(247g) Quantitative Solution-Phase Profiling of Active Transcription Factors in Parallel

Transcription factors (TFs) are key effectors of gene expression. TFs bind specific sites of chromosomal DNA and control the expression of thousands of genes to facilitate proper cell function in response to intracellular and extracellular stimuli. TF networks are highly interconnected so that one TF can be activated by many different stimuli, and, conversely, a single stimulus can activate many TFs. Thus, measuring the level of one TF in response to a stimulus potentially ignores other important events that contribute to the cellular response. Hence, parallel measurements of transcription factors are necessary to capture the whole picture of the instantaneous state of the cell. However, given that there are an estimated 1850 different human TFs, parallel TF profiling still remains an unsolved challenge.

We have sought to develop a new, scalable, flexible, and sensitive approach to analysis of transcription factor levels that complements existing parallel approaches. The goal for our method would be for it to be parallel (up to thousands of TFs simultaneously), quantitative, to have superior dynamic range to existing methods, to require small sample sizes (10⁶ cells or fewer), and requiring no manipulation of the cells being studied prior to the assay. Using magnetic bead affinity separation, we have successfully measured the levels of human TFs, NF-kB and Ap1, in parallel with ~10-fold improved sensitivity over existing approaches. We have also quantified differences in the levels of NF-kB, Ap1 and TFIID in breast cancer cells following TNF-alpha stimulation and IKK inhibitor treatment relative to control cells. Here, we will describe current results along with further directions, including expansion to larger sets of TFs and analysis of complex biological processes.