Current research indicates that expression of many important genes is cyclical, and on the different time scale, such as circadian or ultradian cycles on the scale of hours, or fast and short bursts of transcription on the scale of minutes. The mechanisms of this cycling are poorly understood, but very important to learn about for the correct application of the drugs. 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. Binding of certain TF to their target DNA sequences is highly dynamic, on the scale of seconds. Also, some TF undergo the slow cycling on the scale of minutes, which consist of alternating ON and OFF gene states. It is unclear how this TF cycling is related to the transcriptional cycling. We are interested in molecular mechanisms of the TF cycling on promoters and its correlation with transcription cycles. We use as a model a yeast gene CUP1 encoding metallothionein expressed in response to heavy metal stress. CUP1 is activated by copper-bound TF Ace1p. Our previous studies demonstrated two types of Ace1p cycling at CUP1: fast - on the scale of seconds, and slow - on the scale of minutes. The fast cycling occurs within the slow cycle. The slow cycle of binding may be quantified in individual cells by the regular fluorescence imaging, and the fast cycling - by Single Molecule Tracking (SMT). Previously, we have built a custom instrument for SMT and optimized SMT data interpretation. We have developed methods for SMT in yeast cell nuclei and methods of performing SMT on specific promoters. By SMT, we have measured binding parameters of a chromatin remodeler RSC and TF Ace1p to specific sites (Metal Response Elements, MRE) within CUP1 promoter. Transcription of CUP1 may be observed either by smFISH (Fluorescence In Situ Hybridization) or by live imaging of the fluorescently tagged mRNA. Recently, we correlated the transcriptional activity of CUP1 promoter by smFISH with changes in the search for the binding sites and the specific residence time of TF. We demonstrated that the transient recruitment of TFs is regulated by fast cyclical chromatin remodeling events to ensure the best dynamic transcriptional response. Our current goal is to corroborate these findings with analysis of the transcription in live cells. Preliminary observations indicate that the slow cycle of transcription consists of the fast cycles (bursts) of expression on individual genes. This kind of observations cannot be made by smFISH in fixed cells and provides new information about transcription. This is the first genetic model where the superimposed slow and fast cycles of transcription correlate with superimposed slow and fast cycling of the transcriptional factor. We are working on modeling of the slow and fast transcription cycles by live TS and corroborating the observations by smFISH modeling. Ultimately, these studies will lay the groundwork for the analysis of in vivo interactions of the components of the transcriptional machinery. The technique of correlation of the TF biophysics with transcription modeling that we are in process of developing may be applied to a number of other problems of cellular biology where the information for molecular regulation of transcription is desired.
