Sequential Salt Extractions for the Analysis of Bulk Chromatin Binding Properties of Chromatin Modifying Complexes

Summary

Sequential salt extraction of chromatin bound proteins is a useful tool for determining the binding properties of large protein complexes. This method can be employed to evaluate the role of individual subunits or domains in the overall affinity of a protein complex to bulk chromatin.

Abstract

Elucidation of the binding properties of chromatin-targeting proteins can be very challenging due to the complex nature of chromatin and the heterogeneous nature of most mammalian chromatin-modifying complexes. In order to overcome these hurdles, we have adapted a sequential salt extraction (SSE) assay for evaluating the relative binding affinities of chromatin-bound complexes. This easy and straightforward assay can be used by non-experts to evaluate the relative difference in binding affinity of two related complexes, the changes in affinity of a complex when a subunit is lost or an individual domain is inactivated, and the change in binding affinity after alterations to the chromatin landscape. By sequentially re-suspending bulk chromatin in increasing amounts of salt, we are able to profile the elution of a particular protein from chromatin. Using these profiles, we are able to determine how alterations in a chromatin-modifying complex or alterations to the chromatin environment affect binding interactions. Coupling SSE with other in vitro and in vivo assays, we can determine the roles of individual domains and proteins on the functionality of a complex in a variety of chromatin environments.

Introduction

DNA regulation in eukaryotic cells is an intricate and sophisticated system that is tightly controlled by an assortment of proteins that coordinate responses to intracellular and extracellular stimuli. DNA is wrapped around histone octamers to form nucleosomes, which can be loosely distributed along DNA or compacted into tight coils¹. This structural arrangement of DNA and histones is known as chromatin, which is regulated by a network of proteins that read, write, and erase post translational modifications (PTM) on histones². Some histone PTMs, such as acetylation, change the charge of the amino acid they are deposited ....

Protocol

1. Preparations

1. Prepare 100 mL of hypotonic solution Buffer A: 0.3 M sucrose, 60 mM KCl, 60 mM Tris pH 8.0, 2 mM EDTA, and 0.5% NP-40. Store at 4 ºC.
   NOTE: Some cell lines, such as HEK293T, require less stringent lysing conditions. If nuclei lyse easily, use Modified Buffer A: 25 mM HEPES pH 7.6, 25 mM KCl, 5 mM MgCl₂, 0.05 mM EDTA, 0.1% NP-40, and 10% glycerol.
2. Prepare a 250 mL stock solution of 2x mRIPA solution: 100 mM Tris pH 8.0, 2% NP-40, and 0.5% sodium deoxycholate.
3. Prepare a 100 mL stock solution of 5 M NaCl.
4. Using the 2x mRIPA and 5 M NaCl solutions, prepare 50 mL of 1x mRIPA solution for each ....

Results

In this paper, we demonstrate the advantages and applications of the commonly used sequential salt extraction (SSE) method that we have adapted from the literature⁶. In Figure 1, we compare the reproducibility of SSE to extracting proteins non-sequentially by detecting the elution patterns of ARID1a and PBRM1. We consistently observe that ARID1a, a BAF subunit, elutes primarily at 200 mM NaCl and PBRM1, an exclusive PBAF subunit, elute.......

Discussion

Characterization of protein and chromatin interactions through salt extractions is a common method that has been employed for decades^14,15; however, it has not been systematically optimized before to reveal its full utility. We demonstrate how this sequential method provides a rapid and inexpensive way to distinguish changes in chromatin binding when the protein or the environment is altered. SSE is highly adaptable and optimizable, and importantly, it is technic.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sodium Chloride, Crystal 2.5 Kg AR (ACS)	Avantor/Macron	7581-06
Sucrose, Crystal 500 G AR¨ (ACS)	Avantor/Macron	8360-04
Potassium Chloride, Granular 500 G AR¨ (ACS)	Avantor/Macron	6858-04
Trizma(R) base,Primary Standard and Buffer, >=99.9% (titration), crystalline	Sigma-Aldrich	T1503-1kg
EDTA (Ethylenedinitrilo) Tetraacetic Acid, Disodium Salt, Dihydrate 125 G AR (ACS)	Avantor/Macron	4931-02
NONIDET P-40 SUBSTRATE, 100mL UN3082	Amresco	E109-100ML
HEPES Buffer Solution (1M)	Gibco	15630-080
Magnesium Chloride, 6-Hydrate, Crystal 500 G AR¨ (ACS)	Avantor/Macron	5958-04
GLYCEROL, 1L	Amresco	0854-1L
DEOXYCHOLIC ACID SODIUM SALT, 100g	Amresco	0613-100G
Eppendorf Research plus pipette	Fisher Scientific	13-690-032
Eppendorf 5424 R refrigerated microcentrifuge	Eppendorf	5424 R
Secondary mouse IgG HRP-linked	Cell Signaling	7076
Secondary rabbit IgG HRP-linked	Cell Signaling	7074
BMI-1 antibody	Millipore
PBRM1 antibody	Bethyl	A301-591A
ARID1a antibody	Santa Cruz	sc-32761
BRD4 antibody	Bethyl	A301-9852a
(+) JQ1	Cayman Chemical Company	11187
SAHA	Cayman Chemical Company	10009929
Doxorubicin	AK Scientific	25316-40-9
Dimethyl Sulfoxide (DMSO)	Macron	4948-02
Mini Trans-Blot Cell	BioRad	1703930
Mini Gel Tank	ThermoFisher	A25977
Albumin, Bovine (BSA)	Amresco	0332-100g	Used in as a 5% solution in PBS-T as blocking solution for Western blots
Bolt MOPS SDS Running Buffer (20x)	Life Technologies	B0001
Bolt LDS Sample Buffer (4x)	Life Technologies	B0007
PageRuler Plus Prestained Protein Ladder, 10 to 250 kDa	ThermoFisher	26619
Methyl Alcohol, Anhydrous	Macron	3041-10
Trypsin kEDTA, 1X	Cellgro	25-053-CI
SODIUM AZIDE, 250g UN1687	Amresco	0639-250G
SODIUM DODECYL SULFATE (SDS)500g UN1325	Amresco	0227-500G
Glycine,for electrophoresis, >=99%	Sigma-Aldrich	G8898-1kg
BigTop Microcentrifuge Tubes, Polypropylene	VWR	20170-333
1000 µL Pipet Tips	VWR	83007-382
NuPAGE Bis-Tris Precast Gels 4-12%	Invitrogen	NW04125BOX
Leupeptin Hemisulfate, 5 MG	RPI Research Products	22035-0.005	Used for Protease inhibitor solution.
APROTININ, 10 MG	RPI Research Products	20550-0.01	Used for Protease inhibitor solution.
PEPSTATIN A, 5 MG	RPI Research Products	30100-0.005	Used for Protease inhibitor solution.
OVCA429 cells	Gift from Karen Cowden Dahl. Ph.D.
HEK293T	ATCC	CRL-3216
HeLa cells	ATCC	CCL-2
McCoy's 5A media	Corning Mediatech	10-050-CV
DMEM	Corning Mediatech	10-013-CV
Minimum Essential Medium (MEM), Powder	Corning Mediatech	50-011-PC
MEM NEAA (100X) non-essential amino acid	Gibco	11140-050
FB-11: Fetal Bovine Serum, U.S. Source	Omega Scientific	FB-11
Penicillin-Streptomycin Solution	Life Technologies	15140-122
Glutamax	Life Technologies	35050-061
sodium pyruvate	Life Technologies	11360070
VWR Power Source	VWR	13-690-032