Binding of certain transcription factors to their target DNA sequences is highly dynamic, with residence time on the scale of few seconds. The former head of this group Jim McNally first discovered this phenomenon over fourteen years ago in collaboration with Gordon Hager. Using fluorescence recovery after photobleaching (FRAP), this group had shown that the GFP-tagged glucocorticoid receptor is bound at a specific promoter for at most 60 seconds, even though transcription persists for several hours. For the other TF studied by this group, Ace1p of yeast, the half-time recovery rate by FRAP is 31 s. Similar results have now been observed by FRAP for a number of other transcription factors and for a variety of other nuclear proteins. Lately this group confirmed short residence time of GR on transcription factory by single molecule tracking and found it to be 8.1 s. These in vivo measurements are in many cases very different from measurements made in the test tube, which typically have indicated that nuclear proteins, including transcription factors, are much more stably bound. Thus in vivo direct measurements of the TF binding are calling into question the long held paradigm of stable transcription factor binding derived from in vitro measurements. 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. In order to measure the residence time 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. We use primarily single molecule tracking to analyze transcription factor binding for several different transcription factors and in several different types of cells. We complement the single molecule measurements with FRAP measurements.We are currently performing these measurements on the glucocorticoid receptor in mammalian cells, the heat shock factor (Hsf1p) and the specific transcription activator of the metallothionein genes (Ace1p) in yeast cells. Tracking the TFs in the nucleoplasm we find that the glucocorticoid receptor in mammalian cells and the heat shock factor in yeast cells exhibit transient binding on the order of a few seconds. We need to understand how much of this transient binding reflects non-specific interactions with chromatin which are involved in the search process to locate a target site vs. how much of the binding reflects specific interactions at target sites. For that we are optimizing single molecule tracking of transcription factors on a specific target, an array of the specific promoters, located by binding of a specific fluorescent marker. 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.
