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NIA - National Institute on Aging

PROJECT SUMMARY Despite aging being a major risk factor for lung cancer incidence, most preclinical and clinical studies do not take aging into account. Thus, our understanding of how aging affects lung cancer progression, evolution and therapy response remains limited. The diversity of subpopulations of tumor cells, and their therapeutic responses are shaped by their complex interactions with the host. Our published studies support this, revealing a causal link between the systemic metabolic reprogramming driven by aging and lung cancer progression and immune evasion, putting aging and the changes it causes in the host at the center stage coordinating cancer evolution. Recently, we showed that beyond systemic changes, alterations in the composition and functional state of old lung tumor microenvironment (TME) shape niche signaling and drive divergent lines of lung cancer evolution resulting in age-dependent intrinsic differences in prognosis upon treatment with standard of care therapies. Yet, the age-driven niche-signaling mechanisms that fuel this divergent evolution remain unknown. Importantly, we have identified a damage-associated state of alveolar epithelial cells (alveolar differentiation intermediates, or ADIs) that accumulate within the NSCLC TME and the adjacent lung as a key difference between NSCLC in young versus old animals and patients. Strikingly, ADIs’ preferential accumulation within the old TME dominates the communication between NSCLC cells and the TME through ADI-produced ligands linked to stemness induction. Thus, in this research program we propose that the emergence of ADIs in the old lung TME shapes the niche signaling that NSCLC cells and the other TME cellular components are exposed to. We posit that these changes steer NSCLC evolution towards a primitive stem-like state with higher grade tumors that confer resistance to chemotherapies. We will leverage multiple model systems (human and mouse cancer cells lines, AT2-derived organoids and in vivo mouse models) combined with high throughput single cell technologies and mass spectrometry to characterize the mechanism that underlies the age-induced ADI accumulation within NSCLC TME (Aim 1) and determine the contribution of ADIs to the evolution of the primitive stem-like state of NSCLC (Aim 2). Consequently, the proposed studies will put forward defined roles for aging in NSCLC biology and inducing the evolution of chemoresistance states that confer poor prognosis, thereby bringing awareness to the need of tailoring treatments to the specific age and biology of the patient. Our studies will also unveil for the first-time normal alveolar cells as major regulators of the NSCLC progression. Moreover, they will also put forward age-specific targets and pathways for subsequent studies with the ultimate goal of leveraging this information therapeutically to improve outcomes of the most common and vulnerable NSCLC patient population: the elderly.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

Project Summary/Abstract Epigenetic modifiers govern cell fate transition during animal development and their mutations drive multiple human congenital disorders; however, the molecular mechanisms underlying the roles of epigenetic modifiers in these normal and pathological processes remain poorly understood. It is widely believed that epigenetic modifiers function through the epigenetic marks they catalyze. Nevertheless, the discoveries of catalytic- independent role of epigenetic modifiers challenge this view, raising the question about the biological function of epigenetic marks. Mono-methylation of histone H3 at lysine 4 (H3K4me1) is a reliable mark of enhancers that shape cell identity, and its reconfiguration accompanies the differentiation of pluripotent stem cells, suggesting that the regulation of H3K4me1 plays an instructive role in cell fate transition. To examine this hypothesis, we investigated the catalytic function of LSD1 and LSD2, two paralogous histone demethylases targeting H3K4me1, in regulating gene expression during cell fate transition. Using state-of-the-art approaches such as precise genome engineering, epigenetic and transcriptomic profiling, and stem cell differentiation, we demonstrate functional synergism between the demethylase activity of LSD1 and LSD2 in regulating cellular differentiation. Based on these compelling preliminary data, here we propose to dissect the molecular mechanisms underlying how the demethylase activity of LSD1/2 regulates cell fate transition. The results generated from our proposed studies will not only reveal novel molecular mechanisms underlying the roles of H3K4me1 in gene regulation and cell fate transition, but also provide insights into understanding the pathogenesis of diseases driven by LSD1/2 loss-of-function. This research aligns with the NIH mission to advance our understanding of fundamental biological processes and contribute to knowledge relevant to developmental disorders and regenerative medicine.

NHLBI - National Heart Lung and Blood Institute

ABSTRACT Hematopoietic stem and progenitor cells (HSPCs), which include hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), give rise to all blood and immune cells across the lifespan. HSCs first emerge during gestation and ultimately becoming the adult hematopoietic compartment. Human studies have associated events during pregnancy such as maternal infection, diet, or exposure to microbes with increased risk of immune dysfunction in offspring; however, the mechanisms behind this are unknown. Since hematopoietic progenitors arise during gestation and seed the adult hematopoietic compartment, we hypothesize that prenatal inflammation reprograms the cellular output of distinct fetal HSPCs, thus influencing postnatal immune function. Recent data has suggested that fetal HSCs and fetal MPPs emerge independently from the intra-embryonic aorta during development. This suggests that fetal MPPs and fetal HSCs of distinct origin have specific functions and relative contributions to postnatal blood production at homeostasis and during inflammatory insults, but this has not been directly investigated. Our working hypothesis is that fetal MPPs drive the response to prenatal inflammation in order to preserve the HSC pool, shaping the postnatal hematopoietic compartment. We will test our hypothesis by integrating genetic fate mapping experiments, transplantation assays, transcriptomics, and early life infection models. Our preliminary data suggests independent emergence of fetal MPPs and fetal HSCs from the developing aorta and has revealed the first evidence of functional differences between fetal MPPs and HSCs. We have found that fetal MPPs are the first responders to Type II-IFN mediated prenatal inflammation and expand the pool of downstream myeloid cells, which remain expanded in the postnatal period. Our findings also support the idea that timing of emergence influences the functional output of fetal progenitors at steady state and in response to inflammation. The objective of this proposed work is to comprehensively examine the specific effects inflammation has on fetal HSCs and MPPs and will define the mechanisms by which prenatal inflammation shapes the adult hematopoietic system. We will determine how inflammation experienced in utero influences offspring immunity and response to postnatal immune challenges at the level of fetal HSPCs. We anticipate that the insights gained from this proposed work will help inform underlying causes of disease that may start during development.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY This proposal aims to elucidate how the dynamic oligomeric transitions of Copper Transporter 1 (CTR1) couple copper (Cu) homeostasis with neuronal developmental pathways. Cu is an essential micronutrient for neuronal function, and a deficiency of Cu in early life can have devastating impacts on development. We recently discovered that CTR1 can reversibly transition between trimeric and monomeric states to regulate Cu uptake. Moreover, a CTR1 mutant that fails to undergo these de-oligomerization events impairs growth factor–activated signaling pathways. These findings challenge the conventional view of CTR1 as a stable trimeric transporter and suggest that Cu-induced allosteric changes in CTR1 oligomerization directly influence neuronal health and development. Our primary objectives are to determine the mechanisms driving CTR1’s oligomeric-state transitions and to clarify how these shifts impact CTR1’s function in Cu regulation and stem cell differentiation. Using innovative single-molecule assays, such as single-molecule localization microscopy and in-cell oligomer stoichiometry assays, we will visualize and quantify CTR1’s oligomeric states in situ. Human embryonic stem cell (hESC)-derived neurons will provide a physiologically relevant model for these studies. In addition, we will conduct comprehensive proteomic and biochemical analyses to uncover key protein interaction networks that modulate CTR1 trafficking and function. This proposal aims to address two main research directions: (1) identifying the triggers behind CTR1’s transition from trimeric to monomeric forms under conditions of excess Cu, and (2) exploring CTR1’s role in stem cell differentiation, with emphasis on its interactions with Laloo and SNT1 to regulate neuronal maturation. This multidisciplinary effort will be supported by collaborations with experts in neurobiology, membrane trafficking, Cu homeostasis, and stem cell research, thereby ensuring a robust and integrative approach. The insights gained will provide significant contributions to understanding CTR1’s role in Cu regulation and cell development, as well as elucidating its broader impact on neuronal health and disease. Additionally, the methodologies developed will have broad applicability for examining the dynamics of other membrane proteins. This project is innovative for several reasons: it introduces novel single-molecule assays for in situ studies, uses a more physiologically relevant hESC model, and addresses a critical gap in our understanding of allosteric regulation mediated by CTR1 in neurons. The insights gained from this research could have far-reaching implications in neurobiology and may inform strategies for treating Cu-related neurodegenerative diseases.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Glioblastoma multiforme (GBM) is the most aggressive and common primary brain tumor, posing significant clinical challenges despite multimodal treatments like surgical resection, chemotherapy, and radiation. The prognosis for GBM patients remains poor, primarily due to its inevitable recurrence and resistance to standard therapies. Therefore, identifying novel molecular drivers of GBM progression is essential for developing more effective treatments. Over the past decade, the isolation of glioblastoma stem cells (GSCs) has provided crucial insights into tumor initiation, maintenance, and recurrence, positioning them as a major therapeutic target. Concurrently, advances in genomic research have revealed a vast landscape of long noncoding RNAs (lncRNAs), offering new opportunities to influence gene regulation and cancer biology. Although tens of thousands of lncRNAs have been discovered, only a small fraction have been functionally characterized. Emerging evidence suggests that lncRNAs exhibit cell-type-specific expression, distinct subcellular localization, and critical roles in key oncogenic processes, including proliferation, invasion, and therapy resistance. These properties highlight their potential as both biomarkers and therapeutic targets in GBM. In this study, we propose to characterize the nuclear-retained oncogenic lncRNA, LINC01896, which is significantly upregulated in GSCs. Our preliminary data show that depletion of LINC01896 impairs GSC proliferation and stemness, emphasizing its critical role in GSC maintenance. Furthermore, higher level of LINC01896 correlates with poor overall and disease-free survival in GBM patients, suggesting its potential as both a prognostic biomarker and a therapeutic target. We will investigate how LINC01896 regulates global gene expression in GSCs and identify the molecular pathways driving GSC proliferation and stemness. Additionally, we will examine the functional impact of LINC01896 knockdown in GSCs using antisense oligonucleotides (ASOs) and evaluate these effects in preclinical 3D cerebral organoid model, which mimic the human brain's cellular complexity and tumor microenvironment. Furthermore, we will map the transcriptomic landscape of GSCs and their tumor microenvironment using single-cell RNA sequencing. Together, these studies aim to elucidate the regulatory role of LINC01896 in GSC biology and pave the way for the development of innovative lncRNA-based therapeutic strategies for GBM patients.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT The mammalian heart undergoes profound transcriptional and phenotypical remodeling during postnatal development, a process known as cardiac maturation. However, the molecular mechanisms driving this transition is not fully understood, posing a major challenge in cardiac regenerative medicine, where induced cardiomyocytes from pluripotent stem cell differentiation or non-myocyte reprogramming exhibit an overall immature phenotype that severely limits their application in cell therapy and in vitro disease modeling. In this K99/R00 application, I propose to integrate cutting-edge single cell multiomics with state-of-the-art computational methods to unravel the cell-type-specific gene regulatory networks governing cardiac maturation, and develop a novel dual-reporter system to model and enhance cardiac maturation in vitro and in vivo. During the K99 phase, I will characterize the epigenomic changes of the mouse heart during postnatal development at a single cell resolution using various single cell multiomic technologies (Aim 1), and construct cell-type-resolved gene regulatory networks underlying cardiac maturation using bioinformatic approaches coupled with deep learning (Aim 2). I will also establish cell culture and mouse models with CRISPR-mediated knock-in of dual-fluorescent reporters to track and assess cardiomyocyte maturation (Aim 3a). During the R00 phase, I will experimentally characterize key regulatory elements and novel transcriptional regulators using functional genomic approaches (Aim 3b). I will also leverage these findings to enhance the maturation of in vitro-derived cardiomyocytes for improved therapeutic potential (Aim 3c). The expected outcomes of my proposed research will deepen our understanding of postnatal cardiac development and uncover new therapeutic strategies to improve cardiac function after injury. My career goal is to lead an independent research group that develops and employs innovative technologies to study the regulatory mechanisms underlying cardiac development, regeneration, and disease. In my K99 phase, I will acquire crucial knowledge and skills in advanced single cell genomics and computational biology to complement my previous expertise in developmental biology and cardiac research. My career development will be supported by an exceptional mentoring and advisory committee from UCSD/Salk/HHMI, along with world-class resources, training opportunities, and institutional support at UC San Diego. These elements will provide a strong foundation for my successful transition to an independent tenure- track faculty position.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Post-transcriptional regulation is a critical mechanism controlling cellular differentiation and development, driven by coordinated interactions between RNA-binding proteins (RBPs) and RNA modifications. This proposal investigates the interplay between Quaking (QKI) isoforms and N7-methylguanosine (m7G) RNA modifications. QKI isoforms exhibit distinct subcellular localizations and functions, yet their roles in binding m7G-modified transcripts at 3′ untranslated regions (UTRs) during differentiation remain poorly understood. The central hypothesis is that QKI isoforms regulate differentiation by binding m7G-modified mRNAs at 3′ UTRs, with cytoplasmic QKI6 stabilizing transcripts essential for myeloid differentiation. Furthermore, m7G modifications may independently regulate gene expression in ways yet to be defined. Our preliminary evidence demonstrates that cytoplasmic QKI isoforms mediate myeloid differentiation, highlighting a critical gap in understanding m7G’s functional roles in steady-state cellular processes. Current methods for detecting m7G methylation at single- nucleotide resolution face technical limitations. This project addresses these challenges by employing direct RNA long-read sequencing and orthogonal methods to map m7G modifications with precision and identify QKI isoform-specific mRNA targets. Integrating these approaches will elucidate how QKI-m7G interactions influence myeloid differentiation, with broader implications for RNA modifications in stem cell biology, neurodevelopment, and cancer. Over the next five years, the laboratory’s mission is to (1) develop novel methods for base-resolution m7G detection, (2) define mRNA targets regulated by distinct QKI isoforms, and (3) determine the functional impact of QKI-m7G interactions on myeloid differentiation. These studies will advance understanding of RNA modification-driven gene regulation and may inform therapeutic strategies for diseases such as leukemia. By resolving the interplay between QKI and m7G at 3′UTRs, this work will reveal how their coordination regulates cellular functions across human cell types, directly addressing NIGMS’s mission to support foundational discovery science. The proposed research will provide mechanistic insights into RNA modifications dysregulated in cancer, potentially uncovering new targets for therapeutic intervention in malignancies and developmental disorders.

NCI - National Cancer Institute

Project Summary Despite glioblastoma's reputation as an "immunologically cold" tumor, some glioblastoma patients who received autologous tumor lysate-pulsed dendritic cell (ATL-DC) vaccine have shown remarkable tumor control. While standard of care yields a 5.7% five-year survival rate, the rate for those treated with ATL-DC rises to 13%. In the subset of patients whose tumor exhibit hypermethylation of the MGMT gene, the fraction of patients surviving past 5-year mark nearly doubles to 25%, with their median survival extended from 21 to 33 months after ATLDC therapy. Our team hypothesizes that these long-term survivors harbor more antigen specific, progenitor-exhausted TCF7+ PD1+ CD8 T cells in their tumors. These specialized T cells, maintaining their stemness phenotype, persist longer in the tumor microenvironment and kill more tumor cells throughout their lifetime. We further posit that these CD8 T cells are supported by a T-cell-supportive microenvironment, which is marked by reduced levels of immunosuppressive macrophages commonly found in the brain microenvironment. We propose utilizing spatial transcriptomics and high-plex immunofluorescence to map the immune architecture within tumors from these exceptional responders, comparing them to short-term survivors. In parallel, we will analyze the blood samples of the long-term responders to track systemic immune activation and T cell repertoire dynamics, using single cell sequencing to characterize clonally expanded T cells. Finally, we plan to use two different murine models of glioblastoma, one with a more immune rich tumor microenvironment and one with an immunosuppressive microenvironment, to test any nominated mechanisms from our omics analysis. The use of murine glioblastoma models is necessary to observe the effects of ATL-DC therapy not only in the tumor microenvironment but also in the systemic immunity. Immune system level changes in response to a cellular vaccine therapy such as ATL-DC have not been modeled previously using in vitro models. Through this comprehensive approach, our team aims to uncover the mechanisms behind durable antitumor responses after ATL-DC-based immunotherapy in glioblastoma. If successful, our findings could guide new strategies to extend these benefits more broadly, potentially transforming outcomes for a disease that typically claims lives within two years of its diagnosis.

NHLBI - National Heart Lung and Blood Institute

Project Abstract The alveolar epithelium is composed of two distinct cell types—alveolar epithelial type I (AT1) cells, which facilitate gas exchange, and alveolar epithelial type II (AT2) cells, which act as progenitors for AT1 cells. Successful lung repair following alveolar injuries requires AT2 cell proliferation and differentiation into AT1 cells, a process that involves the restructuring of gene regulatory networks and cell-type specific chromatin landscapes that underly these two cell fates. A dysfunctional regenerative response has been observed in a variety of severe lung diseases, involving AT2 cells acquiring a pathologic, alveolar-basal intermediate (ABI) cell state at the expense of an AT1 fate. The mechanisms that facilitate the cell fate decisions involved in AT2 cell maintenance and differentiation are not well understood, which has resulted in a lack of effective treatments to promote alveolar regeneration. This project aims to identify and characterize regulatory, 3-dimensional “hubs” of chromatin interaction that instruct distinct alveolar epithelial cell fates, and to determine how these hubs and their associated transcription factors regulate the acquisition of healthy and disease-associated states. Using human induced pluripotent stem cell (iPSC) models of AT1- and AT2-like cells (iAT1s and iAT2s), we will apply advanced chromatin mapping techniques to identify cell-type specific enhancer-promoter interactions and to characterize chromatin hubs that potentially regulate normal AT1 and AT2 cell identity. In Aim 1, we will map these interactions in healthy iAT1 and iAT2 cells, comparing their chromatin landscapes to pinpoint regulatory hubs that we hypothesize are responsible for cell-type specific gene expression. In Aim 2, we will explore the effects of haploinsufficiency of the lung lineage transcription factor, NKX2-1, on chromatin topology of iAT2 cells, hypothesizing that reduced NKX2-1 expression disrupts normal AT2 cell identity and favors a pathological ABI state. The findings from this research will enhance our understanding of the chromatin-based mechanisms that control lung cell fate decisions and provide insights into how disruptions of chromatin organization contribute to pulmonary disease.

NIGMS - National Institute of General Medical Sciences

Project Summary/Abstract Congenital heart diseases (CHDs) are the most common birth defect, occurring approximately in 1% of live births. Over 97% of mutations associated with CHD occur within the non-coding genome, potentially disrupting transcription factor (TF) binding to regulatory DNA and dysregulating gene regulation essential for heart development. However, due to their overwhelming numbers, it has remained challenging to identify causal mechanisms between non-coding mutations and human diseases. The long-term goal of the proposed work is to describe the molecular mechanisms by which disease-associated non-coding mutations dysregulate gene expression, leading to human diseases. Herein, we propose a high-throughput biochemical strategy to study the role of CHD-associated non-coding mutations in cardiac TF binding and gene regulation. Our hypothesis is that CHD-associated non-coding mutations will impact cardiac TF- DNA binding and disrupt gene regulation necessary for proper heart development. We have leveraged genome-wide association studies (GWAS) to identify 121 CHD-associated mutations in the non-coding genome. To account for the resolution limitations of GWAS, we performed a linkage disequilibrium expansion on the CHD-associated mutations to include single-nucleotide variants from diverse ancestries, resulting in 3,232 mutations. Additionally, we included all possible alleles for each genomic variant, resulting in 12,928 permutations. In Specific Aim 1, we will measure the impact of ~12,000 single- nucleotide non-coding variants on the binding of three master cardiac developmental TFs GATA4, NKX2-5, and TBX5. We will measure binding aYinities through Single Nucleotide Polymorphism-Systematic Evolution of Ligands by EXponential enrichment (SNP-SELEX) and identify non-coding mutations that decrease or increase cardiac TF binding aYinity. Our approach will allow us to quantitatively measure TF binding aYinities for ~12,000 genomic loci in a single experiment. In Specific Aim 2, we will determine the impact of the ~12,000 CHD-associated non-coding mutations on regulatory element activity during cardiomyocyte diYerentiation. The gene regulatory activity of ~12,000 promoter and enhancer variants will be measured in human embryonic stem cells, cardiac progenitors, and cardiomyocytes by massively parallel reporter assay (MPRA). This integrated approach will train undergraduate and graduate students in high-throughput biochemistry, functional genomics, and large-scale data analysis. Completion of the proposed project will contribute to our understanding of the mechanisms of non-coding mutations in CHDs and can be extended to study gene dysregulation in multiple human diseases.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Abstract Skeletal muscle contains a population of adult stem cells called satellite cells or muscle stem cells (MuSCs) that are responsible for regeneration after injury. MuSCs utilize gene expression programs to maintain quiescence and differentiate after injury and a key regulator of gene expression is splicing, which uniquely changes when transcripts interact with nuclear speckles. Nuclear speckles are membrane-less biomolecular condensates that phase separate proteins, RNAs and chromatin, but how these organelles regulate molecular processes in MuSCs remains unknown. Key experiments from our laboratory provide rigorous support for a role of nuclear speckles and splicing in MuSC function, which were attenuated in old age. The overarching objective of this program is to establish a systems-based approach to understand how nuclear speckles and alternative splicing contribute to MuSC programs of activation and regeneration across lifespan. In Aim 1, we will demonstrate that the loss of a nuclear speckle scaffolding protein, Srrm2, will reduce regenerative potential of muscle stem cells. In Aim 2, we will establish that increases in oxidative stress from old age attenuate nuclear speckles and RNA splicing that regulate muscle stem cell activation and repair of muscle injury. In both Aims, we will use novel transgenic animal models, sophisticated bioinformatics analysis and highly innovative molecular tools to build a comprehensive and new understanding of nuclear speckles influence on alternative splicing and stem cell activity. Successful completion of this program will advance our knowledge of fundamental cell biology for regenerative medicine, and provide a myriad set of insights across molecular, cellular and tissue scales.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

PROJECT SUMMARY/ABSTRACT Very early onset inflammatory bowel disease (VEOIBD) is defined as disease onset at age <6 years and is associated with a more severe and aggressive disease course. Up to 10% of affected children have an underlying disease-causing monogenic immunodeficiency, such as defects in IL10 signaling, but the mechanisms driving disease in the majority of patients remain unknown. Furthermore, there are no biologic or small molecule therapies FDA-approved for use in IBD in this age group and current treatment approaches rely on empiric trials of medications approved for adult IBD. Collectively, these factors contribute to significant treatment challenges in VEOIBD and to substantial cost and morbidity stemming from delays in remission, growth failure, steroid- dependence, hospitalization, and surgical intervention. There is an urgent need to define disease mechanisms that enable biomarker-guided treatment selection in both VEOIBD and IBD more broadly. We have identified a unique subgroup of VEOIBD patients defined by markedly diminished activation of STAT3 in response to multiple cytokines, including IL10 (“STAT3-aberrant” or STAT3-abr, n=7). These patients share a clinical phenotype characterized by severe, refractory colonic disease without an identifiable monogenic basis and a blood transcriptional signature characterized by increased IL23 signaling. Notably, four of these patients have been treated with anti-IL12/23 or anti-IL23 following multiple prior medication failures, and all four achieved rapid, sustained remission, implicating IL23 as a key disease driver and therapeutic target. The overall goals of this proposal are to define the mechanisms underlying this STAT3-abr signaling state and to leverage signatures of this state as biomarker(s) of response to IL23-blocking therapies. Our central hypothesis is that excessive IL23 signaling promotes preferential activation of pro-inflammatory (e.g., IL23) STAT3-dependent programs at the expense of anti-inflammatory (e.g., IL10) STAT3-dependent programs in this STAT3-abr group and a subset of patients with later-onset disease. Aim 1 will define STAT3-dependent signaling and how IL23 modulates that signaling in STAT3-abr patients compared to controls. Aim 2 will examine epigenomic and transcriptional consequences of the STAT3-abr signaling state by examining differences in chromatin accessibility, STAT3-DNA binding, cellular composition, and gene expression in STAT3-abr patients compared to controls. Aim 3 will develop and evaluate a multimodal predictive tool that incorporates STAT3-abr clinical and transcriptional features to predict response to IL23-blocking therapy in pediatric and adult IBD cohorts. Completion of this work will mechanistically define a novel STAT3 signaling state in VEOIBD and develop a biomarker predictive of response to IL23-blocking therapies, impacting personalized medicine opportunities for IBD patients of all ages.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT The following proposal outlines a 5-year career training plan that will prepare Dr. Andrew Berical to be an independent physician-scientist and leader in the field of airway epithelial functional genomics. Motile cilia are found throughout the human body, most notably on multiciliated cells (MCCs) in the conducting airway. Individuals with primary ciliary dyskinesia (PCD) have inherited variants in any one of more than 50 genes that regulate the structure or function of cilia, leading to a lifetime of chronic cough, recurrent infections and respiratory failure. Due to the complexity of the MCC molecular program and limited disease-relevant platforms, there are no targeted therapies available for PCD. An improved understanding of fundamental MCC biology and the availability of a human-based platform would have enormous implications for the PCD field. Dr. Berical’s long-term vision is to utilize pluripotent stem cell-based techniques to understand how specific genes regulate airway epithelial homeostasis and how gene variants lead to the initiation of airway diseases such as PCD, CF, asthma, COPD and IPF. Dr. Berical presents preliminary data suggesting a recently described serine-threonine kinase (STK33) has a fundamental role in the MCC developmental program. STK33 deletion results in 1) fewer MCCs, 2) fewer cilia per cell, 3) an abnormal ciliary structure and 4) reduced ciliary beat frequency. In this proposal, Dr. Berical aims to understand the mechanism by which STK33 effects the MCC molecular program to create this highly irregular phenotype. Leveraging key training opportunities through his collaborators and scientific advisory committee, he will 1) precisely characterize the STK33-dependent MCC defects using time course single cell RNA-sequencing to pinpoint when, during MCC differentiation, STK33 exerts its effect, 2) identify STK33 downstream targets and effector molecules and 3) determine the in vivo ramifications of STK33 loss on the engraftment, differentiation and function of airway epithelial cells. Following this investigation of the STK33-dependent regulation of MCC biology, Dr. Berical then expands these methods to probe the functions of a curated list of high priority ciliary kinases of unknown function. This work will provide much needed insight into the MCC molecular program and develop an essential platform for the interrogation of genes of unknown function in the airway epithelium, applicable to the genetically heterogeneous PCD, as well as other airway diseases. Dr. Berical has 80% protected time from his department to accomplish these aims under the guidance of his mentors Drs. Finn Hawkins and Darrell Kotton at the Center for Regenerative Medicine at Boston University/Boston Medical Center. He has assembled a remarkable team of advisors with diverse expertise to assist in his career development and scientific research. Dr. Berical details a comprehensive training plan that includes experiential training, didactic coursework, attendance and presentation at scientific meetings, preparation of manuscripts and acquiring additional grant support culminating in an R01. Dr. Berical has the commitment of his department to accomplish these goals and transition to an independent physician-scientist position by the end of the award.

NIGMS - National Institute of General Medical Sciences

ABSTRACT All growing cells experience DNA Replication Stress (RS, a slowing of DNA synthesis), which poses a major threat to genome integrity and viability. To safeguard against RS, stalled replication forks trigger DNA Damage Response (DDR) signaling cascades which coordinate resolution of stalled forks with cell cycle progression and other processes to ensure cellular homeostasis and survival. DDR defects can lead to severe human health consequences including stem cell dysfunction, aging, neurodegeneration, neoplasia, immune deficiencies, and developmental syndromes. While DDR signaling is clearly crucial for recovery from RS, the ways in which the many effector branches of the DDR are activated, coordinated with each other, and integrated with other cellular processes to orchestrate cell fate decisions are poorly understood. In exciting recent work we identified the RING finger E3 ligase RNF25 as a major new replication fork-localized DDR factor that is critically required for replicating cells to tolerate RS. Previous studies showed that RNF25 ubiquitylates ribosomal proteins to alleviate ribotoxicity (RNA damage). Remarkably, we demonstrated that RNF25 remediates RS independently of its canonical role in regulating ribosome function. Thus RNF25 is the first example of a `dual stress responder' that mediates responses to both genotoxicity and ribotoxicity. Our discovery of RNF25 as a major new DDR factor reveals large gaps in our knowledge of genome maintenance. The objective of this application is to define how RNF25 is regulated, and how it interfaces with other components of the DDR network and the ribotoxic stress response to alleviate RS. Our separation-of-function RNF25 mutant also provides an outstanding opportunity to determine the relative contribution of genotoxicity vs. ribotoxicity to cell fate decisions. The rationale is that we will mechanistically define a major new effector branch of the DDR (mediated by RNF25), and we will reveal how it is coordinated with other pathways to ensure genome integrity and survival. Our central hypothesis is that RNF25 critically interacts and cooperates with several important DNA repair proteins (REV7, PARPs, and MAGE-A4) to resolve RS. Our Specific Aims (SAs) are: SA1 Define how RNF25 associates with REV7 to orchestrate responses to DNA-damaging agents. SA2 Establish PARPs as mediators of RNF25 signaling in the DDR. SA3 Establish the E3 ligase cofactor MAGE-A4 as a regulator of RNF25 signaling. We will use biochemical approaches to define mechanisms by which RNF25 associates with its partner proteins. We will use unbiased screens to define genetic interactions between RNF25 and other major DDR pathways. We will use phenotypic endpoints (such as DNA synthesis at single DNA fiber resolution, cell cycle progression and viability) to define how RNF25 and its interacting proteins and pathways dictate cell fate. We propose innovative solutions to important problems such as `How do cells activate the DDR?' and `How are different branches of the DDR integrated and coordinated?'. The proposed work is significant because we will mechanistically define an important new branch of the DDR.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY/ABSTRACT Odontogenic pain, also known as toothache, is the most common form of orofacial pain across the world. Most commonly, odontogenic pain stems from inflammation of the tooth pulp following either bacterial infection or iatrogenic damage. While this pain will resolve with clinical intervention that removes the peripheral insult and dental pulp (e.g., root canal or extraction), it may be accompanied by substantial mechanical and thermal allodynia that is not alleviated by local anesthesia. We continue to lack safe approaches to achieve effective, immediate analgesia toward necessary dental treatments to resolve odontogenic pain. To inform the development of non-opioid analgesics we need fundamental knowledge of the neuronal basis and behavioral phenotypes of odontogenic pain. Mammalian teeth are highly innervated by specialized somatosensory neurons, intradental neurons, that detect damaging stimuli and initiate a reflex response to protect the teeth. However, we have yet to define mechanisms by which intradental neurons produce odontogenic pain in the context of damage and inflammation. Here, Dr. Joshua Emrick and his laboratory will investigate the neuronal basis of toothache to provide targets for future development of analgesics. The overall objective of this application is to use mouse models and powerful, cutting-edge approaches to define the neurons, circuits, and phenotypes of odontogenic pain. Aim 1 will reveal peripheral mechanisms whereby intradental trigeminal sensory neurons are activated representing a basis for pulpitis-induced pain. Aim 2 will reveal central mechanisms whereby intradental trigeminal sensory neuron inputs lead to physiological responses. Aim 3 will provide novel preclinical phenotypes of odontogenic pain in mammals to provide objective measures for future evaluation of analgesia. Upon successful completion of this project, we will have a molecularly-defined cellular origin for odontogenic pain from inflammation. Further, we will define the neurons and circuits that relay odontogenic pain in the brainstem. We also will have defined phenotypes for odontogenic pain initiated by the intradental neurons. These outcomes will provide key mechanistic insight into the origin of odontogenic pain toward the long-term development of novel non-opioid analgesics.

NIH

This proposal aims to advance our understanding of clonal hemopoiesis of indeterminate potential (CHIP) in the development of atherosclerotic cardiovascular disease (ASCVD). CHIP is a recently identified acquired risk factor for ASCVD. With aging, hematopoietic stem cells accumulate mutations that can lead to a proliferative advantage resulting in CHIP. Tet Methylcytosine Dioxygenase 2 (TET2) is a commonly mutated gene in CHIP and confers a 50% increased risk for incident coronary disease. How TET2 leads to ASCVD is in humans is not well understood and there is currently no ability to assess whether a specific TET2 mutation is high-risk. The central objective of this proposal is to (1) identify TET2 mutations that are high-risk for developing ASCVD to derive a comprehensive and clinically actionable risk score calculator and (2) identify the aberrant cell states and signaling pathways among TET2 mutated immune cells in the coronary vasculature. To identify high-risk TET2 mutations, the candidate will leverage a population-scale human genetics approach in >1 million people via the Million Veteran Program (MVP). To identify aberrant cell states and signaling pathways, the candidate will deploy their novel single cell lineage tracing protocol in coronary vascular tissue followed by validation experiments via population-based human genetic association studies. The candidate's career goals are to become an independently funded physician scientist focused on developing new ways of treating ASCVD. In addition to the proposed science, the training activities outlined in the candidate's career development plan are focused on the crucial skills and experiences necessary to enable an independent research program. Combined with the direct mentorship of Ors. Brent Ferrell and Adrianna Hung, Tennessee Valley Health System Nashville VAMC represents an ideal environment for the proposed work and leverage some of the world-class strengths of the Veterans Affairs resources. The Ferrell and Hung labs have deep experience in the methods used in this proposal and are prepared to support the candidate throughout the entirety of the grant period. Overall, this VA CDA-2 proposal represents a set of innovative and timely scientific aims combined with a tractable career development plan that will meaningfully contribute to human health research and catalyze the candidate's long-term career goal of developing into an independent investigator.

NINDS - National Institute of Neurological Disorders and Stroke

PROJECT SUMMARY Neural circuit development and function depends on precise interactions between neurons and glia. Astrocytes, the primary peri-synaptic glia, mediate synapse formation, stability, and function. Neuron-astrocyte crosstalk is facilitated by complex protein-protein interactions, and loss of these interactions contributes to circuit instability in many neurological disorders. Thus, understanding the mechanisms that regulate neuron-astrocyte communication is of broad clinical importance. Glycosylation is a posttranslational modification that regulates protein stability and binding through addition of sugar groups to specific amino acids. Mutation of genes in glycosylation pathways cause congenital disorders of glycosylation (CDGs), a group of monogenic disorders associated with neurological dysfunction, including epilepsy, autism, and cerebellar degeneration. The mechanisms underlying neurological dysfunction in CDGs remain unknown. Here, I focus on ALG8, an enzyme in the N-glycosylation pathway. To explore the molecular underpinnings of ALG8-CDG, I first needed to develop models that reflect the patient population. To this end, I generated a predicted null zebrafish line (alg8stl973) and human embryonic stem cell (hESC) lines with a missense mutation (p.Thr47Pro) found in ALG8-CDG patients. My preliminary data revealed a decrease in astrocyte numbers in the brains of alg8 mutant zebrafish with no change in total cells, and reduced proliferation of ALG8 mutant hESC-derived astrocytes. Moreover, in alg8stl973 fish, astrocyte morphological complexity is reduced. As astrocyte-synapse association is necessary for neuronal signaling, I hypothesize that defective glycosylation disrupts specification and maturation of astroglia, which in turn drives circuit imbalance and CDG-associated behavioral deficits. To address this hypothesis, I will leverage preexisting transgenic tools in zebrafish to label astrocytes and test whether changes in proliferation and/or cell death result in reduced astrocytes in alg8stl973 fish (Aim 1). Furthermore, I will use biochemistry and in vivo imaging to characterize how loss of alg8 impacts the glycosylation status of one key regulator of astrocyte morphogenesis: NrCam (Aim 2). Finally, as ALG8 is expressed in all neural cell types, I will use cell-type specific rescue in fish and co-culture of hESC-derived neural cells to determine which cell type(s) drive changes in astrocyte morphology and synaptogenesis in ALG8-CDG (Aim 3). My long-term goal is to define common molecular changes in brain development across distinct CDGs. Critically, various CDG subtypes result in common neurological symptoms, but the cellular and molecular underpinnings of these phenotypes are largely unknown. Similar to my preliminary findings in ALG8-CDG models, recent work indicates that astrogenesis is altered in a mouse model of MGAT5-CDG, a CDG with defective N-glycosylation. Thus, I anticipate that my findings will be broadly applicable to the CDG community and will enhance our fundamental understanding of how glycosylation shapes brain development.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary/Abstract Astroviruses are RNA viruses that commonly cause disease in humans, including gastroenteritis and fatal cases of encephalitis. Despite their broad human impact, astroviruses are understudied and many of the critical steps of the viral lifecycle are poorly characterized. There is limited understanding of host-pathogen interactions that facilitate viral replication, including the role of RNA motifs in the viral genome. Our lab was the first to cultivate astrovirus VA1 (VA1), the most common cause of astrovirus encephalitis to date. In a region overlapping with ORF2 and the 3' untranslated region, VA1 is predicted to encode a stem-loop II motif (s2m). Similar motifs with the same secondary structure have been identified in astroviruses and viruses of other viral families, with its importance in the viral lifecycle being virus-dependent. Using SHAPE-MaP on the full-length VA1 genome, we confirmed the formation of the s2m and its secondary structure. Mutagenesis of the s2m in a novel reverse genetics system for VA1 revealed the s2m to be essential. Deletion of the s2m or mutations that disrupt guanine- cytosine base pairs (GC-bp) that are critical for the secondary structure of the s2m result in virus that cannot be propagated. The mutant genomes can be rescued when complementary mutations are introduced into the s2m that restore GC-bp in the secondary structure. Mutagenesis of a position not involved in GC-bp was important but not essential for the function of the s2m. Capsid expression could not be detected from transfected genomes containing s2m mutations. Translation of capsid was also reduced by mutations of the s2m using a reporter system, and we have identified putative proteins involved in translation that may also bind to the s2m. We are now uniquely positioned to study the mechanism of action for the s2m in promoting the VA1 lifecycle using our novel tools that we have developed. Our central hypothesis is that the s2m facilitates viral translation through RNA-protein interactions, mediated by the s2m sequence, structure, and location in the genome. To test this hypothesis, we will take a combination of genetic and biochemical approaches to mechanistically understand why the s2m is essential. In other viral stem-loop structures, the loop region often serves as an important interaction site. We will define the role of the VA1 pentaloop for the function of the s2m by mutagenesis. Next, we will determine whether the function of the s2m is dependent on location in the genome. We will also assess whether the s2m must be encoded on the expressed RNA strand or if it can function independently. Using an RNA-pulldown, we have identified putative proteins that bind to the s2m that also mediate translation. We will confirm s2m-protein interactions and determine the effects of loss of function of these candidate proteins on the viral lifecycle. The findings of this project will provide important insights into the function of the VA1 s2m, address gaps in our knowledge of the VA1 lifecycle, and contribute to our larger understanding in RNA motifs in viral biology. These results will set the foundation for further dissection of the molecular biology of VA1, ultimately accelerating development of antivirals and vaccine-based approaches.

NIAID - National Institute of Allergy and Infectious Diseases

Project Summary Tissue regeneration represents a frontier in immunology: how immune cells coordinate the rebuilding of complex tissues after injury. While lower vertebrates regenerate entire appendages, mammals heal through inflammatory scarring, a process orchestrated by immune programs that remain poorly defined. This proposal aims to uncover how immune and stem cell interactions determine whether mammalian tissues regenerate or scar, and how these programs deteriorate with age. Our central hypothesis is that the immune system encodes a regenerative “decision code” through temporally and spatially coordinated cellular programs that license stem cells for multi-tissue regrowth. To test this, we will dissect two complementary mammalian models where regenerative outcomes diverge within the same tissue context. Aim 1 will identify the immune circuits that enable digit tip regeneration using intravital two-photon microscopy, single-cell and spatial transcriptomics, and targeted pathway perturbations. Aim 2 will define how age and injury context reshape immune regulation of regeneration in the mouse ear pinna, integrating in vivo photolabeling, optogenetic control of immune gene function, and high-dimensional immune– stromal network mapping. This project introduces a new immunological framework for regeneration, applying next- generation tools including custom-built multiphoton imaging, 4D single-cell profiling, and light- inducible gene editing to visualize and manipulate immune control of tissue repair in living mammals. By defining the immune programs that distinguish regeneration from scarring, this work will establish the immune system as a master regulator of regenerative capacity and reveal molecular targets to restore repair in aged or chronically inflamed tissues. These insights will transform how we understand and therapeutically harness immunity for tissue renewal.

NHLBI - National Heart Lung and Blood Institute

Although squamous and mucous metaplasia are the two cardinal forms of pathologic epithelial injury response in airway disease, the cellular source and molecular mechanisms governing their formation have not been clearly defined. It has long been assumed that both forms of metaplasia arise from a common basal stem cell population biased by specific signaling factors, however, both metaplasias occur together in the same patient in the same signaling milieu. Furthermore, we have previously reported that the distal murine tracheal epithelium is predisposed to mucous metaplasia, while squamous metaplasia tends to form in the dorsal murine and human airway. In parallel, we have reported that specific hillock basal stem cells are found in dorsally located stratified squamous epithelial structures that we named hillocks. In aggregate, these findings suggest the hypothesis that squamous and mucous metaplasia arise from regionally distinct stem cell populations and that these heterogeneous stem cell populations respond differently to common pathologic signaling cascades. With regard to mechanism, high Yap signaling activity has been associated with squamous metaplasia while mucous cell differentiation requires a suppression of Yap activity. As such, we will establish the propensity of anatomically regionalized basal stem cell populations of the mouse and human airway to undergo either squamous or mucous metaplasia including (1) dorsally located murine and human hillock basal stem cells, (2) proximal and (3) distal pseudostratified murine and human basal stem cells populations. Since Yap has been directly associated with mouse and human metaplasia, we will assess the effect of temporally regulated Yap overexpression on the above stem cell populations and the consequences on both squamous and mucous metaplasia. Additionally, using ATAC-Seq and RNA-seq, we will determine the accessibility and expression of the Yap target genes that underpin the differential metaplastic propensities of the above stem cell populations. In contrast to Yap signaling, EGFR signaling activation causes both pathologic mucous and squamous metaplasia. Therefore, we will define the effects of EGFR modulation on both hillock and non-hillock pseudostratified mouse and human basal stem cells. We will also assess whether Yap overexpression will prevent EGFR-induced mucous metaplasia in distal basal stem cell populations and whether suppressing Yap will lead to diminished EGFR-induced squamous metaplasia arising from hillock basal stem cells. Finally, we provide evidence that Yap activity is dramatically upregulated following injury, but this activity subsides as injury resolves. We will define the effects of Yap modulation on the formation of the early post-injury squamous barrier epithelium and injury-associated squamous metaplasia. Understanding how heterogenous stem cell populations of the airway contribute to both squamous and mucous metaplasia and establishing how these stem cells respond to disease-associated signaling pathways will inform strategies to control pathologic metaplasia.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY/ABSTRACT Dilated cardiomyopathy (DCM) is a leading cause of heart failure, with approximately 40% of DCM cases linked to pathogenic genetic variants. Despite advances in genetic testing, there remains a major knowledge gap in how specific variants contribute to the multifaceted clinical manifestations of DCM. A notable example is a pathogenic variant in the phospholamban (PLN) gene resulting from deletion of arginine 14 (PLN-R14del). PLN induces DCM characterized by prominent ventricular arrhythmias and highly variable phenotypes, ranging from severe, early-onset disease to lifelong asymptomatic carriage. Such variability makes it challenging to establish genotype-phenotype relationships in PLN-R14del carriers, highlighting the urgent need for new technologies to bridge this gap. Advances in omics technologies are revolutionizing precision medicine. Among omics methods, top-down proteomics has emerged as a powerful technology for studying post-translational modification (PTMs), genetic variants, and splicing isoforms (collectively known as “proteoforms”). Top-down proteomics is ideally positioned for studying complex genetic diseases like PLN-R14del DCM, providing direct evidence of how genetic mutations affect proteoform compositions and linkage to function and phenotype, thereby bridging genotype-phenotype gap. Our preliminary data show that the PLN-R14del variant is associated with unique changes in cardiac proteoforms, including alterations in critical Ca2+-handling, contractile, and metabolic proteoforms, in both human patient tissue and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Hence, we hypothesize that dysregulation of Ca2+-handling, contractile, and metabolic proteoforms and the corresponding pathways contribute to variability in disease phenotypes and expressivity in PLN-R14del carriers. To test this hypothesis, we will employ a systems biology approach featuring novel multi- omics, incorporating proteomics, metabolomics and lipidomics, in combination with human clinical samples and patient-derived hiPSC-CM cellular models. Specifically, we will carry out multi-omics analysis of myocardial tissue from patients with PLN-R14del DCM, compared with genotype-negative DCM and nonfailing donor tissue as controls. The findings from the omics analyses will be further integrated with clinical data to develop patient- specific disease signatures in PLN-R14del carriers. We will also determine differences between symptomatic and asymptomatic PLN-R14del carriers through multi-omics analysis of patient-derived and isogenic control hiPSC-CMs and link them to changes in contractility/metabolism using functional assays. We will further connect the PLN-R14del variant mechanistically to proteoform alterations and their functional outcomes using gain- and loss-of-function approaches. Successful completion of the proposed study will provide new insights into the mechanisms underlying cardiac dysfunction in PLN-R14del, as well as bridge the genotype-phenotype knowledge gap in familial DCM to improve risk-stratification in variant carriers, advance our understanding of genetic diseases, and facilitate the development of targeted treatments towards precision medicine.

NIA - National Institute on Aging

Project Summary: The overarching goal of this application is to investigate cell mechanics-mediated regulation of hair follicle stem cells (HFSCs) during homeostasis and aging. We propose to study microRNA-205- mediated regulation of extracellular matrix (ECM) and actin cytoskeleton for HFSC quiescence and activation and leverage the ability of microRNA-205 (miR-205) to stimulate HFSC activation to enhance HFSC aging. MicroRNA (miRNA) is a class of small noncoding, regulatory RNAs that play important roles in mammalian development, stem cells, diseases and aging. In our preliminary studies, we have determined mechanical properties of HFSCs during homeostasis and aging. We have revealed that bulge HFSCs reside in a stiff microenvironment with high actomyosin contraction forces. In contrast, hair germ progenitors are relatively soft and undergo periodic enlargement and contraction. Notably, induction of miR-205, one of the most highly expressed miRNAs in HFSCs, downregulates many bona fide targets, which are enriched in the function of ECM, actomyosin cytoskeleton and mechanosensing. And this leads to rapid activation of HFSC cell division and promotes hair regeneration in both young and aged mice. Mechanistically, we have identified Piezo1 as a novel target of miR-205, which functions downstream of miR-205 and translates mechanical cues into a gene expression program to reinforce the mechanical properties and maintain cellular states of quiescent HFSCs. To examine the role of PIEZO1-mediated calcium influx in HFSCs, we have further developed a high-resolution intravital imaging system to accurately record calcium influx in HFSCs over an extended period of time during quiescence and activation. This allows us to quantify cumulative calcium levels and further identify transcription factors, NFATC1 and JUN (AP1), which function downstream of PIEZO1-mediated calcium influx to promote the expression of the ECM and actin cytoskeleton genes. Based on these exciting findings and promising preliminary data, we propose to further elucidate the mechanism of miR-205-mediated HFSC activation and aging through the regulation of ECM and actomyosin contraction forces (Aim 1), determine the regulation of PIEZO1-mediated mechanosensing by miR-205 (Aim 2), and leverage miR-205-induced HFSC activation to improve HFSC functions and hair growth during aging (Aim 3). Together, this application will provide new insights into the mechanisms orchestrating the mechanical properties and stem cell functions of HFSCs. By harnessing the powerful combination of live imaging, cell biology, mouse genetics, and single-cell genomics, we will establish a new paradigm for studying tissue architecture, cell mechanics and underlying mechanisms. These results will lay the foundation for leveraging noncoding, regulatory RNAs to enhance HFSC functions during aging.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

PROJECT SUMMARY Precise regulation of cell fate specification during early embryogenesis is essential for proper tissue and organ formation, and its disruption leads to congenital malformations. However, the gene regulatory pathways controlling these early developmental decisions—particularly in humans—remain poorly understood. This proposal investigates a novel, primate-specific mechanism of cell fate control mediated by the dual-function DNA/RNA-binding protein ILF3. Identified through genome-wide screens in human pluripotent stem cells (PSCs), ILF3 is required for proper exit from pluripotency and lineage specification in human and primate—but not mouse—PSCs. Our data show that ILF3 interacts with and inhibits the RNA editing enzyme ADAR1 to limit adenosine-to-inosine (A-to-I) editing at primate-specific Alu elements, thereby preserving accurate splicing of developmental transcripts. These findings implicate ILF3 as a critical regulator of transcriptome fidelity in early primate development and introduce a novel paradigm where species-specific RNA processing fidelity serves as a developmental checkpoint. To define the developmental and mechanistic roles of ILF3, we propose three integrated aims. In Aim 1, we will use cross-species gastruloid models from human, chimpanzee, rhesus monkey, and mouse to assess ILF3's role in early lineage transitions and test whether it defines a primate- specific pathway in mammalian development. In Aim 2, we will map nascent RNA editing following acute ILF3 depletion using SLAM-seq and identify the protein domains mediating ILF3-ADAR1 interaction, linking RNA editing regulation to cell fate control. In Aim 3, we will define how ILF3 impacts RNA processing at key developmental genes by integrating splicing analysis and quantitative proteomics, uncovering direct effectors of lineage specification. Moreover, we will establish a causal link between expression of mis-edited and mis-spliced developmental regulators and proper gastruloid formation through rescue experiments. This research will uncover a previously unrecognized RNA-based regulatory mechanism controlling early primate development and provide insight into how defects in RNA editing and splicing may contribute to congenital disease. By establishing a functional framework for ILF3 in safeguarding human cell fate transitions, this work will inform future strategies for therapeutic intervention in developmental disorders, directly supporting NICHD's mission to understand and treat the origins of birth defects.

NIGMS - National Institute of General Medical Sciences

The extracellular matrix (ECM) provides essential tissue infrastructure and mechanical cues that regulates cell metabolism. Mechanotransduction is the conversion of mechanical forces from the ECM to intracellular chemical signals and plays a vital role in health and disease through cell-matrix interactions. While evidence supports a ‘mechano-metabolic link’ between mechanotransduction and metabolism, the precise pathway connecting ECM mechanical states to cellular metabolism remains poorly understood due to the ECM’s complexity, including its viscoelastic properties, which exhibit both strain-independent (linear) and strain-dependent (nonlinear) regimes. All tissues exhibit both linear and nonlinear viscoelasticity, typically reported as stiffness and strain-stiffening, respectively; however, a fundamental gap remains in understanding how these distinct mechanical properties counterbalance each other to regulate cell metabolism. Key open questions include how cells engage with nonlinear viscoelastic environments, how mechanotransduction scales with cell and tissue maturity, and how nonlinear viscoelasticity influences cellular uptake and consumption of metabolic biomolecules. To address these gaps, this proposal uses primitive and differentiated induced pluripotent stem cells (iPSCs), both as single-cell and organoid cultures, in a 3D in vitro polymeric hydrogel systems to independently present cell-accessible and cell-inaccessible nonlinear viscoelastic regimes. We combine this with material- and omics-based modeling approaches to define viscoelastic and metabolic signaling regimes. In Project 1, we will use ECM ligand-binding motifs and both primary cells and iPSCs to investigate integrin-mediated cell-matrix interactions across viscoelastic regimes, cell maturity, and tissue complexity. In Project 2, we will examine how nonlinear viscoelasticity influences cellular uptake of metabolic precursors such as lipids and apply model-based approaches to define characteristic metabolic and proteomic signatures associated with linear and nonlinear regimes. The outcomes of this proposal will advance our understanding of mechanosignaling in nonlinear viscoelastic environments and establish a mechanistic model of the mechanical regulation of cellular metabolism. The long-term goal of the lab is to develop complex in vitro models to investigate how physiological processes such as aging and pregnancy induce systemic tissue alterations that drive changes in cell-matrix interactions and mechanosignaling, ultimately influencing cell and tissue function. By uncovering a direct mechano-metabolic connection, this work will have broad implications for fundamental biology while also developing critical tools and workflows for studying cell-matrix interactions. Importantly, while this proposal focuses on fundamental biological processes, the methods and tools developed will be broadly applicable across various cell, tissue, and disease states.