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Enhancers bind transcription factors, chromatin regulators, and non-coding transcripts to modulate the expression of target genes. Here, we report 3D genome structures of single mouse ES cells as they are induced to exit pluripotency and transition through a formative stage prior to undergoing neuroectodermal differentiation. We find that there is a remarkable reorganization of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5–8 distant chromosomal sites that are often separated by many Mb from each other. In the formative state, genes important for pluripotency exit establish contacts with emerging enhancers within these multiway hubs, suggesting that the structural changes we have observed may play an important role in modulating transcription and establishing new cell identities. [Display omitted] • 3D genome structure and H3K27me3 levels are reorganized in formative state pluripotency • Hundreds of multiway chromatin hubs form in each formative state cell • Enhancer-promoter interactions are reconfigured in multiway hubs • The structural reorganization in the formative state is regulated by DNMT3a/b and TET1 Lando, Ma, et al. report 3D genome structures of single mouse ES cells as they begin to differentiate and transition through a formative state. Multiway chromatin hubs form in the formative state, providing a place where genes that are going to be turned on establish long-range contacts with emerging enhancers. [ABSTRACT FROM AUTHOR]

Single-molecule imaging inside living cells has revealed that transcription factors (TFs) bind to DNA transiently, but a long-standing question is how this transient binding is related to transcription activation. Here, we devised a microscopy method to simultaneously measure transient TF binding at a single locus and the effect of these binding events on transcription. We show that DNA binding of the yeast TF Gal4 activates transcription of a target gene within a few seconds, with at least ∼20% efficiency and with a high initiation rate of ∼1 RNA/s. Gal4 DNA dissociation decreases transcription rapidly. Moreover, at a gene with multiple binding sites, individual Gal4 molecules only rarely stay bound throughout the entire burst but instead frequently exchange during a burst to increase the transcriptional burst duration. Our results suggest a mechanism for enhancer regulation in more complex eukaryotes, where TF cooperativity and exchange enable robust and responsive transcription regulation. [Display omitted] • In vivo imaging of transcription factor Gal4 DNA binding and GAL10 transcription • Gal4 association and dissociation activates and deactivates GAL10 transcription rapidly • Gal4 binds DNA surprisingly long, but rarely throughout the entire burst • Gal4 molecules frequently exchange to increase the transcriptional burst duration Pomp et al. use simultaneous imaging of transcription factor DNA binding and transcriptional activity of a single target gene in living cells to understand their kinetic relationship. Transcription factor Gal4 binds DNA transiently but exchanges frequently to prolong transcriptionally active periods. Transcription factor cooperativity and exchange enable robust transcription regulation. [ABSTRACT FROM AUTHOR]

DNA supercoiling has emerged as a major contributor to gene regulation in bacteria, but how DNA supercoiling impacts transcription dynamics in eukaryotes is unclear. Here, using single-molecule dual-color nascent transcription imaging in budding yeast, we show that transcriptional bursting of divergent and tandem GAL genes is coupled. Temporal coupling of neighboring genes requires rapid release of DNA supercoils by topoisomerases. When DNA supercoils accumulate, transcription of one gene inhibits transcription at its adjacent genes. Transcription inhibition of the GAL genes results from destabilized binding of the transcription factor Gal4. Moreover, wild-type yeast minimizes supercoiling-mediated inhibition by maintaining sufficient levels of topoisomerases. Overall, we discover fundamental differences in transcriptional control by DNA supercoiling between bacteria and yeast and show that rapid supercoiling release in eukaryotes ensures proper gene expression of neighboring genes. [Display omitted] • Transcription of neighboring GAL genes is coupled, and supercoils impede coupling • Buildup of supercoils from transcription inhibits transcription at adjacent genes • Inhibition occurs through destabilized DNA binding of the transcription factor • Wild-type yeast contains sufficient topoisomerase levels to prevent inhibition Patel et al. use single-molecule visualization of transcription of neighboring genes at the GAL locus in budding yeast to elucidate how DNA supercoils regulate transcription dynamics in eukaryotes. Accumulation of DNA supercoils from transcription impedes transcription at adjacent genes, but wild-type yeast prevents inhibition by sufficient supercoiling release. [ABSTRACT FROM AUTHOR]

The coactivator p300/CREB-binding protein (CBP) regulates genes by facilitating the assembly of transcriptional machinery and by acetylating histones and other factors. However, it remains mostly unclear how both functions of p300 are dynamically coordinated during gene control. Here, we showed that p300 can orchestrate two functions through the formation of dynamic clusters with certain transcription factors (TFs), which is mediated by the interactions between a TF's transactivation domain (TAD) and the intrinsically disordered regions of p300. Co-condensation can enable spatially defined, all-or-none activation of p300's catalytic activity, priming the recruitment of coactivators, including Brd4. We showed that co-condensation can modulate transcriptional initiation rate and burst duration of target genes, underlying nonlinear gene regulatory functions. Such modulation is consistent with how p300 might shape gene bursting kinetics globally. Altogether, these results suggest an intriguing gene regulation mechanism, in which TF and p300 co-condensation contributes to transcriptional bursting regulation and cooperative gene control. [Display omitted] • Some transcription factors can recruit p300 by co-condensation • Interactions between disordered regions of TF and p300 can drive co-condensation • Co-condensation can promote p300 transactivation and stabilize TF-p300 assembly • Transcriptional bursting and dose-response curve are modulated by co-condensation Ma et al. demonstrate that transcription factor and coactivator p300 can cocondense. They systematically dissect the mechanism and function of co-condensation. The findings offer quantitative insights into how p300 catalytic activity, gene transcriptional bursting, and gene dose-response curve can be regulated by co-condensation. [ABSTRACT FROM AUTHOR]

RNA polymerase II (RNA Pol II) contains a disordered C-terminal domain (CTD) whose length enigmatically correlates with genome size. The CTD is crucial to eukaryotic transcription, yet the functional and evolutionary relevance of this variation remains unclear. Here, we investigate how CTD length and disorder influence transcription. We find that length modulates the size and frequency of transcriptional bursting. Disorder is highly conserved and facilitates CTD-CTD interactions, an ability we show is separable from protein sequence and necessary for efficient transcription. We build a data-driven quantitative model, simulations of which recapitulate experiments and support that CTD length promotes initial polymerase recruitment to the promoter and slows down its release from it and that CTD-CTD interactions enable recruitment of multiple polymerases. Our results reveal how these parameters provide access to a range of transcriptional activity, offering a new perspective for the mechanistic significance of CTD length and disorder in transcription across eukaryotes. • RNA Pol II's C-terminal domain (CTD) length inversely correlates with gene density • CTD length modulates transcriptional bursting and strength of self-interactions • CTD promotes RNA Pol II recruitment via direct and indirect (self-)interactions • Self-interactions link transcription, phase separation, and scaling across genome sizes Quintero-Cadena et al. investigate RNA Pol II's CTD in the context of transcription activation. The authors propose a model to explain the role of CTD-length variation, providing a link between the canonical and phase-separation perspectives of transcription and a potential mechanism for scaling transcription activation across genome sizes. [ABSTRACT FROM AUTHOR]

Formation of self-associating loop domains is a fundamental organizational feature of metazoan genomes. Here, we employed quantitative live-imaging methods to visualize impacts of higher-order chromosome topology on enhancer-promoter communication in developing Drosophila embryos. Evidence is provided that distal enhancers effectively produce transcriptional bursting from target promoters over distances when they are flanked with boundary elements. Importantly, neither inversion nor deletion of a boundary element abrogates this "enhancer-assisting activity," suggesting that they can facilitate intra-domain enhancer-promoter interaction and production of transcriptional bursting independently of topologically associating domain (TAD) formation. In contrast, domain-skipping activity of distal enhancers was lost after disruption of topological domains. This observation raises a possibility that intra-domain and inter-domain enhancer-promoter interactions are differentially regulated by chromosome topology. • Intra-domain enhancer-promoter interaction occurs in the absence of TADs • Topological boundaries increase transcriptional output independently of TAD formation • TAD formation is essential for domain-skipping activity of distal enhancers Yokoshi et al. employ quantitative live-imaging methods to visualize impacts of TAD formation on enhancer-promoter communication. They show that intra-domain enhancer-promoter interaction occurs independently of TAD formation. In contrast, domain-skipping activity of distal enhancers is lost upon TAD disruption, suggesting that intra-domain and inter-domain enhancer-promoter interactions are differentially regulated by chromosome topology. [ABSTRACT FROM AUTHOR]

Summary Transcriptional regulation occurs via changes to rates of different biochemical steps of transcription, but it remains unclear which rates are subject to change upon biological perturbation. Biochemical studies have suggested that stimuli predominantly affect the rates of RNA polymerase II (Pol II) recruitment and polymerase release from promoter-proximal pausing. Single-cell studies revealed that transcription occurs in discontinuous bursts, suggesting that features of such bursts like frequency and intensity could also be regulated. We combined Pol II chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell transcriptional measurements to show that an independently regulated burst initiation step is required before polymerase recruitment can occur. Using a number of global and targeted transcriptional regulatory perturbations, we showed that biological perturbations regulated both burst initiation and polymerase pause release rates but seemed not to regulate polymerase recruitment rate. Our results suggest that transcriptional regulation primarily acts by changing the rates of burst initiation and polymerase pause release. Graphical Abstract Highlights • Burst initiation is required before polymerase recruitment can occur • Biological stimuli changed only burst initiation and polymerase pause release rates • No biological stimuli tested altered polymerase recruitment rate Mammalian genes are transcribed in discontinuous bursts. Using experimental data and computational modeling, Bartman et al. show that the key control points of transcriptional regulation are burst initiation and the release of RNA polymerase II from a paused state, but, unexpectedly, not polymerase recruitment rate. [ABSTRACT FROM AUTHOR]

Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)