Some transactivator-promoter complexes are highly dynamic due to active disruption of the complex by proteolytic or nonproteolytic mechanisms, and this appears to be an important mechanism by which their activity is governed tightly and eventually terminated. However, the generality of these mechanisms is unclear. In this report, we address the dynamics of hypoxiainducible factor-1 (HIF-1) binding to the vascular endothelial growth factor promoter. HIF-1 is a heterodimeric transcription factor whose activity is triggered by an increase in HIF-1␣ levels in hypoxic cells. A "competition ChIP" assay is employed to demonstrate that HIF-1␣ forms a kinetically stable complex with the native vascular endothelial growth factor promoter that has a half-life in excess of 1 h. Thus, HIF-1 activity does not require rapid proteolytic turnover of the promoter-bound transactivator, nor is the activator-promoter complex constantly disassembled by chaperones. However, we do find that after cessation of the inducing signal, HIF-1 activity is slowly returned to basal levels by proteasome-mediated proteolysis of the promoter-bound HIF-1␣ protein.Transcriptional activators are central players in regulating the eukaryotic cellular responses to intracellular and extracellular stimuli. When activated, they bind to specific responsive elements in target promoters and modulate various gene expression events. The kinetics of the association and disassociation of transcriptional activators with their respective responsive elements in the promoters influences the strength, duration, and adaptability of transcriptional events and therefore is an important element in the overall regulation of gene expression (1, 2). Some activators interact with their responsive elements far more dynamically in living cells than would have been predicted from their biochemical half-lives in vitro, arguing that there are activities inside that cell that actively dissociate some activators from promoters (for reviews, see Refs. 3-6). These dissociative events can be either proteolytic and thus irreversible (7-10) or nonproteolytic and reversible (11-14, 23) in nature.The former set of events represents one of the several intimate connections between the ubiquitin-proteasome pathway and RNA polymerase II transcription. Indeed, in several cases, proteasome function has been found to be essential for the full activity of particular activators, and, in a few cases, the available data argue that this reflects a requirement for the periodic clearance of polyubiquitylated activators from the promoter (9, 10, 15, 16), although activator function and turnover can be uncoupled by mutation (17) in the case of the estrogen receptor. In addition to activator turnover, there have been reports of proteasome-mediated turnover of other promoter-bound transcription factors as well, including coactivators and general transcription factors (18,19). These findings and others have resulted in the proposal of the so-called "timer" (6) and "black widow" (3) models, which posit that ac...