Transcriptional activation of euchromatin genes depends on binding of transcriptional activators to sequence-specific DNA binding sites at the promoter regions of genes (1). DNA-bound transcriptional activators recruit coactivators,

From: Methods in Molecular Biology, vol. 338: Gene Mapping, Discovery, and Expression: Methods and Protocols Edited by: M. Bina © Humana Press Inc., Totowa, NJ

Trans activator —

DNA pi

Trans activator —

DNA pi

— Strcpiavidin

— Strcpiavidin

Western analysis

Fig. 1. Schematic illustration of the principle of streptavidin-agarose pulldown assay of protein-DNA binding. Transactivators (shown as footballs) bind to sequence-specific cis-acting elements of a biotinylated double-stranded oligonucleotide 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 suitable for quantitative analysis of not only transactivators but also proteins that interact with transactivators.

corepressors, and associated proteins that interact with basal transcription factors in the transcription machinery and activate the RNA polymerase II to initiate transcription. Mitogenic factors and proinflammatory mediators induce gene expression by enhancing the DNA binding activity of responsive transactivators. Basal gene expression and gene expression induced by exogenous stimuli 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 form 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 beacon assay, chromatin immuno-precipitation (ChIP) assay, and a recently described streptavidin-agarose pulldown assay (SAPA) (2-8). The SAPA assay will be described in detail in this chapter.

The SAPA assay (Fig. 1) takes advantage of an extremely high binding affinity of biotin to avidin (Kd = 10-15 M), which has been widely applied to detecting proteins by enzyme immunoassay and immunohistochemical analysis of proteins (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 biotinylated double-stranded DNA probes in the presence of streptavidin-

conjugated agarose beads. Transactivators in the nuclear extracts bind to sequence-specific binding sites at the biotinylated probe, and the complex binds streptavi-din-agarose 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 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 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 circumstances, a mutant probe should be included as a control. For example, a 24-mer COX-2 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 pulldown by streptavidin-conjugated agarose vs plain agarose beads and found that plain beads did not pull down biotinylated DNA-protein complexes. This control 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 in our experiments to be suitable for analyzing transcriptional factor binding activities in several cell types including human foreskin fibroblasts, human endothelial cells (human umbilical vein endothelial cells, ECV 304 and EA.hy927 cells), and murine RAW 264.7 macrophages (8,11-13). It is reasonable to assume that it is useful in evaluating protein-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 simultaneously all C/EBP isoforms including the C/EBP0 truncated forms, all NF-kB isoforms (P65 RelA, P68 RelB, P75 C-Rel, P50 NF-kB1, and P52 NF-kB2), 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 analyzing 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-inflammatory gene, COX-2. Our results show that it is useful in determining the temporal and spatial relationship between transactivator and p300 coactivator binding to the core COX-2 promoter region and COX-2 transcriptional regulation by proinflammatory mediators.

2. Materials

2.1. Nuclear 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 4°C.

b. 100 mM Phenylmethylsulphonyl fluoride (PMSF; Sigma) solution in isopro-panol, stored at -20°C.

c. 0.1 M Dithiotreitol (DTT; Invitrogen), stored at -20°C.

e. 1.25 M P-Glycerophosphate disodium salt (Sigma), stored at 4°C.

g. 1 M Potassium chloride (KCl; Aldrich, Milwaukee, WI) stored at room temperature (RT).

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.

3. Glycerol (Sigma), stored at RT.

4. PBS buffer containing inhibitors (PBSI): 0.5 mM PMSF, 25 mM P-glycerophos-phate, 10 mM NaF, stored at 4°C.

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 4°C.

6. Buffer B: 20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 2.5% glycerol, stored at 4°C.

7. Buffer D: 20 mM HEPES, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 8% glycerol, stored at 4°C.

8. Cell scraper (Corning Incorporated Life Sciences, Acton, MA).

9. Ultrasonic dismembrator model 500 (Fisher Scientific, Pittsburgh, PA).

10. Microcentrifuge (Eppendorf, 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 hydroxide, and Reagent B containing 4% cupric acid.

2. Albumin standard 2 mg/mL.

3. 96-Well plate (Corning).

4. Microplate spectrophotometer, Benchmark Plus (Bio-Rad, Hercules, CA).

5. Microplate Manager version 5.2 (Bio-Rad).

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