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CPLC Special Seminar:"Mediator and Pol II clusters co-associate in transcription-dependent dynamic condensates in living stem cells."

Event Type
Department of Physics
276 Loomis
Jan 29, 2018   1:00 pm  
Won-Ki Cho, MIT
Marjorie Gamel
Originating Calendar
Physics - Biological Physics (CPLC/iPoLS) Seminar

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell single-molecule imaging approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observed that Pol II cluster lifetime correlates with the β-actin mRNA output. More recently, to investigate biological processes upstream of transcription, we developed an in vivo assay to observe cooperative dynamics of enhancers and transcription sites. Gene activation is thought to involve a multistep process whereby transcription factors bind to distal enhancer sites, recruit the Mediator complex which contacts the pre-initiating Pol II complex assembled at the gene loci. The interaction of Mediator and Pol II has yet to be observed in the nucleus of living cells and the dynamics of this interaction are not yet elucidated. We used quantitative live-cell PALM (photo-activation localization microscopy) and light sheet imaging to study the organization and dynamics of endogenous Mediator and Pol II directly in living mouse embryonic stem cells. In addition to forming transient clusters, we found Mediator and Pol II also form previously uncharacterized large and stable clusters in stem cells. The stable Mediator and Pol II clusters co-localized with each other. Tracking of Mediator and Pol II stable clusters suggests they are chromatin associated and they coalesce upon contact, a property associated with phase separated droplets. We concluded that Mediator and Pol II associate in diffraction-sized condensates on active transcription in living stem cells

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