Altering transcription factor binding reveals comprehensive transcriptional kinetics of a basic gene

Abstract

Transcription is a vital process activated by transcription factor (TF) binding. The active gene releases a burst of transcripts before turning inactive again. While the basic course of transcription is well understood, it is unclear how binding of a TF affects the frequency, duration and size of a transcriptional burst. We systematically varied the residence time and concentration of a synthetic TF and characterized the transcription of a synthetic reporter gene by combining single molecule imaging, single molecule RNA-FISH, live transcript visualisation and analysis with a novel algorithm, Burst Inference from mRNA Distributions (BIRD). For this well-defined system, we found that TF binding solely affected burst frequency and variations in TF residence time had a stronger influence than variations in concentration. This enabled us to device a model of gene transcription, in which TF binding triggers multiple successive steps before the gene transits to the active state and actual mRNA synthesis is decoupled from TF presence. We quantified all transition times of the TF and the gene, including the TF search time and the delay between TF binding and the onset of transcription. Our quantitative measurements and analysis revealed detailed kinetic insight, which may serve as basis for a bottom-up understanding of gene regulation.

Single-molecule tracking (SMT) and localization of SRF and MRTF transcription factors during neuronal stimulation and differentiation

“…Previously, accumulations of long-bound TF molecules in clusters have been suggested to present transcriptional ‘hot-spots’ with high RNA Polymerase II activity [ 7 , 43 , 44 ]. In order to visualize Halo-SRF accumulations in clusters we performed TALM (tracking and localization microscopy; figure 5 ), which allows for high-resolution localization and diffusion analysis of single proteins in living cells [ 33 , 45 , 46 ].…”

Transcription Factor RBPJL Is Able to Repress Notch Target Gene Expression but Is Non-Responsive to Notch Activation

“…Moreover, we compared the binding behaviour of RBPJ and RBPJL in the nucleus of live cells using single-molecule tracking ( Figure 4 C and Methods) [ 31 , 33 ]. To visualize single molecules, we created HeLa cell lines stably expressing RBPJ or RBPJL fused to a HaloTag [ 40 ], which we labeled with the organic dye SiR before imaging [ 41 ].…”

“…Microscope setup: A custom-built fluorescence microscope (as described previously [ 31 ]) was used for single-molecule imaging. It contained a conventional Nikon body (TiE, Nikon) and was equipped with a 638 nm laser (IBEAM-SMART-640-S, 150 mW, Toptica), AOTF (AOTFnC-400.650-TN, AA Optoelectronics) and a high-NA objective (100×, NA 1.45, Nikon).…”

“…The survival-time distributions of each construct were analyzed globally with GRID. GRID uses an inverse Laplace transformation to extract a dissociation rate spectrum from survival-time distributions [ 31 , 34 ]. This spectrum of dissociation rates (“event spectrum”) displays how frequently dissociation from a certain binding state with a corresponding dissociation rate occurs during an observation period.…”

Individual transcription factors modulate both the micromovement of chromatin and its long-range structure

“…Single-particle tracking (SPT) methods have been used to study dynamic TF-DNA interactions and to assess the role of TFs in shaping the chromatin mobility landscape in living eukaryotic cells (19)(20)(21)(22)(23)(24)(25). Most of these studies showed that the residence time of DNA-bound TFs affects the temporal expression patterns of transcription (19,23,24) and the spatial organization of chromatin in live cells (20,22). This shows the importance of residence times as a biophysical characteristic for regulating transcriptional activity and genome organization.…”