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 on, altering interactions between histones and DNA². Histone PTMs also serve to recruit transcriptional regulators, chromatin remodelers, DNA damage repair machinery, and DNA replication machinery to specific regions of the genome³.

Most methods for studying chromatin interactions either probe small scale interactions or involve large genome-wide analyses. In vitro binding studies often utilize individual recombinant domains with histone peptides or DNA in assays such as electrophoresis mobility shift assays (EMSA), isothermal titration calorimetry, fluorescence polarization, and peptide pulldowns. Because these assays typically focus on an individual protein domain, they facilitate the understanding of a small piece of the puzzle, but do not allow us to understand the cooperative nature of multi-domain proteins, let alone their role in multi-protein complexes. Another layer of intricacy is added by the heterogeneous composition of most mammalian chromatin-modifying complexes. This protein heterogeneity, in combination with the dynamic nature of the chromatin landscape, makes it challenging to recapitulate the in vivo binding interactions of chromatin proteins to chromatin in vitro.

In vivo methods have made significant advances; however, they are often expensive, time consuming, and technically challenging. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is very useful for determining the localization of proteins and histone modifications across the genome, however it requires substantial optimization⁴. Proteins are often crosslinked to chromatin to preserve interactions; however, this can produce artificial interactions and may cause epitope masking⁵. Furthermore, the immunoprecipitations (IP) require highly specific antibodies, and extensive optimization of DNA shearing and IP conditions by ChIP-qPCR using a known binding site, which is often not available a priori. After optimization of ChIP conditions, processing of the samples is costly and requires several weeks to months to sequence and analyze. Though this method is invaluable for identifying the localization of chromatin bound proteins across the genome, the cost and time commitment make it prohibitive to use this method to generate hypotheses about how small changes may affect global binding properties.

In this paper, we describe how a sequential salt extraction (SSE) assay can be used to examine global binding profiles of chromatin-bound proteins and distinguish how changes in a protein, complex, or global PTM profile can alter interactions. Though salt extractions are a commonly and broadly used technique, we demonstrate how this sequential method is highly reproducible and versatile. SSE allows us to characterize how a single subunit of a complex or even a single domain contributes to the complex's overall affinity for bulk chromatin. SSE can also be used to determine if the binding of a protein is influenced by changes in chromatin landscape, providing interesting hypotheses for histone mark targeting that can be confirmed using ChIP-seq and other genome wide studies.

We originally adapted this method from Wu et al., to examine of the function of Polybromo1 (PBRM1) in the binding of the PBAF chromatin remodeler^6,7. Using this technique, we determined the role of PBRM1 for chromatin binding within the PBAF chromatin remodeling complex and then determined the relative contribution of the six individual bromodomains to this function⁷.

Here we describe how to optimize this method to explore chromatin binding in different cell types, to assess the relative binding affinity of similar chromatin modifying complexes, to examine the displacement of a protein from chromatin by a chemical inhibitor, and to determine the effects of chromatin binding after alterations to the chromatin landscape.

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 of the six salt concentrations: 0 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM NaCl. Prior to starting the experiment, cool all solutions on ice.
5. Grow cell lines under desired conditions.
   NOTE: When determining the effects of knocking down a protein, SSE for the knockdown line must be compared to wildtype cells. These SSE must be performed side by side. For examples using chemical inhibitors or stimuli, refer to sections 3 - 5.

2. Basic Sequential Salt Extraction

1. Harvest 8 million cells of each condition and wash twice with 5 mL of ice cold PBS.
   NOTE: It is critical to have equal number of cells to have equivalent protein concentrations. The exact number of cells may need to be optimized for individual cell lines or particular proteins. It is important not to have too many, as the increase in protein concentrations will prevent observation of the elution curve, which is dependent on equilibrium binding. However, with too few cells there may not be enough protein to detect the curve, and the chromatin pellet is harder to isolate and can be lost between fractions.
2. Re-suspend cells in 1 mL of Buffer A + protease inhibitors, transfer into 1.5 mL micro centrifuge tubes, and rotate top over bottom at 4 ºC for 10 min.
3. Isolate the nuclei by centrifugation at 6000 x g for 5 min at 4 ºC .
   NOTE: After this step the nuclear envelope should still be intact. If the nuclear envelope is intact, the pellet will re-suspend fully; however, when the envelope is lysed and the chromatin is released, the pellet will not re-suspend. If the nuclear envelope is not intact after Buffer A incubation, use Modified Buffer A.
4. Add 200 µL of mRIPA 0 mM NaCl + protease inhibitors to each nuclei pellet before re-suspending pellet. Homogenize each sample by pipetting 15 times with a 1 mL pipette. The pellet should re-suspend easily, however, the nuclei will lyse as it is pipetted and the sample will become thick and sticky and difficult to pipette.
   1. In order to draw up the pellet into the tip, tap the end of the pipette tip against the bottom of the centrifuge tube. The pellet will not fully dissolve once the DNA is released from the nuclei, but pipetting will break it up.
5. When all the samples have been homogenized, incubate all the samples on ice for 3 min.
   NOTE: The incubation time may need to be optimized depending on the protein of interest.
6. Isolate chromatin pellet by centrifuging the samples for 3 min at 6500 x g at 4 ºC.
7. Transfer supernatant to a clean 1.5 mL centrifuge tube. This will be the 0 mM fraction. This 0 mM mRIPA solution can act as a wash step to lysis the nuclei and remove any loose proteins not associated with chromatin.
8. Add 200 µL of mRIPA 100 mM NaCl + protease inhibitors to each chromatin pellet before re-suspending pellet. Break up the chromatin pellet by pipetting the pellet up and down 15 times.
   NOTE: It is critical to be consistent with the number of times the pellet in pipetted.
9. Incubate on ice for 3 min. This incubation step allows all samples to reach an equilibrium state, which is particularly important when there are multiple samples.
10. Centrifuge at 6500 x g for 3 min at 4 ºC and transfer supernatant to a clean 1.5 mL tube.
11. Repeat 2.6 - 2.10 for the remaining salt concentrations.
    NOTE: After 400 mM NaCl, mRIPA the pellet should be clear and gloopy and will not stay at the bottom of the tube. The pellet can be placed in the lid of the centrifuge tube while the supernatant isolated.
12. Add 70 µL of 4x protein loading dye to each fraction and load 30 µL of each fraction on to an SDS acrylamide gel for western blot analysis.
    NOTE: To determine the binding profile of the protein, it is critical to load equivalent volumes of lysate, rather than loading equal protein concentrations.
13. Perform a standard western blot by transferring onto a membrane and use primary antibodies for proteins of interest.
14. To quantitate the protein eluted from the chromatin, incubate the blot in infrared fluorescence IRDye secondary antibodies and image blot with an imager. While other methods of development can be used, we recommend fluorescence or infrared imaging, as it is more quantitative in nature.
15. Use ImageJ or similar software to calculate the intensity for the protein eluted at each salt concentration. By graphing the band intensity against the salt concentration, the elution pattern of your protein of interest can be determined.

3. Sequential Salt Extraction in the Presence of a "Reader" Inhibitor

1. Harvest two sets of cells (4 million) and isolate the nuclei as in a standard SSE.
2. Re-suspend both sets in 200 µL of mRIPA 0 mM NaCl and incubate for 3 min. This will allow for the removal of any free protein in the nuclei.
3. Add 200 µL mRIPA 0 mM NaCl to each pellet. Add the inhibitor (2 µL of 1 mM (+)JQ1) to one sample and DMSO to the control set.
4. Agitate the pellet by pipetting 15 times and incubate on ice for 5 min.
5. Centrifuge at 6500 x g for 3 min at 4 ºC and transfer supernatant to a clean 1.5 mL tube.
6. Repeat 3.3 - 4 for all the salt concentrations and perform a standard western blot.

4. Sequential Salt Extraction in the Presence of a "Writer" Inhibitor

1. Treat cells with the inhibitor (10 µM SAHA) or DMSO for 3 h.
2. Harvest 4 million cells for each treatment.
3. Perform a standard SSE for the samples with the inhibitor added to all the buffers.

5. Sequential Salt Extraction Following DNA Damage

1. Treat cells with 1 µM doxorubicin for 1 h.
2. Change the media on the cells and allow them to recover for 3 h.
3. Harvest 8 million cells and perform the basic SSE.

6. Non-Sequential Salt Extraction

1. Harvest 12 million cells and wash with PBS.
2. Divide cells so that there are 2 million cells per micro centrifuge tube.
3. Re-suspend in 500 µL of Buffer A and incubate for 10 min.
4. Isolate the nuclei by centrifugation at 6000 x g.
5. Re-suspend pellets in a 200 µL of each of the mRIPA buffers + NaCl.
6. Homogenize each sample by pipetting 15 times with a 1 mL pipette tip.
7. Incubate on ice for 3 min.
8. Isolate the chromatin by centrifugation at 6500 x g and transfer the supernatant to clean tubes. Add 70 µL of loading dye and run 30 µL on a SDS-page gel for western blot analysis.

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