Biology subjects

Darzacq, X.

Publications and source records attributed to Darzacq, X..

At least 19 recordsLinked to original sources

bioRxiv2026

Phenotypic CRISPR screening identifies ZBTB10 as a novel regulator of human trophoblast differentiation

The human placenta is built by trophoblast cells that fuse together, secrete hormones, and invade the uterus, and defects in these processes contribute to pregnancy disorders such as preeclampsia. Because cell-cell fusion and hormone secretion are inherently non-cell-autonomous processes, their regulators have remained inaccessible to conventional pooled CRISPR screens. Here, we developed an arrayed CRISPR screen in fusogenic BeWo trophoblasts that simultaneously quantifies fusion and hCG secretion across 412 gene perturbations. The screen revealed that these two hallmark functions of trophoblast differentiation are genetically separable. We characterized the strongest novel hit, ZBTB10, in trophoblast stem cells, organoids, and placental tissue and find that ZBTB10 is an essential regulator of human trophoblast differentiation. ZBTB10 is required for invasive extravillous trophoblast differentiation and supports syncytiotrophoblast maturation, establishing it as a cross-lineage regulator that both activates and represses distinct trophoblast fate programs. Together, these findings provide a genetic platform and phenotypic dissection of how regulatory networks control human placental development.

cell biology↗

bioRxiv2025

Modulating MyoD1 dosage activates alternate cell fate beyond myogenic differentiation

Transcription factor (TF) upregulation accompanies many cellular state transitions, yet how increased TF abundance impacts gene regulation remains unclear. Two broad models are often invoked, whereby higher TF levels amplify the expression of pre-existing target genes, or, by mass-action binding, expand genome engagement and regulation to lower-affinity sites. We sought to elucidate how these two regulatory modes contribute to cell differentiation in a well characterized myogenic system by upregulating the expression of the myogenic TF MyoD1 in C2C12 myoblasts. Unexpectedly, elevated MyoD1 levels impaired myoblast fusion (a hallmark of myogenic differentiation), yet enabled robust contraction in myotubes that did form. Live-cell single-molecule imaging and CUT&RUN profiling revealed that elevated MyoD1 dosage increased total genome-wide chromatin binding and broadened genome occupancy by preferentially engaging lower-affinity sites. Integrating CUT&RUN with RNA-seq experiments linked expanded MyoD1 binding to upregulation of cell adhesion genes. Cell mixing and fractionated RNA-seq experiments supported a two-population model in which an adhesion-gene-upregulated, unfused myoblast population supported contraction of myotubes formed by fusion-competent cells. Ectopic expression of several individual MyoD1-upregulated cell adhesion genes was sufficient to recapitulate the "off script" myotube contraction phenotype. Together, these results support a MyoD1 dose-dependent "spillover" model, in which increased TF abundance broadens cis-regulatory engagement and produces distinct cell differentiation outcomes. Significance StatementTranscription factor dosage is a central control knob in many biological and disease-associated cell state transitions, but its range of gene regulatory consequences are poorly understood. By inducibly elevating MyoD1 expression in C2C12 cells, we uncovered a paradoxical outcome: elevated MyoD1 levels suppressed myoblast fusion yet enabled robust myotube contraction. Elevated MyoD1 levels expanded MyoD1 genome occupancy through engagement of low-affinity sites and upregulated adhesion genes whose individual ectopic expression was sufficient to recapitulate the contraction phenotype. Mixing experiments indicated a non-cell-autonomous contribution from an adhesion-gene-high subpopulation that supported contraction of myotubes. Our results demonstrate that TF dosage can expand regulatory scope and yield qualitatively different differentiation outcomes.

molecular biology↗

bioRxiv2024

Short activation domains control chromatin association of transcription factors

Transcription factors regulate gene expression with DNA-binding domains (DBDs) and activation domains. Despite mounting evidence to the contrary, it is frequently assumed that DBDs are solely responsible for interacting with DNA and chromatin. Here, we used single-molecule tracking of transcription factors in living cells to show that short activation domains can control the fraction of molecules bound to chromatin. Stronger activation domains have higher bound fractions and longer residence times on chromatin. Furthermore, mutations that increase activation domain strength also increase chromatin binding. This trend was consistent in four different activation domains and their mutants. This effect further held for activation domains appended to three different structural classes of DBDs. Stronger activation domains with high chromatin-bound fractions also exhibited increased binding to the p300 coactivator in proximity-assisted photoactivation experiments. Taken together, these results suggest that activation domains play a major role in tethering transcription factors to chromatin, challenging the traditional view that the DBD is the sole driver of genome binding.

biophysics↗

bioRxiv2024

Single-molecule live imaging of subunit interactions and exchange within cellular regulatory complexes

Cells are built from vast networks of competing molecular interactions, most of which have been impossible to monitor in vivo. We recently devised a new strategy, proximity-assisted photoactivation (PAPA), to detect these interactions at single-molecule resolution in live cells. Here we apply PAPA to visualize the network of interactions that regulate the central transcription elongation factor P-TEFb. PAPA between multiple pairs of endogenous proteins, combined with fast single-molecule tracking (fSMT), revealed that inactive P-TEFb within the 7SK ribonucleoprotein complex is largely unbound to chromatin, that this complex dissociates within minutes of treatment with a P-TEFb kinase inhibitor, and that heterogeneous ribonucleoproteins (hnRNPs) bind 7SK concomitant with P-TEFb release. Unlike 7SK-bound P-TEFb, P-TEFb associated with the coactivator BRD4 exhibited increased binding to chromatin. Our results address longstanding questions about a key transcriptional regulator and demonstrate that PAPA-fSMT can probe subunit interactions and exchange within endogenous regulatory complexes in live cells.

molecular biology↗

bioRxiv2024

Automated live-cell single-molecule tracking in enteroid monolayers reveals transcription factor dynamics probing lineage-determining function

Lineage transcription factors (TFs) provide one regulatory level of differentiation crucial for the generation and maintenance of healthy tissues. To probe TF function by measuring their dynamics during adult intestinal homeostasis, we established HILO-illumination-based live-cell single-molecule tracking (SMT) in mouse small intestinal enteroid monolayers recapitulating tissue differentiation hierarchies in vitro. To increase the throughput, capture cellular features, and correlate morphological characteristics with diffusion parameters, we developed an automated imaging and analysis pipeline, broadly applicable to 2D culture systems. Studying two absorptive lineage-determining TFs, we find an expression level-independent contrasting diffusive behavior: While Hes1, key determinant of absorptive lineage commitment, displays a large cell-to-cell variability and an average fraction of DNA-bound molecules of [~]32%, Hnf4g, conferring enterocyte identity, exhibits more uniform dynamics and a bound fraction of [~]56%. Our results suggest that TF diffusive behavior can indicate the progression of differentiation and modulate early versus late differentiation within a lineage. Highlights- Automated live-cell single-molecule tracking records hundreds of cells in enteroid monolayers - Cellular diffusion clustering and morphological feature correlation reveals subpopulations - Transcription factor dynamics regulate differentiation independent of expression level - Hes1 and Hnf4g display contrasting dynamics assisting early vs. late absorptive differentiation

developmental biology↗

bioRxiv2024

TFEB controls syncytiotrophoblast differentiation

During human development, a subset of differentiating fetal cells form a temporary organ, the placenta, which invades the uterine wall to support nutrient, oxygen, and waste exchange between the mother and fetus until birth. Most of the human placenta is formed by a syncytial villous structure which arises via cell-cell fusion of underlying fetal trophoblast stem cells. Genetic and functional studies have characterized the membrane protein fusogens, Syncytin-1 and Syncytin-2, that are both necessary and sufficient for human trophoblast cell-cell fusion. However, identification and characterization of upstream transcriptional regulators regulating their expression has been limited. Here, using CRISPR knockout in an in vitro cellular model of syncytiotrophoblast development (BeWo cells), we find that the transcription factor TFEB, mainly known as a regulator of autophagy and lysosomal biogenesis, is required for cell-cell fusion of syncytiotrophoblasts. TFEB translocates to the nucleus, exhibits increased chromatin interactions, and directly binds the Syncytin-1 and Syncytin-2 promoters to control their expression during differentiation. While TFEB appears to play an important role in syncytiotrophoblast differentiation, ablation of TFEB largely does not affect lysosomal gene expression or lysosomal biogenesis in differentiating BeWo cells, suggesting that TFEB plays an alternative role in placental cells.

cell biology↗

bioRxiv2023

Oblique Line Scan Illumination Enables Expansive, Accurate and Sensitive Single Protein Measurements in Solution and in Living Cells

Single-molecule localization microscopy (SMLM) techniques, such as single-molecule tracking (SMT), enable in situ measurements in cells from which data-rich metrics can be extracted. SMT has been successfully applied to a variety of biological questions and model systems, aiming to unravel the spatiotemporal regulation of molecular mechanisms that govern protein function, downstream pathway effects, and cellular function. While powerful, SMLM often suffers from low throughput and illumination inhomogeneity, along with microscope and user-induced technical biases. Due to technical limitations in scaling SMLM techniques, a tradeoff between spatiotemporal resolution and throughput has been made historically, restricting broad application of these technologies. Here we address these limitations using Oblique Line Scan (OLS), a robust single-objective light-sheet based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a 250 x 190 m field of view. We demonstrate OLS-enabled SMT on Halo-Tagged proteins in living cells capturing protein motion up to 14 m2 /s. By exploiting the adaptability of the acquisition frame rate and the improved rejection of out of focus light, we extend the utility of OLS beyond cellular compartments with in-solution SMT (isSMT) for single-molecule measurement of ligand-protein interactions and disruption of protein-protein interactions (PPI). We illustrate the versatility of OLS by showcasing two-color SMT, STORM, and single molecule fluorescence recovery after photobleaching (FRAP). OLS expands the range of SMLM applications and paves the way for robust, high-throughput single-molecule investigations of protein dynamics required for drug screening and systems biology studies, both in cells and in solution.

biophysics↗

bioRxiv2023

Double-Strand Break Repair Pathways Differentially Affect Processing and Transduction by Dual AAV Vectors

Recombinant adeno-associated viral vectors (rAAV) are a powerful tool for gene delivery but have a limited DNA carrying capacity. Efforts to expand this genetic payload have focused on engineering the vector components, such as dual trans-splicing vectors which double the delivery size by exploiting the natural concatenation of rAAV genomes in host nuclei. We hypothesized that inefficient dual vector transduction could be improved by modulating host factors which affect concatenation. Since factors mediating concatenation are not well defined, we performed a genome-wide screen to identify host cell regulators. We discovered that Homologous Recombination (HR) is inhibitory to dual vector transduction. We demonstrate that depletion or inhibition of HR factors BRCA1 and Rad51 significantly increase reconstitution of a large split transgene by increasing both concatenation and expression from rAAVs. Our results define new roles for DNA damage repair in rAAV transduction and highlight the potential for pharmacological intervention to increase genetic payload of rAAV vectors.

molecular biology↗

bioRxiv2023

Surprising Features of Nuclear Receptor Interaction Networks Revealed by Live Cell Single Molecule Imaging

Type 2 Nuclear Receptors (T2NRs) require heterodimerization with a common partner, the Retinoid X Receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and over-expression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged retinoid X receptor (RXR) and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

biophysics↗

bioRxiv2023

p300 Is an Obligate Integrator of Combinatorial Transcription Factor Inputs

Transcription coactivators are proteins or protein complexes that mediate transcription factor (TF) function. However, they lack DNA binding capacity, prompting the question of how they engage target loci. Three non-exclusive hypotheses have been posited: coactivators are recruited by complexing with TFs, by binding histones through epigenetic reader domains, or by partitioning into phase-separated compartments through their extensive intrinsically disordered regions (IDRs). Using p300 as a prototypical coactivator, we systematically mutated its annotated domains and show by single-molecule tracking in live cells that coactivator- chromatin binding depends entirely on combinatorial binding of multiple TF-interaction domains. Furthermore, we demonstrate that acetyltransferase activity negatively impacts p300-chromatin association and that the N-terminal TF-interaction domains regulate that activity. Single TF-interaction domains are insufficient for both chromatin binding and regulation of catalytic activity, implying a principle that could broadly inform eukaryotic gene regulation: a TF must act in coordination with other TFs to recruit coactivator activity.

biophysics↗

bioRxiv2023

High-throughput single molecule tracking identifies drug interactions and cellular mechanisms

The regulation of cell physiology depends largely upon interactions of functionally distinct proteins and cellular components. These interactions may be transient or long-lived, but often affect protein motion. Measurement of protein dynamics within a cellular environment, particularly while perturbing protein function with small molecules, may enable dissection of key interactions and facilitate drug discovery; however, current approaches are limited by throughput with respect to data acquisition and analysis. As a result, studies using super-resolution imaging are typically drawing conclusions from tens of cells and a few experimental conditions tested. We addressed these limitations by developing a high-throughput single-molecule tracking (htSMT) platform for pharmacologic dissection of protein dynamics in living cells at an unprecedented scale (capable of imaging > 106 cells/day and screening > 104 compounds). We applied htSMT to measure the cellular dynamics of fluorescently tagged estrogen receptor (ER) and screened a diverse library to identify small molecules that perturbed ER function in real time. With this one experimental modality, we determined the potency, pathway selectivity, target engagement, and mechanism of action for identified hits. Kinetic htSMT experiments were capable of distinguishing between on-target and on-pathway modulators of ER signaling. Integrated pathway analysis recapitulated the network of known ER interaction partners and suggested potentially novel, kinase-mediated regulatory mechanisms. The sensitivity of htSMT revealed a new correlation between ER dynamics and the ability of ER antagonists to suppress cancer cell growth. Therefore, measuring protein motion at scale is a powerful method to investigate dynamic interactions among proteins and may facilitate the identification and characterization of novel therapeutics.

cell biology↗

bioRxiv2021

Proximity-assisted photoactivation (PAPA): Detecting molecular interactions in live-cell single-molecule imaging

Single-molecule imaging provides a powerful way to study biochemical processes in live cells, yet it remains challenging to track single molecules while simultaneously detecting their interactions. Here we describe a novel property of rhodamine dyes, proximity-assisted photoactivation (PAPA), in which one fluorophore (the "sender") can reactivate a second fluorophore (the "receiver") from a dark state. PAPA requires proximity between the two fluorophores, yet it operates at a longer average intermolecular distance than Forster resonance energy transfer (FRET). We show that PAPA can be used in live cells both to detect protein-protein interactions and to highlight a sub-population of labeled protein complexes in which two different labels are in proximity. In proof-of-concept experiments, PAPA detected the expected correlation between androgen receptor self-association and chromatin binding at the single-cell level. These results establish a new way in which a photophysical property of fluorophores can be harnessed to study molecular interactions in single-molecule imaging of live cells.

biophysics↗

bioRxiv2021

Mechanisms Governing Target Search and Binding Dynamics of Hypoxia-Inducible Factors

Transcription factors (TFs) are classically attributed a modular construction, containing well-structured sequence specific DNA-binding domains (DBDs) paired with disordered activation domains (ADs) responsible for protein-protein interactions targeting cofactors or the core transcription initiation machinery. However, this simple division of labor model struggles to explain why TFs with identical DNA binding sequence specificity determined in vitro exhibit distinct binding profiles in vivo. The family of Hypoxia-Inducible Factors (HIFs) offer a stark example: aberrantly expressed in several cancer types, HIF-1 and HIF-2 subunit isoforms recognize the same DNA motif in vitro - the hypoxia response element (HRE) - but only share a subset of their target genes in vivo, while eliciting contrasting effects on cancer development and progression under certain circumstances. To probe the mechanisms mediating isoform-specific gene regulation, we used live cell single particle tracking (SPT) to investigate HIF nuclear dynamics and how they change upon genetic perturbation or drug treatment. We found that HIF- subunits and their dimerization partner HIF-1{beta} exhibit distinct diffusion and binding characteristics that are exquisitely sensitive to concentration and subunit stoichiometry. Using domain-swap variants, mutations, and a HIF-2 specific inhibitor, we found that although the DBD and dimerization domains are important, a major determinant of chromatin binding and diffusion behavior is the AD-containing intrinsically disordered region (IDR). Using Cut&Run and RNA-seq as orthogonal genomic approaches we also confirmed IDR-dependent binding and activation of a specific subset of HIF-target genes. These findings reveal a previously unappreciated role of IDRs in regulating the TF search and binding process that contribute to functional target site selectivity on chromatin.

molecular biology↗

bioRxiv2021

Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription

Gene activation by mammalian transcription factors (TFs) requires dynamic, multivalent, and selective interactions of their intrinsically disordered low-complexity domains (LCDs), but how such interactions mediate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS/FLI1 requires a finely tuned range of LCD-LCD interactions to efficiently activate target genes. Modest or more dramatic increases in LCD-LCD interactions toward putative LLPS repress EWS/FLI1-driven transcription in patient cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS/FLI1 into a bona fide LLPS compartment, the nucleolus, inhibits EWS/FLI1-driven transcription and oncogenic transformation. Our findings reveal fundamental principles underlying LCD-mediated transcription and suggest mislocalizing specific LCD-LCD interactions as a novel therapeutic strategy for targeting disease-causing TFs.

molecular biology↗

bioRxiv2021

Enhancer-promoter interactions and transcription are maintained upon acute loss of CTCF, cohesin, WAPL, and YY1

It remains unclear why acute depletion of CTCF and cohesin only marginally affects expression of most genes despite substantially perturbing 3D genome folding at the level of domains and structural loops. To address this conundrum, we used high-resolution Micro-C and nascent transcript profiling to find that enhancer-promoter (E-P) interactions are largely insensitive to acute (3-hour) depletion of CTCF, cohesin, and WAPL. YY1 has been proposed to be a structural regulator of E-P loops, but acute YY1 depletion also had minimal effects on E-P loops, transcription, and 3D genome folding. Strikingly, live-cell single-molecule imaging revealed that cohesin depletion reduced transcription factor binding to chromatin. Thus, although neither CTCF, cohesin, WAPL, nor YY1 are required for the short-term maintenance of most E-P interactions and gene expression, we propose that cohesin may serve as a "transcription factor binding platform" that facilitates transcription factor binding to chromatin.

molecular biology↗

bioRxiv2021

Recovering mixtures of fast diffusing states from short single particle trajectories

Single particle tracking (SPT) directly measures the dynamics of proteins in living cells and is a powerful tool to dissect molecular mechanisms of cellular regulation. Interpretation of SPT with fast-diffusing proteins in mammalian cells, however, is complicated by technical limitations imposed by fast image acquisition. These limitations include short trajectory length due to photobleaching and shallow depth of field, high localization error due to the low photon budget imposed by short integration times, and cell-to-cell variability. To address these issues, we developed methods to infer distributions of diffusion coefficients from SPT data with short trajectories, variable localization accuracy, and absence of prior knowledge about the number of underlying states. We discuss advantages and disadvantages of these approaches relative to other frameworks for SPT analysis. Significance statementSingle particle tracking (SPT) uses fluorescent probes to track the motions of individual molecules inside living cells, providing biologists with a close view of the cells inner machinery at work. Commonly used SPT imaging approaches, however, result in fragmentation of trajectories into small pieces as the probes move through the microscopes plane of focus. This makes it challenging to extract usable biological information. This paper describes a method to reconstruct an SPT targets dynamic profile from these trajectory fragments. The method builds on previous approaches to provide information about challenging SPT targets without discrete dynamic states while accounting for some known biases, enabling observation of previously hidden features in mammalian SPT experiments.

biophysics↗

bioRxiv2021

RNA polymerase II depletion from the inactive X chromosome territory is not mediated by physical compartmentalization.

Sub-nuclear compartmentalization has been proposed to play an important role in gene regulation by segregating active and inactive parts of the genome in distinct physical and biochemical environments, where transcription and epigenetic factors are either concentrated or depleted. The inactive X chromosome offers a paradigm for studying sub-nuclear compartmentalization. When the non-coding Xist RNA coats the X chromosome, it recruits repressors and chromatin factors that trigger gene silencing, and forms a dense body of heterochromatin from which the transcription machinery appears to be excluded. Phase separation has been proposed to be involved in X-chromosome inactivation (XCI) and might explain exclusion of the transcription machinery by preventing its diffusion into the Xist-coated territory. Here, using quantitative fluorescence microscopy and single particle tracking, we show that RNA polymerase II (RNAPII) freely accesses the Xist territory during initiation of XCI, and that its diffusion is not prevented by biophysical constraints. Instead, the apparent depletion of RNAPII is due to the loss of its chromatin bound fraction. These findings demonstrate that initial exclusion of RNA Pol2 from the inactive X is a consequence of its reduced binding rate at the chromatin and gene level, rather than the biophysical compartmentalization of the inactive X heterochromatin domain. The Xist silent compartment is thus a biochemical rather than a biophysical compartment, at least during initiation of XCI.

developmental biology↗

bioRxiv2021

Structure of the human SAGA coactivator complex

Human SAGA is an essential co-activator complex that regulates gene expression by interacting with enhancer-bound activators, recruiting transcriptional machinery, and modifying chromatin near promoters. Subunit variations and the metazoan-specific requirement of SAGA in development hinted at unique structural features of the human complex. Our 2.9 [A] structure of human SAGA reveals intertwined functional modules flexibly connected to a core that distinctively integrates mammalian paralogs, incorporates U2 splicing subunits, and features a unique interface between the core and the activator-binding TRRAP. Our structure sheds light on unique roles and regulation of human coactivators with implications for transcription and splicing that have relevance in genetic diseases and cancer.

biophysics↗