Robert Tjian

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.

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 preexisting 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 and 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 and RUN with RNA sequencing (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.

How intrinsically disordered regions (IDRs) shape chromatin binding and nuclear organization of transcription factors (TFs) remains unclear. We used proximity-assisted photoactivation (PAPA), a single-molecule protein-protein interaction sensor, to investigate how IDRs might influence TF interactions with each other and with chromatin in live cells. We found that the Sp1 DNA binding domain (DBD) interacted poorly with chromatin and did not colocalize with Sp1. Weak interaction of the isolated IDR with full-length Sp1 was enhanced by fusion to various unrelated DBDs. Live imaging of Drosophila polytene chromosomes confirmed that an IDR could confer sharp locus specificity on an otherwise nonspecific DBD. These findings suggest that TF specificity emerges on chromatin when ensembles of diverse, unstructured interactions are scaffolded by transient DNA contacts.

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 Statement: Transcription 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.

Gene expression is essential for life and development, allowing the cell to modulate mRNA production in response to intrinsic and extracellular cues. Initiation of gene transcription requires a highly regulated molecular process to assemble multisubunit complexes into the preinitiation complex (PIC). Attempts to visualize these processes have been driven largely by electron microscopy, with near atomic-level resolution producing static snapshots complemented by low-resolution fluorescence cell imaging. Here, we review how new advances in superresolution single-molecule imaging in live cells can track transcription across vast spatiotemporal scales. We discuss how recent imaging research has fundamentally recast our understanding of PIC assembly from a stable, ordered process to one constantly in flux, dominated by multivalent weak interactions. We also discuss future advancements that will further expand our ability to measure PIC assembly in concert with cellular behavior, predict complex interactions computationally, and target undruggable transcription factors to treat human disease.

Eukaryotic transcription factors (TFs) contain both structured DNA-binding domains (DBDs) and intrinsically disordered regions (IDRs). While the structures and sequence preferences of DBDs have been extensively characterized, the role of IDR-mediated interactions in chromatin binding and nuclear organization remains poorly understood, in part because these interactions have been difficult to measure in living cells. Here, we use a recently developed single-molecule technique, proximity-assisted photoactivation (PAPA), to investigate how IDRs influence TF associations with each other and with chromatin, focusing on the factors Sp1 and Klf1. We find that the number and patterning of aromatic and basic residues within IDRs govern both TF self-association and chromatin binding. Unexpectedly, the isolated DBD of Sp1 binds chromatin very weakly and non-specifically. The isolated IDR, by contrast, interacts poorly with chromatin-bound wild-type Sp1, yet this interaction is enhanced when even minimal DNA-binding capacity is restored. Strikingly, replacing Sp1’s native DBD with those of heterologous TFs recovers both IDR-mediated interactions and chromatin association, despite divergent sequence preferences. PAPA measurements also reveal extensive heterotypic interactions between wild-type Sp1 and other TFs. Together, these results establish PAPA as a powerful method for studying unstructured interactions in their native context and suggest that IDRs participate in widespread cooperative associations scaffolded by transient DBD-DNA contacts, which concentrate disordered regions along chromatin. In contrast to classical models, we propose that TF specificity in vivo emerges not solely from DBD sequence preferences, but from a constellation of weak, dynamic, and diverse interactions mediated by IDRs.

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 discover 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 roles for DNA damage repair in rAAV transduction and highlight the potential for pharmacological intervention to increase genetic payload of rAAV vectors.

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.