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The packaging of eukaryotic DNA into nucleosomes, the fundamental unit of chromatin, creates a barrier to nuclear processes, such as transcription, DNA replication, recombination, and repair(1). This obstructive nature of chromatin can be overcome by the enzymatic activity of chromatin remodeling complexes which creates a more favorable environment for the association of essential factors and regulators to sequences within target genes. Here we describe a detailed approach for analyzing chromatin architecture and remodeling by restriction endonuclease hypersensitivity assay. This procedure uses restriction endonucleases to characterize changes in chromatin that accompany nucleosome remodeling. The specific experimental example described in this article is the BRG1 complex dependent chromatin remodeling of the steroid hormone-responsive mouse mammary tumor virus promoter. Through the use of these methodologies one is able to quantify changes at specific nucleosomes in response to regulatory signals.
In the eukaryotic nucleus, DNA is packaged with histone and non-histone proteins to form a highly organized chromatin structure(2). This DNA-protein assembly can represents a significant barrier to a number of nuclear processes such as DNA repair, recombination, replication and transcription(3). Often the structural changes in chromatin that accompany transcriptional activation require multi-protein enzymatic complexes that manipulate the nucleosomal architecture(4). Alteration in the chromatin structure by ATP-dependent remodeling complexes is considered a significant initial step in transcriptional regulation(5). Chromatin remodeling complexes to use the energy of ATP-hydrolysis to alter local chromatin structure to facilitate a more “open/active” conformation conducive for the recruitment and binding of essential nuclear factors. A number of chromatin remodeling complexes have been identified that modulate the arrangement and stability of nucleosomes in a non-covalent manner (5). Generally, these ATP-dependent remodeling machines are divided into four major subfamilies, characterized by the identity of their central catalytic subunit, which include BRG1 (or hBrm), ISWI, Mi-2 and Ino80 of their respective complexes SWI/SNF, ISWI, NuRD and INO80 (6, 7)
The SWI/SNF remodeling complex was originally identified in yeast, by mutations in mating type switching (SWI) and sucrose fermentation (SNF), and its function is highly conserved in eukaryotes with homologous found in Drosophila and mammals. Human SWI/SNF is a large multi-protein complex that possesses either BRG1 or hBrm, two ATPases related to yeast SWI2/SNF2 and STH1, and are comprised of about 10 BRG1-associated factors (BAFs) subunits, most of which are orthologous to those found in yeast SWI/SNF and RSC (5).
This article provides a detailed approach for analyzing chromatin architecture and remodeling by the BRG1-SWI/SNF complex upon nuclear receptor signaling (Figure 1). Using the human SW-13 cell line, containing stably integrated mouse mammary tumor virus (MMTV) promoter, as a model system we are able to evaluate chromatin changes upon hormone signaling (8). This procedure allows the use restriction endonucleases to characterize alterations in the chromatin structure that accompany nucleosome remodeling and transcriptional activation (9). The methodology presented will focus on analyzing nucleosomal organization using isolated nuclei, restriction endonucleases, and reiteractive primer extension. All methods described here have been employed extensively to study the activity of BRG1 on chromatin structure and transcriptional regulation from the MMTV promoter (10). Although the focus of this article is on the MMTV promoter in SW-13 cells, these techniques have been applied to a variety of inducible and developmentally responsive gene promoters in higher eukaryotes to analyze changes within chromatin (11-15).
The methodology presented is suitable for the analysis of chromatin structure in vivo and can assist in the mapping of DNA-protein interactions and to elucidate their mechanistic implications regarding various nuclear processes including transcriptional regulation, DNA repair, and replication. The requirements for this procedure include 1) the genomic DNA of interest contains sequence-specific binding sites for and responds to a specific transcription factors and 2) has a restriction enzyme cleavage site located proximal to the binding region and is contained within the confines of a nucleosome or organized as chromatin.
The experimental procedure outlined below is based on studies in human carcinoma cells that were stably transformed to contain multiple copies of the nuclear receptor-dependent MMTV promoter. For the purpose of this protocol, we have selected the human adrenal carcinoma cell line, SW-13, which do not express BRG1 protein (10). The use of this multi-copy promoter cell lines provides a very strong signal-to-noise ratio for the MMTV sequence, as well as, to enhance the ability to define the chromatin structure of the promoter. The use of this cell line has greatly enhanced our ability to study nuclear receptor-initiated cascades that lead to structural changes within chromatin upon receptor binding to specific hormone response elements within target promoters (16, 17). Therefore, these general growth conditions have been optimized for our established SW-13/MMTV cell line. Specific growth requirements may vary from this description depending on cell type used.
This protocol will outline the standard procedure for nuclei isolation from tissue cultured cells. All steps are performed on ice with pre-chilled equipment and solutions at 4°C. Cells were treated with hormone or vehicle prior to nuclei isolation to stimulate glucocorticoid receptor signaling.
The choice of restriction endonuclease to use for this accessibility assay is dependent upon the availability of cleavage sites within the genomic region of interest. Most analysis will require the testing of numerous restriction enzymes found to cleave within the target sequence until an optimal enzyme(s) is identified. The cleavage buffer selected for this assay was chosen because it maintains the structural integrity of the nuclei and is compatible with a broad range of restriction enzymes (see Note 8). The quantity of enzyme to use should be derived empirically and will depend on the efficiency at which the enzyme cleaves DNA when using the buffer recommended for hypersensitivity assays.
The purified in vivo digested genomic DNA is cut to completion with a second restriction endonuclease which recognizes a cleavage site upstream or downstream of the in vivo restriction enzyme site depending on which template strand is to be extended. This in vitro digestion is performed overnight usually with 100U of endonuclease to ensure complete digestion of the target sequence. The in vitro digest serves as an internal control to ensure equal amounts of DNA template was used in the reiterative primer extension reaction.
End-labeling is a rapid and sensitive method for radioactively labeling DNA fragments such as oligonucleotide and is useful for visualizing small amounts of DNA. All of the enzymes employed are specific to either the 3′ or 5′ termini of DNA and will, consequently, only incorporate one radio-phosphate per oligonucleotide. The most common method of radio-labeling oligonucleotides uses polynucleotide kinase (PNK) to transfer a single radioactive phosphate group from γ-32P-ATP to the 5′-end of the oligonucleotide.
The extent of restriction endonuclease hypersensitivity for a given target sequence is determined using reiterative primer extension with Taq polymerase and a template-specific 32P-labeled oligonucleotide. The primer selected to detect restriction endonuclease hypersensitivity regions should be located either upstream or downstream of the transcription factor binding site, ideally 200-300bp from the in vivo cleavage site.
The concentration of Mg and oligonucleotide selected, for primer extension, greatly influences the specificity and yield of the reaction; therefore, the amounts of both should be titrated to achieve optimal results. The primer selected should anneal downstream of the in vivo restriction endonuclease cleavage site and be greater than 18 bases in length with melting temperatures ranging between 45 and 70°C. The reiterative primer extension/PCR reaction, described above, has been optimized for studies involving the MMTV promoter (10). These conditions may be employed for other steroid-responsive promoters although optimization should be performed. (see Note 11)
Primer extension products are resolved on denaturing polyacrylamide gels. Samples should be allowed to electrophorese to yield maximal separation between bands corresponding to the in vivo restriction endonuclease cleavage site and the in vitro extension terminal cleavage site. For the purposes of this procedure, samples were resolved on 38 × 30 cm gels using Sequi-Gen GT nucleic acid sequencing system (see Note 13).
The authors have described the use of restriction enzyme hypersensitivity/ reiterative primer extension for assaying nucleosome remodeling which is often a prerequisite for transcriptional activation (15). This method allows for a high-resolution analysis of changes in the chromatin architecture upon promoter stimulation. The individual example presented here demonstrates the use of restriction enzymes to determine steroid-responsive SWI/SNF-mediated promoter hypersensitivity (Figure 2). This method has also been employed to characterize chromatin remodeling and induction of hypersensitivity regions within endogenous nuclear receptor-dependent promoters. Restriction endonuclease hypersensitivity assays were used to identify differences in the chromatin structure of the IκBα and MMTV promoters upon induction by the glucocorticoid and progesterone receptors (18). Nuclease hypersensitivity has been used to demonstrate that estrogen induction of ERRα involves chromatin remodeling around the multiple hormone-response element (19).
Genome-wide strategies can be used to in concert with restriction endonuclease hypersensitivity to identify regulatory domains and elucidate specificity and function. DNase I hypersensitivity assays can be used to map discrete sites in the genome where changes in chromatin conformation yields DNA hypersensitivity to digest by the endonuclease DNase I. These DNase I hypersensitivity sites have been found in the regulatory domains of promoters, enhancers, silencers, insulators and locus control regions (20). Taken together, the use of these in vivo chromatin analysis techniques can provide insight into the roles structured chromatin and remodeling complexes play in the regulation of nuclear processes such as transcription.
1 The authors do not endorse any products or manufactures listed protocol
2Transfection of BRG1 expression plasmids carried out in order to determine the dependence of SWI/SNF chromatin remodeling on restriction enzyme accessibility. Depending on cell line being used transfection may not be necessary.
3Generally, buffers used for nuclei isolation are not maintained at 4°C for more that two months. The following buffer stock solution may be used in preparation of these solutions. 10X Salt Buffer: 100 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 600 mM KCl (store at room temperature). Spermine (0.15 M) and Spermidine (0.5 M) stock solutions (1000X) should be prepared and added to each buffer just prior to isolation. Store 1000X stock solutions at −20°C.
4Prepare DTT as a 1M (1000X) stock and add prior to use. Proteinase K should be prepared as a 20 mg/ml (100X) stock and added prior to use.
5Sequence specific real-time primer pairs may substitute for 32P-labeled oligonucleotide for real-time PCR analysis of cleavage products.
6Our laboratory uses the following premixed sequencing solutions: SequaGel Sequencing System (National Diagnostics, Atlanta, GA).
7The number of Dounce homogenizer pestle stroke needed for efficient nuclei isolation is cell type specific. It is recommended, after using four pestle strokes, to evaluate extent of nuclei isolation under a light microscope.
8The restriction enzyme buffer supplied by the manufacture may be used for in vivo digest.
9If using a Speed-Vac, be careful not to over dry DNA pellet.
10The recognition sequence for this second restriction enzyme should located upstream of the in vivo restriction enzyme site. This digest serves as an internal standard to ensure equal loading of DNA into the Taq polymerase reiterative primer extension assay. This second digest also makes the sample less viscous and the genomic DNA easier to pipette.
11The PCR conditions described here have been optimized for analysis of stably integrated MMTV promoter. However, these conditions have been used to evaluate in vivo restriction enzyme hypersensitivity from other nuclear receptor-dependent promoters and should prove to be good starting point for analyzing other target sequences.
12Sequencing reactions can be performed to map predicted restriction enzyme cleavage sites using the PCR reactions mix and conditions described above with three deoxynucleotides at 200 μM and the fourth at 100 μM ddGTP/20 μM dGTP, 200 μM ddATP/5 μM dATP, 200 μM ddCTP/ 20 μM dCTP, or 200 μM ddTTP/20 μM dTTP.
13Detection of multiple transcription factor-binding sites can be visualized more effectively suing longer denaturing gels such as 40-50 cm.
14Kodak X-OMAT Blue film can be used for gel exposure if PhosphorImager screen is unavailable.