The aim of my dissertation research was to understand the mechanisms underlying the transcriptional response to stress by the tumor suppressor protein p53 and and their influence on cell fate decisions. This was accomplished by several complementary experimental approaches that compared two p53-mediated transcriptional programs, cell cycle arrest and apoptosis. Since p53 is central to the cellular response to stress signals, it is important to define the events that are involved during p53 activation of cell cycle arrest and pro-apoptotic genes. p53 target promoters are structurally diverse and display pronounced differences in RNA polymerase II (RNAP II) occupancy even in unstressed cells, with higher levels observed on cell cycle arrest genes (p21) compared to apoptotic genes (Fas/APO1). This occupancy correlates well with their ability to undergo rapid or delayed stress-induction. To understand the basis for such distinct temporal assembly of transcription complexes, the role of core promoter structures in this process was examined. My studies revealed that the p21 core promoter directs rapid, TATA box dependent assembly of RNAP II pre-initiation complexes (PIC), but permits few rounds of RNAP II re-initiation. By contrast, PIC formation at the Fas/APO1 core promoter is very inefficient but supports multiple rounds of transcription. A downstream element within the Fas/APO1 core promoter is essential for its activation and binds NF-Y. It is known that NF-Y acts as a bi-functional transcription factor that regulates the expression of Fas/APO1 in vivo. Thus, two critical parameters of the stress-induced p53 transcriptional response are the kinetics of gene induction and duration of expression through frequent re-initiation. These features are intrinsic, DNA-encoded properties of diverse core promoters and may be fundamental to anticipatory programming of p53 response genes upon stress.

Analysis of the p21 and Fas/APO1 promoters by in vitro transcription resulted in the identification of three pharmacologic agents that should aid in further dissecting transcriptional mechanisms employed by p53 target genes and other genes regulated by RNAP II. Three kinase inhibitors (hypericin, rottlerin, and Sp600125), previously not associated with inhibiting transcription, were discovered to block transcription initiation efficiently.