Biology subjects

Munger, C. J.

Publications and source records attributed to Munger, C. J..

4 recordsLinked to original sources

bioRxiv2026

Mapping the regulatory wiring of endothelial cell differentiation reveals a critical role for Hippo mechanosignaling

At each step of development, stem cells encounter a branching decision to differentiate into one type of cell or another. This decision is executed through the action of competing gene programs that specify different cellular identities. Consequently, creating a comprehensive map of the gene programs that drive each branching step of human cell differentiation remains a long-sought goal of developmental and stem cell biology. Here, we introduce a framework combining genome-wide CRISPR screens, single-cell readouts, and gene program inference to systematically discover and characterize regulators of cell differentiation. This framework includes a method for CRISPR perturbations paired with single-cell RNA-seq called circular capture Perturb-seq (CC-Perturb-seq), which improves gRNA detection from split-pool combinatorial indexing protocols and enables perturbing many candidate regulators to map their effects on gene programs. We applied this framework to map how human pluripotent stem cells differentiate into arterial endothelial cells (ECs), and identified 174 regulators that promote or inhibit EC differentiation. By tracking gene programs across three stages of differentiation, we resolved when and how regulators divert cells toward alternative fates. This map revealed an unexpected requirement for the Hippo mechanosignaling pathway in EC specification. During lateral mesoderm differentiation, Hippo mechanosignaling was required to activate ETV2, the master transcriptional regulator of endothelial identity. Inhibiting Hippo blocked ETV2 even in the presence of VEGF and diverted progenitors to a mesenchymal fate in vitro. Likewise, Hippo signaling was required for maximal etv2 expression in developing zebrafish embryos. Conversely, pharmacologically activating the Hippo pathway increased the purity of both arterial and venous EC differentiation in vitro. Our framework thus identifies novel regulators of endothelial cell differentiation, improves stem-cell differentiation protocols via manipulation of mechanosignaling, and provides a generalizable platform for mapping the regulatory wiring of human development.

genomics↗

bioRxiv2026

MARBL: A Live-Cell Method for Profiling Bioenergetic Heterogeneity by Noncanonical Methionine Labeling of the Cell Surface Proteome

Heterogeneity is a hallmark of biological systems, where cell-to-cell variability supports adaptation to changing environments, but also enables maladaptive states such as drug resistance. Many sources of non-genetic variation, particularly bioenergetics and metabolism, remain difficult to measure in living cells and connect to functional outcomes. Here, we introduce MARBL (Methionine Analogues for Ratiometric Bioenergetics in Live cells), a method that encodes translationally-coupled energetic responses to metabolic stress as an internally normalized signal within the surface proteome of living cells. Applying MARBL to primary immune cells reveals that differences in baseline translational activity can underlie apparent metabolic vulnerabilities, underscoring the importance of ratiometric measurements. We demonstrate that MARBL can enrich pathogenic from non-pathogenic TH17 cells based on resilience to bioenergetic stress, which functionally distinguishes cells that produce IFN{gamma} upon restimulation. Overall, MARBL offers a versatile platform to profile metabolic resilience in living cells and link bioenergetic state to cellular function.

cell biology↗

bioRxiv2025

An unbiased survey of distal element-gene regulatory interactions with direct-capture targeted Perturb-seq

Identifying the impact of distal regulatory elements on gene expression is a core challenge in human genetics. Large-scale CRISPR screens have not captured lower effect size element-gene interactions due to selection bias and limited statistical power. We developed a framework for highly powered CRISPR screens, consisting of Direct-Capture Targeted Perturb-seq (DC-TAP-seq), unbiased target selection, and a pipeline accounting for statistical power. Surveying 10,000 random distal element-gene pairs revealed most element-gene interactions have effect sizes <10%, which were virtually undetectable in prior studies. Most interactions occur within 100kb, many elements bind CTCF without classical enhancer chromatin, and housekeeping genes have similar frequencies of distal regulatory elements but with weaker effects. We also highlight limitations of predictive models and suggest that new models consider elements with smaller effect sizes. Our study provides an expanded view of distal regulatory elements and a framework for building more comprehensive maps of distal regulation.

genomics↗

bioRxiv2024

Cohesin-mediated 3D contacts tune enhancer-promoter regulation

Enhancers are key drivers of gene regulation thought to act via 3D physical interactions with the promoters of their target genes. However, genome-wide depletions of architectural proteins such as cohesin result in only limited changes in gene expression, despite a loss of contact domains and loops. Consequently, the role of cohesin and 3D contacts in enhancer function remains debated. Here, we developed CRISPRi of regulatory elements upon degron operation (CRUDO), a novel approach to measure how changes in contact frequency impact enhancer effects on target genes by perturbing enhancers with CRISPRi and measuring gene expression in the presence or absence of cohesin. We systematically perturbed all 1,039 candidate enhancers near five cohesin-dependent genes and identified 34 enhancer-gene regulatory interactions. Of 26 regulatory interactions with sufficient statistical power to evaluate cohesin dependence, 18 show cohesin-dependent effects. A decrease in enhancer-promoter contact frequency upon removal of cohesin is frequently accompanied by a decrease in the regulatory effect of the enhancer on gene expression, consistent with a contact-based model for enhancer function. However, changes in contact frequency and regulatory effects on gene expression vary as a function of distance, with distal enhancers (e.g., >50Kb) experiencing much larger changes than proximal ones (e.g., <50Kb). Because most enhancers are located close to their target genes, these observations can explain how only a small subset of genes -- those with strong distal enhancers -- are sensitive to cohesin. Together, our results illuminate how 3D contacts, influenced by both cohesin and genomic distance, tune enhancer effects on gene expression.

molecular biology↗