Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts.
The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein-DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level.
Egener
Tanja
T
Institute of Biotechnology, University of Lausanne 1015 Lausanne, Switzerland.
Roulet
Emmanuelle
E
Zehnder
Marc
M
Bucher
Philipp
P
Mermod
Nicolas
N
D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism; D002457:Cell Extracts; D045744:Cell Line, Tumor; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015513:Oncogene Proteins / Q000032:analysis*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000032:analysis* / Q000378:metabolism
D002457:Cell Extracts; D015342:DNA Probes; D004268:DNA-Binding Proteins; D015513:Oncogene Proteins; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D004247:DNA