: Assays for Validating Histone Acetyltransferase Inhibitors

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased understanding of the molecular events that give rise to glioblastomas, this cancer still remains highly refractory to conventional treatment. Surgical resection of high grade brain tumors is rarely complete due to the highly infiltrative nature of glioblastoma cells. Therapeutic approaches which attenuate glioblastoma cell invasion therefore is an attractive option. Our laboratory and others have shown that tumor associated macrophages and microglia (resident brain macrophages) strongly stimulate glioblastoma invasion. The protocol described in this paper is used to model glioblastoma-macrophage/microglia interaction using in vitro culture assays. This approach can greatly facilitate the development and/or discovery of drugs that disrupt the communication with the macrophages that enables this malignant behavior. We have established two robust coculture invasion assays where microglia/macrophages stimulate glioma cell invasion by 5 - 10 fold. Glioblastoma cells labelled with a fluorescent marker or constitutively expressing a fluorescent protein are plated without and with macrophages/microglia on matrix-coated polycarbonate chamber inserts or embedded in a three dimensional matrix. Cell invasion is assessed by using fluorescent microscopy to image and count only invasive cells on the underside of the filter. Using these assays, several pharmacological inhibitors (JNJ-28312141, PLX3397, Gefitinib, and Semapimod), have been identified which block macrophage/microglia stimulated glioblastoma invasion.

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Analysis of Histone Antibody Specificity with Peptide Microarrays

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The mass production and distribution of antibodies specific to histone PTMs has greatly facilitated research on these marks. As histone PTM antibodies are key reagents for many chromatin biochemistry applications, rigorous analysis of antibody specificity is necessary for accurate data interpretation and continued progress in the field. This protocol describes an integrated pipeline for the design, fabrication and use of peptide microarrays for profiling the specificity of histone antibodies. The design and analysis aspects of this procedure are facilitated by ArrayNinja, an open-source and interactive software package we recently developed to streamline the customization of microarray print formats. This pipeline has been used to screen a large number of commercially available and widely used histone PTM antibodies, and data generated from these experiments are freely available through an online and expanding Histone Antibody Specificity Database. Beyond histones, the general methodology described herein can be applied broadly to the analysis of PTM-specific antibodies.

This manuscript describes methods for applying peptide microarray technology to specificity profiling of antibodies that recognize histones and their post-translational modifications.

Post-translational modifications (PTMs) on histone proteins are widely studied for their roles in regulating chromatin structure and gene expression. The ...

Biochemistry

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

Chemistry

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of fungal secondary metabolites. Inhibitor screening, however, requires purified enzyme activities. Since class 1 deacetylases exert their function as multiprotein complexes, they are usually not active when expressed as single polypeptides in bacteria. Therefore, endogenous complexes need to be isolated, which, when conventional techniques like ion exchange and size exclusion chromatography are applied, is laborious and time consuming. Tandem affinity purification has been developed as a tool to enrich multiprotein complexes from cells and thus turned out to be ideal for the isolation of endogenous enzymes. Here we provide a detailed protocol for the single-step enrichment of active RpdA complexes via the first purification step of C-terminally TAP-tagged RpdA from Aspergillus nidulans. The purified complexes may then be used for the subsequent inhibitor screening applying a deacetylase assay. The protein enrichment together with the enzymatic activity assay can be completed within two days.

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets to treat fungal infections. Here we present a protocol for the specific enrichment of TAP-tagged RpdA combined with an HDAC activity assay that allows in vitro efficacy testing of histone deacetylase inhibitors.

Class 1 histone deacetylases (HDACs) like RpdA have gained importance as potential targets for treatment of fungal infections and for genome mining of ...

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array of diverse posttranslational modifications (PTMs) that mostly occur in the amino termini of histone proteins (i.e., histone tail) and regulate the accessibility and functional state of the underlying DNA. Histone tails extend from the core of the nucleosome and are subject to the addition of acetyl groups by histone acetyltransferases (HATs) and the removal of acetyl groups by histone deacetylases (HDACs) during cellular growth and differentiation. Specific acetylation patterns on lysine (K) residues on histone tails determine a dynamic homeostasis between transcriptionally active or transcriptionally repressed chromatin by (1) influencing the core histone assembly and (2) recruiting synergistic or antagonistic chromatin-associated proteins to the transcription site. The fundamental regulatory mechanism of the complex nature of histone tail PTMs influences the majority of chromatin-templated processes and results in changes in cell maturation and differentiation in both normal and pathological development. The goal of the current report is to provide novices with an efficient method to purify core histone proteins from cells and brain tissue and to reliably quantify acetylation marks on histones H3 and H4.

The purpose of this article is to provide a comprehensive, systematic guide to the efficient purification of histones H3 and H4 and the quantification of acetylated histone residues.

In all eukaryotic organisms, chromatin, the physiological template of all genetic information, is essential for heredity. Chromatin is subject to an array ...

Assays for Validating Histone Acetyltransferase Inhibitors

Summary

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Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

Single-Step Enrichment of a TAP-Tagged Histone Deacetylase of the Filamentous Fungus Aspergillus nidulans for Enzymatic Activity Assay

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

Purification of H3 and H4 Histone Proteins and the Quantification of Acetylated Histone Marks in Cells and Brain Tissue