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1、Protein DNA Binding Assay281 281 From:Methods in Molecular Biology, vol. 338: Gene Mapping, Discovery, and Expression: Methods and Protocols Edited by: M. Bina Humana Press Inc., Totowa, NJ 20 Analysis of Protein-DNA Binding by StreptavidinAgarose Pulldown Kenneth K. Wu Summary Binding of nuclear tr
2、ansactivators to sequence-specific regulatory elements on the promoter regions is of fundamental importance in gene expression and regulation. DNA- bound transactivators recruit transcription coactivators or repressors and an array of asso- ciated proteins that interact with the basal transcription
3、factors, thereby activating the transcription machinery. Analysis of the large complex of proteins that bind to DNA is an important step in elucidating the mechanisms by which gene expressions are regu- lated. Commonly used techniques to determine DNA-protein binding such as the electro- phoretic mo
4、bility shift assay (EMSA) have limited value for analyzing simultaneously a large number of proteins in the complex. We describe here a streptavidin-agarose pulldown assay that is capable of analyzing quantitatively binding of an array of proteins to DNA probes. The assay is easy to perform and does
5、 not require radiolabeled probes. It involves incubation of nuclear extract proteins with 5biotinylated double-stranded DNA probes and streptavidin-agarose beads. The complex is pulled down, and proteins in the complex are dissociated and analyzed by Western blotting. This method has been shown to b
6、e use- ful in determining the regulation of binding of transactivators, p300/CBP, and associated proteins to the cyclooxygenase-2 (COX-2) promoter. Key Words: Protein-DNA binding; electrophoretic mobility shift assay; transcription factors; streptavidin-agarose; biotinylated oligonucleotide; cycloox
7、ygenase-2; promoter activity. 1. Introduction Transcriptional activation of euchromatin genes depends on binding of tran- scriptional activators to sequence-specific DNA binding sites at the promoter regions of genes (1). DNA-bound transcriptional activators recruit coactivators, 282Wu corepressors,
8、 and associated proteins that interact with basal transcription fac- tors in the transcription machinery and activate the RNA polymerase II to ini- tiate transcription. Mitogenic factors and proinflammatory mediators induce gene expression by enhancing the DNA binding activity of responsive transact
9、i- vators. Basal gene expression and gene expression induced by exogenous stim- uli are regulated by binding of a combination of distinct transactivators to their specific binding sites (enhancer elements) at the promoter region that recruit coactivators or corepressors and associated proteins to fo
10、rm a complex. Thus, quantitative analysis of the complex that binds to specific DNA motifs is crucial for understanding diverse biological functions. Protein-DNA binding may be analyzed by a number of assays including DNase protection assay, electropho- retic mobility shift assay (EMSA), molecular b
11、eacon assay, chromatin immuno- precipitation (ChIP) assay, and a recently described streptavidin-agarose pull- down assay (SAPA) (28). The SAPA assay will be described in detail in this chapter. The SAPA assay (Fig. 1) takes advantage of an extremely high binding affin- ity of biotin to avidin (Kd =
12、 1015M), which has been widely applied to detecting proteins by enzyme immunoassay and immunohistochemical analysis of pro- teins (9,10). The SAPA assay was developed for detecting proteins that bind to oligonucleotides. The principle of the SAPA assay is to incubate nuclear extracts with biotinylat
13、ed double-stranded DNA probes in the presence of streptavidin- Fig. 1. Schematic illustration of the principle of streptavidin-agarose pulldown assay of protein-DNA binding. Transactivators (shown as footballs) bind to sequence-speci- fic cis-acting elements of a biotinylated double-stranded oligonu
14、cleotide probe. Biotin binds streptavidin immobilized on agarose beads. The complex is centrifuged. Trans- activators are dissociated and analyzed by Western blotting. The assay is suitable for studying binding of a single transactivator or multiple transactivators simultaneously. Furthermore, it is
15、 suitable for quantitative analysis of not only transactivators but also proteins that interact with transactivators. Protein DNA Binding Assay283 conjugated agarose beads. Transactivators in the nuclear extracts bind to sequence- specific binding sites at the biotinylated probe, and the complex bin
16、ds streptavi- dinagarose via biotin. The mixture is isolated by centrifugation. Transactivators, coactivators, corepressors, and associated proteins in the complex are dissolved and analyzed by immunoblotting. This technique provides a semiquantitative analysis of an array of proteins that interact
17、with promoter response elements and the transcription machinery. The length of the biotinylated DNA probe may be as short as 20 bp, harboring a single specific binding site, or a 500-bp cyclo- oxygenase-2 (COX-2) core promoter that harbors multiple binding sites. Several internal controls should be
18、included in this pulldown assay. The most crucial is the inclusion of a probe that does not harbor any recognizable enhancer element. We have routinely used a 21-mer as a universal control (8). Under special circum- stances, a mutant probe should be included as a control. For example, a 24-mer COX-2
19、 promoter fragment containing a C/EBP binding site (5-ACCGGCTTA CGCAATTTTTTTAAG-3) and a C/EBP mutant (5-ACCGGCGCGATAGTTT TTTTAAG-3) are used to illustrate the specific binding of C/EBP isoforms to this regulatory element (11). In our initial experiments, we compared the pull- down by streptavidin-c
20、onjugated agarose vs plain agarose beads and found that plain beads did not pull down biotinylated DNA-protein complexes. This con- trol may be unnecessary for each experiment but should be included when a new batch of streptavidin-conjugated agarose beads is used. This binding assay has been shown
21、in our experiments to be suitable for analyzing transcriptional factor binding activities in several cell types includ- ing human foreskin fibroblasts, human endothelial cells (human umbilical vein endothelial cells, ECV 304 and EA.hy927 cells), and murine RAW 264.7 macro- phages (8,1113). It is rea
22、sonable to assume that it is useful in evaluating pro- tein-DNA interaction in any cell type. This assay is suitable for analyzing multiple transcription factors that bind to a promoter. By using a 500-bp COX-2 promoter probe, we have shown that this assay is useful in identifying and quantifying si
23、multaneously all C/EBP isoforms including the C/EBP truncated forms, all NF-B isoforms (P65 RelA, P68 RelB, P75 C-Rel, P50 NF-B1, and P52 NF-B2), CREB/ATF isoforms, C-Jun and C-Fos isoforms, p300/CBP, PCAF, TFIIB, Med7, and Srb7 to the COX-2 promoter (8). The assay appears to be versatile in analyzi
24、ng all the proteins in the complex as long as suitable specific antibodies with reasonable affinity for the candidate proteins are available. The assay is relatively simple and does not require radiolabeled probes. We have tested its feasibility for studying promoter regulation in a prototypic pro-
25、inflammatory gene, COX-2. Our results show that it is useful in determining the temporal and spatial relationship between transactivator and p300 coactivator 284Wu binding to the core COX-2 promoter region and COX-2 transcriptional regula- tion by proinflammatory mediators. 2. Materials 2.1. Nuclear
26、 Extract Isolation 1. Phosphate-buffered saline (PBS), pH 7.4 (Sigma, St. Louis, MO). 2. The following reagents are prepared and stored in stock concentrations: a. 0.5 M Sodium fluoride (NaF; Sigma), stored at 4C. b. 100 mM Phenylmethylsulphonyl fluoride (PMSF; Sigma) solution in isopro- panol, stor
27、ed at 20C. c. 0.1 M Dithiotreitol (DTT; Invitrogen), stored at 20C. d. 1 mg/mL Leupeptin (Sigma), stored at 20C. e. 1.25 M -Glycerophosphate disodium salt (Sigma), stored at 4C. f. 1 M Sodium vanadate (Sigma), stored at 20C g. 1 M Potassium chloride (KCl; Aldrich, Milwaukee, WI) stored at room tem-
28、perature (RT). h. 1 M HEPES (Sigma), stored at 4C. i. 1 M Magnesium chloride hexahydrate (MgCl2, Sigma), stored at RT. j. 2 M Sucrose (Sigma), stored at RT. k. 10% Igepal CA-630 (NP-40; Sigma), stored at RT. l. 5 M Sodium chloride (NaCl; Sigma), stored at RT. m.0.5 M EDTA (Invitrogen), stored at RT.
29、 3. Glycerol (Sigma), stored at RT. 4. PBS buffer containing inhibitors (PBSI): 0.5 mM PMSF, 25 mM -glycerophos- phate, 10 mM NaF, stored at 4C. 5. Buffer A: 10 mM HEPES, pH 7.9, 1.5 mM MgCl2,10 mM KCl, 300 mM sucrose, 0.5% NP-40, stored at 4C. 6. Buffer B: 20 mM HEPES, pH 7.9, 1.5 mM MgCl2,420 mM N
30、aCl, 0.2 mM EDTA, 2.5% glycerol, stored at 4C. 7. Buffer D: 20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 8% glycerol, stored at 4C. 8. Cell scraper (Corning Incorporated Life Sciences, Acton, MA). 9. Ultrasonic dismembrator model 500 (Fisher Scientific, Pittsburgh, PA). 10. Microcentrifuge (Eppendo
31、rf, Hamburg, Germany, cat. no. 5415D). 11. Temperature-controlled room (Biocold Environmental, Fenton, MO). 2.2. Protein Assay 1. BCA Protein Assay Reagent Kit (Pierce, Rockford, IL): Reagent A containing sodium carbonate, sodium bicarbonate, bicinchoninic acid, and sodium tartrate in 0.1 M sodium h
32、ydroxide, and Reagent B containing 4% cupric acid. 2. Albumin standard 2 mg/mL. 3. 96-Well plate (Corning). Protein DNA Binding Assay285 4. Microplate spectrophotometer, Benchmark Plus (Bio-Rad, Hercules, CA). 5. Microplate Manager version 5.2 (Bio-Rad). 2.3. Binding Assay Stepwise, the assay is div
33、ided into (1) the pulldown procedure; (2) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); and (3) West- ern blot analysis. Materials for each procedure are described separately. 2.3.1. Pulldown Procedure 1. Streptavidin immobilized on 4% beaded agarose activated by cyanogen bro
34、mide (Sigma). 2. Single-stranded 5-biotinylated oligonucleotides (Integrated DNA Technologies, Coralville, IA) (see Note 1). 3. PBSI buffer. 4. Rocking platform labquake (Barnstead International, Dubuque, IA). 5. Microcentrifuge. 2.3.2. SDS-PAGE 1. 2X Laemmli sample buffer: 950 L of Laemmli sample b
35、uffer mixed with 50 L of 2-mercapthoethanol (Bio-Rad). 2. 4 to 15% Gradient polyacrylamide gel in Tris-HCl (Bio-Rad). 3. 10X Running buffer containing 250 mM Tris, 1.92 M glycine, 1% SDS (all from Sigma). 1X Running buffer is prepared by diluting 100 mL of 10X buffer with 900 mL deionized water. 4.
36、10X Transfer buffer containing 250 mM Tris-HCl, 1.92 M glycine (both from Sigma). Stored at RT. 1X Transfer buffer is prepared by mixing 100 mL of 10X transfer buffer with 200 mL of methanol and 700 mL of deionized water. 5. Methanol (AAPER Alcohol and Chemicals Company, Shelbyville, KY). 6. Prestai
37、ned broad-range SDS-PAGE standards (Bio-Rad). 7. Mini-Trans Blot Electrophoretic Transfer Cell (Bio-Rad). 8. Minigel holder cassette (Bio-Rad). 9. Fiber pads (Bio-Rad). 10. Trans-Blot Transfer Medium Supported Nitrocellulose Membrane, 0.2 m (Bio-Rad). 11. Filter paper (Bio-Rad). 2.3.3. Western Blot
38、1. Rabbit affinity-purified polyclonal antibodies specific for transactivators and coac- tivators (various sources). Nonimmune IgG control (various sources) (see Note 2). 2. IgG-horseradish peroxidase-conjugated polyclonal antibody (see Note 3). 3. Blotting grade blocker nonfat dry milk (Bio-Rad). 4
39、. PBS with Tween 0.05% (PBST) (Sigma). 5. Rocking platform Labquake (Barnstead International). 6. Materials for detection of proteins by chemiluminescence: 286Wu a. Kodak X-Omatic Cassette (Eastman Kodak, Rochester, NY). b. Kodak BioMax MS film (Eastman Kodak). c. X-ray film processor Konica SRX-201
40、-A (Source One Healthcare Technologies, Mentor, OH). d. SuperSignal West Pico Chemiluminescent Substrate kit (Pierce) (see Note 4). e. Rocking platform Labquake (Barnstead International). f. Plastic Sheet (Reports Cover, C-Line Products, Mount Prospect, IL) (see Note 5). 3. Methods 3.1. Nuclear Extr
41、act Isolation 1. All procedures are done on ice or in a controlled temperature room at 4C. 2. Remove medium from cultured cells and wash them once with cold PBS. Add PBSI buffer (1 mL/10-cm dish) and harvest cells with a rubber scraper. Cells are collected into a 50-mL conical tube, which is centrif
42、uged at 550g for 5 min (see Note 6). 3. Remove supernatant. Transfer the pellet to a 1.5-mL microcentrifuge tube, and centrifuge at 1500g for 30 s. 4. To buffers A, B, and D, add the following inhibitors: 0.5 mM PMSF, 1 mM Na3VO4, 0.5 mM DTT, 1 g/mL leupeptin, 25 mM -glycerophosphate, 10 mM NaF. 5.
43、Remove supernatant and resuspend the pellet in 2 package cell volume of buffer A with inhibitors. Keep on ice for 10 min. Vortex briefly, and centrifuge at 2600g for 30 s. 6. Remove supernatant and resuspend the pellet in 2/3 package cell volume of buffer B with inhibitors. 7. Sonicate the mixture f
44、or 5 s. Centrifuge at 10,400g for 5 min (see Note 7). 8. Dilute the supernatant isovolumetrically with buffer D with inhibitors. Aliquot and store at 80C (see Note 8). 3.2. Protein Assay 1. Prepare standard albumin concentrations at 50, 100, 250, 500, 750, 1000, 1500, and 2000 g/mL. 2. Pipet 10 L of
45、 standards and samples each in duplicate onto a 96-well plate. 3. Mix 50 parts of buffer A with 1 part of buffer B. 4. Add 200 L of mixed buffer A and B to standards and samples. 5. Incubate plate at 37C for 30 min. 6. Read the protein content at 562 nm on a microplate spectrophotometer. 3.3. Bindin
46、g Assay 3.3.1. Pulldown Procedure 1. Dissolve single-stranded biotinylated oligonucleotides in deionized sterile water. The final concentration is 1 g per 1 to 2 L. 2. Add an equal quantity of sense and antisense biotinylated oligonucleotide solu- tion to a microfuge tube, mix, and place it in a 100
47、C water bath for 1 h. Remove the tube and allow it to cool down at RT (see Note 9). Protein DNA Binding Assay287 3. Gently mix the streptavidin-agarose bead suspension with a vortex and place the suspension in a 1-mL (tuberculin) syringe. Pull the syringe piston forward and backward gently two to th
48、ree times to ensure an even suspension (see Note 10). 4. Add 2 drops of the streptavidin-agarose bead suspension to a mixture of 400 g of nuclear extract proteins and 4 g of double-strand biotinylated oligonucleotides in 500 L of PBSI buffer (see Note 11). 5. Place the mixture on a rocking platform
49、at RT and rock the mixture at a gentle speed for 2 h (see Note 12). 6. Centrifuge the mixture at 550 g for 1 min (see Note 13). 7. Discard the supernatant. 8. Wash the pellet with PBSI for three times. 9. Suspend the pellet in 40 L of 2X Laemmli sample buffer. 10. Incubate the sample at 95C for 5 min (see Note 14). 11. Centrifuge the sample at 7000g for 30 s in a microfuge and collect the supernatant (see Note 15). 3.3.2. SDS-PAGE 1. Remove 4 to 15% gradient “ready to use” gel from plastic, remove the comb, and cut the tape along the black