Biophysical Society Thematic Meeting| Les Houches 2019

Multiscale Modeling of Chromatin: Bridging Experiment with Theory

Tuesday Speaker Abstracts

SUPERRESOLUTION IMAGING OF THE START TRANSCRIPTION FACTORS Labe Black 1 ; Sylvain Tollis 2 ; Jean-Bernard Fiche 3 ; Savanna B Dorsey 1 ; Jing Cheng 2 ; Marcelo Nollmann 3 ; Mike Tyers 2 ; Catherine A Royer 1 ; 1 Rensselaer Polytechnic Institute, Biological Sciences, Albany, New York, United States 2 Université de Montréal, Institute for Research in Immunology and Cancer, Montreal, Quebec, Canada 3 CNRS, INSERM, Université de Montpellier, Centre de Biochimie Structurale, Montpellier, Hérault, France An outstanding aspect of how the main G1/S transcription factors (TFs) in budding yeast, called SBF and MBF, function to regulate the commitment to division (Start) is the spatial organization of the ~200 G1/S promoters as cells progress through G1-phase. Here we have used super- resolution Photo-Activatable Localization Microscopy (PALM) to map the static and dynamic positions of mEos3.2 fusions of the G1/S TF components, Swi4, Mbp1 and Swi6, expressed from their natural loci in fixed and live G1-phase yeast cells. We found that 85% of each TF subunit is organized into a few nuclear clusters and that each cluster contains a cell-size independent number of TF copies (~8). The number of clusters increased during G1 phase from ~5 in small cells to ~30 in large cells, concordant with a size-dependent increase in TF copy number. This small maximum number of clusters compared to the ~200 target promoters implies close spatial proximity of several promoter sites within each cluster. Using live cell single particle tracking (spt)-PALM we observed slow and fast dynamic modes for each TF, which likely correspond to binding/dissociation from specific and non-specific DNA target sites, respectively, combined with diffusion on or off DNA. These results suggest that the promoters of the G1/S regulon are spatially organized into clusters that are titrated temporally in a hierarchical manner by increasing G1/S TF copy number as cells grow.

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