Assays for Validating Histone Acetyltransferase Inhibitors

Summary

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

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

Lysine acetyltransferases (KATs) catalyze the acetylation of lysine residues on both histone and non-histone proteins^1,2,3,4. Recent research reveals that KATs and their acetyltransferase function can promote solid tumor growth^4,5,6,7,8,9. For example, CREB-binding protein (CBP)/p300 are two paralogous KATs that regulate numerous signaling pathwa....

Protocol

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 a.......

Discussion

Lysine acetyltransferases (KATs) acetylate several lysine residues on histone tails and transcription factors to regulate gene transcription^2,3. 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 types^4,5,9,15

Materials

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