Abstract
Transcription is a fundamentally important process for determining cell type identity, growth, and function. A central regulatory step occurs during the formation of the transcriptional preinitiation complex (PIC) at promoter sites. While the PIC consists mainly of a set of general transcription factors (GTFs) and RNA polymerase II (Pol II), there are many other factors and complexes that recruit the machinery and/or control chromatin structure at promoters to facilitate PIC assembly and activity. Although much is known about the binding locations of these factors, their in vivo dynamics remain largely unknown. Recent studies indicate that binding of factors in vivo is a dynamic process, but techniques for measuring chromatin interaction dynamics have in general been limited either by insufficient time resolution or by the inability to monitor binding to single copy genes. Our lab developed the crosslinking kinetics (CLK) assay, which measures binding dynamics based on the formaldehyde crosslinking-time dependent nature of the measured chromatin immunoprecipitation (ChIP) signal. We updated the crosslinking and quenching conditions to improve the quench efficiency and measured TATA-binding protein (TBP), a component of the PIC, at select promoters. We found that there is a wide range of formaldehyde crosslinking rates, and crosslinking time-dependent changes in ChIP signal can be described by factor-limited or crosslink-limited models. The residence time of TBP was ~2 minutes at the promoters measured, while the fractional promoter occupancy varied from ~0.05 to 0.7. We have also adapted the Anchor Away approach, a nuclear depletion technique, to estimate the stability of a chromatin-factor interaction by elucidating the off-rate and residence time as the nuclear factor is depleted. This data is approaching agreement with the CLK data at some loci, but in disagreement at others. A previous study developed the Competition ChIP approach, where a diploid strain with differentially tagged copies of the target allele, one under the control of an inducible promoter, can measure residence time of a factor at specific sites based on the exchange of tagged protein as determined by ChIP. The CLK, Anchor Away, and Competition ChIP data for TBP at select loci are beginning to converge. By comparing binding dynamics measured with these three techniques, the in vivo binding dynamics of PIC components can be determined to understand PIC assembly pathways and transcriptional regulation at individual promoters. Additionally, we measured binding dynamics of the activator Gal4 at several of the GAL genes. A residence time of ~14 minutes is in agreement with the previous CLK study as well as previous results obtained using competition ChIP. Our collaborators have measured real-time transcriptional output at two GAL genes. Comparison of the results from different approaches will be valuable in correlating transcription factor dynamics with gene expression and better understanding the molecular mechanisms of complex assembly and regulator activity in vivo.
