Cellular Arrays for Dynamic Large-scale Quantification of Transcription Factor Activity

Numerous signal transduction pathways interact to form a complex signaling network, whose net output determines cellular response. Characterizing the regulation of this network will aid in our basic understanding of cell function, and the molecular basis of disease. Current methods of identifying cellular regulation are typically based on high-throughput technologies that rely on single time point measurements of abundance of mRNA or protein; however, abundance does not always correlate with functional activity, and signaling is a dynamic process that can change over time. Transcription factors (TFs) directly modulate gene expression, are powerful regulators of cell function and, as the termination points of signaling pathways, are the outputs of the signaling network. Therefore, the quantification of TF activity within cells can be used to characterize cell function. The primary objective of the research outlined in this dissertation is to develop a cell-based assay to quantify large-scale dynamic changes in TF activity over time. TF-specific luminescent reporter plasmids are applied within the TF activity array and bioluminescence imaging (BLI) is used as a rapid and non-invasive method of quantifying TF activity within the array. The array provides a platform that produces consistent levels of reporter gene expression to enable the quantification of the TF activity. After initial validation of imaging consistency, the functional output of the TF activity array is validated using more traditional lysed-cell assays. The array is then applied to measure the response to external stimulation of 32 TFs within a breast cancer cell line. The utility of the array is then expanded to incorporate the analysis of cell growing within 3D culture and time course measurements. The TF activity array is able to capture differences in cellular regulation upon the addition of stimuli as function of the dimensionality of culture as the cells proliferated both on a 2D surface and form more complex structures within a 3D matrix. Our TF activity array has the potential to provide large-scale analysis of signaling activity of cells cultured within a physiologically relevant context, thus paving the way for more detailed study of disease progression.