Sequence-specific DNA binding transcription factors are the key elements in interpreting and transmitting the genetic DNA sequence information to the RNA polymerase II transcriptional machinery (1). Their functionality depends on the cell-specific expression, nuclear translocation and modification in response to signaling events, and binding to specific DNA sequences as well as the chroma-tin remodeling state of a target promoter and interaction with other regulatory proteins. Several useful databases of transcription factor (TF) binding motifs exist, together with analytical tools that model protein-DNA interactions (reviewed in ref. 2). However, the current databases are limited by the lack of quantitative surveys of TF binding affinities to multiple sequence variants. For example, TRANSFAC NF-kB binding profile (based on published dichotomous data) was a poor predictor of quantitative binding to variant DNA motifs (3). Moreover, the data in existing databases may be subject to sampling biases if investigators

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

focused on particular motifs and often are not freely available but must be purchased through subscription.

The established technique of systematic in vitro binding site selection (SELEX [4]) provides little information about low- and medium-affinity sites, which are required to generate accurate quantitative models of binding. The SELEX-SAGE method addresses the issue of low- and medium-affinity sites (5), but it is laborious and involves several steps that are not scaleable for the analysis of hundreds of transcription factors. An alternative approach involves microarrays of double-stranded DNA molecules that allow the quantification of protein binding to DNA (6). In fact, a systematic profiling of in vitro TF binding to yeast intergenic DNA sequences using this approach has shown significantly stronger correlation with genome localization data (7). We have described a number of key improvements to the microarray technology that improve its specificity, reproducibility, and sensitivity and make it suitable for assaying many TF families (8).

The quantitative data generated by this assay can be used for analyzing the binding specificities of any TF using various statistical approaches and for large-scale analysis of potential TF binding sites predicted from genomic DNA.

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