Whitehead Institute For Biomedical Research

MECHANISM OF BREAST DEVELOPMENT AND CARCINOGENESIS

CENTER FOR GENOME EDITING AND RECORDING

TRANSCRIPTIONAL REGULATORY NETWORKS IN LIVING CELLS

GENOMIC STUDIES OF SEX CHROMOSOMES

THE PURPOSE OF THIS COOPERATIVE AGREEMENT IS TO FUND RESEARCH IN THE AMOUNT OF 1,638,000 TO RECIPIENT TO CARRY OUT PUBLIC PURPOSE OF SUPPORT OR STIMU

IN VITRO REPROGRAMMING OF SOMATIC CELLS INTO PLURIPOTENT ES-LIKE CELLS

GENETICALLY ENGINEERED HUMAN PLURIPOTENT STEM CELLS AS A PLATFORM TO DEFINE THE B

EPI-GENETIC PROGRAMS IN CANCER PROGRESSION

MOLECULAR ANALYSIS OF KINETOCHORE FUNCTION

ADIPONECTIN AND CTRP9 SIGNALING IN MUSCLE AND LIVER

POST-TRANSCRIPTIONAL GENE REGULATION

GENETIC CONTROL OF NUTRITION STARVATION IN YEAST

CELL GROWTH SIGNALING IN CANCER DEVELOPMENT

MICRORNA GENES AND THEIR FUNCTIONS

REGULATION OF THE MTOR GROWTH PATHWAY BY NUTRIENTS

CATALYTIC SUBUNIT OF THE TELOMERASE GENE HEST2

MICORRNAS AND HEMATOPOIETIC DIFFERENTIATION

AN IPSC BASED PLATFORM FOR FUNCTIONALLY ASSESSING GENETIC AND ENVIRONMENTAL RISK

NOVEL COMPONENTS OF THE MTORC1 AND MTORC2 PATHWAYS

STEM CELL AND REGENERATION REGULATORY GENES IN PLANARIANS

PROGRAMMING AND REPROGRAMMING HUMAN CELLS

CHAPERONE PROTEIN AND PROTEIN CONFORMATIONAL SWITCHES

TRANSCRIPTIONAL REGULATION IN MAMMALIAN CELLS - PROJECT SUMMARY/ABSTRACT  TRANSCRIPTION IS A FUNDAMENTAL CELLULAR PROCESS WHOSE PROPER REGULATION IS ESSENTIAL TO ESTABLISHMENT AND MAINTENANCE OF HEALTHY CELL STATES. AS WITH MANY REGULATORY PROCESSES IN THE CELL, TRANSCRIPTION IS NOW UNDERSTOOD TO INVOLVE THE DYNAMIC FORMATION AND DISSOLUTION OF LARGE ASSEMBLIES OF PROTEIN AND RNA MOLECULES CALLED BIOMOLECULAR CONDENSATES. OUR RESEARCH PROGRAM IS FOCUSED ON THREE GOALS AT THE INTERSECTION OF TRANSCRIPTION AND CONDENSATES THAT WE BELIEVE WILL PROVIDE IMPORTANT NEW INSIGHTS INTO GENE REGULATION AND FILL IMPORTANT GAPS IN OUR UNDERSTANDING OF CONDENSATES AND THEIR REGULATION. GOAL 1) WE WILL TEST THE HYPOTHESIS THAT MANY LONG NONCODING RNAS (LNCRNAS) REGULATE TRANSCRIPTIONAL CONDENSATES AT NEARBY GENES. CONDENSATES ARE FORMED BY AN ENSEMBLE OF LOW-AFFINITY MOLECULAR INTERACTIONS AND RNA CAN BE A POWERFUL REGULATOR OF CONDENSATE DYNAMICS. THOUSANDS OF LNCRNA SPECIES ARE EXPRESSED IN ANY ONE CELL TYPE, BUT THE FUNCTIONS OF THE VAST MAJORITY OF THESE RNA MOLECULES ARE NOT KNOWN. MOST LNCRNAS ARE TRANSCRIBED WITHIN 10KB OF PROTEIN CODING GENES AND APPEAR TO ACCUMULATE AT THOSE LOCI, SUGGESTING THAT MANY OF THESE RNAS FUNCTION TO TUNE THE EXPRESSION OF LOCAL PROTEIN CODING GENES BY AFFECTING THE DYNAMICS OF LOCAL CONDENSATE FORMATION AND DISSOLUTION. GOAL 2) WE WILL TEST THE HYPOTHESIS THAT CONDENSATE IMMISCIBILITY CONTRIBUTES TO THE FUNCTIONAL SEPARATION OF ACTIVE AND SILENT CHROMATIN. THE NUCLEAR ARCHITECTURE OF A CELL INVOLVES TRANSCRIPTIONALLY ACTIVE AND INACTIVE COMPARTMENTS, AND CURRENT EVIDENCE INDICATES THAT THE TWO COMPARTMENTS FORM SEPARATE CONDENSATES. WE HAVE OBSERVED THAT CONDENSATES FORMED BY REGULATORS OF ACTIVE AND SILENT GENES ARE IMMISCIBLE AND POSTULATE THAT THIS PROPERTY CONTRIBUTES TO THE FUNCTIONAL SEPARATION OF ACTIVE AND INACTIVE COMPARTMENTS IN THE NUCLEUS OF MAMMALIAN CELLS. GOAL 3) WE WILL EXPLORE THE PHYSICOCHEMICAL ENVIRONMENTS OF NUCLEAR CONDENSATES WITH THE GOAL OF DETERMINING THE TYPES OF CHEMISTRIES THAT DISTINGUISH DIVERSE CONDENSATES. A MAJOR ISSUE IN CONDENSATE BIOLOGY IS THE EXTENT TO WHICH THE CHEMICAL ENVIRONMENTS OF DIVERSE CONDENSATES ENABLE BIOLOGICAL SPECIFICITY. OUR EVIDENCE INDICATES THAT SMALL MOLECULES CAN BE USED TO PROBE THE INTERNAL CHEMICAL ENVIRONMENT THAT GOVERNS THE BEHAVIOR OF CONDENSATES AND THUS TEACH US ABOUT THE INTERNAL CHEMISTRY OF DIVERSE CONDENSATES THAT MAY ENABLE BIOLOGICAL SPECIFICITY. THIS INFORMATION MAY ALSO PROVIDE INSIGHTS INTO THE CHEMICAL FEATURES THAT SELECTIVELY CONCENTRATE SMALL MOLECULES IN SPECIFIC CONDENSATES, WHICH MAY ENABLE FUTURE ADVANCES IN DRUG DESIGN FOR TARGETS THAT RESIDE IN SPECIFIC CONDENSATES. WHILE CONDUCTING THESE STUDIES, WE WILL CONTINUE TO IDENTIFY PROTEIN AND RNA COMPONENTS OF EUCHROMATIC AND HETEROCHROMATIN CONDENSATES AND TO INVEST IN ASSAYS OF CONDENSATE DYNAMICS AND TRANSCRIPTIONAL OUTPUT. WE WILL ALSO CONTINUE TO TRAIN AND MENTOR DIVERSE YOUNG SCIENTISTS IN AN ENVIRONMENT THAT FACILITATES COLLABORATION WITH LEADING EXPERTS IN BIOCHEMISTRY, CHEMISTRY AND PHYSICS.

ASSEMBLY OF MHC CLASS I MOLECULES IN VITRO AND IN VIVO

BIOLOGICAL FUSIONS CONJUGATIONS IN YEAST

TRANSCRIPTIONAL REGULATORY NETWORKS IN LIVING CELLS

MOLECULAR ANALYSIS OF THE KINETOCHORE-MICROTUBULE INTERFACE

DEVELOPMENT AND MAINTENANCE OF CHRONIC TOXOPLASMOSIS - 7. PROJECT SUMMARY/ABSTRACT CHRONIC TOXOPLASMA GONDII INFECTIONS ARE WIDESPREAD AND THEIR REACTIVATION CAN CAUSE LIFE-THREATENING DISEASE IN IMMUNOCOMPROMISED INDIVIDUALS AND RECURRENT OCULAR LESIONS IN THE IMMUNOCOMPETENT. THE RECENTLY IDENTIFIED MASTER REGULATOR OF CHRONIC DIFFERENTIATION, BFD1, PROVIDES AN UNPRECEDENTED OPPORTUNITY TO INVESTIGATE THE MOLECULAR EVENTS THAT ESTABLISH AND MAINTAIN CHRONIC T. GONDII INFECTIONS. BFD1 IS NECESSARY FOR CHRONIC DIFFERENTIATION IN CELL CULTURE AND IN MOUSE MODELS OF INFECTION, AND ITS EXPRESSION IS SUFFICIENT TO INDUCE CHRONIC DIFFERENTIATION. CONSISTENT WITH A SUSTAINED REQUIREMENT FOR BFD1 DURING CHRONIC STAGE MAINTENANCE, THE DIFFERENTIATION PROGRAM IS REVERSED UPON CONDITIONAL DOWN-REGULATION OF BFD1. PRELIMINARY RESULTS INDICATE THAT BFD1 IS POST-TRANSCRIPTIONALLY CONTROLLED THROUGH ITS 5' UTR, LEADING TO THE HYPOTHESIS THAT TRANSLATIONAL REGULATION OF BFD1 IS FUNDAMENTAL TO THE DEVELOPMENT AND MAINTENANCE OF CHRONIC T. GONDII STAGES. THIS PROPOSAL SEEKS TO INTEGRATE BFD1 INTO A BROADER REGULATORY NETWORK THROUGH THREE COMPLEMENTARY AIMS. AIM 1 WILL EXAMINE THE SEQUENCE ELEMENTS, SECONDARY RNA STRUCTURES, AND RIBOSOMAL OCCUPANCY DYNAMICS THAT MEDIATE BFD1 TRANSLATIONAL REGULATION. AIM 2 WILL USE CONDITIONAL DEPLETION OF BFD1 TO CHARACTERIZE TRANSCRIPTIONAL AND PROTEOMIC CHANGES THAT MEDIATE REACTIVATION, AND THE MOLECULAR CIRCUITS THAT MAINTAIN THE DIFFERENTIATED STATE. FINALLY, AIM 3 WILL EXTEND THE REGULATORY PATHWAYS THAT CONTROL DIFFERENTIATION BY SCREENING FOR GENES INVOLVED IN THE TRANSLATIONAL REGULATION OF BFD1 AND FURTHER EXAMINING THE FUNCTION OF TRANSCRIPTION FACTORS DIRECTLY REGULATED BY BFD1. THE OVERARCHING GOAL OF THIS COMPREHENSIVE ANALYSIS IS TO UNDERSTAND THE CONDITIONS THAT PROMOTE CHRONIC DIFFERENTIATION AND LICENSE THE DEVELOPMENT OF CURATIVE THERAPIES AGAINST TOXOPLASMOSIS.

DIFFERENTIAL DNA REPLICATION IN DROSOPHILA DEVELOPMENT

EPIGENOMIC MAPPING IN HUMAN TUMOR STEM CELLS

BIOLOGY AND APPLICATIONS OF MAMMALIAN HIBERNATION-LIKE STATES - PROGRAM DIRECTOR/PRINCIPAL INVESTIGATOR (LAST, FIRST, MIDDLE): HRVATIN, SINISA PROJECT SUMMARY: LIFE ON EARTH HAS EVOLVED FASCINATING ADAPTATIONS SUCH AS TORPOR AND HIBERNATION TO SURVIVE EXTREME ENVIRONMENTS. THESE EXTRAORDINARY ADAPTATIONS ARE CHARACTERIZED BY PROFOUNDLY DECREASED PHYSIOLOGICAL FUNCTIONS, INCLUDING METABOLIC RATE, BODY TEMPERATURE, CIRCULATION, BREATHING, AND HEART-RATE. HOW WARM-BLOODED ANIMALS ENTER, REGULATE, AND SURVIVE THESE STATES REMAINS ONE OF THE MOST FASCINATING MYSTERIES OF HOMEOTHERM BIOLOGY, THE UNDERSTANDING OF WHICH COULD HAVE PROFOUND IMPLICATIONS ON HUMAN MEDICINE. INVESTIGATING, FOR EXAMPLE, THE MECHANISMS THAT ADAPTIVELY MODULATE METABOLISM COULD PROVIDE NEW APPROACHES TO REGULATE HUMAN ENERGY BALANCE AND TREAT METABOLIC DISEASES. AN INDUCED HYPOTHERMIC AND HYPOMETABOLIC STATE COULD SLOW DOWN DISEASE PROGRESSION, FOR EXAMPLE CANCER GROWTH. THE PATHWAYS THAT ENABLE CELLS AND ORGANS IN TORPOR AND HIBERNATION TO SURVIVE HYPOXIC AND HYPOTHERMIC STRESS MIGHT ALSO BE HARNESSED TO FACILITATE CELL SURVIVAL DURING TRAUMA, STROKE, CARDIAC ARREST, CHEMOTHERAPY, OR EVEN NEURODEGENERATION. AMONG THE SPECIES NATURALLY CAPABLE OF ENTERING THESE STATES ARE LABORATORY MICE. MICE PLACED IN ENVIRONMENTS DEVOID OF FOOD INITIATE TORPOR - A BEHAVIOR CHARACTERIZED BY BOUTS OF GREATLY REDUCED CORE BODY TEMPERATURE, MOVEMENT, AND METABOLIC RATE, LASTING SEVERAL HOURS. RECENTLY, EMPLOYING NOVEL TRANSGENIC TOOLS AND SEQUENCING APPROACHES, WE EXAMINED THIS COMPLEX BEHAVIOR AND DISCOVERED THAT MOUSE TORPOR IS REGULATED BY A DISTINCT POPULATION OF NEURONS IN THE HYPOTHALAMUS. INHIBITING THESE NEURONS PREVENTS NATURAL TORPOR AND STIMULATING THEM RAPIDLY DECREASES METABOLIC RATE AND BODY TEMPERATURE, INDUCING A TORPOR-LIKE STATE. THIS DISCOVERY FORMS THE FOUNDATION FOR FUTURE EXPLORATIONS OF MECHANISMS REGULATING TORPOR AND HIBERNATION, ENABLING FOR THE FIRST TIME GENETIC ACCESS TO MONITOR, INITIATE, MANIPULATE, AND STUDY THESE BEHAVIORS. IN THIS PROPOSAL, WE INVESTIGATE KEY UNANSWERED QUESTIONS IN THE FIELD OF TORPOR AND HIBERNATION AND EXPLORE POTENTIAL APPLICATIONS FOR INDUCED HIBERNATION-LIKE STATES IN CANCER BIOLOGY. SPECIFICALLY, WE EXAMINE THE NEURONAL CIRCUIT THAT REGULATES THE DECREASE IN BODY TEMPERATURE AND METABOLIC RATE AND EXPLORE THE ROLE OF CIRCULATING FACTORS IN INDUCING THIS ORGANISM-WIDE STATE. WE PIONEER A NEW APPROACH TO INDUCE A LONG-TERM HIBERNATION-LIKE STATE IN MICE, EXPLORE MAMMALIAN PHYSIOLOGY IN THIS STATE, AND EXAMINE THE IMPACT OF THIS STATE ON CANCER GROWTH AND PROTECTION FROM CHEMOTHERAPY. FINALLY, WE INVESTIGATE THE EVOLUTIONARY CONSERVATION OF TORPOR-REGULATING NEURONS ACROSS SPECIES INCLUDING IN NON-HUMAN PRIMATES AND HUMAN TISSUES AND EXAMINE WHETHER A TORPOR-LIKE STATE CAN BE INDUCED IN A NON-HUMAN PRIMATE, PAVING THE WAY TO POTENTIAL HUMAN APPLICATIONS. THIS PROPOSAL IS BOLD AND AMBITIOUS; HOWEVER, EACH OF THE PROPOSED PROJECTS CONTAINS CLEARLY DEFINED AND FEASIBLE EXPERIMENTS WHOSE RESULTS HAVE THE POTENTIAL TO GREATLY ADVANCE, IF NOT TRANSFORM, OUR UNDERSTANDING OF TORPOR, HIBERNATION, AND HOMEOTHERM BIOLOGY. THE INNOVATIVE AND EARLY-STAGE NATURE OF THIS WORK, ALONG WITH ITS POTENTIAL TO ADVANCE MEDICAL TREATMENT, MAKES IT IDEALLY SUITED FOR THE NEW INNOVATOR AWARD.

SYSTEMATIC RECONSTRUCTION OF EPITHELIAL-MESENCHYMAL COMMUNICATION IN ORGAN DEVELOPMENT - PROJECT SUMMARY MESENCHYMAL CELLS ARE MAJOR COMPONENTS OF TISSUE STROMA AND BEST KNOWN FOR THEIR ROLE IN SUPPORTING THE PHYSICAL STRUCTURE OF ORGANS. THEY JUXTAPOSE TO AND COMMUNICATE WITH EPITHELIAL CELLS, TO CONTROL ORGAN DEVELOPMENT IN EMBRYOS, AND TO REGULATE TISSUE INFLAMMATION, INJURY REPAIR AND CANCER PROGRESSION IN ADULTS. TRADITIONALLY, MESENCHYMAL CELLS IN DIFFERENT ORGANS WERE THOUGHT TO PERFORM SIMILAR FUNCTIONS. HOWEVER, RECENT STUDIES HAVE REVEALED UNEXPECTED DIVERSITY OF GENE EXPRESSION SIGNATURES AMONG MESENCHYMAL CELLS FROM DIFFERENT ORGANS. SUCH DIVERSITY IS LIKELY TO PLAY A CRUCIAL ROLE IN REGULATING MESENCHYMAL-EPITHELIAL INTERACTIONS AND THUS ORGAN DEVELOPMENT AND PHYSIOLOGY. THE OVERARCHING GOAL OF THIS PROPOSAL IS TO UNDERSTAND HOW EPITHELIAL-MESENCHYMAL INTERACTIONS CONTROL ORGANOGENESIS ACROSS DIFFERENT ORGANS. TO ACHIEVE THIS GOAL, WE WILL FIRST ADDRESS THE QUESTION OF HOW MESENCHYMAL DIVERSITY ARISES AS THE CONSEQUENCE OF DISTINCT SIGNALING HISTORIES. WE WILL DEVELOP A NOVEL HIGH-THROUGHPUT SYSTEM TO SIMULTANEOUSLY TEST TENS OF THOUSANDS OF SIGNAL COMBINATIONS. WE WILL THEN INVESTIGATE HOW EPITHELIAL-MESENCHYMAL INTERACTIONS CONTROL TISSUE MORPHOGENESIS, BY RECONSTITUTING AND GENETICALLY PERTURBING SUCH INTERACTIONS EX VIVO. WE ARE ESPECIALLY INTERESTED IN COMPARING THE SPECIFICITY AND PLASTICITY OF EPITHELIAL-MESENCHYMAL INTERACTIONS ACROSS DIFFERENT ORGANS, AND IDENTIFYING THE ROLE OF EXTRACELLULAR MATRICES THAT ARE SHARED BY EPITHELIAL AND MESENCHYMAL CELLS IN CONTROLLING MORPHOGENESIS. WE WILL CLOSELY INTEGRATE EXPERIMENTAL MEASUREMENTS WITH COMPUTATIONAL MODELS TO PRODUCE A QUANTITATIVE FRAMEWORK FOR UNDERSTANDING ORGAN MORPHOGENESIS. BY INTEGRATING TOOLS AND MATERIALS FROM DIVERSE FIELDS, WE WILL GAIN A FUNDAMENTAL UNDERSTANDING ABOUT THE DEVELOPMENTAL ORIGIN AND FUNCTIONAL IMPLICATIONS OF THE LONG-OVERLOOKED MESENCHYMAL CELLS. THESE INSIGHTS AND TOOLS WILL ENABLE THE ENGINEERING OF ORGANOIDS TO BETTER MODEL DEVELOPMENT AND DISEASES, AND WILL ADVANCE THE FIELD OF TISSUE ENGINEERING.

DELINEATING GENETIC RISK TO ADDICTION VIA ANALYSIS OF 3D CHROMATIN ARCHITECTURE

MAKING STRUCTURALLY COMPLEX GENOMIC REGIONS ACCESSIBLE

EPIGENETICS, STEM CELLS, AND CANCER

GERM CELL MIGRATION IN DROSOPHILA.

CONTROL OF PARASITE INVASION BY A MICRONEME PROTEIN COMPLEX CONSERVED IN APICOMPLEXANS

DISSECTING ESSENTIAL SIGNALING PATHWAYS IN APICOMPLEXAN PARASITES

QUANTITATIVE APPROACHES TO REVEAL THE HOMEOSTATIC CONTROL MECHANISMS OF STRESS RE

RNA CONFORMATION AND CATALYSIS

CELL CYCLE CONTROL IN EARLY DROSOPHILA DEVELOPMENT

STEM CELL AND REGENERATION REGULATORY MECHANISMS IN PLANARIANS - PROJECT SUMMARY THE CAPACITY TO REGULATE STEM AND PROGENITOR CELLS FOR REGENERATION IS WIDESPREAD IN THE ANIMAL KINGDOM AND HAS ATTRACTED INVESTIGATION FOR CENTURIES. EVOLUTION HAS SELECTED FOR MECHANISMS INVOLVED IN WOUND REPAIR AND TISSUE REGROWTH THAT ARE THE DREAMS OF REGENERATIVE MEDICINE. UNCOVERING THE PRINCIPLES OF REGENERATION IN CASE STUDY ORGANISMS SHOULD IDENTIFY PROCESSES THAT NATURALLY PROMOTE OR LIMIT REGENERATION, ENABLING FUTURE DEVELOPMENT OF THERAPEUTIC APPROACHES TO TISSUE DAMAGE REPAIR. PLANARIANS ARE FLATWORMS CAPABLE OF REGENERATING ANY MISSING BODY PART, INCLUDING NEW HEADS. THEIR REGENERATIVE POWERS HAVE COMBINED WITH EASE OF EXPERIMENTATION TO MAKE THEM A CLASSIC REGENERATION MODEL. PLANARIAN REGENERATION INVOLVES ADULT STEM CELLS CALLED NEOBLASTS, WHICH WE PREVIOUSLY SHOWED CAN DISPLAY PLURIPOTENCY AT THE SINGLE CELL LEVEL (CNEOBLASTS). A MAJOR DIRECTION OF OUR RESEARCH ADDRESSES HOW FATE SPECIFICATION OCCURS IN REGENERATION. EXTENSIVE WORK INDICATES THAT FATE CHOICES ARE MADE WITHIN THE NEOBLASTS (CALLED SPECIALIZED NEOBLASTS), WITH THE REGENERATION OUTGROWTH AT THE WOUND (A BLASTEMA) BEING A COMPOSITE OF DIFFERENT FATE-SPECIFIED CELLS. THIS HIGHLIGHTS THE STEP OF FATE-CHOICE IN NEOBLAST STEM CELLS AS CENTRAL FOR UNDERSTANDING THE MECHANISTIC BASIS FOR PLANARIAN REGENERATION. WE SEEK TO UNDERSTAND "SPECIFICITY" IN REGENERATION, IN WHICH DIVERSE INJURIES APPEAR TO RESULT IN RESPONSES TAILORED TO THE IDENTITY OF MISSING TISSUE. WE AIM TO DISTINGUISH BETWEEN THE POSSIBLE EXISTENCE OF SURVEILLANCE SYSTEMS INDICATING THE PRESENCE OR ABSENCE OF DIFFERENTIATED CELL TYPES AND WHAT WE NAMED TARGET-BLIND REGENERATION. IN TARGET-BLIND REGENERATION, PROGENITOR PRODUCTION OCCURS AT A LOW BASAL RATE SUFFICIENT FOR REPAIR FROM SMALL WOUNDS, WITHOUT NEEDING TISSUE SURVEILLANCE; WE HYPOTHESIZE TISSUE-SPECIFIC PROGENITOR PRODUCTION IS PRIMARILY REGULATED BY WOUND-INDUCED PROLIFERATION COMBINED WITH POSITIONAL INFORMATION. WE AIM TO UNDERSTAND HOW POSITIONAL INFORMATION REGULATES STEM CELL FATE CHOICE DURING TISSUE TURNOVER, AND DYNAMICALLY DURING REGENERATION. OUR PRIOR WORK ON A PLANARIAN WHOLE-ANIMAL CELL-TYPE TRANSCRIPTOME ATLAS INDICATES THE EXISTENCE OF OVER 100 ADULT CELL TYPES. WE SEEK TO UNDERSTAND WHAT PROCESSES WITHIN NEOBLAST STEM CELLS REGULATE HOW THEY MAKE ANY ONE OF SO MANY POSSIBLE CHOICES. WE WILL STUDY THE PATTERN OF FATE CHOICES USING SPATIAL TRANSCRIPTOMICS AND SEEK TO DISTINGUISH BETWEEN A HIGHLY REGULATED FATE- SPECIFICATION PROCESS, SUCH AS BY EXTRINSIC LOCAL TISSUE CUES, AND A MORE STOCHASTIC PROCESS INTERNAL TO THE STEM CELLS. FINALLY, WE WILL INVESTIGATE HOW ADULT PROGENITORS GENERATED FROM STEM CELLS BRING ABOUT THE RESTORATION OF TISSUE ARCHITECTURE IN REGENERATION. OUR PRIOR WORK INDICATES THAT MIGRATORY TARGETING BY EXTRINSIC CUES COMBINES WITH SELF-ORGANIZATION OF PROGENITORS WITH THEIR TARGET TISSUE TO GENERATE AND MAINTAIN TISSUE PATTERN IN REGENERATION. WE WILL STUDY THE MOLECULAR BASES FOR THESE PROCESSES, WHICH WILL BE CRITICAL TO ELUCIDATE FOR UNDERSTANDING THE BASIS FOR TISSUE REPAIR AND REGENERATION.

FUNCTION AND MECHANISMS OF EPIGENETIC STABILITY AND DYNAMICS IN ARABIDOPSIS - PROJECT SUMMARY/ABSTRACT EPIGENETIC MODIFICATIONS REGULATE GENOME FUNCTION. MY RESEARCH IS FOCUSED ON DEFINING THE SCOPE, MECHANISMS, AND FUNCTIONAL CONSEQUENCES OF EPIGENOME DYNAMICS DURING DEVELOPMENT, USING THE POWERFUL GENETIC MODEL SYSTEM ARABIDOPSIS THALIANA. DNA METHYLATION (5- METHYLCYTOSINE) IS A HERITABLE EPIGENETIC MARK ASSOCIATED WITH HIGHLY INTERTWINED PROCESSES INCLUDING TRANSCRIPTIONAL GENE SILENCING, TRANSPOSABLE ELEMENT (TE) REPRESSION, AND REGULATION OF GENOMIC IMPRINTING. DNA METHYLATION IS CONCENTRATED IN TES TO PREVENT THEIR TRANSCRIPTION AND PROLIFERATION, BUT THIS GENOME DEFENSE SYSTEM COMES AT A POTENTIAL COST – THE METHYLATION OF TES OR THEIR REMNANTS CAN INAPPROPRIATELY SILENCE THE TRANSCRIPTION OF PROXIMAL PROTEIN-CODING GENES. ACTIVE DNA DEMETHYLATION BY 5-METHYLCYTOSINE DNA GLYCOSYLASES (ALSO REFERRED TO AS DNA DEMETHYLASES) FUNCTIONS TO COUNTERACT DNA METHYLTRANSFERASES. BUT HOW THESE TWO ANTAGONISTIC ACTIVITIES ARE BALANCED TO MAINTAIN THE GENOME IN A STABLE EPIGENETIC STATE – WHERE TRANSPOSABLE ELEMENTS ARE KEPT METHYLATED AND SILENCED AND GENES ARE KEPT RELATIVELY FREE OF METHYLATION AND CAN BE EXPRESSED – IS A MAJOR QUESTION ACROSS EUKARYOTES AND IS ONE FOCUS OF OUR CURRENT RESEARCH. OUR EFFORTS IN THIS AREA WILL SIGNIFICANTLY INCREASE UNDERSTANDING OF HOW THE ACTIVITY OF EPIGENETIC PATHWAYS IS MODULATED IN RESPONSE TO THE STATE OF THE EPIGENOME AND HOW THIS INFORMATION IS INTEGRATED OVER CELLULAR, DEVELOPMENTAL, AND GENERATIONAL TIME SCALES. ALTHOUGH EPIGENETIC HOMEOSTASIS APPEARS TO BE A MAJOR FORCE IN THE SOMA, DEVELOPMENTALLY- REGULATED EPIGENETIC REPROGRAMMING IS ESSENTIAL FOR SUCCESSFUL REPRODUCTION. DNA DEMETHYLATION IN THE FEMALE GAMETE ESTABLISHES IMPRINTED GENE EXPRESSION IN A PLACENTA-LIKE EXTRA-EMBRYONIC SEED TISSUE AFTER FERTILIZATION. A CRITICAL UNANSWERED QUESTION IS HOW EPIGENETIC CHANGES THAT ARE INITIATED BY DNA DEMETHYLATION ARE SUBSEQUENTLY MAINTAINED, OR NOT, WHEN MATERNAL AND PATERNAL GENOMES ARE COMBINED AFTER FERTILIZATION AND AS CELLS TAKE ON DISTINCT IDENTITIES. MY LABORATORY APPLIES DIVERSE GENETIC, GENOMIC, COMPUTATIONAL, AND COMPARATIVE EVOLUTIONARY APPROACHES TO ADDRESS THESE FUNDAMENTAL QUESTIONS IN EPIGENETICS.

EXPLOITING MITOCHONDRIAL HETEROPLASMY FOR CANCER CHEMOTHERAPY

REGULATION OF DNA METHYLATION HOMEOSTASIS IN ARABIDOPSIS

RAPAMYCIN-INSENSITIVE SIGNALING BY RICTOR-MTOR

CAREER: REGULATION AND FUNCTION OF IMPRINTED GENE EXPRESSION IN SEEDS

PROTECTING AND SUSTAINING GERM CELL IDENTITY - PROJECT SUMMARY/ABSTRACT GERM CELLS HAVE THE EXTRAORDINARY POTENTIAL TO GENERATE EVERY CELL TYPE IN THE BODY. YET, THE MOLECULAR PROGRAM THAT PROTECTS AND SUSTAINS THIS PROMISE FOR TOTIPOTENCY IN GERM CELLS, WHICH CAN LAST MORE THAN FORTY YEARS IN HUMANS, IS POORLY UNDERSTOOD. TO FACE THIS CHALLENGE, GERMLINE REGULATORS ASSUME DUAL ROLES IN MAINTAINING GERM CELL IDENTITY: ACTIVATING A GERMLINE TRANSCRIPTIONAL PROGRAM WHILE SIMULTANEOUSLY PROTECTING GERM CELLS FROM REPROGRAMMING TO A SOMATIC CELL FATE. INDEED, GENES INVOLVED IN GERMLINE SPECIFICATION, SUCH AS THE CONSERVED RNA-BINDING PROTEIN NANOS AND THE TRANSPOSABLE ELEMENT (TE) REGULATOR PIWI, ARE ERRONEOUSLY UPREGULATED IN CERTAIN HUMAN TUMORS. THESE TUMORS ARE THOUGHT TO UNDERGO SOMA-TO-GERMLINE TRANSFORMATIONS TO ACQUIRE A MORE IMMORTAL, GERMLINE-LIKE STATE. HOWEVER, A SPECIFIC PROGRAM FOR GERM CELL TRANSCRIPTIONAL ACTIVATION HAS YET TO BE DESCRIBED, MAINLY BECAUSE A ‘MASTER-REGULATOR TRANSCRIPTION FACTOR’ FOR GERM CELL FATE IS MISSING. THIS PROPOSAL USES A MULTIPRONGED APPROACH TO SYSTEMATICALLY CHARACTERIZE THE GENE EXPRESSION PROGRAM IN PRIMORDIAL GERM CELLS (PGCS) AND IDENTIFY PRIME REGULATORS OF GERM CELL FATE. THE GOAL IS TO DISTINGUISH BETWEEN A ‘DEFAULT’ MODEL, BY WHICH REPRESSION OF THE SOMATIC PROGRAM ‘ALLOWS’ THE PGC PROGRAM, AND AN ‘INSTRUCTIVE’ MODEL, BY WHICH PGC SPECIFICATION IS ACTIVELY CONTROLLED. IN AIM1, WE PROBE THE INSTRUCTIVE MODEL TO UNCOVER THE GERMLINE TRANSCRIPTIONAL PROGRAM. BY SINGLE-CELL RNA SEQUENCING, WE HAVE DETECTED SPECIFIC, TEMPORALLY-REGULATED GERMLINE GENES. TO IDENTIFY THE TRANSACTING REGULATORY FACTORS, WE WILL USE ATAC-SEQUENCING, CHARACTERIZE KNOWN FACTORS WITH MATCHING BINDING ACTIVITIES, AND IDENTIFY POTENTIAL NEW REGULATORY CANDIDATES FOR GERMLINE GENES. IN AIM2, WE ADDRESS THE FUNCTION OF A CRITICAL REGULATOR OF GERM CELL FATE, THE CONSERVED RNA REGULATOR NANOS. USING THE RNA TARGET IDENTIFICATION METHOD HYPER-TRIBE AND RIBOSOME PROFILING, WE WILL IDENTIFY NANOS TARGETS AND FUNCTIONALLY DISTINGUISH BETWEEN THOSE TARGETS THAT PROMOTE THE GERMLINE PROGRAM AND/OR REPRESS THE SOMATIC PROGRAM. AIM3 FOCUSES ON THE PIRNA PATHWAY, WHICH PROVIDES MATERNALLY INHERITED IMMUNITY AGAINST TE ACTIVITY BY CONTROLLING THE TRANSCRIPTION OF TE ELEMENTS, SPECIFICALLY IN GERM CELLS. USING A NOVEL DEGRADATION STRATEGY TO SPECIFICALLY DEGRADE COMPONENTS OF THE PIRNA PATHWAY IN PGCS WITHOUT IMPACTING OOGENESIS, WE WILL DETERMINE THE ROLE OF THIS PATHWAY FOR EMBRYONIC PGC GENE EXPRESSION BEYOND TE CONTROL. BY INTEGRATING MULTIPLEX FINDINGS, THIS PROPOSAL WILL UNCOVER THE REGULATORY LANDSCAPE OF EARLY GERM CELLS, THEREBY PROVIDING A NECESSARY UNDERSTANDING OF THE PROCESS OF GONADOGENESIS AND, ULTIMATELY, THE SURVIVAL OF THE SPECIES.

INHIBITING THE HEAT SHOCK FACTOR 1-REGULATED TRANSCRIPTIONAL PROGRAM IN CANCER

CHEMICAL APPROACHES TOWARDS THE STUDY DEUBIQUITINATING ENZYMES

THE FUNCTION AND PROPERTIES OF RNA POLYMERASE IV IN SEED DEVELOPMENT

ENDOSOMAL TLRS AND THEIR ACCESSORY PROTEINS: CELL BIOLOGY AND BIOCHEMISTRY

ELUCIDATING THE MOLECULAR AND CELLULAR FUNCTIONS OF POLYAMINES - PROJECT SUMMARY POLYAMINES ARE SMALL ORGANIC COMPOUNDS WITH TWO OR MORE AMINE GROUPS. THESE EVOLUTIONARILY ANCIENT METABOLITES ARE ESSENTIAL FOR A VARIETY OF CELLULAR PROCESSES, INCLUDING CELL GROWTH AND SURVIVAL, AND ARE PRESENT AT MILLI-MOLAR CONCENTRATIONS IN MAMMALIAN CELLS. CELLULAR POLYAMINE CONCENTRATIONS ARE TIGHTLY REGULATED, AND CHANGES IN POLYAMINE LEVELS DISRUPT NUMEROUS CELLULAR PROCESSES, INCLUDING DNA COMPACTION, TRANSCRIPTION, TRANSLATION, AUTOPHAGY, AND STRESS RESPONSE. DISRUPTION OF POLYAMINE METABOLISM IS ALSO OBSERVED IN SEVERAL DISEASES. GENETIC MUTATIONS IN POLYAMINE METABOLIC PATHWAYS ARE LINKED WITH LEARNING DISABILITY IN SNYDER- ROBINSON SYNDROME AND ARE A KNOWN RISK FACTOR FOR PARKINSON'S DISEASE. POLYAMINE LEVELS ARE ELEVATED IN MANY CANCERS, AND THERE IS ENORMOUS INTEREST IN USING THIS PATHWAY AS A CHEMOTHERAPEUTIC TARGET. MECHANISTICALLY, HOW DISRUPTION OF POLYAMINE METABOLISM AFFECTS CELL FUNCTION AND PRODUCES DISEASE IS NOT KNOWN. THE PRECISE BIOCHEMICAL FUNCTIONS OF POLYAMINES IN THE CELL REMAIN MYSTERIOUS. THIS GAP IN OUR KNOWLEDGE STEMS FROM INTRINSIC TECHNOLOGICAL DIFFICULTIES IN PROBING POLYAMINES. THIS PROJECT WILL DELIVER NEW METHODS AND CONCEPTUAL FRAMEWORKS TO ELUCIDATE THE MOLECULAR FUNCTIONS OF POLYAMINES. ONE, WE WILL DEVELOP NEW QUANTITATIVE ASSAYS TO MEASURE POLYAMINE CONCENTRATIONS IN LIVING CELLS. TWO, WE WILL INVESTIGATE HOW MAMMALIAN CELLS MAINTAIN POLYAMINE HOMEOSTASIS AND IDENTIFY THE PROTEINS INVOLVED IN THEIR UPTAKE. THIRD, WE WILL EXAMINE HOW CHANGES IN CELLULAR POLYAMINE LEVELS AFFECT RNA LOCALIZATION, STRUCTURE, AND TRANSLATION. BY DELIVERING NEW TECHNOLOGIES AND FUNCTIONAL FRAMEWORKS, THIS WORK WILL ADVANCE OUR UNDERSTANDING OF THE ROLES OF POLYAMINES IN THE CELL, AND MAY HELP UNLOCK THE PHARMACOLOGICAL POTENTIAL OF THESE METABOLITES IN HEALTH AND CURING DISEASE.

BEYOND THE REPRODUCTIVE TRACT: THE FUTURE OF Y CHROMOSOME RESEARCH

PRODUCING, PROVISIONING, AND PROTECTING THE EGG: REGULATION OF DNA REPLICATION, MRNA TRANSLATION, AND PROTEOLYSIS FOR THE TRANSITION FROM OOCYTE TO E

REGULATION OF GLIAL CELL SIZE

GENOMIC IMPRINTING AND THE CLONING OF MICE

HARNESSING EVOLUTION TO REVEAL THE MOLECULAR LOGIC OF KINETOCHORE WIRING

GROWTH FACTORS AND ENGINEERED STROMA FOR HEMATOPOIETIC STEM CSLL (HSC) EXPANSION

MOLECULAR CONTROL OF CENTROMERE SPECIFICATION AND KINETOCHORE ASSEMBLY

HEAT SHOCK FACTOR (HSF1) AS A MODIFIER OF NF1-ASSOCIATED TUMORIGENESIS AND A POTENTIAL THERAPEUTIC TARGET

PATIENT-SPECIFIC IPS CELLS TO STUDY MYELOPROLIFERATIVE DISEASE.

LEVERAGING CELL-DERIVED BIOPARTICLES FOR MACROMOLECULAR DELIVERY - 7. PROJECT SUMMARY/ABSTRACT MANY EMERGING THERAPEUTIC STRATEGIES EMPLOY MACROMOLECULES, LIKE PROTEINS OR RNAS, TO MANIPULATE INTRACELLULAR CONTENTS AND PROCESSES, THUS MITIGATING DISEASE. THE SUCCESS OF THESE THERAPEUTIC STRATEGIES REQUIRES THE ABILITY TO SAFELY AND EFFICIENTLY DELIVER MACROMOLECULES INTO CELLS WITHIN THE BODY, WHICH POSES A SIGNIFICANT CHALLENGE. TO OVERCOME THE BIOLOGICAL BARRIERS TO MACROMOLECULAR DELIVERY, RESEARCHERS HAVE DEVELOPED VARIOUS DELIVERY VEHICLES, SUCH AS VIRAL VECTORS AND NANOPARTICLES, THAT CAN PACKAGE AND DELIVER MACROMOLECULAR CARGOS INTO DESIRED TARGET CELLS. RECENTLY, CELL-DERIVED BIOPARTICLES, INCLUDING VIRUS-LIKE PARTICLES AND EXTRACELLULAR VESICLES, HAVE EMERGED AS PROMISING DELIVERY VEHICLES THAT COMBINE KEY BENEFITS OF BOTH VIRAL- AND NANOPARTICLE-BASED DELIVERY METHODS. HOWEVER, A MAJOR CHALLENGE ASSOCIATED WITH THESE NEW METHODS IS THEIR POOR MANUFACTURABILITY, WHICH SEVERELY LIMITS THEIR PROSPECTS FOR CLINICAL TRANSLATION. THE PROPOSED WORK SEEKS TO UNLOCK THE FULL POTENTIAL OF CELL-DERIVED BIOPARTICLES FOR THERAPEUTIC MACROMOLECULE DELIVERY BY DEVELOPING NEW STRATEGIES FOR HIGHLY EFFICIENT BIOPARTICLE PRODUCTION. INITIAL EFFORTS WILL FOCUS ON IDENTIFYING, UNDERSTANDING, AND MANIPULATING GENES IN BIOPARTICLE PRODUCER CELLS THAT INFLUENCE BIOPARTICLE PRODUCTION EFFICIENCY. TO DETERMINE GENES THAT REGULATE BIOPARTICLE PRODUCTION, WE WILL ASSESS THE EFFECTS OF THOUSANDS OF DIFFERENT GENETIC PERTURBATIONS ON BIOPARTICLE PRODUCTION BY PERFORMING POOLED GENOME- WIDE KNOCKDOWN AND ACTIVATION SCREENS IN PRODUCER CELLS. THESE SCREENS WILL UTILIZE A UNIQUE DESIGN IN WHICH SEQUENCING A GIVEN BIOPARTICLE’S CONTENTS WILL IDENTIFY THE PERTURBATION IN THE CELL THAT PRODUCED THAT BIOPARTICLE. WE WILL PERFORM SEPARATE SCREENS TO INTERROGATE (1) THE PRODUCTION OF VIRUS-LIKE PARTICLES THAT PACKAGE RIBONUCLEOPROTEIN CARGOS AND (2) THE PRODUCTION OF EXTRACELLULAR VESICLES THAT PACKAGE MESSENGER RNA CARGOS, YIELDING INSIGHTS INTO TWO DISTINCT TYPES OF CELL-DERIVED BIOPARTICLES AND TWO IMPORTANT CLASSES OF THERAPEUTICALLY RELEVANT MACROMOLECULAR CARGOS. THESE RESULTS WILL NOT ONLY ENHANCE OUR UNDERSTANDING OF THE MECHANISMS OF CELL-DERIVED BIOPARTICLE FORMATION BUT ALSO REVEAL NEW WAYS TO MANIPULATE PRODUCER CELLS TO IMPROVE PARTICLE PRODUCTION. IN PARALLEL, WE WILL ESTABLISH A NEW PARADIGM FOR DIRECTLY TRANSFERRING THERAPEUTIC BIOPARTICLES FROM PRODUCER CELLS INTO TARGET CELLS VIA CELL-TO-CELL CONTACT, A STRATEGY THAT HAS THE POTENTIAL TO SUBSTANTIALLY IMPROVE DELIVERY POTENCY RELATIVE TO EXISTING METHODS. THIS INVESTIGATION WILL LAY A FOUNDATION FOR POTENTIALLY TRANSFORMATIVE THERAPEUTIC APPROACHES IN WHICH PATIENT-DERIVED CELLS ARE ENGINEERED INTO BIOPARTICLE PRODUCER CELLS AND TRANSPLANTED INTO THE BODY, WHERE THEY WOULD SUBSEQUENTLY ENGAGE DESIRED TARGET CELLS TO DELIVER THERAPEUTICS VIA LOCALLY RELEASED BIOPARTICLES. COLLECTIVELY, THESE STUDIES WILL DRAMATICALLY EXPAND THE UTILITY OF CELL-DERIVED BIOPARTICLES AS DELIVERY VEHICLES FOR MACROMOLECULAR THERAPEUTICS.

USING NATURAL VARIATION TO PROBE THE EVOLUTION, MECHANISMS AND FUNCTION OF IMPRINTING IN PLANTS

HSF1 ENABLES THE EVOLUTION OF AGGRESSIVE BREAST CANCERS

CONTROL OF HOST INVASION BY PROTEIN COMPLEXES CONSERVED ACROSS APICOMPLEXAN PARASITES - 7. PROJECT SUMMARY/ABSTRACT HOW APICOMPLEXAN PARASITES RECOGNIZE HOST CELLS TO INVADE THEM REMAINS A MYSTERY. SINCE THESE PARASITES ONLY REPLICATE INTRACELLULARLY, INVADING HOST CELLS IS ESSENTIAL TO THEIR SURVIVAL. INVASION RELIES ON THE COORDINATED SECRETION OF PROTEINS FROM TWO DISTINCT TYPES OF ORGANELLES: MICRONEMES AND RHOPTRIES. RHOPTRIES ARE EXCLUSIVELY DISCHARGED UPON HOST-CELL CONTACT; HOWEVER, THE MOLECULAR EVENTS THAT TRIGGER THIS PROCESS REMAIN COMPLETELY UNKNOWN. UNDERSTANDING THE FACTORS REQUIRED FOR RHOPTRY DISCHARGE CAN HELP US UNCOVER THE BASIS OF HOST-CELL RECOGNITION. TWO CONSERVED MICRONEME COMPLEXES—THE CLAMP AND CRMP COMPLEXES—HAVE RECENTLY BEEN SHOWN BY THE LOURIDO AND LEBRUN LABS TO BE REQUIRED FOR RHOPTRY DISCHARGE, MAKING THEM LIKELY MEDIATORS OF HOST-CELL RECOGNITION. OUR PRELIMINARY STUDIES HAVE IDENTIFIED N-GLYCOSYLATION PATHWAYS AS KEY HOST REQUIREMENTS FOR RHOPTRY DISCHARGE. LOSS OF HOST N-GLYCOSYLATION SIGNIFICANTLY REDUCED RHOPTRY DISCHARGE, WHILE BUFFERING ANY ADDITIONAL EFFECT FROM KNOCKING DOWN THE CLAMP OR CRMP COMPLEXES. BASED ON THESE OBSERVATIONS, WE HYPOTHESIZE THAT THE CLAMP AND CRMP COMPLEXES RECOGNIZE HOST CELLS THROUGH N-GLYCANS TO MEDIATE RHOPTRY DISCHARGE. THIS PROPOSAL BRINGS TOGETHER THE TWO TEAMS THAT DISCOVERED THESE CRITICAL COMPLEXES TO EXAMINE HOW THEY RECOGNIZE HOST N-GLYCOSYLATION AND MEDIATE RHOPTRY DISCHARGE. AIM 1 WILL DETERMINE THE GLYCAN SPECIFICITY OF THE CLAMP AND CRMP COMPLEXES THROUGH TRADITIONAL OR LIQUID GLYCAN ARRAYS. AIM 2 WILL CHARACTERIZE HOW N-GLYCOSYLATION ALTERS THE VARIOUS STAGES OF INVASION USING HIGH-SPEED LIVE-CELL IMAGING TO DISSECT THE MULTI-STAGE INVASION PROCESS AND PINPOINT DEFECTS TO RHOPTRY DISCHARGE. AIM 3 WILL CHARACTERIZE THE SIGNALING EVENTS TRIGGERED BY HOST-CELL RECOGNITION THROUGH PHOSPHOPROTEOMICS AND PROXIMITY-LABELING. TAKEN TOGETHER THE PROPOSED EXPERIMENTS WILL UNCOVER HOW APICOMPLEXANS RESPOND TO HOST CONTACT TO DISCHARGE RHOPTRIES AND INITIATE INVASION. BEYOND ITS FUNDAMENTAL IMPORTANCE, THIS INFORMATION CAN BE USED TO DESIGN INHIBITORS OF HOST CELL ENTRY THAT WOULD BLOCK PARASITE INFECTION.

RNA GELATION IN REPEAT EXPANSION DISORDERS

CREATION OF ADULT EPITHELIAL STEM CELLS FROM DIFFERENTIATED EPITHELIAL

SPATIO-TEMPORAL SIGNALING DYNAMICS IN NEURONAL CELL FATE DECISION AND PATTERNING

IN VITRO REPROGRAMMING OF SOMATIC CELLS INTO PLURIPOTENT ES-LIKE CELLS

IDENTIFYING CYTOKINES THAT ENHANCE MACROPHAGE IMMUNOTHERAPY OF COLORECTAL CANCER

INVOLVEMENT OF THE EMT IN INVASION, METASTASIS, AND THE FORMATION OF CANCER STEM CELLS

ELUCIDATION AND ENGINEERING OF COMPLETE FIREFLY LUCIFERIN BIOSYNTHESIS

ZEISS LSM 980 WITH AIRYSCAN 2 CONFOCAL MICROSCOPE SYSTEM - PROJECT SUMMARY/ABSTRACT THE WHITEHEAD INSTITUTE REQUESTS $600,000 FOR THE PURCHASE OF A CRITICALLY-NEEDED ZEISS LSM 980 WITH AIRYSCAN 2 LASER SCANNING CONFOCAL MICROSCOPE (“ZEISS 980AS2”), TO BE LOCATED IN THE W.M. KECK FACILITY FOR BIOLOGICAL IMAGING (“KECK FACILITY”) AT THE WHITEHEAD INSTITUTE FOR BIOMEDICAL RESEARCH (“WHITEHEAD”). THE INSTRUMENT REQUESTED IS OF GREAT IMPORTANCE, AS IT WILL REPLACE A NINE YEAR OLD ZEISS LSM 710 NLO (NON-LINEAR OPTICS; “ZEISS710”) THAT IS CURRENTLY THE MOST HEAVILY USED MICROSCOPE IN THE KECK FACILITY AND PROVIDE TRANSFORMATIVE NEW IMAGING TECHNOLOGIES THAT ARE CURRENTLY UNAVAILABLE AT WHITEHEAD OR IN THE KECK FACILITY. FOR EACH OF THE PAST 3 YEARS, THE ZEISS710 AVERAGED 1400 HOURS OF USE BY 40 RESEARCHERS FROM 25 DIFFERENT LABS. ZEISS HAS INFORMED US THAT GUARANTEED SERVICE FOR THE ZEISS710 WILL END IN 2022. REPLACEMENT OF THIS CRITICAL INSTRUMENTATION PRIOR TO THAT TIME IS IMPORTANT TO MINIMIZE INTERRUPTIONS CAUSED BY EXCESSIVE MAINTENANCE ISSUES. OTHER MICROSCOPY FACILITIES IN OUR AREA ARE UNABLE TO ACCOMMODATE THE SUBSTANTIAL AMOUNT OF USE THE ZEISS710 CURRENTLY SUPPORTS, AND ALSO LACK THE CRITICAL NEW CAPABILITIES REQUIRED FOR THE PROPOSED RESEARCH PROJECTS. THE ZEISS 980AS2 WILL PROVIDE ROBUST CONFOCAL CAPABILITIES FOR THE DIVERSE RESEARCH PROJECTS DESCRIBED IN THIS PROPOSAL, SUCH AS ENABLING OPTICAL SECTIONING TO GENERATE HIGH-CONTRAST IMAGES IN THICK SPECIMENS, GENERATING 3D DATASETS, AND CONDUCTING PHOTOMANIPULATION TO ANALYZE MOLECULAR DYNAMICS, SUCH AS FRAP (FLUORESCENCE RECOVERY AFTER PHOTOBLEACHING). CRITICALLY, THE REQUESTED ZEISS 980AS2 WILL PROVIDE NEW CUTTING-EDGE TECHNOLOGIES ESSENTIAL FOR ADDRESSING MANY OUTSTANDING QUESTIONS FROM THE 10 NIH-FUNDED RESEARCHERS PRESENTED IN THIS PROPOSAL. THE AIRYSCAN 2 DETECTOR AND SOFTWARE WILL BRING THE FIRST SUPER-RESOLUTION CAPABILITIES TO THE KECK FACILITY. THE 32-CHANNEL SPECTRAL DETECTOR WILL ALLOW SIMULTANEOUS IMAGING OF UP TO TEN FLUOROPHORES, ENABLING STUDIES THAT INVESTIGATE CELL TYPE DIFFERENTIATION IN ORGANOIDS AND TISSUES. THE HIGH-EFFICIENCY GAASP DETECTORS WILL ENABLE IMAGING OF DIM SAMPLES AND REDUCE PHOTOBLEACHING AND PHOTOTOXICITY DURING LIVE-CELL IMAGING. THE AIRYSCAN 2 8Y MULTIPLEX OPTION AND Z-PIEZO STAGE ALLOW FOR EXTREMELY FAST IMAGE COLLECTION, ENABLING LARGER SAMPLE SIZES FOR LARGE VOLUME SCANNING. THE ZEISS ZEN BLUE SYSTEM SOFTWARE THAT CONTROLS THE ZEISS 980AS2 WILL SUBSTANTIALLY SIMPLIFY CURRENTLY ARDUOUS TASKS, SUCH AS MULTI-POSITION TILE SCANNING AND FINDING RARE EVENTS. BY EXPANDING THE EXPERIMENTAL APPROACHES AND CAPABILITIES AVAILABLE TO RESEARCHERS, THE ZEISS 980AS2 WILL GREATLY ENHANCE AND FACILITATE THE SUCCESS OF MANY NIH-FUNDED PROJECTS, INCLUDING THE ANALYSIS OF NEURAL DEGENERATION, CANCER, GERM CELL DIFFERENTIATION, METABOLIC REGULATION, CELL DIVISION, AND STEM CELL IDENTIFICATION. ACQUISITION OF THE ZEISS LSM 980 WITH AIRYSCAN 2 WILL ALLOW RESEARCHERS TO ADDRESS THESE IMPORTANT BIOMEDICAL ISSUES, AND THE OUTCOMES FROM THIS RESEARCH WILL INCLUDE NEW DIAGNOSTICS AND THERAPEUTICS, IN ACCORDANCE WITH THE MISSION OF THE NIH.

BIOPHYSICS OF THE BACTERIAL NUCLEOID: STRUCTURE, REPLICATION, AND SEGREGATION OF THE E. COLI CHROMOSOME

ROLE OF LYSOSOMAL TRANSPORTERS IN PROMOTING THE GROWTH OF CLEAR CELL RENAL CELL CARCINOMA AND OTHER TUMOR TYPES

GENERATION OF BREAST CANCER STEM CELLS BY THE EMT

HUNTING RARE HUMAN VARIATIONS USING DNA SUDOKU

DESIGNING NOVEL STRATEGIES TO TARGET TUMOR-PROMOTING FUNCTIONS OF PANCREATIC CANCER FIBROBLAST SUBTYPES

BASAL CONSTRICTION, AND THE ROLE OF NON-CANONICAL WNT SIGNALING

HIV-1 RNA STRUCTURE EFFECTS ON LATENCY REVERSAL

INVESTIGATING ABETA AND ALPHA-SYNUCLEIN TOXICITY BY ANALYZING SINGLE-CELL DYNAMIC

A NEW SYSTEM FOR MAMMALIAN CELL GENETICS USING A HAPLOID GENOMIC CONTEXT

TRANSDUCTION OF CELLS WITH TRANSCRIPTION FACTORS TO ACHIEVE NUCLEAR REPROGRAMMING

IDENTIFICATION OF NOVEL TOXOPLASMA GENES INVOLVED IN HOST-PARASITE INTERACTIONS

DEFINING MOLECULAR SIGNATURES UNDERLYING LYSOSOMAL DYSFUNCTION IN ALZHEIMER?S DISEASE - LYSOSOMES ARE MEMBRANE-BOUND DEGRADATIVE COMPARTMENTS THAT BREAK DOWN MACROMOLECULES FROM ENDOCYTIC, PHAGOCYTIC AND AUTOPHAGIC PATHWAYS, AND SERVE THE ROLE OF KEY METABOLIC AND SIGNALING HUBS. ACCUMULATING EVIDENCE SUGGESTS THAT IN ALZHEIMER'S DISEASE AND OTHER NEURODEGENERATIVE DISORDERS LYSOSOMES FAIL TO CORRECTLY PERFORM THEIR FUNCTIONS. HOWEVER, DUE TO THE PAUCITY OF TOOLS TO STUDY ORGANELLES IN VIVO, SO FAR THERE HAS BEEN NO SYSTEMATIC ASSESSMENT OF LYSOSOMAL ALTERATIONS DURING PROGRESSION OF ALZHEIMER'S DISEASE, AND THE EXACT MOLECULAR NATURE OF THE PROPOSED IMPAIRMENTS IS NOT KNOWN. OUR UNDERSTANDING OF THE INVOLVEMENT OF THE LYSOSOME IN THE DISEASE IS FURTHER LIMITED BECAUSE LYSOSOMES ARE RARE, CONSTITUTING <3% OF THE CELL. HERE, WE SEEK TO COMBINE POWERFUL, STATE-OF-THE-ART APPROACHES INCLUDING RECENTLY DEVELOPED RAPID LYSOSOMAL ISOLATIONS (LYSOIPS) AND UNBIASED PROTEOMIC AND METABOLOMIC ANALYSES TO DETERMINE IF AND HOW LYSOSOMES CHANGE IN VIVO IN MURINE MODELS OF ALZHEIMER'S DISEASE. WE PROPOSE TO FOCUS ON LYSOSOMES ISOLATED FROM NEURONS AND MICROGLIA WHICH WE EXPECT TO BE CRITICAL TO THE PATHOLOGY OF THE DISEASE. COMPLEX RECIPROCAL INTERACTIONS BETWEEN NEURONS AND MICROGLIA ARE ESSENTIAL FOR REGULATION OF THE MOST IMPORTANT ASPECTS OF BRAIN FUNCTION, AND WE HYPOTHESIZE THAT ALTERATIONS OF THE ENDOLYSOSOMAL SYSTEMS IN THESE TWO CELL TYPES COMPROMISE THE INTEGRITY OF THE CENTRAL NERVOUS SYSTEM. HERE, IN AIM I WE PROPOSE TO DEFINE LYSOSOMAL ALTERATIONS IN NEURONS AND MICROGLIA OVER A TIME COURSE OF ALZHEIMER'S DISEASE PROGRESSION GENERATING A DYNAMIC ATLAS OF LYSOSOMAL PROTEINS AND METABOLITES IN THESE CELLS. THIS AIM WILL GENERATE NOVEL MOUSE MODELS AND ROBUST PROTOCOLS ENABLING RAPID LYSOSOMAL ISOLATIONS FROM NEURONS AND MICROGLIA. IN AIM 2 WE WILL VALIDATE BIOINFORMATICALLY FILTERED CANDIDATES FROM AIM 1, PAVING THE WAY FOR FUTURE MECHANISTIC DISSECTIONS. THE PROPOSED RESEARCH UTILIZES INNOVATIVE TECHNOLOGIES AND CONCEPTS TO ADDRESS FUNDAMENTAL MOLECULAR ASPECTS OF PATHOBIOLOGY OF ALZHEIMER'S DISEASE, BUILDING A COMPREHENSIVE ATLAS OF IN VIVO LYSOSOMAL CHANGES IN NEURONS AND MICROGLIA. WE BELIEVE THAT THIS WORK WILL SHED LIGHT ON NOVEL ASPECTS OF LYSOSOMAL BIOLOGY IN THE BRAIN AND HAS THE POTENTIAL TO TRANSFORM OUR UNDERSTANDING OF THE MECHANISTIC BASIS OF ALZHEIMER'S DISEASE, INFORMING FUTURE DEVELOPMENTS IN THE TREATMENT OF THIS DEVASTATING DISORDER.

NON-INVASIVE IMAGING OF THE IMMUNE RESPONSE BASED ON THE USE OF ISOTOPICALLY LABELED SINGLE DOMAIN ANTIBODY FRAGMENTS

MOLECULAR DISSECTION OF SYNUCLEINOPATHIES USING YEAST, RODENT AND HUMAN IPS CELLS

ELUCIDATING A MECHANISM OF MTORC1 ACTIVATION INDEPENDENT OF AMINO ACIDS SIGNALING

ENZYMATIC MODIFICATION OF ANTI-DEC205 TO MANIPULATE ITS IMMUNOGENIC PROPERTIES

REGULATION OF CHROMOSOME DYNAMICS AND SEGREGATION IN DROSOPHILA

ILLUMINA GENOME ANALYZER IIX

ZEISS LSM710 SCANNING CONFOCAL MICROSCOPE

DECODING FUNCTIONAL CELL-CELL COMMUNICATION TO UNCOVER CELLULAR STATE MODULATION, TISSUE HOMEOSTASIS, AND DISEASE GENETICS - 7. PROJECT SUMMARY/ABSTRACT ALTHOUGH RECENT ADVANCEMENTS IN SINGLE CELL AND SPATIAL TRANSCRIPTOMICS HAVE PROVIDED UNPRECEDENTED RESOLUTION TO STUDY INTERCELLULAR COMMUNICATION, THERE ARE CURRENTLY STILL NO COMPREHENSIVE METHODS TO FULLY DECIPHER THE COMPLEXITY OF TRULY FUNCTIONAL CELL-CELL COMMUNICATION AND TO INVESTIGATE ITS IMPLICATIONS FOR HUMAN HEALTH AND DISEASE. THE LONG-TERM GOAL IS TO REVEAL THE COMPLEX NETWORKS OF CELL-CELL COMMUNICATION TO BETTER UNDERSTAND THEIR ROLES IN MAINTAINING TISSUE HOMEOSTASIS, CONTRIBUTING TO DISEASE MECHANISMS, AND ULTIMATELY GUIDING THE DEVELOPMENT OF NOVEL THERAPEUTIC STRATEGIES. THE OVERALL OBJECTIVES IN THIS APPLICATION ARE TO 1) DEVELOP COMPUTATIONAL METHODS TO SYSTEMATICALLY DECIPHER FUNCTIONAL CELL-CELL COMMUNICATION, AND 2) INVESTIGATE THE REGULATORY ROLES OF CELL-CELL COMMUNICATION IN CELLULAR STATES, TISSUE ORGANIZATION, AND DISEASE GENETICS. THE RATIONALE FOR THIS PROJECT IS THAT WHILE CELL-CELL COMMUNICATION IS RECOGNIZED AS A FUNDAMENTAL ASPECT OF TISSUE BIOLOGY, ITS FULL COMPLEXITY, ESPECIALLY IN THE CONTEXT OF DISEASE, REMAINS INCOMPLETELY UNDERSTOOD. BY ADVANCING COMPUTATIONAL METHODOLOGIES AND INTEGRATING MULTI-DIMENSIONAL DATA, WE CAN PROVIDE DEEPER INSIGHTS INTO THE MECHANISMS GOVERNING INTERCELLULAR COMMUNICATION, WHICH ARE CRUCIAL FOR BOTH NORMAL TISSUE FUNCTION AND DISEASE PROGRESSION. THE CENTRAL CHALLENGES WILL BE ADDRESSED BY PURSUING THREE SPECIFIC AIMS: 1) DEVELOP INNOVATIVE COMPUTATIONAL APPROACHES TO ACCURATELY DEFINE AND CATEGORIZE ACTIVE CELL- CELL COMMUNICATION NETWORKS WITHIN TISSUES; 2) SYSTEMATICALLY EXPLORE THE IMPACT OF THESE COMMUNICATION NETWORKS ON CELLULAR STATES AND TISSUE ORGANIZATION; AND 3) ELUCIDATE THE FUNCTIONAL CONTRIBUTIONS OF CELL-CELL COMMUNICATION IN THE CONTEXT OF DISEASE GENETICS. THE RESEARCH PROPOSED IN THIS APPLICATION IS INNOVATIVE, IN THE APPLICANT’S OPINION, BECAUSE IT LEVERAGES CUTTING-EDGE COMPUTATIONAL AND EXPERIMENTAL TECHNIQUES TO INTEGRATE SPATIAL AND SINGLE-CELL DATA, PROVIDING A MULTI-LAYERED UNDERSTANDING OF CELL-CELL COMMUNICATION. THIS APPROACH ALLOWS FOR THE CREATION OF DETAILED COMMUNICATION MAPS AND THE IDENTIFICATION OF KEY REGULATORS THAT COULD SERVE AS TARGETS FOR NOVEL THERAPEUTIC INTERVENTIONS. THIS RESEARCH IS SIGNIFICANT BECAUSE IT ADDRESSES A CRITICAL GAP IN OUR UNDERSTANDING OF HOW CELL-CELL COMMUNICATION INFLUENCES TISSUE FUNCTION AND DISEASE SYSTEMATICALLY. BY PROVIDING A COMPREHENSIVE FRAMEWORK TO STUDY THESE COMMUNICATIONS, THIS PROJECT HAS THE POTENTIAL TO TRANSFORM OUR APPROACH TO UNDERSTANDING COMPLEX GENETIC DISEASES AND TO IDENTIFY NEW THERAPEUTIC TARGETS. ULTIMATELY, SUCH KNOWLEDGE HAS THE POTENTIAL TO OFFER NEW OPPORTUNITIES FOR THE DEVELOPMENT OF INNOVATIVE THERAPIES TO TREAT GENETIC DISEASES, CONTRIBUTING TO THE BROADER FIELD OF PRECISION MEDICINE.

A NOVEL CHIP-SPEC TECHNOLOGY TO ISOLATE PROTEIN COMPLEXES AT UNIQUE GENOMIC LOCI

RAPID SELECTION AND EVALUATION OF CYCLIC PEPTIDES IN PARKINSON'S DISEASE MODELS

SORTASE-MEDIATED INSTALLATION OF RECOGNITION MODULES ON T CELLS FOR REDIRECTED KI

IMPACT OF AGING ON INTESTINAL TUMORIGENESIS

BRAIN VENTRICLE DEVELOPMENT AND MENTAL HEALTH

THIOL-BASED REDOX SWITCH IN PLANT FLAVONOID BIOSYNTHESIS

IDENTIFICATION OF THERAPEUTIC TARGETS FOR VIRAL HEMORRHAGIC FEVER VIRUSES

THE ROLE OF LYSOSOMAL DEREGULATION IN CANCER PROGRESSION

TARGETING PATHWAY STHAT PROCESS ENDOGENOUS TAXIC METABOLITES IN PANCREATIC CANCERS

NEURONAL PATHWAYS REGULATING METABOLIC ADAPTATION

LYSOMAL METABOLOMICS: A NOVEL APPROACH TO MTOR ACTIVATION AND METABOLIC DISEASES

DEFINING THE ROLE OF THE PERK PATHWAY IN REDOX MOMEOSTASIS AND THERAPEUTIC RESISTANCE IN BREAST CANCER

DEVELOPMENT AND CHARACTERIZATION ANIMAL MODELS FOR FILOVIRUSES

DEVELOPMENT AND CHARACTERIZATION ANIMAL MODELS FOR ARENAVIRUSES

METABOLIC RECYCLING AND COMPARTMENTALIZATION IN TUMOR PROGRESSION

ESTABLISHMENT OF THE MEIOTIC CELL CYCLE PROGRAM THROUGH POST-TRANSCRIPTIONAL REGULATION BY MEIOC AND YTHDC2

MOLECULAR CONTROL OF OOCYTE ARREST, MEIOSIS, AND THE TRANSITION TO DEVELOPMENT

COMBINATORIAL SIGNAL INTEGRATION IN THE MAINTENANCE AND RENEWAL OF ADULT GERMLINE STEM CELL FATE - PROJECT SUMMARY/ABSTRACT THROUGHOUT AN ORGANISM’S LIFETIME, ADULT STEM CELLS (ASCS) PLAY A PIVOTAL ROLE IN MAINTAINING TISSUE HOMEOSTASIS. ASCS POSSESS THE REMARKABLE ABILITY TO UNDERGO ASYMMETRIC DIVISIONS, SIMULTANEOUSLY RENEWING THEMSELVES AND PRODUCING DIFFERENTIATING DAUGHTER CELLS. UNLIKE ASCS, THOSE DAUGHTER CELLS LACK THE CAPACITY FOR SELF-RENEWAL AND INSTEAD UNDERGO TERMINAL DIFFERENTIATION. SUCH DIVERGENT CELL BEHAVIORS – ASYMMETRIC DIVISION AND SELF-RENEWAL BY ASCS VERSUS SYMMETRIC DIVISION AND DIFFERENTIATION BY THEIR DAUGHTERS – SUGGEST THAT ASCS AND THEIR DAUGHTERS ARE FUNDAMENTALLY DISTINCT CELL TYPES. HOWEVER, CONTRARY TO THIS MODEL, DAUGHTER CELLS CAN DEDIFFERENTIATE TO FULLY REGAIN ASC IDENTITY, INCLUDING A RETURN TO STEM CELL POTENCY. INDEED, DEDIFFERENTIATION IS FREQUENTLY REQUIRED TO REESTABLISH ASC POPULATIONS AFTER LOSS. THESE PHENOMENA PRESENT A FUNDAMENTAL PARADOX: HOW CAN STEM CELLS AND THEIR DIFFERENTIATING PROGENY MAINTAIN DIFFERENT BEHAVIORS, YET PRESERVE THE SAME POTENTIAL? NOTABLY, BOTH THESE EXCLUSIVELY PROPERTIES ARE CLEARLY PRESENT IN GERMLINE STEM CELL (GSC) POPULATIONS, AND ARE STRICTLY REQUIRED TO MAINTAIN SPECIES INTEGRITY. DROSOPHILA MALE GSCS ARE AN EXCEPTIONALLY TRACTABLE MODEL OF ASC BIOLOGY. IN PRELIMINARY STUDIES WITH THIS MODEL, I DISCOVERED THAT COMBINATORIAL INTEGRATION OF TWO INDEPENDENT SIGNALING FACTORS ENABLES CELLS TO ACHIEVE DISTINCT BEHAVIORS WHILE MAINTAINING EQUIVALENT POTENCY. I REFER TO THIS CONCEPT AS “TWO-FACTOR AUTHENTICATION,” ANALOGIZING STEM CELL MAINTENANCE TO CYBERSECURITY. BUILDING ON THESE PRELIMINARY DISCOVERIES, THIS PROPOSAL AIMS TO COMPREHENSIVELY CHARACTERIZE HOW ADULT STEM CELL SYSTEMS BALANCE REGENERATIVE PLASTICITY WITH THE MAINTENANCE OF DISCRETE CELL BEHAVIORS. IN AIM 1, I WILL DETERMINE HOW COMBINATORIAL SIGNALS FROM THE STEM CELL NICHE YIELD DISTINCT CELLULAR STATES, INCLUDING SELF-RENEWAL, DIFFERENTIATION, AND DEDIFFERENTIATION. IN AIM 2, I WILL DETERMINE IF GSCS ASYMMETRICALLY RETAIN A STEMNESS SIGNATURE THAT MAINTAINS THEIR UNIQUE FEATURES. FINALLY, IN AIM 3, I WILL EXAMINE THE FACTORS INVOLVED IN REESTABLISHMENT OF GSC FATE DURING DEDIFFERENTIATION IN MULTIPLE CONTEXTS. EXPANDING MY PRIOR EXPERTISE IN ORGANISMAL STEM CELL BIOLOGY WITH ADVANCED BIOINFORMATIC AND GENETIC TOOLS WILL ENABLE THE COMPLETION OF EACH OF THESE AIMS. TOGETHER, THESE EXPERIMENTS DEFINING GSC REGULATION (AIM 1), IDENTITY (AIM 2), AND REGENERATION (AIM 3) WILL ALLOW FOR A COMPREHENSIVE ASSESSMENT OF THE CORE REQUIREMENTS TO SET AND MAINTAIN THESE EXCEPTIONAL STEM CELLS.

ADIPOSE-CARCINOMA CELL INTERACTIONS DURING BREAST CANCER PROGRESSION

METABOLISM AND PHOSPHATASE REGULATION OF THE TOR PATHWAY

TUMOR AND IMMUNE CELL DYNAMICS DURING IMMUNOTHERAPY AND CANCER PROGRESSION - PROJECT SUMMARY TREATMENT OF CANCER HAS BEEN TRANSFORMED BY IMMUNOTHERAPIES THAT AIM TO REACTIVATE TUMOR-SPECIFIC IMMUNE CELL RESPONSES, IN PARTICULAR CHECKPOINT BLOCKADE THERAPIES THAT TARGET INHIBITORY RECEPTORS ON T CELLS. ALTHOUGH THESE THERAPEUTICS HAVE ACHIEVED DURABLE CLINICAL RESPONSES, MANY PATIENTS DO NOT RESPOND OR SHORTLY RELAPSE. RATIONALE DESIGN OF EFFECTIVE IMMUNOTHERAPY STRATEGIES REQUIRES A DETAILED UNDERSTANDING OF THE DYNAMICS BETWEEN TUMORS AND THE IMMUNE SYSTEM. MY GOAL IS TO DECODE THESE DYNAMIC INTERACTIONS USING A COMBINATION OF IN VIVO MODELS, GENOMIC TECHNOLOGIES, AND LARGE-SCALE ANALYSES OF PATIENT DATA TO GAIN NOVEL INSIGHTS INTO THE BIOLOGICAL PROCESSES THAT UNDERLIE CANCER PROGRESSION AND RESPONSE TO IMMUNOTHERAPY. IN THE F99 PHASE, I AIM TO IDENTIFY TO ORIGIN OF IMMUNE CELLS THAT RESPOND TO CHECKPOINT BLOCKADE. AN OUTSTANDING QUESTION IS WHETHER THE T CELL RESPONSE TO CHECKPOINT BLOCKADE RELIES ON REINVIGORATION OF PRE-EXISTING TUMOR-INFILTRATING LYMPHOCYTES OR ON RECRUITMENT OF NOVEL T CELLS. IN MY DISSERTATION WORK SO FAR, I HAVE USED SINGLE CELL RNA AND T CELL RECEPTOR (TCR) SEQUENCING OF SITE-MATCHED TUMOR BIOPSIES BEFORE AND AFTER PD-1 BLOCKADE TO PROFILE CLONAL T CELL DYNAMICS IN RESPONSE TO IMMUNOTHERAPY. I FOUND THAT T CELLS THAT CLONALLY EXPAND IN RESPONSE TO PD-1 BLOCKADE ARE ENRICHED FOR NOVEL CLONES THAT HAVE NOT BEEN PREVIOUSLY OBSERVED IN THE SAME TUMOR, A PHENOMENON WE TERM CLONAL REPLACEMENT. HOWEVER, THE SOURCE OF NOVEL T CELLS AS WELL AS THE ROLE OF OTHER IMMUNE CELL POPULATIONS IN THIS PROCESS REMAIN UNCLEAR. I HYPOTHESIZE THAT NOVEL T CELLS ARE DERIVED FROM SECONDARY LYMPHOID ORGANS AND THAT OVERCOMING DEFICIENCIES IN T CELL PRIMING BY ANTIGEN PRESENTING CELLS IS REQUIRED FOR CLONAL REPLACEMENT FOLLOWING PD-1 BLOCKADE. I WILL USE A COMBINATION OF FLOW CYTOMETRY, SINGLE CELL SEQUENCING, AND BULK TCR SEQUENCING OF IN VIVO SYNGENEIC MOUSE TUMOR MODELS TO DETERMINE THE ORIGIN OF T CELLS THAT RESPOND TO PD-1 AND CTLA-4 BLOCKADE. FURTHER, I WILL USE GENETIC MOUSE MODELS TO DETERMINE THE ROLE OF PD-L1 EXPRESSION ON ANTIGEN PRESENTING CELLS, IN PARTICULAR CONVENTIONAL TYPE 1 DENDRITIC CELLS, ON PD-1 BLOCKADE EFFICIENCY. IN THE K00 PHASE, I AIM TO ELUCIDATE THE RELATIONSHIP BETWEEN EXTRACHROMOSOMAL DNA AMPLIFICATION, INNATE IMMUNE SIGNALING, AND THE EFFICACY OF CHECKPOINT BLOCKADE. COPY NUMBER ALTERATIONS LEADING TO ONCOGENE AMPLIFICATION FREQUENTLY OCCURS ON CIRCULAR EXTRACHROMOSOMAL DNA (ECDNA), WHICH ARE COMMONLY FOUND IN THE CYTOPLASM DUE TO LACK TO CENTROMERES AND ASSOCIATE WITH LOSS OF CYTOSOLIC DNA SENSING THROUGH THE CGAS-STING PATHWAY. FURTHER, INNATE IMMUNE SIGNALING THROUGH CGAS-STING HAS BEEN SHOWN TO IMPROVE THE EFFICACY OF CHECKPOINT BLOCKADE. I PROPOSE THAT LOSS OF CYTOSOLIC DNA SENSING IS PERMISSIVE FOR EXTRACHROMOSOMAL DNA PRODUCTION, WHICH PROMOTES TUMOR PROGRESSION NOT ONLY THROUGH ONCOGENE AMPLIFICATION BUT ALSO THROUGH IMPAIRED INNATE IMMUNE SIGNALING, LIMITING IMMUNE SURVEILLANCE AND THE EFFICACY OF CHECKPOINT BLOCKADE. TOGETHER, THIS WORK WILL PROVIDE NOVEL INSIGHTS INTO IMMUNE AND TUMOR DYNAMICS THAT UNDERLIE CANCER PROGRESSION AND RESPONSE TO IMMUNOTHERAPY.

"NEW DRUGS FOR ANEMIA TREATMENT BASED ON A NEW UNDERSTANDING OF THE MECHANISMS OF STRESS ERYTHROPOIESIS"

THE ROLE OF TUMOR-ASSOCIATED MACROPHAGES IN PROMOTING THE EPITHELIAL-MESENCHYMAL TRANSITION IN BREAST CANCER

REGULATION OF THE INTESTINAL STEM CELL NICHE IN AGING

THE WORLD’S SMALLEST VERTEBRATE BRAIN: A NEW MODEL FOR NERVOUS SYSTEM FUNCTION AND REGENERATION RESEARCH - PROJECT SUMMARY / ABSTRACT REGENERATION IS WIDESPREAD AND VARIABLE IN THE ANIMAL KINGDOM. REGENERATIVE CAPACITY OF THE HUMAN CENTRAL NERVOUS SYSTEM (CNS) FOLLOWING INJURY OR DISEASE IS POOR, YET SEVERAL MECHANISMS EXIST IN SEVERAL VERTEBRATE ORGANISMS THAT LEAD TO FUNCTIONAL REGENERATION OF THE CNS. WHAT IS DIFFERENT BETWEEN WHAT HAPPENS AT INJURY SITES IN THESE ORGANISMS AND IN HUMANS? ARE LOST NEURON TYPES AND NEURONAL WIRING PATTERNS RESTORED UPON REGENERATION OF NEURAL CIRCUITRY IN ANIMALS THAT CAN ACHIEVE THIS FEAT? I PROPOSE TO DEVELOP A NOVEL EXPERIMENTAL VERTEBRATE REGENERATION SYSTEM THAT WILL ENABLE IDENTIFICATION OF MECHANISMS THAT NATURALLY PROMOTE NERVOUS SYSTEM REGENERATION. SPECIFICALLY, I PLAN TO FOCUS ON DANIONELLA CEREBRUM, A MINIATURE AND TRANSPARENT FISH SPECIES THAT HAS THE SMALLEST VERTEBRATE BRAIN ON RECORD, WITH ~650,000 NEURONS IN TOTAL. BECAUSE OF ITS MINUTE SIZE, OPTICAL TRANSPARENCY, COMPLEX BEHAVIORAL REPERTOIRE, AND GENETIC TRACTABILITY DANIONELLA CEREBRUM IS POTENTIALLY UNPARALLELED AS A VERTEBRATE FOR TISSUE-WIDE SINGLE-CELL RNA SEQUENCING (SCRNA-SEQ), AND FOR WHOLE- BODY AND -BRAIN IMAGING APPLICATIONS TARGETED TO EVALUATE CELLULAR DYNAMICS IN THE ADULT STATE. I HAVE TWO MAIN AIMS: IN AIM 1, I PLAN TO GENERATE A COMPLETE DANIONELLA CEREBRUM SCRNA-SEQ ATLAS THAT INCLUDES HOMEOSTATIC AND REGENERATING STATES. GENERATION OF A SINGLE-CELL TRANSCRIPTOME ATLAS OF THE WHOLE BODY OF A VERTEBRATE WILL BE A POWERFUL RESOURCE FOR A MYRIAD OF CENTRAL PROBLEMS IN VERTEBRATE BIOLOGY AND NEUROSCIENCE. THE PROPOSED RESOURCE COMBINED WITH THE SMALL SIZE OF THE DANIONELLA NERVOUS SYSTEM WILL ENABLE UTILIZATION OF SCRNA-SEQ EXPERIMENTS IN THE FUTURE TO PROBE A HOST OF PROBLEMS THAT INVOLVE BODY-WIDE MANIPULATION OF NEURAL DEVELOPMENT AND CIRCUIT ACTIVITY, AND RECOVERY FOLLOWING INJURY. THIS DATASET WILL ALSO ALLOW CHARACTERIZATION AND MANIPULATION OF CIRCUIT COMPONENTS WITHIN DISTINCT AREAS OF THE CENTRAL AND PERIPHERAL NERVOUS SYSTEM, AND WILL IDENTIFY GENES WITH APPROPRIATE CELL-SPECIFIC EXPRESSION TO ENABLE GENETIC CELL ABLATIONS, CELL-SPECIFIC LABELING, AND IMAGING OF REGENERATING NEURAL CIRCUITS. BECAUSE OF ITS OPTICAL TRANSPARENCY, I HAVE COMPLETE ACCESS TO THE DANIONELLA NERVOUS SYSTEM FOR VALIDATION AND FUTURE FUNCTIONAL IMAGING STUDIES. IN AIMS 2 AND 3, I PROPOSE TO DEVELOP STRATEGIES TO STUDY AND MANIPULATE NEURAL DYNAMICS DURING REGENERATION, AND IN THE ABSENCE OF IT (I.E. FOLLOWING INHIBITION OF REGENERATION). THIS AIM WILL INCLUDE GENERATION OF CELL TYPE-SPECIFIC NITROREDUCTASE EXPRESSION-BASED GENETIC CELL ABLATION LINES, SURGICAL INJURY STRATEGIES, LEARNING AND MEMORY PARADIGMS, AND A CALCIUM IMAGING PLATFORM TO ENABLE CHARACTERIZATION OF FUNCTIONAL REGENERATION IN THE NERVOUS SYSTEM. SUCCESS WITH THIS AIM WILL ENABLE FUTURE WORK ELUCIDATING THE “RULES” FOR FUNCTIONAL INTEGRATION OF NEW NEURONS FOLLOWING ACUTE AND CHRONIC ABLATION IN THE NERVOUS SYSTEM. THE MOLECULAR AND CELLULAR INSIGHTS GAINED FROM THE PROPOSED AIMS WILL ACCELERATE DISCOVERY AND UNDERSTANDING OF FUNDAMENTAL ASPECTS OF NEURAL REGENERATION WITH POTENTIAL FOR DEVELOPING APPROACHES THAT COULD BE TAKEN TO GENERATE THERAPEUTIC INTERVENTION IN THE CASES OF NERVOUS SYSTEM INJURY AND DEGENERATION.

UNCOVERING THE MECHANISMS THAT PROMOTE AND MAINTAIN GERM CELL IDENTITY - PROJECT SUMMARY/ABSTRACT GERM CELLS HAVE THE POTENTIAL TO GENERATE EVERY CELL TYPE IN THE BODY. SPECIFIED IN THE EMBRYO, GERM CELLS FACE THE CHALLENGE OF PROTECTING AND MAINTAINING THIS POISED TOTIPOTENCY FOR THE ENTIRETY OF AN ORGANISM’S REPRODUCTIVE LIFESPAN. DESPITE THE FUNDAMENTAL IMPORTANCE OF THIS PROTECTION FOR THE FERTILITY AND HEALTH OF THE NEXT GENERATION, THE MOLECULAR PROGRAMS THAT PROMOTE AND PROTECT GERM CELL IDENTITY ARE POORLY UNDERSTOOD. A SPECIFIC PROGRAM FOR GERM CELL TRANSCRIPTIONAL ACTIVATION HAS YET TO BE DESCRIBED, LARGELY BECAUSE A ‘MASTER REGULATOR TRANSCRIPTION FACTOR’ FOR GERM CELL FATE HAS NOT BEEN IDENTIFIED IN ANY ORGANISM. INSTEAD, PRIMORDIAL GERM CELLS (PGCS) RELY ON CONSERVED NETWORKS OF RNA REGULATORS THAT REPRESS SOMATIC DIFFERENTIATION PROGRAMS AND PROTECT THE GERMLINE GENETIC PROGRAM, A STRATEGY THAT IS COMMON TO PGCS OF WORMS, FLIES, MICE, AND HUMANS. YET HOW THESE RNA REGULATORS INTERSECT WITH THE GENE REGULATORY NETWORK OF PGCS IS UNKNOWN. MY PROPOSED RESEARCH PROGRAM SEEKS TO UNCOVER THE MOLECULAR MECHANISMS THAT INITIATE AND MAINTAIN GERM CELL IDENTITY BY REVEALING THE PGC GENE REGULATORY NETWORK. THE GOAL OF THE RESEARCH PROPOSED HERE IS TO (A) UNCOVER MECHANISMS THAT ‘REPRESS’ THE SOMATIC PROGRAM THEREBY ALLOWING THE PGC PROGRAM TO DEVELOP AND (B) IDENTIFY THE ‘INSTRUCTIVE’ CUES THAT ACTIVELY CONTROL PGC FATE. IN AIM 1, I FOCUS ON DISCOVERING THE DYNAMICS OF GERMLINE GENE ACTIVATION DURING EMBRYOGENESIS BY IDENTIFYING SPECIFIC, TEMPORALLY REGULATED SETS OF GENES THAT DEFINE THE PGC TRANSCRIPTIONAL PROGRAM USING SINGLE-CELL RNA SEQUENCING. USING TISSUE-SPECIFIC INTERFERENCE, I WILL DISTINGUISH BETWEEN CELL- INTRINSIC AND NON-AUTONOMOUS SIGNALING MECHANISMS REGULATING THE PGC TRANSCRIPTIONAL PROGRAM. IN AIM 2, I WILL PROBE THE ‘INSTRUCTIVE’ CUES OF GERM CELL IDENTITY BY UNCOVERING PGC-SPECIFIC CIS-REGULATORY SEQUENCES USING ATAC-SEQ. I WILL IDENTIFY TRANSCRIPTIONAL REGULATORS (SUCH AS TRANSCRIPTION FACTORS/CHROMATIN FACTORS) THAT BIND THESE CIS-REGULATORY SEQUENCES AND TEST THEIR ROLE IN THE PGC TRANSCRIPTIONAL PROGRAM. IN AIM 3, I ADDRESS THE ‘REPRESSIVE’ MODEL BY EXAMINING THE FUNCTION OF A CRITICAL REGULATOR OF GERM CELL FATE, THE CONSERVED RNA-BINDING PROTEIN NANOS. USING THE RNA TARGET IDENTIFICATION METHOD HYPERTRIBE, I WILL IDENTIFY NANOS TARGETS AND ASK WHETHER THESE TARGETS PROMOTE THE GERMLINE PROGRAM AND/OR REPRESS SOMATIC PROGRAMS. I WILL USE DROSOPHILA AS MY MODEL SYSTEM, EXPANDING MY PRIOR TRAINING IN BIOCHEMISTRY AND MOLECULAR BIOLOGY TO ENCOMPASS ORGANISMAL BIOLOGY, DEVELOPMENT, AND GENETICS AND GAINING EXPERTISE IN BIOINFORMATICS AND GENE REGULATION. TOGETHER, THIS WORK WILL UNCOVER THE REGULATORY LANDSCAPE OF PRIMORDIAL GERM CELLS, PROVIDING A FUNDAMENTAL UNDERSTANDING OF THE MECHANISMS THAT SPECIFY GERM CELL FATE, PROMOTE FERTILITY, AND ULTIMATELY PROTECT THE SURVIVAL OF THE SPECIES.

THE ROLE OF IME4 AND IME2 IN DROSOPHILA GAMETOGENESIS

UNDERSTANDING THE HIGHLY DIVERGENT MITOCHONDRIAL ATP SYNTHASE IN T. GONDII

THE ROLE OF EPITHELIAL MESENCHYMAL TRANSITION IN THE FORMATION OF NORMAL AND NEOPLASTIC MAMMARY EPITHELIAL STEM CELLS

USING MITOCHONDRIAL DNA MUTATIONS TO UNDERSTAND LIMITATIONS OF MITOCHONDRIAL QUALITY CONTROL

THE ROLE OF FUNGAL ADHESINS IN THE MODULATION OF THE INNATE IMMUNE RESPONSE

SPATIALLY-RESOLVED, INTEGRATED CELL STATE AND LINEAGE TRACING TO DEFINE PROGENITOR CELL DYNAMICS IN EARLY MOUSE DEVELOPMENT - PROJECT SUMMARY: MAMMALIAN DEVELOPMENT IS A PROBABILISTIC AND ROBUST PROCESS WHERE PROGENITOR CELLS MUST INTEGRATE GENETIC, EPIGENETIC, AND ENVIRONMENTAL INFORMATION TO GENERATE A COMPLEX BODY PLAN. THE GOAL OF THIS WORK IS TO DELINEATE HOW PROGENITOR BEHAVIORS ELICIT SPECIFIC PHENOTYPES IN HEALTH AND DISEASE. TO DO SO, I WILL LEVERAGE A TECHNOLOGY I DEVELOPED IN MY POSTDOCTORAL WORK IN THE WEISSMAN LAB, TERMED PETRACER, A PRIME EDITING-BASED TOOL THAT ENABLES THE 1) ASSESSMENT OF CELL STATE AND LINEAGE USING BOTH IMAGING- AND SEQUENCING- BASED PROFILING METHODS, INCLUDING SPATIALLY-RESOLVED TECHNIQUES, 2) PRECISE PERTURBATION OF GENOMES FOR TESTING HYPOTHESES ABOUT GENE FUNCTION, 3) STUDYING THESE PROCESSES AT SCALE IN COMPLEX TISSUES, IN 4) A MANNER THAT MINIMALLY AFFECTS NORMAL CELL FUNCTION. TO SHOWCASE THE PETRACER SYSTEM, I USED IT TO STUDY A XENOGRAFT MODEL OF BREAST CANCER WHERE I RECONSTRUCTED THE 3-DIMENSIONAL GROWTH PATTERNS OF A PRIMARY TUMOR AND THEN UNCOVERED HOW EACH CELL’S INTERACTIONS WITH ITS NEIGHBORS AND MICROENVIRONMENT INFLUENCED ITS PROPENSITY TO SEED METASTASES. THESE IMPORTANT DISCOVERIES BREAK NOVEL GROUND IN CANCER BIOLOGY AND STAND TO INFORM THERAPEUTIC DEVELOPMENT. HERE, I PROPOSE TO LEVERAGE THIS TECHNOLOGY IN ENGINEERED MOUSE EMBRYONIC STEM CELLS AND MOUSE MODELS TO CREATE THE FIRST INTEGRATED MAPS OF CELL STATE AND LINEAGE DURING NORMAL DEVELOPMENT, BIOLOGICAL CIPHERS THAT COULD BE USED TO UNDERSTAND HOW TISSUES ARE ASSEMBLED AND MAINTAINED AND HOW THESE PROCESSES GO AWRY IN DISEASE. I WILL USE IN VITRO STEM CELL-BASED GASTRULOID MODELS WHERE HIGH REPLICATION POWER ENABLES A STATISTICAL VIEW OF DEVELOPMENTAL PROCESSES AND ALLOWS FOR PERTURBATION STUDIES TO PROBE THE GENETIC DETERMINANTS OF CELL STATE AND LINEAGE DECISIONS IN DEVELOPMENT. I WILL ALSO USE MOUSE MODELS TO STUDY THESE QUESTIONS IN THE COMPLETE CONTEXT OF A DEVELOPING ORGANISM. IN THE K99 TRAINING PHASE, I WILL DEVELOP EXPERTISE IN STEM CELL BIOLOGY, LEARN HOW TO ENGINEER MOUSE MODELS, AND SHARPEN COMPUTATIONAL SKILLS NEEDED TO ANALYZE THE UNIQUE DATASETS THESE PROJECTS GENERATE. I WILL LEARN THESE TECHNICAL SKILLS FROM MY BRILLIANT CO-MENTORS, DR. JONATHAN WEISSMAN AND DR. ZACHARY SMITH, AND MY PIONEERING COLLABORATORS. FURTHER, I WILL GAIN CRITICAL EXPERIENCE MANAGING A GROWING RESEARCH PROGRAM DURING THIS TIME. THE STEM CELL AND MOUSE MODELS I ENGINEER DURING MY K99 PHASE WILL BE FOUNDATIONAL FOR MY R00 PHASE AND BEYOND WHERE I WILL STUDY PROGENITOR CELL DYNAMICS DURING MAMMALIAN DEVELOPMENT USING BOTH CUTTING-EDGE IN VITRO AND IN VIVO MODEL SYSTEMS. I AIM TO STUDY HOW THE BEHAVIORS AND HISTORIES OF SINGLE CELLS LEAD TO COMPLEX CELL STATES AND CHARACTERISTICS. THIS WORK WILL PROVIDE A FOUNDATION FOR MY FUTURE RESEARCH PROGRAM AS AN INDEPENDENT INVESTIGATOR AT A MAJOR RESEARCH INSTITUTION WHERE I WILL USE THE SPATIALLY-RESOLVED CELL STATE AND LINEAGE MAPS OF PROGENITOR CELLULAR DYNAMICS IN NORMAL DEVELOPMENT THAT I GENERATE THROUGH THIS K99/R00 AWARD TO UNDERSTAND HOW THESE SAME PROCESSES GO AWRY IN DEVELOPMENTAL DISORDERS AND DISEASES. THIS WORK THAT MAY UNLOCK A NEW ERA OF QUANTITATIVE GENOMICS IN DEVELOPMENTAL BIOLOGY AND GUIDE FUTURE TRANSLATIONAL RESEARCH.

GENETIC AND BIOPHYSICAL ANALYSIS OF MORPHOGEN GRADIENT FORMATION - PRINCIPAL INVESTIGATOR: SCHLISSEL, GAVIN PROJECT SUMMARY ANIMAL DEVELOPMENT AND PHYSIOLOGY REQUIRE REGULAR COMMUNICATION AMONG CELL TYPES EMBEDDED IN TISSUES. CELLULAR COMMUNICATION COMMONLY RELIES ON SIGNALING PROTEINS, WHICH CAN TRANSIT THE SPACE BETWEEN CELLS AND RELAY INFORMATION ACROSS A WIDE RANGE OF SPATIAL SCALES FROM NANOMETERS TO METERS. PROPER CONTROL OF SIGNALING RANGE IS STRICTLY NECESSARY DURING ANIMAL DEVELOPMENT, AND DYSREGULATION OF SIGNALING RANGE CAN RESULT IN A SPECTRUM OF EMBRYONIC LETHAL CONDITIONS OR DEVELOPMENTAL DISORDERS. THE PATTERNING GRADIENT FORMED BY A SIGNALING PROTEIN REFLECTS THE SIGNALING PROTEIN’S ABILITY TO TRAVEL THROUGH THE EXTRACELLULAR MATRIX, WHICH IS AN AMALGAM OF PROTEIN, SUGAR AND LIPIDS THAT ORGANIZE CELLS IN NATURAL TISSUES. ALTHOUGH SIGNALING PROTEINS ARE THOUGHT TO DIFFUSE FROM THEIR SOURCE TO THEIR TARGET, MANY PROTEINS VIOLATE THE ASSUMPTIONS OF FREE DIFFUSION AND INSTEAD SHOW CONTEXT-DEPENDENT DIFFERENCES IN THEIR SIGNALING RANGE. FOR EXAMPLE, SONIC HEDGEHOG FAMILY DEVELOPMENTAL MORPHOGENS FORM SIGNALING GRADIENTS OVER ~10ΜM IN THE TESTES, ~50ΜM IN THE DEVELOPING NEURAL TUBE OR IN ADULT HAIR FOLLICLES, AND ~300ΜM IN DEVELOPING LONG BONES. NOTABLY, TISSUES IN WHICH SONIC HEDGEHOG FORMS LONGER SIGNALING GRADIENTS TEND TO EXPRESS SCUBE FAMILY EXTRACELLULAR MATRIX PROTEINS, AND SCUBE FAMILY PROTEINS CAN DRAMATICALLY EXTEND SONIC HEDGEHOG SIGNALING GRADIENTS IN CELL CULTURE. I SUSPECT THAT TISSUE-SPECIFIC DIFFERENCES IN SIGNALING RANGE MIGHT REFLECT DIRECT REGULATION OF SONIC HEDGEHOG’S DIFFUSION RATE, AND THAT REGULATED DIFFUSION OF HEDGEHOG MIGHT REFLECT A BROADLY USED STRATEGY TO CONTROL THE SIZE OF SIGNALING GRADIENTS IN ANIMALS. TO UNDERSTAND HOW MORPHOGENS AND THE EXTRACELLULAR MATRIX INTERACT TO GENERATE APPROPRIATELY SIZED SIGNALING GRADIENTS, I WILL MEASURE VARIATION IN PROTEIN DIFFUSION BOTH BETWEEN DIVERSE SIGNALING PROTEINS, AND BETWEEN DISTINCT EXTRACELLULAR ENVIRONMENTS. I WILL APPLY THIS MECHANISTIC UNDERSTANDING TO DISCOVER HOW BIOCHEMICAL FEATURES OF SIGNALING PROTEINS AND THE EXTRACELLULAR MATRIX RESULT IN SIZE VARIATION AMONG SIGNALING GRADIENTS AS WELL AS MORPHOLOGICAL VARIATION AMONG THE ANATOMICAL FEATURES THAT THEY PATTERN. TO THAT END, I PROPOSE THE FOLLOWING SPECIFIC AIMS: 1) UNDERSTAND HOW SCUBE FAMILY PROTEINS MODIFY HEDGEHOG DIFFUSION BY TRACKING SINGLE PARTICLES OF SONIC HEDGEHOG DIFFUSING THROUGH THE EXTRACELLULAR MATRIX. 2) DISCOVER WHICH BIOCHEMICAL FEATURES OF A SIGNALING PROTEIN AFFECT ITS DIFFUSION RATE THROUGH THE EXTRACELLULAR MATRIX BY DEVELOPING SYNTHETIC MORPHOGENS, IN WHICH DIFFUSION CAN BE UNCOUPLED FROM DOWNSTREAM SIGNAL TRANSDUCTION. 3) IDENTIFY EXTRACELLULAR MATRIX MODIFIERS OF PROTEIN DIFFUSION THAT CONTRIBUTE TO TISSUE- OR ORGANISM-SPECIFIC SIGNALING GRADIENT SIZE DISCREPANCIES BY GENETICALLY SIMULATING TISSUE-SPECIFIC EXTRACELLULAR MATRIX VARIATION K99/R00 FELLOWSHIP APPLICATION OCTOBER 2022

MECHANISMS GOVERNING POST-TRANSCRIPTIONAL REGULATION OF NEURONAL MICRORNAS - PROJECT SUMMARY MANY ASPECTS OF NEURAL PLASTICITY REQUIRE CHANGES IN GENE EXPRESSION, EITHER BY ALTERING TRANSCRIPTION IN THE NUCLEUS OR BY TARGETING RNA MOLECULES POST-TRANSCRIPTIONALLY. POST-TRANSCRIPTIONAL REGULATION MAY BE PARTICULARLY IMPORTANT FOR ALLOWING NEURONS TO REGULATE PROTEIN PRODUCTION IN A RAPID AND COMPARTMENT-SPECIFIC MANNER. MICRORNAS (MIRNAS) INFLUENCE POST-TRANSCRIPTIONAL GENE EXPRESSION BY TARGETING MRNA MOLECULES FOR TRANSLATIONAL REPRESSION AND DEGRADATION AND ARE KNOWN TO INFLUENCE NEURAL DEVELOPMENT AND FUNCTION. A GROWING BODY OF EVIDENCE ALSO SUGGESTS THAT POST-TRANSCRIPTIONAL REGULATION OF MIRNAS CONTRIBUTES TO EXPERIENCE-DEPENDENT SYNAPTIC PLASTICITY. HOWEVER, MUCH REMAINS UNKNOWN ABOUT THE POST-TRANSCRIPTIONAL MECHANISMS THAT CONTROL MIRNA EXPRESSION. ONE SUCH MECHANISM IS TARGET-DIRECTED MIRNA DEGRADATION (TDMD), A PHENOMENON IN WHICH TRIGGER RNA MOLECULES DIRECT THE DEGRADATION OF SPECIFIC MIRNA MOLECULES. IT WAS RECENTLY SHOWN THAT TDMD IN MAMMALS IS MEDIATED BY THE ZSWIM8 UBIQUITIN LIGASE. PRELIMINARY DATA INDICATE THAT DOZENS OF MIRNAS ARE ZSWIM8 SENSITIVE IN THE MAMMALIAN BRAIN, INCLUDING SEVERAL MIRNAS THAT HAVE PREVIOUSLY BEEN LINKED TO SYNAPTIC PLASTICITY, SUGGESTING THAT THE TDMD PATHWAY IS POISED TO HAVE WIDESPREAD EFFECTS ON THE NERVOUS SYSTEM. MICE WITH ALTERED ZSWIM8 EXPRESSION ALSO EXHIBIT PERTURBED EXPRESSION OF MANY PHYSIOLOGICALLY RELEVANT MRNAS, INCLUDING GENES ENCODING ION CHANNELS AND SYNAPTIC ADHESION MOLECULES, FURTHER SUPPORTING THE HYPOTHESIS THAT TDMD AFFECTS NEURAL DEVELOPMENT AND PHYSIOLOGY. THIS PROPOSAL WILL ASSESS HOW TDMD AFFECTS NEUROBIOLOGY BY (1) DETERMINING HOW ZSWIM8 INFLUENCES ACTIVITY- DEPENDENT GENE EXPRESSION; (2) IDENTIFYING TDMD TRIGGER RNAS THAT DRIVE MIRNA DEGRADATION IN MOUSE AND HUMAN NEURONS; AND (3) ESTABLISHING HOW TDMD INFLUENCES NEURONAL ACTIVITY AND THE MORPHOLOGY AND PLASTICITY OF DENDRITIC SPINES. COLLECTIVELY, THIS WORK WILL EXPAND OUR UNDERSTANDING OF THE REGULATORY PATHWAYS CONTROLLING NEURONAL GENE EXPRESSION AND THEIR DOWNSTREAM CONSEQUENCES FOR NEURAL FUNCTION.

DETERMINING THE CRITICAL IN VIVO ROLES OF MTOR IN CANCER

NANOSTRING INSTRUMENTATION

THE CONTROL OF THE BREAST CANCER STEM CELL STATE BY SNAIL-DRIVEN EMT PROGRAM

MICRORNAS AND HEMATOPOIETIC DIFFERENTIATION

ELUCIDATING THE STRESS RESPONSE REGULATORY NETWORKS THAT ENABLE MALIGNANCY

ROLE OF THE RETT SYNDROME-CAUSING GENE MECP2 IN 3D CHROMOSOMAL ORGANIZATION AND RESCUE OF CELLULAR DISEASE PHENOTYPES

IDENTIFICATION OF METABOLIC LIABILITIES IN BREAST CANCER

REGULATION OF ERYTHROID TERMINAL DIFFERENTIATION BY LONG NONCODING RNAS

EFFECTS OF SEX CHROMOSOME CONSTITUTION ON GLOBAL GENE EXPRESSION

MATERNAL ORGANELLE CONTRIBUTION TO OFFSPRING GERMLINE HEALTH - PROJECT SUMMARY ORGANELLES ENGAGE IN HOMEOSTATIC PROCESSES TO IMPROVE CELLULAR HEALTH. HOWEVER, SOME ORGANELLE PROTEINS IN THE SOMA CAN SURVIVE MANY MONTHS, MAKING THEM SUSCEPTIBLE TO DAMAGE DURING AGING. IN THE GERMLINE, GAMETES CAN BE ARRESTED FOR A PROLONGED PERIOD BEFORE THEY PASS DOWN THEIR ORGANELLE CONTENT TO THE NEXT GENERATION. HAVING DAMAGE-PRONE PROTEINS PERDURE IN THE GERMLINE WOULD THEREFORE BE PROBLEMATIC AND CHALLENGE THE ABILITY OF GERM CELLS TO PASS DOWN HEALTHY MATERIAL FROM GENERATION TO GENERATION. UPON OOCYTE MATURATION AND FERTILIZATION, CERTAIN MATERNAL PRODUCTS ARE DEGRADED TO INITIATE THE MATERNAL TO ZYGOTIC TRANSITION IN THE DEVELOPING EMBRYO, THOUGH LITTLE IS KNOWN HOW MATERNALLY DERIVED ORGANELLES ARE TURNED OVER AND REPLACED WITH ZYGOTIC ORGANELLES. INTRIGUINGLY, QUALITY CONTROL EVENTS HAVE BEEN SHOWN TO TAKE PLACE IN SINGLE CELL ORGANISMS AND DURING ASYMMETRIC DIVISIONS TO ENSURE THE ELIMINATION OF ORGANELLE LONG-LIVED PROTEINS (LLPS). HOWEVER, WHETHER SPECIALIZED ORGANELLE QUALITY CONTROL TAKES PLACES TO REMOVE MATERNALLY DERIVED LLPS IN DEVELOPING PROGENY, AND HOW SUCH A PROCESS WOULD TAKE PLACE IN A MULTI-CELLULAR ORGANISM REMAINS POORLY UNDERSTOOD. THROUGH MOLECULAR AND GENETIC ANALYSIS, THE RESEARCH PROPOSED IN THIS FELLOWSHIP WILL INVESTIGATE HOW MATERNAL ORGANELLE INPUT DERIVED FROM DROSOPHILA OOCYTES INFLUENCES GERMLINE HEALTH OF THEIR OFFSPRING. FIRST, I WILL ENDOGENOUSLY TAG WELL CHARACTERIZED LLPS TO VISUALIZE THE ZYGOTIC POOL OF PROTEINS ALONG WITH THE MATERNALLY DERIVED LONG-LIVED POOL OF THE SAME PROTEIN ACROSS EMBRYONIC, LARVAL AND ADULT PROGENY. BY DIFFERENTIALLY TAGGING THE MATERNAL AND ZYGOTIC POOL, I WILL COMPARE PROTEIN LEVELS IN THE PROGENY GERMLINE TO ASSESS IF IT SELECTIVELY PREVENTS MATERNALLY DEPOSITED ORGANELLE LLPS TO BE TRANSGENERATIONALLY INHERITED. NEXT, I WILL PERTURB OOCYTE HEALTH TO INVESTIGATE HOW MATERNALLY DAMAGED LLPS CONTRIBUTES TO OFFSPRING VIABILITY. FINALLY, I WILL IDENTIFY ADDITIONAL LONG-LIVED PROTEINS THAT ARE DERIVED FROM MATERNAL ORGANELLES USING A BIORTHOGONAL AMINO ACID LABELING APPROACH AND COMPARE THEIR FATES IN THE PROGENY GERMLINE WITH WELL CHARACTERIZED LLPS. THE PROPOSED EXPERIMENTS WILL HIGHLIGHT HOW MATERNALLY DERIVED LLPS IMPACT ORGANELLE FUNCTION IN THE GERMLINE OF ENSUING PROGENY. IN ADDITION, THESE STUDIES WILL SHED LIGHT ON HOW ORGANELLE CONTINUITY IS MAINTAINED BETWEEN GENERATIONS AND AIM TO REVEAL SPECIALIZED QUALITY CONTROL PATHWAYS IN THE GERMLINE.

X CHROMOSOMAL STUDIES OF SPERMATOGENIC FAILURE

MECHANISMS OF GENE EXPRESSION REGULATION IN PANCREATIC CANCER - PROJECT SUMMARY PANCREATIC DUCTAL ADENOCARCINOMA (PDAC) IS PREDICTED TO BECOME THE SECOND DEADLIEST CANCER WITHIN THE NEXT 10 YEARS. INVESTIGATIONS INTO NOVEL MECHANISMS OF PDAC TRANSFORMATION ARE URGENTLY NEEDED AS NO EFFECTIVE PDAC TREATMENT CURRENTLY EXISTS. TRANSCRIPTIONAL REGULATION DURING TRANSFORMATION IS MULTIFACETED WITH GENETIC MUTATIONS AND EPIGENETIC ALTERATIONS PLAYING VITAL INTERCONNECTED ROLES. A KEY UNELUCIDATED FACET OF GENE REGULATION IN THE CONTEXT OF TRANSFORMATION IS THE MOLECULAR MECHANISM WHICH MEDIATES CHANGES IN CHROMATIN REORGANIZATION. CHIP-SEQ AND CHIP-PCR STUDIES IN AN INDUCIBLE MODEL OF ONCOGENIC KRAS, A KEY INITIATING FACTOR IN PDAC, DEMONSTRATE A RELOCATION OF HETEROCHROMATIN TO THE NUCLEAR PERIPHERY. THESE LAMINA ASSOCIATED DOMAINS (LADS) ARE ENRICHED IN H3K9ME2 AND POSITIONED AT THE NUCLEAR LAMINA. CHIP-SEQ AND RNA-SEQ IDENTIFIED LOSS OF ACTIVE ENHANCER REGIONS INCORPORATED INTO LADS AND DOWNREGULATION OF LAD ASSOCIATED GENES, RESPECTIVELY. TO INVESTIGATE THE MECHANISM REGULATING THE ASSEMBLY OF LADS DOWNSTREAM OF ONCOGENIC KRAS WE PERFORMED SEVERAL MOLECULAR AND BIOCHEMICAL STUDIES. WE CONDUCTED A SCREENING BIOID UTILIZING LAMIN A, A CORE COMPONENT OF THE NUCLEAR LAMINA KNOWN TO INTERACT WITH LADS, AS BAIT. LAMIN A BIOID IDENTIFIED A NOVEL MYOSIN, MYOSIN 18A (MYO18A), ENRICHED AT THE NUCLEAR LAMINA UNDER ONCOGENIC KRAS SIGNALING. IMMUNOCYTOCHEMISTRY REVEALED INCREASED NUCLEAR LOCALIZATION OF MYO18A AND ENRICHMENT AT THE LAMINA UPON KRAS ACTIVATION. BIOCHEMICAL ANALYSIS VALIDATED MYO18A-LAMIN A INTERACTION AND CONFIRMED MYO18A INTERACTION WITH CHROMATIN. SIRNA KNOCKDOWN FOR MYO18A RESCUED EXPRESSION OF GENES ASSOCIATED WITH ONCOGENIC KRAS-MEDIATED LADS. THESE DATA LEAD US TO OUR CENTRAL HYPOTHESIS THAT MYO18A ACTS AS A DOWNSTREAM EFFECTOR OF KRAS TO MODULATE CHROMATIN POSITIONING AT THE NUCLEAR LAMINA TO SILENCE ONCOGENIC GENE EXPRESSION. TO TEST THIS HYPOTHESIS, WE WILL CONDUCT CHIP-SEQ AND RNA-SEQ EXPERIMENTS IN PARALLEL TO DETECT MYO18A ASSOCIATED LADS IN IN VITRO MODELS OF PDAC. LAD ASSEMBLY WILL THEN BE INVESTIGATED IN THE PRESENCE/ABSENCE OF MYO18A. WE WILL ALSO DEVELOP MYO18A GENETIC ENGINEERED MOUSE MODELS TO EXPLORE THE ROLE OF MYO18A AND NUCLEAR MYO18A IN PDAC DEVELOPMENT AND PROGRESSION. FURTHER STUDY INTO THIS MECHANISM CAN PROVIDE VALUABLE INSIGHT INTO GENE REGULATION IN THE TRANSFORMATION PROCESS AND WILL IDENTIFY POTENTIALLY DRUGGABLE TARGETS.

HUMAN INDUCED PLURIPOTENT STEM CELL MODELING OF GENETIC RISK FACTORS FOR ALZHEIMER'S DISEASE

INNOVATIVE IN VIVO MODELING OF NEUROFIBROMATOSIS BY HUMAN-MOUSE NEURAL CREST CHIMERA

RNA-MEDIATED FEEDBACK CONTROL OF ONCOGENIC TRANSCRIPTION

TOWARDS PHARMACOLOGICAL RESCUE OF TSC LOSS OF FUNCTION

STRUCTURAL BASIS OF MICRORNA BIOGENESIS

REGULATION OF METAZOAN DNA REPLICATION FORK PROGRESSION, STABILITY AND COMPOSITIO

EXPLORING TRANSCRIPTIONAL ADAPTATION: THERAPEUTIC POTENTIAL AND IMPACT ON HUMAN GENETICS - PROJECT SUMMARY/ABSTRACT MUTATIONS CAN LEAD TO A WIDE-VARIETY OF GENETIC DISEASES. RECENT SEQUENCING STUDIES, HOWEVER, HAVE IDENTIFIED LOSS-OF-FUNCTION MUTATIONS, INCLUDING WITHIN DISEASE-RELEVANT GENES, IN APPARENTLY HEALTHY INDIVIDUALS, REVIVING THE INTEREST IN THE CONCEPT OF GENETIC ROBUSTNESS. IN PREVIOUS STUDIES, WE IDENTIFIED TRANSCRIPTIONAL ADAPTATION (TA), A NOVEL MECHANISM THROUGH WHICH CELLS CAN COMPENSATE FOR NONSENSE MUTATIONS BY INCREASING THE EXPRESSION LEVELS OF FUNCTIONALLY RELATED GENES (E.G., PARALOGS, OR THE WILD-TYPE (WT) ALLELE IN CASE OF HETEROZYGOUS MUTATIONS), IN A MANNER DEPENDENT ON MUTANT MRNA DECAY. WE PROPOSED A MODEL WHEREBY FOLLOWING MUTANT MRNA DEGRADATION, MRNA DECAY INTERMEDIATES ARE TRANSLOCATED BACK TO THE NUCLEUS TO INDUCE THE UPREGULATION OF GENES EXHIBITING SEQUENCE SIMILARITY. IN MY POSTDOCTORAL WORK, I IDENTIFIED ILF3 AS THE KEY RNA BINDING PROTEIN MODULATING THE TRANSPORT OF MRNA DEGRADATION INTERMEDIATES FROM THE CYTOPLASM TO THE NUCLEUS TO INDUCE TA. ADDITIONALLY, I DEVELOPED A SCREENING STRATEGY THAT IDENTIFIED ILF3-ASSOCIATED RNA FRAGMENTS, TERMED “TRIGGER RNAS,” WHICH CAN ACTIVATE GENE EXPRESSION WHEN TRANSFECTED INTO CELLS. TRIGGER RNAS HOLD THE POTENTIAL TO BE A NEW CLASS OF GENE-AUGMENTING THERAPEUTIC RNAS. IN THIS K99/R00 APPLICATION, MY FIRST AIM IS TO CHARACTERIZE THE MOLECULAR CHARACTERISTICS OF TRIGGER RNAS, INCLUDING THEIR SEQUENCE AND STRUCTURAL REQUIREMENTS. THESE STUDIES WILL ENABLE BETTER DESIGN OF TRIGGER RNAS, WHICH CAN BE BENEFICIAL FOR BOTH GENETIC AND NON-GENETIC DISEASES. MY SECOND AIM IS TO EXPLORE THE BROADER PHYSIOLOGICAL ROLE OF TA BY INVESTIGATING HOW TA INFLUENCES THE LANDSCAPE OF GENETIC DISEASES. USING A COMBINATION OF FUNCTIONAL GENOMICS, AND POPULATION GENETICS DATA ANALYSIS, I AIM TO IDENTIFY GENES WHOSE PERTURBATION PHENOTYPE IS INFLUENCED BY TA. SUCH GENES COULD REPRESENT A CLASS OF GENETIC DISEASES THAT CAN BE THERAPEUTICALLY TARGETED THROUGH HARNESSING TA. IN ADDITION, THE STUDIES WILL ALLOW FOR BETTER UNDERSTANDING OF HOW DIFFERENT VARIANTS INFLUENCE DISEASE OUTCOMES. MY THIRD AIM WILL BE TO INVESTIGATE THE PREVALENCE OF DOSAGE COMPENSATION TO HETEROZYGOUS MUTATIONS, AND UNDERSTAND HOW TA CONTRIBUTES TO TOLERANCE OF HETEROZYGOUS MUTATIONS IN MAMMALS. MY BACKGROUND IN RNA BIOLOGY AND GENE EXPRESSION REGULATION, AND HAVING BEEN A LEAD AUTHOR IN THE DISCOVERY OF TA, PUTS ME IN A UNIQUE POSITION TO ACCOMPLISH THIS PROPOSAL. DURING THE K99 PHASE, I WILL BE SUPPORTED BY AN OUTSTANDING TEAM OF MENTORS AND ADVISORS (DRS. JONATHAN WEISSMAN, MARK DALY, OLIVIA CORRADIN, KONRAD KARCZEWSKI AND HARVEY LODISH) WITH EXPERTISE IN ALL ASPECTS AND METHODOLOGIES REQUIRED FOR THE COMPLETION OF THE PROPOSED RESEARCH. I WILL ACQUIRE NEW SKILLS IN POPULATION GENETICS AND BIG DATA ANALYSIS. I SHALL ALSO PROMOTE MY SOFT SKILLS, INCLUDING GRANT WRITING, LEADERSHIP AND MANAGEMENT SKILLS, THROUGH FORMAL COURSEWORK AND TRAINING AND INSTITUTIONAL SUPPORT FROM THE WHITEHEAD INSTITUTE. THE COMBINATION OF MENTORED SUPPORT, TRAINING AND DATA OBTAINED IN THE K99 PHASE WILL ACT AS A STEPPING STONE TOWARDS ACHIEVING INDEPENDENCE AS AN INVESTIGATOR IN THE R00 PHASE AND BEYOND, AND LEADING A SUCCESSFUL LAB.

DISSECTING TRANSLATIONAL REGULATION BY GENOME-WIDE MAPPING OF INITIATION FACTORS

SYSTEMATIC IDENTIFICATION AND MECHANISTIC CHARACTERIZATION OF NOVEL OXIDATIVE PROTECTIVE GENES TO PREVENT RPE DEGENERATION - PROJECT SUMMARY / ABSTRACT AGE-RELATED MACULAR DEGENERATION (AMD) AFFECTS APPROXIMATELY 196 MILLION PEOPLE GLOBALLY AND IS PRIMARILY CAUSED BY OXIDATIVE STRESS IN THE RETINAL PIGMENT EPITHELIUM (RPE) CELLS. THESE CRUCIAL CELLS LIE BETWEEN THE OXYGEN-RICH CHOROID AND THE PHOTO-OXIDATIVELY STRESSED RETINA, MAKING THEM HIGHLY SUSCEPTIBLE TO DAMAGE FROM REACTIVE OXYGEN SPECIES (ROS). GIVEN THE PREVALENCE OF AMD AND THE LACK OF HIGHLY EFFECTIVE TREATMENTS, THERE IS A PRESSING NEED FOR NOVEL INTERVENTIONS THAT ENHANCE THE OXIDATIVE DEFENSE MECHANISMS OF RPE CELLS. IN THIS PROJECT, I HAVE INITIATED, TO MY KNOWLEDGE, THE FIRST-EVER GENOME-WIDE OVEREXPRESSION SCREEN SPECIFICALLY IN RPE CELLS, IDENTIFYING TEN NOVEL GENE CANDIDATES THAT ENHANCE CELL SURVIVAL AGAINST OXIDATIVE STRESS, PARTICULARLY NAIO3 WHICH IS KNOWN TO CAUSE MITOCHONDRIAL DYSFUNCTION. AMONG THESE, THE CARDIAC-SPECIFIC TRANSCRIPTION FACTOR NKX2-5 EMERGED AS A STANDOUT DUE TO ITS POTENTIAL TO CONFER HEART-LIKE ANTIOXIDATIVE PROPERTIES TO RPE CELLS. PRELIMINARY DATA SUGGEST THAT NKX2-5 CAN SIGNIFICANTLY AMELIORATE OXIDATIVE STRESS IN THESE CELLS, THEREBY POTENTIALLY MITIGATING AMD PROGRESSION. THE PROJECT IS STRUCTURED INTO THREE PRIMARY AIMS: AIM 1 FOCUSES ON ELUCIDATING THE MECHANISTIC PATHWAYS THROUGH WHICH NKX2-5 CONFERS OXIDATIVE PROTECTION, INCLUDING DIRECT ROS REDUCTION, ENHANCEMENT OF MITOCHONDRIAL BIOGENESIS, AND ACTIVATION OF ANTI-APOPTOTIC PATHWAYS. THIS WILL BE ASSESSED USING IPSC-DERIVED RPE CELLS EXPRESSING NKX2-5, WHERE CELLULAR ROS LEVELS, MITOCHONDRIAL METRICS, AND GENOMIC CHANGES WILL BE EVALUATED. AIM 2 TESTS THE HYPOTHESIS THAT NKX2-5 CAN IMPROVE BOTH THE MORPHOLOGY AND FUNCTION OF RPE CELLS IN VIVO. THIS WILL INVOLVE DEVELOPING A NEW ADENO-ASSOCIATED VIRUS (AAV) CONSTRUCT FOR INDUCIBLE NKX2-5 EXPRESSION SPECIFICALLY IN RPE CELLS, WHICH WILL THEN BE ADMINISTERED IN BOTH AN ACUTE MODEL OF RPE DEGENERATION AND IN AGED MICE TO ASSESS IMPROVEMENTS IN RPE HEALTH AND VISUAL FUNCTION. AIM 3 EXPANDS THE INVESTIGATION TO INCLUDE ALL IDENTIFIED GENES FROM THE SCREEN, ASSESSING THEIR ABILITY TO PROTECT AGAINST VARIOUS OXIDATIVE STRESSORS RELEVANT TO AMD, SUCH AS OXIDIZED-LDL AND CIGARETTE SMOKE EXTRACT. THE ULTIMATE GOAL IS TO IDENTIFY GENE COMBINATIONS THAT PROVIDE THE BROADEST OXIDATIVE PROTECTION. THESE COMBINATIONS WILL BE TESTED IN A COMPREHENSIVE MOUSE MODEL OF AMD THAT INCORPORATES GENETIC RISK FACTORS, A HIGH-FAT DIET, AND AGING INFLUENCES. THROUGHOUT THIS PROJECT, I AIM TO GAIN A DEEP UNDERSTANDING OF CELLULAR DEFENSE MECHANISMS AGAINST ROS, PAVING THE WAY FOR NOVEL GENE THERAPY APPROACHES TO TREAT AMD AND POTENTIALLY OTHER OCULAR DISEASES INFLUENCED BY OXIDATIVE STRESS.

ROLES FOR MRNA METHYLATION AND LNCRNA IN CANDIDA MATING AND PATHOGENICITY

LINKING MAINTENANCE OF CHROMOSOME STRUCTURE TO TRANSCRIPTIONAL REGULATION

TARGETING DEHYDROGENASES IN CANCER METABOLISM

FETAL LIVER STROMAL CELLS FOR HEMATOPOIETIC STEM CELLS

RECOGNITION AND PHAGOCYTOSIS OF CANDIDA ALBICANS BY THE INNATE IMMUNE SYSTEM

UNDERSTANDING GENETICS OF SPERMATOGENIC FAILURE BY RESEQUENCING THE SEX CHROMOSOME

ROLE OF MICRORNAS IN MALIGNANT PROGRESSION

GENERATION OF PLURIPOTENT CELLS BY REPROGRAMMING OF THE SOMATIC EPIGENOME.

MECHANISTIC INSIGHTS INTO LYSOSOMAL NUTRIENT EFFLUX IN CANCER - ABSTRACT MANY RAS-TRANSFORMED AGGRESSIVE CANCER CELLS ARE ABLE TO ESCAPE CYTOTOXIC CHEMOTHERAPY AND SURVIVE IN NEAR-STARVATION CONDITIONS. ONE ADAPTATION MAKING THEM HARD TO KILL IS THEIR ABILITY TO SCAVENGE EXTRACELLULAR PROTEINS AND RECYCLE THE CELLULAR COMPONENTS USING AUTOPHAGY, BOTH OF WHICH ARE THEN DIGESTED IN LYSOSOMES TO RECOVER FREE AMINO ACIDS. THE PROCESS OF SCAVENGING AND INTERNALIZATION IS KNOWN AS MACROPINOCYTOSIS, AND CANCER CELLS AQUIRE MUTATIONS TO UPREGULATE IT WHEN FACED WITH NUTRIENT-POOR CONDITIONS. TO FIGHT SUCH CANCERS, RESEARCHERS ARE CURRENTLY TARGETING THE MACROPINOCYTOSIS MACHINERY; HOWEVER, BECAUSE THIS PROCESS IS NOT VERY WELL STUDIED, AND LIKELY INVOLVES HUNDREDS OF PROTEINS WITH REDUNDANT FUNCTIONS, SUCH THERAPY MIGHT PROVE CHALLENGING TO EXECTUTE WITHOUT INVOLVING MULTIPLE DRUGS. WE PROPOSE A BETTER WAY OF TARGETING NUTRIENT-SCAVENGING CANCERS BY FOCUSING ON A DOWNSTREAM PROCESS OF RELEASING DIGESTED NUTRIENTS FROM LYSOSOMES TO CYTOSOL. THE SABATINI LAB SHOWED THAT THE RELEASE OF DIGESTED AMINO ACIDS FROM LYSOSOMES IS ORCHESTRATED BY THE MTORC1 PATHWAY, AND SPECIFICALLY BY SLC38A9. THIS LYSOSOMAL MEMBRANE PROTEIN SENSES THE RISING LEVELS OF DIGESTED AMINO ACIDS IN LYSOSOMES BY DIRECTLY BINDING ARGININE. OUR LAB FOUND THAT THIS SENSING IS COUPLED TO ACTIVATION OF THE TRANSPORTER FUNCTION, AND RESULTS IN THE EFFLUX OF ESSENTIAL NON-POLAR AMINO-ACIDS, SUCH AS LEUCINE, FROM LYSOSOMES TO CYTOSOL. IMPORTANTLY, RAS-TRANSFORMED PANCREATIC CANCER CELLS THAT FEED ON EXTRACELLULAR PROTEIN WERE UNABLE TO EFFICIENTLY FORM TUMORS IN THE ABSENCE OF SLC38A9. THESE RESULTS PRESENT A NOVEL THERAPEUTIC IDEA OF TARGETING A METABOLIC VULNERABILITY IN CANCERS TRANSFORMED BY ONCOGENIC RAS SIGNALING. IN THIS FIVE-YEAR PROJECT WE WILL ELUCIDATE THE MOLECULAR MECHANISM OF RELEASING DIGESTED AMINO ACIDS FROM LYSOSOMES TO CYTOSOL VIA SLC38A9, AND THEREFORE PROVIDE A RATIONAL APPROACH TO DRUG DISCOVERY. IN PARALLEL TO THAT, WE WILL SCREEN FOR SMALL MOLECULES THAT SPECIFICALLY BIND TO SLC38A9, AND DEVELOP THEM INTO CHEMICAL PROBES THAT MODULATE ITS TRANSPORT ACTIVITY. IMPAIRED EFFLUX FUNCTION OF SLC38A9 WILL LEAD TO ENTRAPMENT OF MACROPINOCYTOSIS-DERIVED AMINO-ACIDS WITHIN THE LYSOSOMES, AND OUR EXPECTATION IS THAT THIS TREATMENT WILL IMPAIR THE GROWTH OF RAS-MUTANT AND OTHER TUMORS ADDICTED TO PROTEIN SCAVENGING, WHILE SPARING NORMAL CELLS THAT LACK THIS REQUIREMENT. OVER THE FIRST TWO YEARS OF THE MENTORED PHASE, I WILL BE BASED AT THE WHITEHEAD INSTITUTE, WHERE I WILL LEARN CELL SIGNALING AND METABOLOMICS APPROACHES FROM THE EXPERTS IN THE FIELD. I WILL ALSO VENTURE INTO A COMPLETELY NEW RESEARCH AREA TO ME, CHEMICAL BIOLOGY, WORKING WITH EXPERTS AT THE BROAD INSTITUTE. AFTER THE COMPLETION OF MY K99 TRAINING, MY ASPIRATION IS TO LEAD A LABORATORY THAT COMBINES CELL SIGNALING, STRUCTURAL BIOLOGY, AND CHEMICAL BIOLOGY TO STUDY MEMBRANE TRANSPORTERS AND THEIR ROLE IN CANCER METABOLISM. IN PARALLEL TO UNDERSTANDING BASIC BIOLOGY, I WANT MY LAB TO DEVELOP SPECIFIC SMALL-MOLECULE MODULATORS THAT ADJUST TRANSPORT ACTIVITIES OF THOSE PROTEINS, FACILITATING FURTHER RESEARCH IN THE FIELD, AND IN LONG TERM – NEW MEDICINES.

INVESTIGATING THE ROLE AND MECHANSIMS OF MIRNA DECAY

HERBAL MEDICINE HAS BEEN DEVELOPED AND PRACTICED BY MULTIPLE INDIGENOUS CULTURES AROUND THE WORLD FOR MILLENNIA. IN MODERN MEDICINE, OF THE 175 THERAPEUTIC SMALL MOLECULES APPROVED BY FDA FOR TREATING CANCER SINCE 1940, 131, OR 74.8%, ARE OTHER THAN SYNTHETIC, WITH 85, OR 48.6%, ACTUALLY BEING EITHER NATURAL PRODUCTS OR DIRECTLY DERIVED THEREFROM. AMONG NATURAL PRODUCTS-DERIVED MODERN MEDICINES, 25% COME FROM PLANTS. RECENT ADVANCES IN FUNDAMENTAL KNOWLEDGE OF PLANT SPECIALIZED METABOLISM AND THE ABILITY TO ENGINEER PLANT NATURAL PRODUCT BIOSYNTHESIS IN HETEROLOGOUS HOSTS GREATLY EXPEDITE OUR ABILITY TO HARNESS THE MEDICINAL VALUES OF PLANTS TO TREAT HUMAN DISEASES. IN THE PROJECT, WE FOCUS ON TWO RELATED CLASSES OF MEDICINAL PEPTIDE NATURAL PRODUCTS INITIALLY ISOLATED FROM THE CHINESE MEDICINAL PLANT LYCIUM BARBARUM (GOJI BERRY) AND DENDROCNIDE MOROIDES (AUSTRALIAN STINGING TREE). BY ELUCIDATING AND ENGINEERING THE BIOSYNTHETIC PATHWAYS FOR THESE NATURAL PRODUCTS, THIS RESEARCH WILL OPEN UP TREMENDOUS NEW OPPORTUNITIES FOR GREATLY EXPANDING THE CHEMICAL SPACE OF CYCLIC PEPTIDES THROUGH THE MEANS OF SYNTHETIC BIOLOGY APPROACH FOR BOTH AGRONOMICAL AND PHARMACEUTICAL APPLICATIONS.

REPROGRAMMING TOWARDS A GERMLINE STEM CELL FATE - PROJECT SUMMARY GERMLINE CELLULAR IMMORTALITY UNDERLIES THE CONTINUATION OF ALL SEXUALLY REPRODUCING SPECIES. RECENT WORK HAS IDENTIFIED FREQUENT EXAMPLES OF INTERCONVERSION BETWEEN GERMLINE AND SOMATIC FATES, SUGGESTING PLASTICITY, RATHER THAN A STRICT DICHOTOMY, BETWEEN GERMLINE AND SOMA. IN ADULTS, PRODUCTION OF MALE GAMETES IS DRIVEN BY GERMLINE STEM CELL (GSC) DIVISION. IN MY POSTDOCTORAL WORK, I PROPOSE TO USE THE POWERFULLY TRACTABLE SYSTEM OF THE DROSOPHILA MELANOGASTER MALE GERMLINE TO IDENTIFY NECESSARY FACTORS OF IMMORTAL GERMLINE FATE. IN ADDITION TO UNDERGOING NORMAL SELF-RENEWAL AND DIFFERENTIATION, GSCS CAN BE GENERATED THROUGH DEDIFFERENTIATION OF SPERMATOGONIA, AND CAN BE LOST THROUGH BOTH FORCED DIFFERENTIATION TO GERM CELLS AND TRANSDIFFERENTIATION TO SOMA. FIRST, USING SINGLE-CELL RNA SEQUENCING ON TESTES CELLS IN WHICH THE GERMLINE IS UNDERGOING DEDIFFERENTIATION, I WILL RECONSTRUCT LINEAGE TRANSITIONS TO IDENTIFY GENES UNDERLYING GSC FORMATION FROM A DIFFERENTIATED CELL, INCLUDING BOTH GERMLINE CELL- AUTONOMOUS FACTORS AND NON-AUTONOMOUS SIGNALING FACTORS. I WILL VALIDATE THESE DATA USING BOTH IN VIVO EXPRESSION CHARACTERIZATION AND ASSAYS OF GENE FUNCTION. NEXT, I WILL DESCRIBE THE TRANSCRIPTIONAL CHANGES NECESSARY FOR TRANSDIFFERENTIATION OF GSCS TO SOMATIC CELLS, IDENTIFYING THE COMPONENTS THAT MAINTAIN GERMLINE IDENTITY. FINALLY, I WILL LEVERAGE DISCOVERY OF FACTORS INVOLVED IN BOTH A) THE CREATION OF GSCS FROM DIFFERENTIATING CELLS, AND B) THE LOSS OF GSC IDENTITY THAT RESULTS IN SOMATIC CELL FORMATION, IN ORDER TO DEVELOP A NOVEL MODEL FOR INDUCIBLE SOMATIC REPROGRAMMING TO A GERMLINE FATE. THIS WORK WILL SIGNIFICANTLY FURTHER OUR UNDERSTANDING OF THE FACTORS THAT CONFER CELLULAR IMMORTALITY, AND WILL AID IN THE DEVELOPMENT OF THERAPIES FOR DIVERSE HUMAN DISEASES.

IMPACT OF AGING ON INTESTINAL TUMORIGENESIS

REGULATION OF THE CELL CYCLE DURING MEIOTIC PROPHASE I BY MEIOC

REGULATORY ROLES OF MIRNAS DURING EARLY ARABIDOPSIS EMBRYOGENESIS

A PLATFORM FOR GENOME-WIDE DISCOVERY OF SYNTHETIC LETHAL INTERACTIONS IN CANCER

SUBSTRATES AND FUNCTIONS OF THE SIDEROFLEXIN MITOCHONDRIAL TRANSPORTER FAMILY

SYTEMIC LIPID MOBILIZATION AND BREAST CANCER PROGRESSION

TARGETING ONE-CARBON METABOLISM IN BREAST CANCER

MAMMALIAN TARGET OF RAPAMYCIN (MTOR) SIGNALING IN HEALTH AND LONGETIVITY

SIGNALING MISSING TISSUE: UNCOVERING THE PATHWAY OF PLANARIAN REGENERATION

EXPLOITING THE EPITHELIAL-TO-MESENCHYMAL TRANSITION FOR THE DIFFERENTIATION OF CANCER STEM CELLS

MECHANISMS OF MITOCHONDRIAL INHERITANCE - PROJECT SUMMARY THE SURVIVAL OF EUKARYOTIC SPECIES DEPENDS ON THE FAITHFUL TRANSMISSION OF BOTH NUCLEAR AND MITOCHONDRIAL GENOMES. MUTATIONS IN MITOCHONDRIAL DNA (MTDNA) CAUSE NEURODEGENERATIVE AND NEUROMUSCULAR DISEASES IN HUMANS. STRIKINGLY, THOUGH MITOCHONDRIA ARE INHERITED EXCLUSIVELY THROUGH THE MATERNAL LINEAGE, RAPID CHANGES IN MTDNA ALLELE FREQUENCY CAN OCCUR, RESULTING IN SEVERE MITOCHONDRIAL DISEASE IN A SUBSET OF OFFSPRING DUE TO AN INCREASED MUTATIONAL LOAD. THE LONG-TERM GOAL OF THIS PROJECT IS TO DECIPHER THE MOLECULAR MECHANISMS REGULATING MITOCHONDRIAL SEGREGATION IN THE GERMLINE. TO ACHIEVE THIS GOAL, I WILL TAKE A MULTIDISCIPLINARY APPROACH COMBINING GENETICS, PROTEOMICS, BIOCHEMISTRY, AND HIGH-RESOLUTION QUANTITATIVE MICROSCOPY USING THE MODEL ORGANISM, DROSOPHILA MELANOGASTER. THE FOLLOWING AIMS WILL BE PURSUED: (1) ANALYZE MTDNA ALLELE FREQUENCY IN GAMETE PRECURSOR CELLS TERMED PRIMORDIAL GERM CELLS (PGCS). DURING EMBRYOGENESIS, A SMALL SUBSET OF MITOCHONDRIA IS PERMANENTLY SEPARATED FROM THE REST OF THE OOCYTE INTO PGCS, RESULTING IN AN ~1000-FOLD REDUCTION IN MTDNA CONTENT. TO EXAMINE THE CONSEQUENCE OF THIS MITOCHONDRIAL POPULATION BOTTLENECK ON THE SEGREGATION OF MTDNA ALLELES, I WILL USE A HETEROPLASMIC FLY STRAIN HARBORING BOTH WILD-TYPE AND MUTANT MITOCHONDRIAL GENOMES. I WILL DETERMINE MTDNA ALLELE FREQUENCY IN INDIVIDUAL PGCS USING HIGH-RESOLUTION IMAGING OF SINGLE MTDNA MOLECULES AND QUANTITATIVE PCR AND WILL EXAMINE HOW THESE RATIOS CHANGE WHEN THE SIZE OF THE BOTTLENECK IS GENETICALLY CONSTRICTED. (2) DETERMINE THE NETWORK OF LONG OSKAR INTERACTING PROTEINS. LONG OSKAR IS THE MASTER REGULATOR OF MITOCHONDRIAL INHERITANCE. TO RECRUIT MITOCHONDRIA TO THE SITE OF PGC FORMATION, LONG OSKAR STIMULATES F-ACTIN REORGANIZATION, BUT IT DOES NOT CONTACT MITOCHONDRIA DIRECTLY. TO IDENTIFY PROTEINS DOWNSTREAM OF LONG OSKAR, I WILL USE PROXIMITY LABELLING AND TANDEM MASS SPECTROMETRY. I WILL THEN MAP LONG OSKAR-BINDING REGIONS ON DIRECT BINDING PARTNERS. (3) IDENTIFY NUCLEAR-ENCODED MITOCHONDRIAL PROTEINS REQUIRED FOR MITOCHONDRIAL INHERITANCE. CURRENTLY, OUR UNDERSTANDING OF HOW MITOCHONDRIA ARE TARGETED TO SITES OF PGC FORMATION IS LIMITED BY AN INCOMPLETE PARTS LIST OF THE MITOCHONDRIAL SEGREGATION MACHINERY. I WILL PERFORM A COMPREHENSIVE RNAI SCREEN OF MITOCHONDRIAL MEMBRANE-ASSOCIATED PROTEINS TO IDENTIFY THOSE REQUIRED FOR MITOCHONDRIAL LOCALIZATION. TOGETHER, THESE AIMS WILL REVEAL HOW THE MITOCHONDRIAL BOTTLENECK IMPACTS THE SEGREGATION OF MTDNA ALLELES AND WILL LIKELY INFORM ON THE POPULATION RISK OF MITOCHONDRIAL ASSOCIATED DISEASES. IN ADDITION, THESE EXPERIMENTS WILL IDENTIFY MOLECULAR COMPONENTS OF THE MTDNA SEGREGATION MACHINERY THAT IS USED TO TRANSMIT MITOCHONDRIA TO GERMLINE CELLS DURING EARLY DROSOPHILA EMBRYOGENESIS. TOGETHER, THESE RESULTS HAVE THE POTENTIAL TO SHED LIGHT ON HOW SIMILAR EVENTS MAY OCCUR IN PRE-IMPLANTATION HUMAN EMBRYOS.

EVALUATING SMALL MOLECULES IN NEURONAL AND YEAST MODELS OF TDP-43 PROTEINOPATHY

INVESTIGATING DIFFERENTIATION, MAINTENANCE, AND REACTIVATION OF THE TOXOPLASMA CHRONIC STAGE - 7. PROJECT SUMMARY/ABSTRACT TOXOPLASMA GONDII IS ONE OF THE MOST UBIQUITOUS PARASITES OF HUMANS, CHRONICALLY INFECTING ONE QUARTER OF THE WORLD’S POPULATION. THOUGH TYPICALLY ASYMPTOMATIC IN IMMUNOCOMPETENT HOSTS, INFECTION CAN GIVE RISE TO SEVERE CLINICAL COMPLICATIONS IN THE IMMUNOCOMPROMISED OR DURING PERIODS OF WEAKENED IMMUNE FUNCTION. PARASITES THAT MAINTAIN THE CHRONIC INFECTION ARE TRANSCRIPTIONALLY DISTINCT FROM THE ACUTE STAGES THAT CAUSE PATHOLOGY, AND ARE RESISTANT TO TREATMENT WITH ANTIPARASITICS. UNDERSTANDING REGULATION OF THE T. GONDII CHRONIC STAGE IS THEREFORE NECESSARY TO PREVENT AND ERADICATE PERSISTENT RESERVOIRS OF DISEASE. OUR LAB RECENTLY IDENTIFIED TWO CORE COMPONENTS OF THE CHRONIC DIFFERENTIATION PROGRAM. FIRST, WE FOUND THAT A SINGLE TRANSCRIPTION FACTOR, BFD1, IS NECESSARY AND SUFFICIENT FOR STAGE CONVERSION. MORE RECENTLY, I DISCOVERED A CYTOSOLIC RNA-BINDING PROTEIN (BFD2) THAT FUNCTIONS IMMEDIATELY UPSTREAM OF BFD1 TO REGULATE ITS EXPRESSION. PARASITES LACKING EITHER FACTOR ARE NOT ABLE TO PRODUCE CHRONIC STAGES, EITHER IN MICE OR IN RESPONSE TO ALKALINE STRESS—A STANDARD IN VITRO TRIGGER OF DIFFERENTIATION. MY WORK SUGGESTS THAT BFD2 FUNCTIONS AS A TRANSLATIONAL ACTIVATOR OF BFD1 UNDER STRESS, AND THAT THIS REQUIRES BINDING OF BFD2 TO BFD1 MRNA. IN TURN, BFD1 PROMOTES BFD2 TRANSCRIPTION, OUTLINING A FEEDBACK LOOP. THIS RECIPROCAL POSITIVE REGULATION PROVIDES THE DRIVING FORCE TO COMMIT PARASITES TO THEIR CHRONIC DIFFERENTIATION PROGRAM; YET, PRECISELY HOW THE BFD1-BFD2 CIRCUIT IS TRIGGERED BY STRESS, AND LATER OVERTURNED DURING REACTIVATION, REMAINS TO BE EXPLORED. IN THE PROPOSED STUDIES, I WILL UNCOVER THE MOLECULAR BASIS OF BFD2 REGULATION THROUGH THREE COMPLIMENTARY AIMS. AIM 1 WILL INVESTIGATE THE MECHANISM OF TRANSLATIONAL ACTIVATION BY BFD2, AND HOW THIS IS INFLUENCED BY PROTEIN-PROTEIN INTERACTIONS. AIM 2 WILL ASSESS THE ROLE OF POST-TRANSLATIONAL MODIFICATION IN LICENSING STRESS-RESPONSIVE BFD2 ACTIVITY. AIM 3 WILL EMPLOY A NOVEL GAIN-OF-FUNCTION SCREENING APPROACH TO IDENTIFY THE FACTORS RESPONSIBLE FOR ANTAGONIZING THE BFD1-BFD2 CIRCUIT DURING CHRONIC-STAGE REACTIVATION. TOGETHER, THESE STUDIES WILL INTEGRATE THE GENETIC MECHANISMS UNDERLYING ENTRY AND EXIT FROM THE T. GONDII CHRONIC STAGE WITH THE SIGNALS THAT TRIGGER STAGE CONVERSION, WHICH MAY INFORM THE DESIGN OF THERAPIES TO COMBAT THIS IMPORTANT HUMAN PATHOGEN.

QUANTITATIVE ANALYSIS OF THE EVOLVING GENOTYPE-TO-PHENOTYPE MAP

DECIPHERING TRANSLATIONAL CONTROL IN STEM CELLS - PROJECT SUMMARY TRANSLATIONAL CONTROL IS CRITICAL FOR A VARIETY OF CELLULAR PROCESSES SUCH AS HOMEOSTASIS AND DIFFERENTIATION. DYSREGULATION OF TRANSLATION HAS BEEN IMPLICATED IN MANY HUMAN DISEASES, INCLUDING CANCER. STEM CELLS ARE CHARACTERIZED BY LOW GLOBAL PROTEIN SYNTHESIS RATES, DESPITE HIGH LEVELS OF RIBOSOME BIOGENESIS, SUGGESTING THAT STEM CELLS ACTIVELY REPRESS TRANSLATION. INDEED, PRECISE TRANSLATIONAL CONTROL HAS BEEN SHOWN TO BE ESSENTIAL FOR MAINTAINING PLURIPOTENCY, AS PERTURBATIONS THAT INCREASED TRANSLATION RATES INDUCED DIFFERENTIATION IN VARIOUS TYPES OF STEM CELLS. ALTHOUGH THERE ARE SEVERAL EXAMPLES OF HOW TRANSLATION IS REGULATED, THE MOLECULAR MECHANISMS THAT MODULATE TRANSLATION, PARTICULARLY IN STEM CELLS, ARE LARGELY UNKNOWN. TO ACHIEVE A COMPREHENSIVE UNDERSTANDING OF TRANSLATIONAL CONTROL, I PROPOSE TO SYSTEMATICALLY INVESTIGATE THE REGULATORY PATHWAYS THAT MEDIATE TRANSLATION IN STEM CELLS AND ACROSS CELL TYPES. I WILL BEGIN BY DEVELOPING A GENOME-SCALE GENETIC SCREENING PLATFORM THAT COMBINES CRISPR SCREENING WITH POLYSOME PROFILING TO IDENTIFY GENETIC PERTURBATIONS THAT ALTER TRANSLATION. COMPARED TO PREVIOUS APPROACHES THAT RELIED ON REPORTER PROTEIN EXPRESSION, THE PROPOSED SCREENING APPROACH MEASURES PRESENCE OF RIBOSOMES ON MRNA, WHICH MORE DIRECTLY AND ACCURATELY QUANTIFIES TRANSLATION RATES. THIS SCREENING APPROACH IS ALSO MORE VERSATILE AND CAN BE ADAPTED FOR STUDYING OTHER MODES OF TRANSLATION BY EXCHANGING THE REPORTER GENE IN THE VECTOR. I WILL THEN APPLY THIS SCREENING APPROACH TO SYSTEMATICALLY DISCOVER GENES THAT REGULATE GLOBAL TRANSLATION LEVELS IN STEM CELLS AND FIBROBLASTS AND INVESTIGATE ASSOCIATED MECHANISMS. FINALLY, I WILL FOCUS ON GENES THAT ARE SELECTIVELY TRANSLATED USING RIBOSOME PROFILING AND CHARACTERIZE THE PATHWAYS THAT MEDIATE SELECTIVE MRNA TRANSLATION. TOGETHER, MY PROPOSED WORK ESTABLISHES A FRAMEWORK FOR SYSTEMATIC EXPLORATION OF TRANSLATIONAL CONTROL NETWORKS AND, IN CONCERT WITH ITS APPLICATION IN OTHER CONTEXTS, WILL CONTRIBUTE TO A COMPREHENSIVE UNDERSTANDING OF THE PROCESSES CONTROLLING TRANSLATION THAT ARE RELEVANT IN DEVELOPMENT AND DISEASE.

UNDERSTANDING MIRNA-TARGET RECOGNITION THROUGH COMPREHENSIVE AFFINITY AND REPRESSION MEASUREMENTS

SEX CHROMOSOME CONTROL OF CHROMATIN IN THE GAMETES

A NOVEL IN VIVO MODEL SYSTEM OF MELANOMA USING A HUMAN-MOUSE NEURAL CREST CELL CHIMERA

POST-TRANSCRIPTIONAL GENE REGULATION DURING EARLY DROSOPHILA DEVELOPMENT

EPITHELIAL-MESENCHYMAL TRANSITION IN STEM CELL INDUCTION AND MAINTENANCE

INVESTIGATING ALPHA-SYNUCLEIN OLIGOMER TOXICITY IN SYNUCLEINOPATHIES.

UNDERSTANDING THE MECHANISMS OF AGE-DEPENDENT DECLINE OF INTESTINAL STEM CELL FUNCTION

VARIATIONS IN LIPID HOMEOSTASIS AND SIGNALING AFFECT ALPHA-SYNUCLEIN PATHOBIOLOGY

INVESTIGATING THE PRION FORMATION OF NEURONAL CPEB

THE IN VIVO REGULATION OF GLUCOSE HOMEOSTASIS AND LIFESPAN BY MTORC2

MOLECULAR MECHANISM AND IN VIVO STUDY OF ER ASSOCIATED DEGRADATION

DISCOVERY OF SMALL MOLECULE INHIBITORS OF C-MYC/MAC DIMERIZATION AND DNA BINDING

IDENTIFICATION OF COMPOUNDS THAT REVERSE CELLULAR TOXICITY OF A BETA PEPTIDE IN A

IDENTIFYING SPECIES-SPECIFIC ANTI-MALARIAL HSP90 INHIBITORS USING GENETICALLY ENG

IDENTIFICATION OF MALARIA HSP40 CHAPERONE INHIBITORS IN YEAST

INHIBITORS OF SERINE BIOSYNTHESIS

FOLDING OF N-RICH ERYTHROCYTE REMODELING PROTEINS OF MALARIAL PARASITE

METAL ION TRANSPORT IN PARKINSON'S DISEASE

YEAST MITOCHONDRIAL ENGINEERING: A NEW TECHNOLOGY FOR SYNTHETIC BIOLOGY AND METAB

FUNCTIONAL ANALYSIS OF MDIA FORMINS IN HEMATOPOIETIC STEM CELL ENGRAFTMENT AND MI

MODELING DNA METHYLATION CHANGES IN ALZHEIMER'S DISEASE USING INDUCED HUMAN PLURIPOTENT STEM CELLS.

UTILIZING SINGLE CELL APPROACHES TO DECIPHER THE MECHANISM UNDERLYING SOMATIC CEL

PREDICTIVE MODELING OF MAMMALIAN CELL FATE TRANSITIONS OVER TIME AND SPACE WITH SINGLE-CELL GENOMICS - PROJECT SUMMARY DESPITE REMARKABLE ADVANCES IN SINGLE-CELL PROFILING, MACHINE LEARNING AND SYSTEMS BIOLOGY, OUR ABILITY TO EXPLOIT THESE MEASUREMENTS IS LIMITED BY THE LACK OF AN APPROPRIATE FRAMEWORK TO MODEL AND ANALYZE THEM. IN THIS APPLICATION, I PROPOSE AN ORGANIC SYNTHESIS OF EXPERIMENTAL TECHNOLOGICAL DEVELOPMENT, MATHEMATICAL MODELING, AND MACHINE LEARNING ALGORITHM INNOVATIONS TO MOVE BEYOND CONVENTIONAL DESCRIPTIVE AND MERELY STATISTICAL ANALYSES OF SINGLE CELLS TO MECHANISTIC AND PREDICTIVE MODELING OF CELL FATE TRANSITION OVER TIME AND SPACE, AND ACROSS TRANSCRIPTOMIC, EPIGENETIC AND PROTEOMIC LEVELS. FIRSTLY, IN ORDER TO UNVEIL THE REGULATORY NETWORKS THAT GOVERN THE MAINTENANCE OF STEM CELLS AND PROGENITORS, I WILL EXTEND THE DYNAMO FRAMEWORK THAT PUBLISHED RECENTLY TO PREDICT KEY REGULATORS THAT STABILIZE OR DESTABILIZE CELLS STATES, E.G. THE HEMATOPOIETIC STEM CELL STATE, VIA SENSITIVITY ANALYSES OF THE RECONSTRUCTED VECTOR FIELD. IN ADDITION, I WILL BUILD UPON THE CURRENT SUCCESS OF PREDICTING A BROAD RANGE OF HEMATOPOIETIC CELL FATE TRANSITIONS WITH OUR LEAST ACTION PATH APPROACH TO EXTEND IT TO STUDY OTHER BIOLOGICAL SYSTEMS, SUCH AS PANCREATIC ENDOCRINOGENESIS. TO VALIDATE THESE PREDICTIONS, I WILL CONTINUE MY ONGOING COLLABORATION WITH DR. VIJAY SANKRAN’S LAB (CO-MENTOR LAB) TO FIRST IMPLEMENTED METABOLIC LABELING BASED SCRNA-SEQ WITH THE 10X CHROMIUM SYSTEM AND INTEGRATE IT WITH PERTURB-SEQ THAT CHAMPIONED BY THE WEISSMAN LAB (MY MENTOR LAB) TO TEST THE PREDICTED FACTORS’ EFFICACY IN MAINTAINING THE HSC STATE. SECOND, I WILL DEVELOP NEW APPROACHES TO SEAMLESSLY INTEGRATE MULTI-OMICS AND HARMONIZE SHORT-TERM RNA VELOCITIES WITH LONG-TERM LINEAGE TRACING. BY DOING SO, WE CAN ENABLE EVEN MORE ACCURATE MODELING OF SINGLE CELL FATE TRANSITIONS THAT CONSIDER LINEAGE-RESOLVED, EPIGENETIC, PROTEOMIC KINETICS, OFFERED BY CUTTING-EDGE SINGLE-CELL GENOMIC TECHNOLOGIES AND CUTTING-EDGE DEEP LEARNING METHODS. LASTLY, I WILL TAKE ADVANTAGE OF MY EARLY ACCESS OF MOUSE EMBRYOGENESIS DATASET PROFILED WITH THE POWERFUL STEREO-SEQ THROUGH MY CLOSE COLLABORATION WITH BGI RESEARCH TO BUILD 3D IN SILICO SPATIOTEMPORALLY MODELS OF MAMMALIAN ORGANOGENESIS. I WILL ALSO TRAIN MYSELF TO STUDY OTHER STATE-OF-THE-ART IN-SITU SEQUENCING APPROACHES, FOR EXAMPLE THE STAR-MAP METHOD FROM MY COLLABORATOR, DR. XIAO WANG FROM BROAD. THROUGH THE K99 PHASE OF THIS PROPOSED CAREER DEVELOPMENT PLAN, I WILL DEVELOP NEW COMPUTATIONAL TOOLKITS AND FURTHER STRENGTHEN MY EXPERIMENT SKILLS, BOTH IN HUMAN HEMATOPOIESIS, PERTURB-SEQ AND SPATIAL TRANSCRIPTOMICS. WHEN COMBINING THESE NEW SKILLS WITH MY RIGOROUS TRAINING IN SYSTEMS BIOLOGY, AND SINGLE CELL GENOMICS, I WILL BE BETTER PREPARED TO TRANSITION INTO AN INDEPENDENT INVESTIGATOR IN A TOP-TIER RESEARCH UNIVERSITY. UNDOUBTEDLY, MY RESEARCH AND CAREER DEVELOPMENT DURING BOTH K99 PHASE AND MY TRANSITION TO R00 PHASE WILL BE GREATLY FACILITATED THANKS TO THE EXCELLENT RESEARCH ENVIRONMENT IN WHITEHEAD INSTITUTE, BROAD AND HARVARD STEM CELL INSTITUTE. TO SUM UP, MY PROPOSED STUDY WILL PAVE THE ROAD TO LAUNCH MY FUTURE INTERDISCIPLINARY TEAM THAT AIMS AT BUILDING MECHANISTIC AND PREDICTIVE MODELS OF CELL FATE TRANSITIONS WITH A FOCUS IN HUMAN HEMATOPOIESIS.

DEVELOPIN IN-VITRO MODELS OF A BETA TOXICITY: COMPLEMENTARY YEAST AND HUMAN STEM CELL APPROACHES