At the transcription level gene expression is controlled by the accessibility of the regulatory elements within DNA - promoters and enhancers. Research in yeast and mammalian cells indicates that modulation of chromatin accessibility occurs through interaction of transcription factors (TF) and chromatin remodelers. It has been shown that binding of certain TF to their target DNA sequences is highly dynamic, with residence time on the scale of few seconds, as observed by biophysical methods and directly, by single molecule tracking. These observations were recently supported by evidence from a ChIP assay with subsecond temporal resolution (CLK) and from high-resolution footprinting by deep sequencing. Slow cycles of the TF on the scale of a few minutes were also observed for several model mammalian genes. Our previous studies in yeast demonstrated that fast cycling occurs independently from slow cycling as they may coexist at the same promoter. Furthermore, we demonstrated that fast cycling of TF Ace1p on promoters of CUP1 are productive. We are interested in mechanisms of the rapid exchange on promoters exhibited by numerous TF and in the physiological significance of this fast cycle. Our working hypothesis is that TF factors are transiently recruited to the promoters and assist in loading of the co-factors. Fast cycling of the TF on the promoters is functional and essential for the optimization of the gene expression. We predict that the chromatin remodelers control the fast cycle by physically interacting with TA and removing them from the DNA target. Simultaneously, dynamic interaction of remodelers with TF controls the accessibility to chromatin, which is the pre-requisite to fast cycling. We predict that the residence time of each TF is optimized by chromatin remodelers for the best dynamic transcriptional response. The crucial question is to understand how TF residence times on chromatin relate to the amount of transcript produced from genes to which the transcription factor binds. We apply new method to measure residence times of transcription factors on chromatin within live cells: Single Molecule Tracking (SMT). This method allows direct evaluation of diffusion and binding in the nucleus. Using this approach, we direct our attention to how transcription factor residence times affect transcription. We optimized SMT data interpretation by developing a benchmark for nonspecific binding. To address this issue, we took advantage of the power of molecular genetics of yeast. We developed methods for SMT in yeast cell nuclei. We showed that the estimated residence time of the short-residence molecules is essentially the same for Hht1p (histone H3), and transcriptional factors Ace1p and Hsf1p, equalling 0.12- 0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (a) short-residence molecules are bound to DNA non-specifically, and (b) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. Currently we measure residence time of the transcription activator of the metallothionein genes (Ace1p) on specific promoters. We correlate the transcriptional activity of promoters in individual wild type cells and in cells defective for specific chromatin remodelers with changes in the residence time of TF. We observe the dynamics of transcription in live individual cells by live fluorescent marking of mRNA and in cell populations by single-molecule FISH. Additional important information about the dynamics of the TF interaction on specific promoter may be extracted from SMT data if two TF with related functions may be tracked simultaneously. We are developing and adapting instrumentation and technology to be able to track simultaneously two interacting functionally related molecules at a time and to visualize the chromatin target with a third fluorescent label. Ultimately, these studies will lay the groundwork for the analysis of in vivo interactions of the components of the transcriptional machinery. SMT technique that we are in process of developing may be applied to a number of other problems of cellular biology where the information for molecular interactions is desired.
