Multilevel Analysis of Eukaryotic Transcription Regulation

Abstract

Animal development depends on spatially and temporally precise control of gene expression. This control occurs at multiple levels, and is fundamentally linked to transcription regulation. Cell type-specific transcription is controlled by the binding of sequence-specific transcription factors (TFs) to DNA regulatory elements. Although significant progress has been made in identifying elements and TFs, their functional characterization is incomplete. Traditional experiments to study regulatory elements rely on time- and labor-intensive reporter assays in whole organisms, or employ high-throughput cell culture-based approaches, which may lack the appropriate trans environment. To address these limitations, I developed a scalable, quantitative approach that analyzes hundreds of regulatory elements simultaneously within a population of Drosophila embryos. Using this approach, I investigated the role of uncharacterized DNA motifs in mesodermal transcription regulation and identified combinatorial interactions between these motifs. My results provide insight into novel regulators of transcription in Drosophila embryonic mesoderm, identify complex regulatory interactions between these factors, and this method will enable future investigations of developmental regulatory networks and models of enhancer activity. In addition, I investigated bispecific DNA binding of the human TF FoxN3, providing the first in vivo evidence for the use of two distinct DNA motifs by a single TF. These results complement crystallographic evidence that FoxN3 binds each consensus site using the same DNA-contacting residues and challenge the current model of DNA recognition as occurring through a single primary motif. As increasing evidence implicates regulatory element mutations and disruption or hyperactivation of TF function in developmental disorders and cancer, further study of regulatory elements and interacting TFs will provide critical insight into transcription regulation and disease etiology.