Claire Dugast-Darzacq

Real-Time Dynamics of RNA Polymerase II Clustering in Live Human Cells

Pol II Micro Clusters In higher eukaryotes, messenger RNA (mRNA) synthesis is thought to involve foci of clustered RNA polymerase II (Pol II) called transcription factories. However, clustered Pol II have not been resolved in living cells, raising the debate about their existence in vivo and what role, if any, they play in nuclear organization and regulation of gene expression. Cisse et al. (p. 664, published online 4 July; see the Perspective by Rickman and Bickmore) developed single-molecule in vivo analyses revealing the distribution and dynamics of Pol II clustering in living cells. Pol II clusters were smaller than the diffraction limit (<250 nm). Transient dynamics of the Pol II clusters, and correlation with changes in transcription, pointed to a role in transcription initiation rather than in elongation. A single-cell quantitative method reveals changes in the distribution of proteins with single-molecule sensitivity. [Also see Perspective by Rickman and Bickmore] Transcription is reported to be spatially compartmentalized in nuclear transcription factories with clusters of RNA polymerase II (Pol II). However, little is known about when these foci assemble or their relative stability. We developed a quantitative single-cell approach to characterize protein spatiotemporal organization, with single-molecule sensitivity in live eukaryotic cells. We observed that Pol II clusters form transiently, with an average lifetime of 5.1 (± 0.4) seconds, which refutes the notion that they are statically assembled substructures. Stimuli affecting transcription yielded orders-of-magnitude changes in the dynamics of Pol II clusters, which implies that clustering is regulated and plays a role in the cell’s ability to effect rapid response to external signals. Our results suggest that transient crowding of enzymes may aid in rate-limiting steps of gene regulation.

Single-molecule tracking in live cells reveals distinct target-search strategies of transcription factors in the nucleus

Gene regulation relies on transcription factors (TFs) exploring the nucleus searching their targets. So far, most studies have focused on how fast TFs diffuse, underestimating the role of nuclear architecture. We implemented a single-molecule tracking assay to determine TFs dynamics. We found that c-Myc is a global explorer of the nucleus. In contrast, the positive transcription elongation factor P-TEFb is a local explorer that oversamples its environment. Consequently, each c-Myc molecule is equally available for all nuclear sites while P-TEFb reaches its targets in a position-dependent manner. Our observations are consistent with a model in which the exploration geometry of TFs is restrained by their interactions with nuclear structures and not by exclusion. The geometry-controlled kinetics of TFs target-search illustrates the influence of nuclear architecture on gene regulation, and has strong implications on how proteins react in the nucleus and how their function can be regulated in space and time. DOI: http://dx.doi.org/10.7554/eLife.02230.001

Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher

Many cellular functions rely on DNA-binding proteins finding and associating to specific sites in the genome. Yet the mechanisms underlying the target search remain poorly understood, especially in the case of the highly organized mammalian cell nucleus. Using as a model Tet repressors (TetRs) searching for a multi-array locus, we quantitatively analyse the search process in human cells with single-molecule tracking and single-cell protein–DNA association measurements. We find that TetRs explore the nucleus and reach their target by 3D diffusion interspersed with transient interactions with non-cognate sites, consistent with the facilitated diffusion model. Remarkably, nonspecific binding times are broadly distributed, underlining a lack of clear delimitation between specific and nonspecific interactions. However, the search kinetics is not determined by diffusive transport but by the low association rate to nonspecific sites. Altogether, our results provide a comprehensive view of the recruitment dynamics of proteins at specific loci in mammalian cells.

Imaging dynamic and selective low-complexity domain interactions that control gene transcription

Phase separation and gene control Many components of eukaryotic transcription machinery—such as transcription factors and cofactors including BRD4, subunits of the Mediator complex, and RNA polymerase II—contain intrinsically disordered low-complexity domains. Now a conceptual framework connecting the nature and behavior of their interactions to their functions in transcription regulation is emerging (see the Perspective by Plys and Kingston). Chong et al. found that low-complexity domains of transcription factors form concentrated hubs via functionally relevant dynamic, multivalent, and sequence-specific protein-protein interaction. These hubs have the potential to phase-separate at higher concentrations. Indeed, Sabari et al. showed that at super-enhancers, BRD4 and Mediator form liquid-like condensates that compartmentalize and concentrate the transcription apparatus to maintain expression of key cell-identity genes. Cho et al. further revealed the differential sensitivity of Mediator and RNA polymerase II condensates to selective transcription inhibitors and how their dynamic interactions might initiate transcription elongation. Science, this issue p. eaar2555, p. eaar3958, p. 412; see also p. 329 Low-complexity domains of eukaryotic transcription factors form hubs via dynamic, multivalent, sequence-specific interactions. INTRODUCTION DNA binding transcription factors (TFs) are quintessential regulators of eukaryotic gene expression. Early studies of TFs revealed their well-structured DNA binding domains (DBDs) and identified functionally critical activation domains (ADs) required for transcription. It later became evident that many ADs contain intrinsically disordered low-complexity sequence domains (LCDs), but how LCDs activate transcription has remained unclear. Although it is known that transcriptional activation by LCDs requires selective interaction with binding partners, it has been challenging to directly measure selective LCD-LCD recognition in vivo and unravel its mechanism of action. RATIONALE Traditional biochemical reconstitution and genetics studies have identified most of the molecular players central to transcription regulation. However, the mechanism by which weak, dynamic protein-protein interactions drive gene activation in living cells has remained unknown. Advances in live-cell single-molecule imaging have opened a new frontier for studying transcription in vivo. In this study, we used synthetic LacO (Lac operator) arrays as well as endogenous GGAA microsatellite loci to study LCD-LCD interactions of TFs such as EWS/FLI1, TAF15, and Sp1 in live cells. To probe the dynamic behavior of TF LCDs at target genomic loci, we have combined CRISPR-Cas9 genome editing, mutagenesis, gene activation, cell transformation assays, and various high-resolution imaging approaches including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, lattice light-sheet microscopy, three-dimensional DNA fluorescence in situ hybridization, and live-cell single-particle tracking. RESULTS Live-cell single-molecule imaging revealed that TF LCDs interact to form local high-concentration hubs at both synthetic DNA arrays and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic (seconds to minutes), selective for binding partners, and differentially sensitive to disruption by hexanediols. These findings suggest that under physiological conditions, rapid, reversible, and selective multivalent LCD-LCD interactions occur between TFs and the RNA Pol II machinery to activate transcription. We observed formation of functional TF LCD hubs at a wide range of intranuclear TF concentrations. Although we detected apparent liquid-liquid phase separation with gross overexpression of LCDs, transcriptionally competent TF LCD hubs were observed at physiological TF levels at endogenous chromosomal loci in the absence of detectable phase separation. In addition, mutagenesis, gene expression, and cell transformation assays in Ewing’s sarcoma cells revealed a functional link between LCD-LCD interactions, transactivation capacity, and oncogenic potential. CONCLUSION The use of various imaging methods in live cells powerfully complements in vitro studies and provides new insights into the nature of LCD interactions and their role in gene regulation. We propose that transactivation domains function by forming local high-concentration hubs of TFs via dynamic, multivalent, and specific LCD-LCD interactions. It also seems likely that weak, dynamic, and transient contacts between TFs play a role in disease-causing dysregulation of gene expression (i.e., EWS/FLI1 in Ewing’s sarcoma), suggesting that LCD-LCD interactions may represent a new class of viable drug targets. Although we examined a small subset of TF LCDs, the principles uncovered regarding the dynamics and mechanisms driving LCD-LCD interactions may be applicable to other classes of proteins and biomolecular interactions occurring in many cell types. From hubs to phase separation: Activation occurs in a wide range of TF concentrations. In vivo LCD-dependent transactivation occurs in hubs formed over a broad range of TF concentrations (100 nM to 100 μM) and time scales (<1 s to minutes). At endogenous concentrations, TF LCDs form transactivation hubs at native genomic loci without undergoing evident phase separation. Upon TF LCD overexpression, phase separation is observed at synthetic TF binding site arrays. Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.

RNA polymerase II clustering through CTD phase separation

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters at active genes through interactions between CTDs and with activators, and that CTD phosphorylation removes Pol II enzymes from clusters for transcription elongation.

Differential effects of Mxi1-SRalpha and Mxi1-SRbeta in Myc antagonism.

Mxi1 belongs to the Myc‐Max‐Mad transcription factor network. Two Mxi1 protein isoforms, Mxi1‐SRα and Mxi1‐SRβ, have been described as sharing many biological properties. Here, we assign differential functions to these isoforms with respect to two distinct levels of Myc antagonism. Unlike Mxi1‐SRβ, Mxi1‐SRα is not a potent suppressor of the cellular transformation activity of Myc. Furthermore, although Mxi1‐SRβ exhibits a repressive effect on the MYC promoter in transient expression assays, Mxi1‐SRα activates this promoter. A specific domain of Mxi1‐SRα contributes to these differences. Moreover, glyceraldehyde‐3‐phosphate dehydrogenase interacts with Mxi1‐SRα and enhances its ability to activate the Myc promoter. Our findings suggest that Mxi1 gains functional complexity by encoding isoforms with shared and distinct activities.

Master regulators in primary skin fibroblast fate reprogramming in a human ex vivo model of chronic wounds.

Fibroblasts are important players in regulating tissue homeostasis. In the dermis, they are involved in wound healing where they differentiate into contractile myofibroblasts leading to wound closure. In nonhealing chronic wounds, fibroblasts fail to undertake differentiation. We established and used a human ex vivo model of chronic wounds where fibroblasts can undergo normal myofibroblast differentiation, or take on a nondifferentiable pathological state. At the whole genome scale, we identified the genes that are differentially regulated in these two cell fates. By coupling the search of evolutionary conserved regulatory elements with global gene network expression changes, we identified transcription factors (TF) potentially involved in myofibroblast differentiation, and constructed a network of relationship between these key factors. Among these, we found that TCF4, SOX9, EGR2, and FOXS1 are major regulators of fibroblast to myofibroblast differentiation. Conversely, down‐regulation of MEOX2, SIX2, and MAF causes reprogramming of fibroblasts to myofibroblasts even in absence of TGF‐β, the natural inducer of myofibroblast differentiation. These results provide insight into the fibroblast differentiation program and reveal a TF network essential for cellular reprogramming. They could lead to the development of new therapeutics to treat fibroblast‐related human pathologies.

RNA polymerase II clustering through carboxy-terminal domain phase separation

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation, with the shorter yeast CTD forming less-stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association, whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters or hubs at active genes through interactions between CTDs and with activators and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.In vitro assays and high-resolution microscopy show that Pol II CTD undergoes length-dependent liquid phase separation and controls Pol II clustering and mobility in human cells.

mRNA expression-mediated gene repression in human cells

We recently discovered a common mode of gene regulation in budding yeast, by which mRNA production represses protein expression. Whether this regulatory mechanism is conserved was unknown. Here we find that a similar mechanism regulates the human oncogene MDM2, which is transcribed from two promoters. Transcription from the distal MDM2 promoter produces a poorly translated mRNA isoform and transcription from the proximal promoter produces a well-translated transcript. Remarkably, we find that down-regulation of transcription from the distal promoter markedly up-regulates expression from the proximal promoter and results in the loss of histone H3K36 trimethylation marks. Moreover, we observe transcript toggling between the two different MDM2 isoforms as a natural part of two distinct human embryonic stem cell differentiation programs. We conclude that the integrated gene repression mechanism recently identified in yeast is conserved in human cells.We previously discovered a new mode of gene regulation in budding yeast, by which mRNA production represses protein expression through a cis -acting transcriptional and translational interference mechanism (Chen et al. 2017; Chia et al., 2017). Whether this regulatory mechanism is conserved in other eukaryotes was unknown. Here we found that a similar mechanism regulates the human oncogene MDM2 , which is transcribed from two different promoters. Transcription from the distal MDM2 promoter produces a poorly translated mRNA isoform, which establishes repressive histone H3K36 trimethylation marks at the proximal MDM2 promoter. In this manner, production of the 5′-extended transcript interferes with the expression of the MDM2 transcripts that are well translated. Accordingly, downregulation of transcription from the distal promoter up-regulates MDM2 protein levels. We conclude that this non-canonical mechanism, first defined in yeast, is conserved in human cells. We propose that a similar mechanism may modulate the expression of other mammalian genes with alternative promoters and differentially translated mRNA isoforms.

Transient DNA Binding Induces RNA Polymerase II Compartmentalization During Herpesviral Infection Distinct From Phase Separation

During lytic infection, Herpes Simplex Virus 1 generates replication compartments (RCs) in host nuclei that efficiently recruit protein factors, including host RNA Polymerase II (Pol II). Pol II and other cellular factors form hubs in uninfected cells that are proposed to phase separate via multivalent protein-protein interactions mediated by their intrinsically disordered regions. Using a battery of live cell microscopic techniques, we show that although RCs superficially exhibit many characteristics of phase separation, the recruitment of Pol II instead derives from nonspecific interactions with the viral DNA. We find that the viral genome remains nucleosome-free, profoundly affecting the way Pol II explores RCs by causing it to repetitively visit nearby binding sites, thereby creating local Pol II accumulations. This mechanism, distinct from phase separation, allows viral DNA to outcompete host DNA for cellular proteins. Our work provides new insights into the strategies used to create local molecular hubs in cells.