Abstract: Recent advances in single-molecule imaging technologies have made it possible to study gene expression dynamics at unprecedented resolution. In this talk, I will describe two projects that use this technology to visualize, quantify, and model gene expression at different levels. The first project involves the study of RNAP2 phosphorylation at a single-copy gene. Here, I combined three-color fluorescent microscopy with antibody-based probes that bind the different phosphorylated forms of endogenous RNAP2. Applying this methodology together with computational modeling, I found evidence that genes contain a heterogeneous distribution of RNAP2 along their length, with a large cluster of RNAP2 phosphorylated in Serine 5 predominantly forming near the promoter. Additionally, RNAP2 fluctuations are temporally correlated from those of the nascent mRNA, with RNAP2 being phosphorylated in the Serine 5 within 6 seconds, escaping the promoter within ~ 1.5 minutes, and completing transcription in 5 minutes. The second project uses single-molecule inexpensive fluorescence in situ hybridization (smiFISH) with automated image processing to visualize and quantify the transcription dynamics of the endogenous pro- and counter-inflammatory genes (DUSP1, IkBα, and COX-2) in hundreds of cells. Here I determined the temporal gene expression in the nuclear and cytoplasmic compartments, the number of active transcription sites, and their spatial localization. Finally, I will discuss how new single-molecule imaging technologies are opening new avenues for dissecting the signaling sequence of events from gene activation to mRNA synthesis and its chromatin landscape, and their potential implications in health and disease.
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JSCBB A115 Butcher Auditorium
Jennie Smoly Caruthers Biotechnology Building (JSCBB)
3415 Colorado Ave.
Boulder, CO 80303