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Event Type:  Seminar (This is an NIH Science event)
Annual Lecture:  Other
Title:  Single-Molecule Imaging and the Future of Simultaneous Tracking of Multiple Transcription Factors
Description:  Population-based assays have been employed extensively to investigate the interactions of transcription factors (TFs) with chromatin and are often interpreted in terms of static and sequential binding. However, fluorescence microscopy techniques reveal a more dynamic binding behavior of TFs in live cells. With recent advances in fluorescence imaging, it has become possible to track individual TF molecules in single live cells. In particular, single-molecule tracking (SMT) approaches allow one to follow individual protein molecules in single live cells. These technological advances now provide the means for the visualization and the measurement of the in vivo behavior of TF-binding events at chromatin targets such as enhancers and core promoters. Steroid receptors are a class of ligand-inducible TFs that respond to environmental stimuli and mediate the expression of genes involved in metabolic, developmental, and inflammatory pathways. Their hormone-dependent nature makes these proteins ideal for studying transcription factor dynamics.
We have utilized highly inclined and laminated optical sheet (HILO) illumination, HaloTag and SNAP-tag labelling system, and Janelia Fluor (JF) fluorescent dyes to characterized the single-molecule action of several steroid receptors (GR, ER, PR, AR), other TFs (FoxA1, CREB1, AP-1) and cofactors (GRIP1, BRG1). Our results indicate that TF residence time at specific response elements ranges between 6 and 14 sec. In addition to short residence time at chromatin, our data also indicates that only a small proportion of TFs and cofactors are functionally bound at any given time. These results support transient rather than stable TF-chromatin interactions.
The experiments above were performed with single-color HILO microscope. To take the next step in understanding real-time dynamics of TF action, we are currently utilizing 3-color HILO microscope to simultaneously track multiple TF and cofactors in living cells. I will discuss several cha
Series Name:  Light Microscopy Interest Group Seminar
Videocast:  Event will not be videocast
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Tuesday, April 18, 2017   11:00am - 12:00pm Add To Outlook Calendar     Add To iCal Calendar     Add To Entourage Calendar
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Name:   Ville Paakinaho
Title:   Dr
Organization:   NCI/NIH
City/Province:   Bethesda
State:   Maryland
Country:   USA

Organization(s):  [NIH] Light Microscopy Interest Group

Location:  On the main NIH Campus
Building:  37
Room:  4107/4041
Street Address:  37 Convent Drive
City:  Bethesda
State:  Maryland
Zip Code:  20892

Name:   Tatiana Karpova
Phone:   240-760-6637
Fax:   240-541-4450
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