Dynamics of transcriptional regulation Chromatin and associated regulatory protein complexes are known to play an active role in the gene activation. Recent live cell studies have revealed that many regulatory proteins are highly mobile, and exist in a rapid equilibrium with response elements within cells. The long term goal of this project is to understand the purpose of dynamic transcription complexes and the mechanisms that promote dissociation of structures employed to initiate transcription events. As a first step toward understanding the transcriptional dynamics, I will focus on HSF1-mediated transcription. HSF1 belongs to a family of heat shock transcription factors (HSFs) and is a key regulator of a heat-shock response in mammalian cells. Using biochemical, molecular biological and cellular biological assays, I will test the hypothesis that the p23 molecular chaperone disengages DNA-bound transcription factors and that the longevity of the dissociation is mediated through reversible acetylation/deacetylation of an affected DNA binding domain. To address this premise, I plan to execute the three following aims:
Aim 1. Define the effects of p23 and GCN5 on promoter protein recruitment. The roles of p23 and GCN5 in regulating transcription protein complex recruitment and in influencing chromatin assembly at the heat-activated hsp70 promoter will be established using a combination of in vitro immobilized template and in vivo quantitative ChIP assays.
Aim 2. Determine the functional overlap between p23, GCN5, and HDAC1 activities. I will explore the operational relationship between these proteins and basal/induced transcription using biochemical tactics such as DNA binding, in vitro transcription, and protein-protein interaction assays.
Aim 3. Establish the effects of p23, GCN5, and HDAC1 on transcription factor dynamics in vivo. The fluorescence recovery after photobleaching (FRAP) technique will be used in conjunction with engineered cell lines that contain hsp70 loci arrays. The mobility of HSF1 will be determined in parental live cells or following changes to p23, GCN5, or HDAC1 using available mutants and RNAi treatments. Together, these studies will provide new mechanistic insights into the functional interactions between the p23 molecular chaperone and cellular acetylase/deacetylase machinery and therefore will lead to a better understanding of the events that control transcription factor DNA dynamics. Since alternations in gene expression are known to contribute to a number of human diseases, including cancer, the information obtained from the proposed studies is expected to facilitate development of new therapeutics to treat these abnormalities.
Transcription is a first and a key step in regulation of gene expression. Misregulation of this process has been linked to the development of many of the human pathologies, including cancer. Hence, investigation of molecular mechanisms of transcriptional control will potentially facilitate design of drug-and gene-based therapeutic strategies to treat these abnormalities.