Rockefeller University

NIH Research Projects · FY 2025 · 2015-03

ABSTRACT Nuclear pore complexes (NPCs) are huge macromolecular assemblies that serve as the only conduit for bidirectional transport between the nucleus and cytoplasm. We have determined the constituents, architecture, and detailed high precision structure of the archetypal yeast NPC. However, despite our increasing structural information on NPCs, we still lack a fundamental understanding of the mechanics of numerous of its functions. With our detailed maps in hand, we are, for the first time, in a unique position to map and reveal the structural changes associated with functional states that throw light on mechanisms underlying critical aspects of NPC function. Our hypothesis is that, despite some overlap, discrete and distinct structural stages and states are associated with NPCs’ varied functions. We have therefore established a powerful pipeline for analyzing NPCs and their vicinal associated complexes both structurally and functionally in defined functional states onto which we map quantitative phenotypic information. This information will allow us to move from static models of NPCs to working models of the machine in action, breathing life into our NPC maps and dissecting out how particular functionalities are mechanistically supported at the structural level. We focus on two such functionalities that are central to nuclear function at two related levels: first, as a regulator of transport, NPCs control mRNA packaging and export to the cytoplasm to both mediate and regulate gene expression; and second, NPCs directly control genes by binding chromatin and its regulators to alter expression states epigenetically. Both processes are incompletely understood at the molecular level, and have profound effects on cellular function as evidenced by the fact that disruptions of NPC-associated proteins associated with these functions lead to many human diseases. For Aim 1, we will determine the molecular machinery of NPC-mediated mRNP export by studying NPCs effectively “frozen” in defined intermediate stages of mRNP export. For Aim 2, we will determine the molecular machinery of NPC-mediated chromatin organization, specifically focusing on subtelomeric gene silencing. Using our established pipeline, we will identify and structurally characterize these NPC stages and states and their vicinal interactomes. Realizing these Aims will generate NPC structure-function maps in unprecedented detail, which will be of great use to the field to understanding how the mRNP export and chromatin remodeling machineries act in concert with different parts of the NPC to enable their functionalities and will shed light on the nature of numerous disorders associated with dysfunction in these processes. The resulting structure-function NPC maps promise to set the stage for tapping the NPC’s tremendous potential as a drug target for many human conditions ranging from cancers to infectious diseases to developmental and neurological disorders. 1

NIH Research Projects · FY 2026 · 2014-04

Project Summary The aim of this proposal is to hold an annual, five-day course in advanced gene mapping at The Rockefeller University in New York. The course is directed towards advanced researchers who are familiar with the fundamental aspects of statistical genetics but who need to become more proficient in the analysis of complex traits. Forty-five students will be accepted to each course. Travel cost will be covered as well as a per diem to cover the cost of hotel and board will be provided to participants from outside of the New York City area. The course consists of two components: lectures on important current topics in gene mapping, as well as hands-on exercises to be performed with the latest freeware software programs using Docker containers and Jupyter Notebooks. The emphasis of the course is analyses of sequence and other omics data. The next Advanced Gene Mapping course will be held April 22-26, 2024. The two MPIs and three instructors who are experts in their respective fields will teach on a variety of topics that include: data quality control; qualitative and quantitative trait (population and family-based data) association analysis of whole-genome data (genotype, sequence, and imputed); controlling for population admixture and substructure; meta-analysis; sample size estimation and power calculations; detecting gene x gene interactions; analysis of RNAseq data; performing TWAS studies; estimating heritability, imputation of genotypes and their analysis, elucidating pleiotropy; functional prediction and variant annotation; estimation of polygenic risk scores; Mendelian randomization and mediation analysis; analysis of epigenomic data, multi-trait colocalization, fine mapping. Course lectures include examples of genomic studies performed in non-European populations, in addition to using non-European datasets in the computer exercises to aid researchers in analyzing data from diverse populations. An additional instructor will provided a lecture on responsible conduct of reasearch that includes the following topics: conflict of interest, research ethics, protection of human subjects, and data management and security. A variety of freely available software including ANNOVAR, GCTA, FaST-LMM, LDScore regression, MR-base, MultiPhen, PLINK, R, REGENIE, SuSiE, LDpred2, and VCFtools will be implemented to perform practical exercises. Since the methods used in gene mapping are constantly changing, the topics and analytic programs will be updated annually to reflect the latest developments in the field of statistical genetics. Given the vast amounts of generated genetic data, it is essential to train researchers and give them the necessary information and tools for data analysis to elucidate the genetic etiology of complex traits.

NIH Research Projects · FY 2026 · 2013-01

Project Summary The highly specialized intestinal immune system is charged with maintaining tolerance to harmless stimuli from commensal bacteria and food, while providing protective immunity against pathogens. Dysregulation of this critical balance can lead to inflammatory bowel disease, food allergy, or increased susceptibility to enteric pathogens. CD4+ T cells are key players in intestinal homeostasis, finely tuning responses at the level of antigen recognition and functional differentiation. In the intestinal epithelium (IE) and underlying lamina propria (LP), tissue adapted pro-inflammatory (ie. Th17, Th1) and regulatory (Treg) and intraepithelial (CD8aa+ CD4IEL) CD4+ T cells coordinate immunity and tolerance to diverse intestinal stimuli. Nevertheless, it remains to be defined how TCR repertoire and its specificity translate to function of both LP and IELs in response to microbial antigens. Additionally, very little is known about the characteristics of dietary antigen-specific intestinal T cells. Oral tolerance, a critical mechanism of gut homeostasis whereby oral administration of antigen results in both local and systemic tolerance to that antigen, is known to depend on Tregs. However, the clonal dynamics of polyclonal T cell responses in the setting of tolerance remain unclear. Finally, outside the context of immunization or allergy, T cells that specifically recognize food protein have yet to be identified. Based on data recently published as well as preliminary data obtained during the current funding cycle, we hypothesize that tissue cues combined with TCR-driven signals from commensal microbes and food dictate intestinal T cell functional differentiation in steady state, while disruption of normal T cell responses by infections or allergens results in inappropriate clonal dynamics including expansion of pathological T cells. Aim 1 will define how stimulation by microbiota or enteric viruses impact TCR repertoire and functional differentiation of intestinal CD4 T cells. Aim 2 will characterize the role of dietary antigens in the context of tolerance, enteric infections, or food allergy, affect TCR repertoire and functional differentiation of intestinal CD4 T cells. We combine single cell transcriptomics with a novel fate- mapping strategy to selectively label peripheral T cells that are recruited to the intestine under various microbial and dietary challenges. A recently developed LIPSTIC tool that allows intestinal epithelial cells (IECs) labeling of interacting cells will be utilized to define potential mechanisms of IEL recruitment/expansion in response to nonself stimuli. Recently generated murine strains with fixed TCR Vb chain, or that carry microbiota-specific TCRs, combined with complementary tetramer and gnotobiotic strategies will be used to specifically track and define recognition properties during T cell migration and differentiation in the gut. Novel “super-tetramer” and dietary manipulations, combined with inflammatory challenges will be used to define food antigen-specific T cells during steady state and how intestinal CD4+ T cell responses to dietary antigen are altered in a food allergic or infection context. By combining these innovative approaches and concepts in mucosal immunology, this proposal will help better defining the role of nonself antigen stimulation in T cell repertoire and function in the intestine.

NIH Research Projects · FY 2025 · 2011-05

Project Summary Mendelian susceptibility to mycobacterial disease (MSMD) is a genetic and selective predisposition to clinical disease caused by weakly virulent mycobacteria, such as Bacillus Calmette-Guérin (BCG) vaccines and environmental mycobacteria (EM). Patients with MSMD are occasionally vulnerable to other intra-macrophagic pathogens (e.g. salmonella). The pathogenesis of MSMD remained unclear until 1996, when its first genetic etiology was deciphered in children with interferon-γ receptor 1 (IFN-γR1) deficiency. Genetic studies over the last 25 years have identified 16 MSMD-causing genes, including 14 autosomal (IFNG, IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, IL12RB2, IL23R, IRF8, SPPL2A, RORC, ISG15, TYK2, JAK1) and 2 X-linked genes (NEMO, CYBB). The high level of allelic heterogeneity at these loci has defined 31 distinct disorders. There is however physiological homogeneity, as all disorders impair IFN-γ immunity. Mutations in 5 genes (RORC, ISG15, TYK2, JAK1, STAT1) can underlie an atypical, syndromic form of MSMD, with an associated phenotype. With hindsight, MSMD is a misnomer, as most genetic etiologies show incomplete penetrance for MSMD. This serendipitously led to the discovery of genetic etiologies of bona fide tuberculosis. Remarkably, only about half of the 900 international patients studied in our lab carry MSMD-causing lesions in the exons and flanking intron regions at any of these 16 loci. In this renewal application, we hypothesize that unexplained MSMD cases can result from novel monogenic inborn errors of immunity, possibly but not necessarily involving IFN-γ mediated immunity. We aim to identify new MSMD-causing genes by following a genome-wide (GW) approach, based primarily but not exclusively on whole-exome sequencing (WES). We will enroll at least 50 MSMD patients each year. We will search for novel genetic etiologies by testing a hypothesis of genetic homogeneity, i.e. searching for genes mutated in two or more families. We will also test a hypothesis of genetic heterogeneity, i.e. searching for genes mutated in a single family. This search will benefit from our 12-year-long development of computational tools to analyze WES. Causal relationships between candidate genotypes and MSMD will be established experimentally in great mechanistic depth at the molecular, cellular, and immunological levels, taking advantage of cutting-edge technologies and our 25-year-long study of MSMD. In patients without candidate genotypes by WES, we will search for candidate regulatory variations in known and unknown MSMD-causing genes by whole genome sequencing (WGS). Our preliminary results are exciting, as we have identified MSMD-causing mutations in genes known to be crucial for IFN-γ immunity (TBX21, IRF1) and in other genes that probably disrupt IFN-γ immunity by novel mechanisms (ZNFX1, MCTS1). From an immunological standpoint, this research will provide novel insights into the mechanisms of human immunity to mycobacteria. From a medical standpoint, this work will provide molecular diagnoses for MSMD patients and genetic counseling for families, while offering the use of therapeutic IFN-γ, at least in patients whose genetic disorder does not abolish cellular responses to IFN-γ.

NIH Research Projects · FY 2026 · 2010-04

PROJECT SUMMARY Herpes simplex virus 1 (HSV-1) encephalitis (HSE) is the most common sporadic viral encephalitis in Western countries. Its prevalence peaks in individuals < 18 and > 50 yo. While infection with HSV-1 is innocuous in most people, HSE is devastating. No vaccine against HSV-1 is available, and acyclovir-treated survivors of HSE often suffer from neurological sequelae. The lesions typically affect the forebrain (95%), and rarely the brainstem (5%). Its pathogenesis remained unclear until we showed from 2003 onward that ~10% of HSE children carry inborn errors of immunity (IEI) to HSV-1 in the central nervous system (CNS). Following a candidate gene approach, in 2010 we initiated a genome-wide search for novel HSE-causing genes (NIH R01AI088364). By 2019, we had found 8 genetic etiologies, most of which impaired TLR3-dependent type I IFN immunity in children with forebrain HSE and DBR1-dependent immunity in children with brainstem HSE. Over the last five years, we discovered 6 novel genetic etiologies of forebrain HSE, with mutations in IFNAR1, STAT2, TYK2, SNORA31, RIPK3, and TMEFF1. The last three disorders impair cell-intrinsic immunity to HSV-1 by new IFN-independent mechanisms. We also found that auto-antibodies (Abs) neutralizing type I IFNs (AAN-I-IFNs) can underlie 40% of cases of West Nile virus encephalitis. No genetic etiology has yet been identified for 424 of the 460 HSE children we study. Moreover, adult HSE remains unexplained. We hypothesize that new monogenic inborn errors of CNS- intrinsic immunity to HSV-1, or AAN-I-IFNs, can underlie forebrain or brainstem HSE in children or adults. We will expand our unique international cohort of children and adults with HSE, and search for AAN-I-IFNs in their plasma. We will use integrative next-generation sequencing (NGS) approaches, including whole exome or genome sequencing (WES, WGS) and RNAseq, to search for novel genetic etiologies. We will analyze the NGS data at both the population and individual levels, testing models of physiological or genetic homogeneity and heterogeneity. To establish causality between genotype and phenotype, all candidate genotypes will be characterized at the molecular and cellular levels by in-depth mechanistic studies using ad hoc cell types. This renewal application is highly innovative but supported by exciting preliminary data. We have established a unique international cohort of 690 children and 320 adults with HSE. We have already identified biallelic mutations in MEX3B and MEX3C (type I IFN immunity), RIPK1 (cell death pathway), and ATG4C and ATG4D (autophagy pathway) among children with HSE. Finally, we also found that about 5% of children with HSE carry AAN-I-IFNs, suggestive of an underlying IEI of tolerance to self as the root cause of HSE. The genetic analysis of HSE is at the forefront of our paradigm-shifting search for human genetic determinants of sporadic, severe infectious diseases striking an isolated organ in otherwise healthy individuals. This project will expand the groundbreaking notion that HSE can result from defective CNS-resident cell-intrinsic immunity in both children and adults. Understanding the mechanism of HSE paves the way for new diagnostic, preventive, and therapeutic strategies.

NIH Research Projects · FY 2025 · 2008-07

Project Summary/Abstract: This application is a resubmission of a renewal for a pre-doctoral training program in Immunity and Infectious Disease at The Rockefeller University, an institution with a rich history in these areas. The Immunity and Infectious Disease Training Program is specifically designed for education in immunology and infectious disease, and incorporates required coursework, professional skills development, rotations, and extensive research opportunities. Trainees will complete thesis work comprising a body of novel scientific work in immunology and infectious disease. The 21 faculty trainers are accomplished scientists, including 8 members of the US National Academy of Sciences and one Nobel laureate, with a shared interest and experience in graduate education. The faculty has expertise in a very broad range of immunology and infectious disease, and the program encourages trainees to perform collaborative work in various areas with different faculty. We propose to support 6 pre-doctoral trainees during years 2-4 of graduate study. The applicant pool is outstanding, including a large number of students with accomplished undergraduate records, extensive research experience and a strong interest in immunology and infectious disease. Trainees would be mentored by the Program Director; members of the university Dean’s Office, a Program Advisory Committee of selected faculty for general curriculum and research advice; and a Faculty Advisory Committee, specifically designed for each trainee to provide detailed experimental guidance. An External Advisory Committee will evaluate the effectiveness of the program and provide advice on new initiatives. Finally, the University provides extensive support for the graduate program in general, which benefits the Immunity and Infectious Disease training program. The confluence of these attributes defines a specific training program that will prepare trainees with the educational background, analytical abilities, and experimental expertise for exciting career pathways in immunology and infectious disease.

NIH Research Projects · FY 2026 · 2004-02

Project Summary/Abstract The global objective of this research is to elucidate the mechanisms underlying tissue homeostasis and regeneration in mammalian skin and to understand how this process goes awry in human disorders, including cancers. Central to achieving this goal is the characterization of the different stem cells (SCs) within skin, determining their relative contributions to tissue homeostasis and wound-repair, and elucidating how changes in their niche microenvironments impact these events. Past AR050452 research led to purification of hair follicle (HF) bulge and basal inter-follicular epidermal (Epd) cells and established them as long-lived, self-renewing SCs that function in tissue regeneration and wound-repair. However, both in their biology and their tissue regenerative tasks, these SCs display distinct behaviors predicated by their unique microenvironments (niches). The field still lacks a comprehensive knowledge of the constituents of these niches, the nature of SC:niche interactions, and how they help SCs cope with stressful situations. Past AR050452 research set the foundations to tackle the next key questions: (1). What are dynamics in bulge niche components that drive HFSC behavior during the hair cycle? (2) What are the key niche:HFSC interactions that maintain quiescence and drive tissue (hair) regeneration and how do they differ from the niche: short-lived progeny interactions that drive hair differentiation? (3) How are HFSCs spared during the destructive phase of the cycle when hair growth ceases and most follicle cells below the bulge apoptose? Does eating confer increased SC fitness? (4) How do HFSC:niche interactions change when skin is injured and the SCs become repurposed to repair the wound? How do SCs protect themselves against immune/pathogen attack so that they can orchestrate the re-epithelialization process? (5) How does the natural process of wound-repair differ from the behavior of a SC when it acquires an oncogenic mutation that will ultimately lead it down a path to cancer? To answer these questions, we'll use FACS, single cell spatial transcriptomics and chromatin landscaping, conditional gene knockout and RNAi screens in vivo and employ these methods to explore skin stem cells in their native, wound-induced and tumorigenic environments.

NIH Research Projects · FY 2025 · 1999-05

This long-term project is focused on replicative senescence, the process whereby telomere shortening limits the proliferation of human cells. In the past, our work on this project showed how telomeres are protected by shelterin and how loss of shelterin-mediated protection can induce senescence. Recently, we have focused more narrowly on aspects of telomere biology specifically relevant to replicative senescence of primary human cells. Our preliminary data has revealed that replicative senescence is induced by the activation of the ATM (not ATR) kinase at critically-short telomeres that have become deprotected because they lack sufficient TRF2. Furthermore, we have addressed the question of why cells grown at 3% (physiological) oxygen levels show an extended replicative life span compared to cultures in normoxia. In both conditions, ATM inhibition (ATMi) or overexpression of TRF2 resulted in life-span extension. Life-span extension at 3% oxygen was not due to a difference in telomere shortening rates or altered expression of TRF2 or ATM pathway factors. Instead, we found that the ATM kinase is less responsive to DSBs and damaged telomeres at 3% oxygen, leading to a greater tolerance for short telomeres and an extension of replicative life span. These observations form the basis for AIM 1, which is focused on further defining the mechanisms of proliferative senescence, understanding how ATM is regulated by oxygen levels, and determining what aspect of TRF2-mediated protection is lacking at critically-short telomeres. As part of our long-term interest in understanding how TRF2 protects telomeres, we have obtained preliminary data that formation of the t-loop, which represses ATM signaling, involves tetramerization of TRF2. We found that TRF2 binds to telomeric DNA as a tetramer whereas its paralog TRF1, which does not protect telomeres, binds as a dimer. A dimeric form of TRF2 is defective in telomere protection but could be partially restored by enforced tetramerization. Furthermore, an engineered TRF1 tetramer formed t-loops and protected telomeres in vivo. In AIM 2, we will further test the model that t-loop formation requires TRF2 tetramers. We recently reported that the highly conserved iDDR domain of TRF2, which binds to the Rad50 subunit of the Mre11/Rad50/Nbs1 (MRN) complex, prevents MRN from associating with CtIP and thus blocks it from becoming an active endonuclease. Interestingly, the iDDR is functionally (but not structurally) similar to MRN inhibitory modules in yeast telomeric proteins, pointing to convergent evolution. Our preliminary data suggest that the iDDR also interferes with the ability of MRN to active the ATM kinase. In AIM 3, we will determine the mechanism of ATM inhibition and explore the physiological role of the iDDR, seeking to explain its evolutionary conservation. Together, these experiments will provide insights into the ATM kinase, its regulation, and its repression by TRF2, revealing mechanistic insights into how shortening telomeres limit the proliferation of human cells.

NIH Research Projects · FY 2025 · 1983-01

Project Summary Our global objective is to elucidate the mechanisms underlying tissue homeostasis and regeneration in mammalian skin and to understand how this process goes awry in inflammation and cancers. Central to achieving this goal is to determine how stem cells sense and tailor their programs of gene expression in order to perform their particular tasks during homeostasis and wound repair and survive under stressful situations. Past AR31737 research has revealed that adult epidermal and hair follicle (HF) stem cells originate from a common embryonic skin progenitor, but in adult skin, they reside in distinct microenvironments. While maintaining some commonalities, their different niches endow stem cells with distinct molecular properties and instructions to perform separate tasks. Upon injury, the nearest stem cells must respond and adopt a plasticity that enables them to regenerate either epidermis or hair follicles irrespective of the niche from whence they came. We have learned that this `dual lineage' plasticity, transient in a wound state, is hijacked and becomes constitutive in cancer. We've also learned that at the heart of stem cell identity and responsiveness are niche-specific transcription factors (TFs) that act in concert with DNA effectors of environmental signals (e.g. pSMAD1/4 for BMP signaling and LEF1/TCF1 for WNT/β-catenin signaling) to regulate key genes whose expression must rapidly change with the environment. In yrs 39-44 of AR31737 research, we'll now address: What are the chromatin dynamics that enable skin stem cells to choose between epidermal and HF fates? What are the key TFs involved and how do they drive the epigenetics needed to make these fate decisions? How do HF stem cells maintain their fate during homeostasis? How do these stem cells enter a plastic state following injury and then change their identity to epidermal stem cells when they find themselves in the epidermis after repair is complete? Finally, we have discovered that chromatin remodeling in epidermal stem cells is rapid after injury and other types of inflammatory stimuli, but once tissue homeostasis is restored and inflammation has waned, some of these changes resolve slowly. What are the mechanisms underlying this epigenetic memory, and how does it affect tissue fitness? To answer these questions, we will couple in vivo high throughput technologies with our functional interrogation methods, involving in utero lentiviral delivery of epigenetic reporter genes, inducible genes, RNAis and Crispr/CAS guide RNAs to selectively target the skin's stem cells. At the conclusion of this research, we expect to have advanced our knowledge of tissue homeostasis, wound-repair, inflammatory disorders and malignancies and provided new insights to therapeutic strategies.

NIH Research Projects · FY 2025 · 1980-12

Project Summary Our global objective is to develop a molecular understanding of how during development a population of stem cells (SCs) are set aside to produce, maintain and regenerate our tissues. As our longstanding AR27883 research on skin has shown, our basic science approach has led to advances in regenerative medicine and our understanding of human syndromes. Our skin epidermis is our body's barrier to the outside world: it must keep harmful microbes out and retain essential body fluids. Our current research centers on how emerging tissue SCs sense and interact with their surroundings (cells, signals and mechanical forces) not only to achieve a balance of proliferation and differentiation, but also to eliminate underperforming tissue cells as they arise. Understanding how healthy epidermal SCs arise and how they perform their duties is prerequisite to unraveling how cellular organization goes awry in inflammatory disorders and cancers of the skin. During skin development, stratified epidermis (and its hair follicle appendages) forms from a single layer of unspecified progenitors. As morphogenesis proceeds, resident epidermal SCs are set aside so that in adult skin, these self-renewing progenitors maintain and repair the skin's barrier. To what extent do self and non-self epidermal SC neighbors, and the mechanical forces they generate, participate in regulating SC behavior during normal homeostasis? What is the molecular nature of this communication circuitry that balances epidermal growth and differentiation? In the next 5 years, we'll: (1). Spatially map the temporal dynamics of cells and their transcriptomes as embryonic skin progenitors interact with newly emerging self and/or non-self neighbors, which together shape the niches that allow progenitors to behave as mature SCs with defined tasks. (2). Identify key signaling inputs that orchestrate the continual flux of epidermal progenitors and differentiating progeny that maintains and rejuvenates the body's barrier. (3). Elucidate how epidermis eliminates poorly performing cells for the sake of tissue fitness. (4). Elucidate how key signaling pathways and transcriptional events drive different regional mechanical forces during skin development and contribute to distinct morphologies and tissue functions. We'll apply the knowledge gained to determine how deviations in stem cell crosstalk with neighbors (`niche') lead to disease. To meet our aims, we and our collaborators developed unprecedented tools to a) spatiotemporally landscape gene expression across the skin, b) interrogate how mechanical forces impact tissue biology and c) track and characterize SCs as they sense and eliminate aberrant neighbors to preserve tissue fitness. While promising for regenerative medicine, adult tissue stem cells are long-lived and can also accumulate mutations that compromise tissue fitness and enhance cancer susceptibility. By unraveling how healthy SCs eliminate aberrantly performing cells, and yet over time, allow phenotypically silent oncogenic clonal diversity to accumulate, we aim to advance therapeutics to halt cancers before they appear. Our work on how mechanical forces impact SC behavior will be equally key in understanding how aggressive cancers arise.

Other NSERC · FY 2024

chromatin biology, chromatin associated RNA, linker histone, genome architecture, genomics, non-coding RNA, epigenomics