Abstract
Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays.
Highlights
Since the complete sequencing of the human genome in 2001 [1,2], a wealth of DNA sequences has been available via online databases [1,3,4,5,6,7]
Activating protein 2 alpha (AP2a) binding assay on 6000 human genomic sequences We first aimed at identifying potential target genes of the AP2a oncogene/tumor suppressor protein by assessing its binding to double-stranded DNA sequences immobilized on a microarray
Expressed and purified AP2a protein was incubated with the microarray, and its binding to particular sequences was assessed using Cy3-labeled fluorescent antibodies and microarray scanning
Summary
Since the complete sequencing of the human genome in 2001 [1,2], a wealth of DNA sequences has been available via online databases [1,3,4,5,6,7]. Human diseases like cancer have often been linked to the improper interplay of proteins involved in the transcriptional control of cells and tissues, as illustrated by the prominent role of oncogenes in regulating gene transcription and chromatin structure [8,9]. Several laboratory techniques have been devised for large scale identification of transcription factor target sites, either in vitro or using cellular assays [10]. One such assay relies on proteinbinding microarrays (PBM) that bear immobilized doublestranded DNA molecules to which the binding of regulatory proteins can be probed. The actual binding of the transcription factors to the predicted site must be confirmed experimentally, as it may be occluded by chromatin or DNA modification or by other proteins binding overlapping DNA sequences, while synergistic binding may occur on non-canonical sites that are not detected by in silico predictions
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