Towards a ‘Spot On’ Understanding of Transcription in the Nucleus

The Mediator kinase module: an interface between cell signaling and transcription

“…Because enhancer-promoter looping is transient and varies from cell-to-cell [106], population-based methods such as 3C or Hi-C are limited in their ability to resolve stimulus-specific or Mediator-dependent architectural changes. Live cell imaging experiments currently lack the spatial resolution required to confirm direct enhancer-promoter loops [107]. Based upon structural data of the human PIC [41][42][43], direct enhancer-promoter-PIC interaction may separate these sequences by 20-40 nm, which is beyond current resolution limits for fluorescence microscopy [108].…”

“…Based upon structural data of the human PIC [41][42][43], direct enhancer-promoter-PIC interaction may separate these sequences by 20-40 nm, which is beyond current resolution limits for fluorescence microscopy [108]. Moreover, live cell imaging requires signal averaging over time, which may preclude detection of transient enhancerpromoter interactions [107]. Nevertheless, chromosome conformation methods and live cell imaging experiments have markedly advanced our understanding of pol II transcription in cells and we anticipate that methodological improvements will yield new discoveries about enhancer-promoter dynamics and potential CDK-Mediator-dependent regulation.…”

The Mediator complex as a master regulator of transcription by RNA polymerase II

“…Many live-cell imaging experiments also rely on fluorophores tethered to 5′ ends of nascent RNA transcripts, which may diffuse away from Pol II, and this complicates interpretation of data. Moreover, imaging transcription in live cells requires averaging fluorescent signals over time frames of seconds to minutes, which may prevent detection of transient or infrequent enhancer–promoter interactions 183 .…”

Following the tracks: How transcription factor binding dynamics control transcription

“…Improvements of photostable dyes and thin optical sectioning techniques have enhanced the signal-to-noise ratio to allow researchers to directly observe and track the position of individual biomolecules with high spatiotemporal resolution in vivo, a technique called single-molecule tracking (SMT) ( 8 , 9 , 10 , 11 ). SMT has been an essential tool for understanding the real-time TF dynamics in living cells and has transformed our understanding of the molecular processes underlying gene regulation ( 12 , 13 , 14 , 15 , 16 ).…”