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 two different methods to measure residence times of transcription factors on chromatin within live cells: fluorescence recovery after photobleaching data (FRAP), and Single Molecule Tracking (SMT). A limitation of FRAP is that it is an indirect method that relies on mathematical models to describe changes in fluorescence intensity that arise due to at least two underlying processes, diffusion and binding. Neither process can be directly visualized by FRAP, so an incorrect assumption about how diffusion occurs can lead to an error in the estimates of binding. To evaluate more directly how diffusion and binding occur in the nucleus this group developed methods for single molecule tracking of transcription factors in live cell nuclei. This approach makes it easier to distinguish diffusion from binding since single molecules bound to chromatin move much less than molecules that diffuse through the nucleoplasm. Using this approach, we direct our attention to how transcription factor residence times affect transcription. The powerful technique of Single Molecule Tracking still lacks a solid foundation for distinguishing between specific and non-specific interactions.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. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. 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. We plan to measure residence time of the heat shock factor (Hsf1p) and the specific transcription activator of the metallothionein genes (Ace1p) on specific promoters. Currently we are able to track the molecules of only one TF at a time. 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. We will 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. For that we will observe the dynamics of transcription in live individual cells by live fluorescent marking of mRNA and we will observe transcripts in cell populations by single-molecule FISH. 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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Scientific Cores Intramural Research (ZIC)
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Paakinaho, Ville; Presman, Diego M; Ball, David A et al. (2017) Single-molecule analysis of steroid receptor and cofactor action in living cells. Nat Commun 8:15896
Presman, Diego M; Ball, David A; Paakinaho, Ville et al. (2017) Quantifying transcription factor binding dynamics at the single-molecule level in live cells. Methods 123:76-88
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