Faculty Bibliography

Nuclear factor I (NFI) misexpressions in humans are associated with severe brain malformations, yet the underlying mechanisms remain poorly understood. Here, we show that NFIs regulate the broad lineage progression and lifespan of radial glial progenitors (RGPs), thereby bidirectionally controlling neocortical development. Human cerebral organoids carrying patient-mimicking NFI mutations exhibit expression-level-dependent bidirectional impairments in RGP temporal development, coinciding with patient phenotypes. In mouse models, selective removal of NFIs leads to a dramatic protraction of RGP lineage progression and lifespan, excessive progeny output, and cortical overgrowth and abnormal folding, whereas overexpression of NFIs accelerates RGP lineage progression, resulting in developmental-stage-dependent precocious production of diverse neural progenies. Moreover, NFIs exhibit positive autoregulation and progressive increase in expression and regulate distinct temporal-specific targets underlying RGP lineage progression. These results suggest that NFIs act as evolutionarily conserved key global temporal regulators of RGP lineage progression and neocortical development.

Multidrug efflux pumps are major drivers of antibiotic resistance, yet progress in understanding and inhibiting these transporters has been limited by a lack of selective chemical probes and inhibitor-bound structures. Here we report IMP-2380, a potent and selective chemical probe targeting the clinically important Staphylococcus aureus efflux pump NorA. A phenotypic high-throughput screen monitoring suppression of the ciprofloxacin-induced SOS DNA damage response identified a chemical series that selectively inhibits NorA and was optimized to yield IMP-2380. The probe restores ciprofloxacin susceptibility in methicillin-resistant S. aureus, delivering low-nanomolar potentiation in vitro and robust efficacy in an in vivo infection model. Cryo-electron microscopy at 2.52-Å resolution revealed the structure of NorA bound to a small-molecule inhibitor. IMP-2380 binds an 'outward-open' transporter conformation, occluding the cytosolic substrate-binding cavity and preventing antibiotic efflux. IMP-2380 provides a high-quality probe for dissecting multidrug efflux and establishes a structural framework for restoring antibiotic efficacy through efflux pump inhibition.

Classic models of the French flag problem depict sharp cell type boundaries emerging from threshold responses to morphogen gradients. How discrete cell type boundaries arise from morphogen signals that vary continuously across developing tissues remains incompletely understood. We use hair follicle dermal condensate (DC) formation to study a sharp developmental transition in which proliferative progenitors undergo cell cycle exit concurrent with molecular differentiation. Using genetic and genomic approaches, we show that Wnt and Hedgehog signaling coordinate separable cellular events during DC commitment. Elevated Wnt signaling promotes cell cycle exit through reduced chromatin binding of the Hedgehog mediator GLI3, while Hedgehog signaling induces differentiation genes in a Wnt-dependent manner and simultaneously elevates Wnt activity. When these responses coincide, differentiation and cell cycle exit occur together, limiting the duration and abundance of intermediate states and producing a sharp boundary. When they do not, intermediate states persist and expand, producing a graded boundary. Thus, a sharp boundary can emerge from a continuous transition that is compressed in time and space.

Complex human diseases exhibit substantial clinical heterogeneity driven by poorly understood molecular mechanisms, while many also lack sufficient molecular and omics data for mechanistic investigation, hindering therapeutic development. We introduce PiMInfer, a "phenotype-to-mechanism" framework that leveraged largely available real-world clinical data-based deep phenotypic characterizations with a biomedical knowledge graph approach to resolve disease clinical heterogeneity into phenotype-informed molecular modules, thereby accelerating therapeutic target discovery. We applied PiMInfer to investigate Hidradenitis Suppurativa (HS), an autoimmune skin disease with poorly understood pathogenesis and limited treatment options. PiMInfer identified a coherent, phenotype-informed HS gene module (PiHSM) and functional endotypes, which were validated using multimodal evidence. In silico drug repurposing using PiHSM prioritized Carfilzomib, targeting the immunoproteasome subunit PSMB9, essential for MHC Class I antigen presentation. Preclinical testing using human patient lesional skin explants confirmed its anti-inflammatory activity and demonstrated a significant downregulation of IFN-γ, IL-17, and mTOR signaling pathways within HS lesional microenvironment through single-cell RNA sequencing. PiHSM-based network predictions further suggest a potential enhanced efficacy of combining Carfilzomib with approved HS agents. Collectively, PiMInfer provides a scalable framework that bridges real-world phenome-wide comorbid associations to mechanism-anchored therapeutic discovery, enabling a paradigm shift in precision medicine approaches for complex diseases with limited molecular characterization and in need of better therapeutic strategies.

Age is the greatest risk factor for mortality and morbidity. We previously reported premature ageing in PKP2 arrhythmogenic cardiomyopathy (PKP2-ACM). However, depiction of cardiomyocyte molecular anatomy in premature ageing remains incomplete. Furthermore, the relationship between myocyte premature ageing and the molecular components of electrical homeostasis in PKP2-deficient hearts remains understudied. Therefore, we aimed to identify molecular anatomical changes of premature ageing in PKP2-deficient adult cardiomyocytes, the corresponding gene cohort and its imprint in the human electrocardiogram (ECG). We used a murine model of cardiomyocyte-specific PKP2 knockout. Molecular anatomy was resolved by expansion and structured illumination microscopy, allowing 3D-nanometric visualization, and by serial block-face scanning electron microscopy. We used omics databases to distill a subset of genes related to ageing and PKP2 deficiency containing single nucleotide polymorphisms (SNPs) with an ECG-relevant genomic signature. We found increased DNA damage and reduced abundance of transcriptionally repressed heterochromatin at the lamin-associated domain (LAD), and disrupted mitochondria ultrastructure at the intercalated disc. Genes dysregulated in ageing hearts and PKP2-deficient cells were distilled to identify those in the PKP2 human left ventricular gene network (by GTEx) that contain SNPs with an imprint in the human ECG (based on ECG GWAS). These findings indicate that in PKP2 deficiency, transcriptionally repressed heterochromatin in the LAD is vulnerable to damage, and erasing of otherwise transcriptionally inaccessible genomic regions, consistent with premature cellular ageing and transcriptional reprogramming. Multi-omics analysis indicates that reduced PKP2 expression accelerates cardiomyocyte ageing, and the age-related gene network contributes to dysfunction of cell metabolism and arrhythmia risk in PKP2-ACM. KEY POINTS: We used advanced imaging technology to unveil DNA damage and loss of heterochromatin architecture in the lamin-associated domain (LAD), and mitochondrial remodelling at the intercalated disc, as features of premature myocyte ageing in adult PKP2-deficient murine cardiomyocytes. Multi-omics analysis identified a network of genes dysregulated by ageing and by PKP2 deficiency. Cross-correlation with the ECG GWAS database found a subset of single nucleotide polymorphisms with an imprint in the human electrocardiogram. These results provide novel insight into the mechanisms of transcriptional remodelling and take an initial step toward a better understanding of the oligogenic bases of arrhythmia risk in PKP2-deficient hearts.

VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) is a severe adult-onset inflammatory disease caused by somatic mutations that reduce cytoplasmic activity of UBA1, the primary initiating enzyme for ubiquitylation. How this hypomorphic state drives cell-intrinsic immune activation in mature myeloid cells is unknown. Using unbiased multi-omic, biochemical, and cell biological analyses of model systems and patient-derived cells, we show that loss of cytoplasmic UBA1 activity convergently disrupts endoplasmic reticulum-associated degradation (ERAD) and mitochondrial homeostasis. ERAD failure arises from preferential under-charging of ERAD E2 enzymes, explaining hallmark VEXAS features, including ER-derived vacuoles and unfolded protein response activation, and promotes accumulation of the ERAD substrate STING. Simultaneously, mitochondrial dysfunction drives cytosolic leakage of mitochondrial DNA, inducing cGAS-dependent STING signaling and inflammatory cytokine production. STING inhibition or reversal of mitochondrial DNA leakage resolves multi-cytokine inflammation in VEXAS models and patient myeloid cells, establishing the cGAS-STING pathway as a therapeutically actionable vulnerability.

Autotomy is a unique phenotype whereby an animal sheds a body part to escape predation^1-3. The timing and location of autotomy are tightly regulated by preformed planes of weakness (aka fracture planes) which facilitate tissue loss. While autotomy is often followed by regeneration, these phenotypes are rarely reported in mammals^4-9. A notable exception are spiny mice (Acomys) which exhibit skin autotomy and more remarkably, complete tissue regeneration^10-14. Presently, mechanisms underlying autotomy and complete regeneration in Acomys skin remain elusive. Here, we report the discovery of a honeycomb-like fracture lattice in Acomys skin whose design directs tissue destruction but also facilitates regenerative healing. Unlike the single continuous surface of a fracture plane, this fracture lattice consists of a three-dimensional array of hexagonal units whose boundaries guide tissue breakage. Moreover, we identify collagen VI as the main constituent of the fracture lattice and find that it is distinctly arranged to initiate fracturing and propagation of skin tearing. By preconditioning the tissue for autotomy, the fracture lattice dampens the damage-induced inflammatory response but also upregulates a pro-regenerative gene signature, accelerating skin appendage regeneration. Lastly, we discovered the key role of spiny hairs in fracture lattice formation, as inhibiting their development leads to abnormal pattern formation and changes in skin fracture mechanics. Our results present a novel example of a uniquely evolved structural adaptation in mammalian skin that links tissue patterning, autotomy and regeneration. We expect that the application of a modular compartment structure to artificial skin and other organ engineering may enhance resilience to injury and facilitate efficient regeneration.

The human sodium-citrate cotransporter NaCT imports various tri- and di-carboxylates into the cell as TCA cycle intermediates. This substrate uptake process is driven by an inward sodium gradient. The protein is a member of the divalent anion-sodium symporter (DASS) family. Whereas extensive biochemical and structural studies have been carried out for NaCT, how the substrate binding and translocation is coupled to the sodium gradient remains unclear. Here using single particle cryo-electron microscopy, we determined the structures of the human NaCT protein in three states: sodium-free, in the presence of sodium, and bound to sodium and a substrate mimicking inhibitor. These structures suggest a simultaneous binding mechanism for sodium-substrate coupling, distinct from the sequential binding, conformational selection mechanism previously observed for the bacterial DASS protein VcINDY.

Temporal patterning is an evolutionarily conserved mechanism to produce neuronal and glial diversity from common cells of origin during neurodevelopment. This process is controlled by a series of temporal transcription factors that are transiently expressed and drive the sequential production of specific progeny subtypes. Intermediate neural progenitors (INPs) and optic lobe neural stem cells (OL NSCs) share striking similarities in temporal factor expression despite divergent cells of origin. Tailless (Tll) is a terminal temporal factor in the visual system, in OL NSCs. Tll expression coincides with the termination of neurogenesis and onset of gliogenesis. Here, we report that Tll also acts as a terminal factor in INPs, demonstrating functional conservation. Tll expression is activated by the preceding temporal factor, Scarecrow, and represses odd-paired and hamlet. tll also plays a partial role in promoting gliogenesis in gliogenic NSCs. We performed genome-wide binding analysis of Tll in the OL NSCs and INPs by Targeted DamID, revealing both conserved and divergent targets, reflecting differences in regulatory outcomes. We show that temporal patterning mechanisms are conserved between different brain regions, whilst facilitating lineage-specific outputs.