Stowers Institute For Medical Research

ISOLATION AND CHARACTERIZATION OF INTESTINAL STEM CELLS

GENETIC MAPPING OF FUNCTIONAL VOMERONASAL CIRCUIT

MECHANISMS OF TRANSCRIPTION REGULATION IN CHROMATIN

INVESTIGATION OF EXPERIENCE-DEPENDENT POST TRANSCRIPTIONAL REGULATION OF DROSOPHI

ZEBRAFISH SENSORY HAIR CELL REGENERATION

THE MOLECULAR BASIS OF PLANARIAN REGENERATION

STARVATION RESISTANCE AND RESILIENCE OF METABOLIC DYSFUNCTION IN CAVEFISH

INVESTIGATING DEVELOPMENTAL POTENTIAL BASED ON GENOME-WIDE CHROMATIN STATUS

MODELS OF SELFISHNESS: MOLECULAR AND EVOLUTIONARY ANALYSES OF THE WTF MEIOTIC DRIVERS

INTRINSIC AND EXTRINSIC REGULATION OF CRANIAL MESODERM

A TRANSPOSASE SYSTEM FOR INTEGRATIVE CHIP-EXO AND ATAC-SEQ ANALYSIS AT SINGLE-CELL RESOLUTION

STRUCTURE AND FUNCTION OF MAMMALIAN CPEB2 AGGREGATES IN NORMAL AND AD BRAIN - PROJECT SUMMARY THE AGGREGATE FAILURE OF PRE-CLINICAL AND CLINICAL TRIALS IN AD HAS DEMONSTRATED THAT AN IMPROVED FUNDAMENTAL UNDERSTANDING OF MEMORY AND HOW THE MOLECULAR COMPONENTS OF MEMORY ARE ALTERED IN THE AD DISEASE PROCESS IS NECESSARY TO DEVELOP EFFECTIVE TREATMENT. THE BROAD OBJECTIVE OF THE PROJECT IS TO IDENTIFY THE BIOCHEMICAL SUBSTRATES OF LONG-LASTING MEMORIES IN MAMMALS. THE CURRENT PROPOSAL FOCUSES ON A FAMILY OF RNA-BINDING, CYTOPLASMIC POLYADENYLATION ELEMENT BINDING PROTEIN (CPEB), THAT STABILIZES MEMORY IN INVERTEBRATES AND MICE. REMARKABLY, CPEB FAMILY PROTEIN FORMS NON-DISEASE-CAUSING AMYLOIDOGENIC AGGREGATES AND AGGREGATION OF CPEB IS NECESSARY TO STABILIZE MEMORY. AS AMYLOIDS ARE TYPICALLY LINKED TO DISEASE STATES, THE QUESTION REMAINS HOW SIMILARLY STRUCTURED ASS42 OR TAU PROTEINS CAN HAVE OPPOSING EFFECTS ON MEMORY. THEREFORE, TO DEVELOP A BETTER UNDERSTANDING OF THE RELATIONSHIP BETWEEN AMYLOIDS THAT SUPPORT MEMORY AND AMYLOIDS THAT DISRUPT MEMORY, WE WILL USE A VARIETY OF TECHNIQUES TO SOLVE THE STRUCTURE AND FUNCTION OF THE CPEB FAMILY MEMBERS, CPEB2 AND CPEB3, IN HUMAN AND MICE. IN AIM 1, WE WILL USE CRYO- ELECTRON MICROSCOPY TO SOLVE THE STRUCTURE OF CPEB AGGREGATES FROM FRESH HUMAN FRONTOTEMPORAL LOBE TISSUE COLLECTED FROM 25-50-YEAR-OLD HUMAN SUBJECTS UNDERGOING TISSUE REMOVAL UNDER THE STANDARD OF CARE FOR THEIR DISEASE. THESE TISSUES WOULD HAVE BEEN OTHERWISE DISCARDED. IN AIM 2, MICE LACKING THE ABILITY TO FORM AGGREGATES OF CPEB2 AND CPEB3 WILL BE TRAINED AND TESTED IN A ONE-TRIAL INHIBITORY AVOIDANCE TASK TO ASSAY THEIR ABILITY TO FORM, MAINTAIN, AND RECALL MEMORY. IN AIM 2 WE WILL ALSO INVESTIGATE THE CONSEQUENCE OF CPEB2 AND CPEB3 AGGREGATION IN TRANSLATION OF MRNA ENCODING SYNAPTIC PROTEINS. THE RESULTS WOULD BE THE FIRST TO PROVIDE DIRECT STRUCTURAL ANALYSIS OF A FUNCTIONAL AMYLOID LINKED TO MEMORY IN MAMMALS, THE STRUCTURAL DISTINCTIONS, IF ANY, BETWEEN FUNCTIONAL AND TOXIC AMYLOID IN THE HUMAN BRAIN, AND PRECISELY LINK CPEB2 AND CPEB3 AGGREGATION AND ACTIVITY TO ANIMALS’ ABILITY TO FORM OR STABILIZE MEMORY. THIS KNOWLEDGE WOULD PROVIDE THE FOUNDATION TO INVESTIGATE IN THE FUTURE HOW TOXIC AMYLOIDS OF ASS42 OR TAU SPECIFICALLY PERTURB MEMORY.

SMALL TRANSLATED ORFS IN THE 3'UTR ENHANCE TRANSLATION IN VERTEBRATES - PROJECT SUMMARY THE PREVAILING DOCTRINE THAT MESSENGER RNAS (MRNAS) IN HIGHER ORGANISMS ENCODE FOR A SINGLE PROTEIN HAS UNDERGONE A DRAMATIC REVISION IN RECENT YEARS. RIBOSOME AND PROTEOMIC PROFILING HAVE REVEALED A LARGE NUMBER OF SMALL TRANSLATED OPEN READING FRAMES (ORF) WITHIN PREVIOUSLY DESCRIBED “UNTRANSLATED REGIONS” (UTRS) AND LONG NON-CODING RNAS. INDEED, SOME OF THE PEPTIDES DERIVED FROM SMALL ORFS HAVE BEEN IMPLICATED IN VARIOUS FUNDAMENTAL PROCESSES (E.G., DEVELOPMENT). TRANSLATION OF SMALL ORFS IN THE 5’UTR, KNOWN AS UPSTREAM-ORFS (UORFS), HAS BEEN SHOWN TO HAVE A PROFOUND REGULATORY EFFECT ON GENE REGULATION, INDEPENDENT OF THE ENCODED PEPTIDE. FURTHER, TRANSLATION OF UORFS VARY UNDER PATHOLOGIC CONDITIONS SUCH AS CANCER, AND MUTATIONS AFFECTING UORFS ARE ASSOCIATED WITH VARIOUS HUMAN DISEASES. WE AND OTHERS HAVE ALSO INDICATED THE EXISTENCE OF TRANSLATED SMALL ORFS IN THE 3’UTR KNOWN AS DOWNSTREAM OPEN READING FRAMES (DORFS) IN HUMAN CELLS AND ZEBRAFISH EMBRYOS. HOWEVER, CONTRARY TO UORFS, THERE HAS BEEN NO SYSTEMATIC STUDY OF DORF FUNCTIONS, AND THEIR RELATIONSHIP TO HUMAN HEALTH AND DISEASE REMAINS UNTESTED. FURTHER, GIVEN THEIR LOCATION IN THE 3’UTR, THE MOLECULAR MECHANISM BY WHICH DORFS ENGAGE THE TRANSLATIONAL MACHINERY REMAIN COMPLETELY UNKNOWN. OUR LONG-TERM GOAL IS TO UNDERSTAND HOW POST-TRANSCRIPTIONAL REGULATION (MRNA HALF-LIFE AND TRANSLATION) SHAPES GENE EXPRESSION IN VERTEBRATES, AND ITS IMPACT ON HUMAN DISEASE. THE CENTRAL HYPOTHESIS OF THIS APPLICATION IS THAT TRANSLATION OF DORFS REGULATES GENE EXPRESSION. OUR PRELIMINARY DATA STRONGLY INDICATE THAT, CONTRARY TO UORFS, DORFS STRONGLY ENHANCE TRANSLATION OF THE CANONICAL ORF AND EMERGE AS AN UNCHARACTERIZED AND POTENT REGULATORY MECHANISM ACROSS VERTEBRATES. THE OBJECTIVES ARE TO: 1) IDENTIFY FACTORS INVOLVED IN ENHANCING TRANSLATION OF THE MAIN ORF. 2) DISSECT THE REGULATORY INFORMATION DRIVING DORF TRANSLATION, AND 3) CHARACTERIZE THE BIOLOGICAL IMPACTS OF DORF-MEDIATED REGULATION. THE RATIONALE FOR THE PROPOSED RESEARCH IS TO GAIN A MECHANISTIC UNDERSTANDING OF DORF-MEDIATED REGULATION IN ORDER TO ASSESS THE POSSIBLE BIOLOGICAL IMPORTANCE OF DORF DYSREGULATION UNDER STRESS OR DISEASE CONDITIONS. THIS PROPOSAL IS CONCEPTUALLY INNOVATIVE AS IT IS BASED ON THE EXPLORATION OF A NOVEL, YET WIDESPREAD AND POTENT TRANSLATION REGULATORY MECHANISM CONSERVED ACROSS VERTEBRATES. TECHNICALLY, THIS PROPOSAL WILL COMBINE GENOMIC PROFILES (RNA-SEQ, RIBOSOME PROFILING); REPORTER (CYTOMETRY); BIOCHEMISTRY TOOLS: RNA PULLDOWNS FOLLOW BY PROTEOMICS, CRISPR-CAS-9 AND - 12A (TO EDIT) AND OUR NOVEL CAS13D TOOL (KNOCK-DOWN IN EMBRYOS); COMBINING HUMAN CELL AND ZEBRAFISH EMBRYOS. THE OUTCOMES FROM THIS PROJECT WILL HELP UNDERSTAND HOW DORFS ARE TRANSLATED, SHAPE GENE EXPRESSION AND GENERATE PHENOTYPES. THIS NOVEL FUNCTION OF THE RIBOSOME ADDS TO THE RECENTLY EMERGING REGULATORY EFFECTS OF TRANSLATION ON GENE EXPRESSION (E.G. UORF, CODON OPTIMALITY). UNDERSTANDING DORF BIOLOGY WILL PROVIDE AN ENTRY POINT AND PERHAPS EVEN A DIAGNOSTIC TOOL TO ASSOCIATE MUTATIONS WITH HUMAN DISEASES. IDENTIFYING THE MOLECULAR MACHINERY INVOLVED IN THIS PATHWAY MIGHT PROVIDE TARGETS FOR THERAPEUTIC INTERVENTIONS.

HOW CELLS MONITOR THE INTEGRITY OF THEIR TRANSLATION APPARATUS

MAINTAINING THE INTEGRITY OF A GENOME - PROJECT SUMMARY/ABSTRACT IN ORDER TO FULLY GRASP THE MOLECULAR ORIGINS OF GENOME INSTABILITY, THE FIELD MUST UNDERSTAND AT A MOLECULAR LEVEL HOW CENTROMERES WORK TO PROMOTE THE STABLE TRANSMISSION OF CHROMOSOMES. GENOME INSTABILITY UNDERLIES A VARIETY OF HUMAN PATHOLOGIES, INCLUDING CANCER AND REPRODUCTIVE AGING. OUR LONG-TERM GOAL IS TO DETERMINE HOW THE EVOLUTIONARILY CONSERVED COHESIN COMPLEX MAINTAINS GENOME INTEGRITY THROUGH ITS ROLES IN CHROMOSOME SEGREGATION, CHROMOSOME ORGANIZATION, AND DOUBLE-STRAND BREAK REPAIR. LOSS OF SISTER CHROMATID COHESION IS SPECULATED TO BE A MAJOR CONTRIBUTOR TO CHROMOSOME INSTABILITY. THE OBJECTIVE OF THIS APPLICATION IS TO PRODUCE A MOLECULAR MODEL FOR HOW COHESIN OPERATES AT INDIVIDUAL HUMAN CENTROMERES TO ACHIEVE CENTROMERIC COHESION AND ACCURATE CHROMOSOME SEGREGATION. THE CENTRAL HYPOTHESIS IS THAT COHESIN AND DNA CATENATION TOGETHER CREATE CENTROMERE-UNIQUE LANDSCAPES OF SISTER CHROMATID COHESION TO PREVENT CHROMOSOME INSTABILITY. THE VARIATION IN HUMAN CENTROMERES AND CENTROMERIC COHESION MAY THEREFORE IMPACT THE TRANSMISSION OF EACH CHROMOSOME. WE WILL TEST THE IDEA THAT CENTROMERE-SPECIFIC COHESION MUST BE CONSIDERED AS A GENETIC DETERMINANT OF SISTER CHROMATID COHESION AND SEGREGATION IN ORDER TO HAVE A COMPLETE MODEL FOR HOW CHROMOSOMAL INSTABILITY OCCURS THROUGH TWO SPECIFIC AIMS: 1) DISCOVER THE LANDSCAPE OF CENTROMERIC COHESION AT INDIVIDUAL HUMAN CENTROMERES AND 2) EXAMINE HOW CHROMOSOME CENTROMERIC COHESION MAINTAINS EUPLOIDY. UNDER THE FIRST AIM, CALIBRATED PAIRED-END CHIP SEQ WILL BE USED TO MAP COHESIN BINDING RELATIVE TO KINETOCHORE PROTEINS AND HUMAN CENTROMERIC ARRAYS IN HUMAN TISSUE CULTURE CELLS. THIS APPROACH WILL BE COMPLEMENTED BY SUPERRESOLUTION IMAGING OF THE SAME THREE COMPONENTS (CENTROMERES, KINETOCHORES, AND COHESIN) IN CELLS, AND WILL INCLUDE IMAGING-BASED DETERMINATION OF CENTROMERE-SPECIFIC COHESION. TOGETHER THESE APPROACHES WILL PRODUCE A LINEAR AND 3D MAP OF COHESION WITHIN AND AROUND INDIVIDUAL HUMAN CENTROMERES. IN THE SECOND AIM WE WILL EXAMINE HOW CENTROMERE-SPECIFIC PATTERNS OF CENTROMERIC COHESION PREVENT CHROMOSOME MISSEGREGATION EVENTS IN CULTURED CELLS AND IN XENOGRAFT TUMOR TISSUE. THE OUTCOME WILL BE FUNDAMENTAL PRINCIPLES OF CENTROMERIC ARRAY-BASED COHESION FATIGUE AND RESULTING PATTERNS OF CHROMOSOME INSTABILITY. THE RESEARCH IS INNOVATIVE BECAUSE IT INCORPORATES THE LATEST INFORMATION ON HUMAN CENTROMERIC DNA ARRAYS, A NEW WORKING MODEL FOR THE ORGANIZATION OF CENTROMERIC DNA BY COHESION, AND NEW QUANTITATIVE MOLECULAR, GENOMIC, AND IMAGING TOOLS TO PROBE HOW CENTROMERIC COHESION ENFORCES ACCURATE SISTER CHROMATID SEGREGATION. THE PROPOSED RESEARCH IS SIGNIFICANT BECAUSE CENTROMERIC ARRAYS MAY BE UNRECOGNIZED GENETIC DETERMINANTS OF CHROMOSOME INSTABILITY. MANY TYPES OF CANCER ARE ASSOCIATED WITH SEEMINGLY RANDOM PATTERNS OF INSTABILITY THAT MAY HAVE MOLECULAR ORIGINS IN UNIQUE CENTROMERIC COHESION PROFILES. FURTHERMORE, MANY CANCERS ARE ASSOCIATED WITH MUTATIONS THAT IMPACT CHROMOSOME SEGREGATION MACHINERY, SUCH AS COHESIN. THE OUTCOME OF THIS PROJECT WILL BE A MORE COMPLETE PICTURE OF THE MECHANISMS UNDERLYING CHROMOSOMAL INSTABILITY.

PHYSIOLOGICAL ROLE OF MLL AND ELL PROTEINS IN LEUKEMIA

IDENTIFICATION OF GENES ASSOCIATED WITH MYOBLAST FUSION

IN VIVO ANALYSIS OF THE MECHANISMS OF NEURAL CREST MIGRATION

CONTRIBUTIONS OF PROTEIN AGGREGATION TO GENE REGULATION AND PHENOTYPIC DIVERSITY

INHIBITOR REPROGRAMMING OF A HUMAN HISTONE DEACETYLASE PROTEIN INTERACTION NETWORK

BIOCHEMISTRY OF EUKARYOTIC MESSENGER RNA SYNTHESIS

MOLECULAR MECHANISMS OF CHROMOSOME SEGREGATION IN YEAST

MECHANISMS OF TRANSCRIPTIONAL REGULATION IN CHROMATIN

MITOTIC ROUNDING AND PLANAR SPINDLE ALIGNMENT IN PROLIFERATING EPITHELIA

MOLECULAR MECHANISMS AND EVOLUTIONARY IMPACTS OF THE WTF MEIOTIC DRIVERS - PROJECT SUMMARY/ABSTRACT GENOMES ARE PLAGUED BY PARASITIC DNA SEQUENCES THAT DO NOT PROMOTE HEALTH OR FERTILITY. MEIOTIC DRIVE GENES ARE ONE SUCH CLASS OF GENETIC PARASITE THAT SELFISHLY MANIPULATE GAMETOGENESIS TO INCREASE THEIR OWN TRANSMISSION INTO THE NEXT GENERATION. RATHER THAN BEING PASSED TO HALF OF AN INDIVIDUAL’S OFFSPRING, LIKE REGULAR ALLELES, MEIOTIC DRIVERS MANIPULATE GAMETOGENESIS TO ENSURE THEIR TRANSMISSION TO MOST, OR EVEN ALL, THE OFFSPRING. DRIVERS ARE FOUND THROUGHOUT EUKARYOTES, INCLUDING HUMANS, BUT MANY MORE LIKELY REMAIN TO BE IDENTIFIED. CHARACTERIZING MEIOTIC DRIVERS IS IMPORTANT BECAUSE THESE PARASITES CAN HAVE MAJOR IMPACTS ON FERTILITY AND HEALTH. MEIOTIC DRIVERS CAN CONTRIBUTE TO INFERTILITY DIRECTLY BY DISRUPTING MEIOTIC CHROMOSOME SEGREGATION OR BY DESTROYING GAMETES THAT INHERIT THE COMPETING ALLELE. MEIOTIC DRIVERS CAN ALSO CAUSE INFERTILITY OR OTHER DISEASE STATES INDIRECTLY BY PROMOTING THE SPREAD OF LINKED DELETERIOUS (E.G., DISEASE-ASSOCIATED) ALLELES. DESPITE THEIR LARGE IMPACT, THERE IS RELATIVELY LITTLE MOLECULAR UNDERSTANDING OF MEIOTIC DRIVERS OR THEIR EVOLUTIONARY IMPACTS. HOWEVER, THERE ARE EMERGING THEMES THAT UNITE MANY KNOWN DRIVE SYSTEMS, INCLUDING THE USE OF A POISON/ANTIDOTE MECHANISM AND THE ASSOCIATION OF MEIOTIC DRIVE GENES WITH DISTRIBUTED DNA SEQUENCE REPEATS. THIS PROPOSAL EXPLOITS A HIGHLY TRACTABLE MODEL, THE WTF GENE FAMILY FOUND IN FISSION YEASTS, TO INVESTIGATE THE MOLECULAR MECHANISMS AND EVOLUTIONARY IMPACTS OF DRIVE SYSTEMS. THE WTF GENES ENACT DRIVE BY DESTROYING THE WTF- GAMETES PRODUCED BY WTF+/WTF- HETEROZYGOTES. EACH WTF DRIVER ENCODES BOTH A POISON PROTEIN AND A SEPARATE ANTIDOTE PROTEIN ON OVERLAPPING CODING SEQUENCES. ALL DEVELOPING GAMETES ARE POISONED, BUT THOSE THAT INHERIT THE WTF+ ALLELE ARE RESCUED BY THE ANTIDOTE. THIS PROPOSAL AIMS TO UNDERSTAND THE MECHANISMS UNDERLYING THE TOXICITY OF WTF POISON PROTEINS AND HOW THE WTF ANTIDOTE PROTEINS RESCUE THAT TOXICITY. ANALOGOUS TO OTHER DRIVE SYSTEMS, THE WTF GENES ARE FLANKED BY SHORT REPETITIVE DNA SEQUENCES (TRANSPOSON-DERIVED REPEATS OR 5S RDNA GENES). THE PROPOSED WORK WILL TEST THE IDEA THAT THE REPEATS FLANKING WTF GENES AFFECT THEIR EVOLUTION BY PROMOTING NON-ALLELIC GENE CONVERSION. FINALLY, THE PROPOSED RESEARCH PROGRAM WILL EXPLORE THE EVOLUTIONARY IMPACT OF THE WTF GENES ON THE FLANKING 5S RDNA GENES. SPECIFICALLY, WE WILL TEST THE HYPOTHESIS THAT THE WTF DRIVERS HAVE PROMOTED THE MAINTENANCE OF DELETERIOUS VERSIONS OF THE 5S RDNA GENES, WHICH ENCODE AN ESSENTIAL COMPONENT OF RIBOSOMES. THIS RESEARCH PROGRAM WILL GREATLY EXPAND OUR UNDERSTANDING OF THE MOLECULAR MECHANISMS AND MOLECULAR EVOLUTION OF MEIOTIC DRIVE SYSTEMS. THIS KNOWLEDGE WILL HELP GUIDE THE SEARCH FOR AND THE MOLECULAR CHARACTERIZATION OF MEIOTIC DRIVERS IN MORE COMPLEX SYSTEMS, INCLUDING HUMANS. MORE BROADLY, THIS WORK WILL EXPAND OUR UNDERSTANDING OF DNA PARASITES AND HOW THEY CAN DIRECTLY AND INDIRECTLY IMPACT HEALTH, PARTICULARLY INFERTILITY. THIS EXPANDED UNDERSTANDING SHOULD ULTIMATELY LEAD TO IMPROVED REPRODUCTIVE OUTCOMES IN HUMANS.

ASSEMBLY AND REGULATION OF YEAST SPINDLE POLES

ELUCIDATING MECHANISMS OF AMYLOID NUCLEATION IN VIVO

MECHANISMS OF TRANSCRIPTIONAL REGULATION IN CHROMATIN

ANALYSIS OF METAZOAN SAGA COMPLEX FUNCTION IN GENE EXPRESSION

INVESTIGATION OF NOTCH SIGNALING IN THE REGULATION OF CILIARY BODY DEVELOPMENT AND FUNCTION

ANALYSIS OF HAIR CELL REGENERATION IN ZEBRAFISH

MOLECULAR MECHANISMS REGULATING DROSOPHILA OVARIAN GERMLINE STEM CELLS

BIOCHEMISTRY OF EUKARYOTIC MESSENGER RNA SYNTHESIS

MECHANISMS OF MEIOTIC DRIVE AND THE FUNCTIONAL CONSEQUENCES OF RAPID GENOME EVOLUTION

GENE REGULATORY NETWORKS OF SYNAPTIC SPECIFICITY - PROJECT SUMMARY/ABSTRACT NEURONAL TYPE IDENTITY IS CENTRAL TO THE DEVELOPMENT AND FUNCTION OF NEURAL CIRCUITS, AS IT INSTRUCTS BOTH THE CONNECTIVITY OF NEURONS AS WELL AS THEIR SYNAPTIC AND ELECTROPHYSIOLOGICAL PROPERTIES. NEURONAL FATES ARE THOUGHT TO BE CONTROLLED BY COMBINATIONS OF TRANSCRIPTION FACTORS (TF) KNOWN AS TERMINAL SELECTORS, BUT VERY LITTLE IS KNOWN ABOUT THE GENE REGULATORY MECHANISMS THAT LINK DIFFERENTIAL TF EXPRESSION TO SPECIFIC NEURONAL FEATURES. THE DROSOPHILA VISUAL SYSTEM, A WELL-CHARACTERIZED BRAIN REGION THAT HAS AN ORGANIZATION ANALOGOUS TO THE VERTEBRATE RETINA AND CORTEX, PROVIDES THE IDEAL BALANCE OF COMPLEXITY AND ACCESSIBILITY TO INVESTIGATE THESE MECHANISMS. THE FIRST AIM OF THIS PROJECT WILL BE TO DECIPHER THE TERMINAL SELECTOR TF CODES THAT ESTABLISH AND MAINTAIN THE UNIQUE IDENTITY OF APPROXIMATELY 200 NEURONAL TYPES THAT MAKE THE DROSOPHILA OPTIC LOBES. USING A SINGLE-CELL RNA SEQUENCING (SCRNA-SEQ) DATASET I GENERATED FROM DEVELOPING OPTIC LOBES, I IDENTIFIED THE COMBINATIONS OF TFS THAT ARE STABLY MAINTAINED IN EACH NEURONAL TYPE THROUGHOUT THEIR DIFFERENTIATION. UNDER THE MENTORSHIP OF CLAUDE DESPLAN (K99 PHASE), I WILL TEST THE HYPOTHESIS THAT THESE TFS FUNCTION AS TERMINAL SELECTORS BY MODIFYING THE TF CODES OF SPECIFIC OPTIC LOBE NEURONS IN VIVO, WITH THE GOAL OF PREDICTABLY TRANSDIFFERENTIATING THEM INTO OTHER CELL TYPES. THIS WILL DEMONSTRATE THE SUFFICIENCY OF TERMINAL SELECTORS TO CONFER NEURONAL IDENTITY AND BENEFIT THE FIELD OF REGENERATIVE MEDICINE. THE CONSERVED MECHANISMS IN MAMMALIAN SYSTEMS COULD BE EXPLOITED TO INDUCE DIFFERENTIATION OF PLURIPOTENT CELLS INTO SPECIFIC NEURONS THAT COULD BE TRANSPLANTED TO TREAT BLINDNESS OR NEURODEGENERATION. THE SECOND AIM OF THIS PROJECT WILL LINK THE TERMINAL SELECTOR TFS TO THEIR DOWNSTREAM TARGETS. IN COLLABORATION WITH RICHARD BONNEAU, I WILL LEARN TO USE THE “INFERELATOR” ALGORITHM TO GENERATE COMPUTATIONAL MODELS OF GENE REGULATORY NETWORKS BY COMBINING MY EXISTING SCRNA-SEQ DATA WITH NEW CHROMATIN ACCESSIBILITY (SCATAC-SEQ) DATA I WILL ACQUIRE. DURING THE K99 PHASE, I WILL TEST THE EFFECTS OF PERTURBATING KEY PREDICTED DOWNSTREAM EFFECTORS ON THE MORPHOLOGY AND CONNECTIVITY OF A SELECT GROUP OF NEURONS TO ESTABLISH PROOF-OF- CONCEPT. I WILL THEN GENERALIZE THIS APPROACH IN THE R00 PHASE BY INFERRING GENE REGULATORY NETWORKS FOR ALL OPTIC LOBE NEURONS AT MULTIPLE DEVELOPMENTAL STAGES. THE THIRD AIM WILL BE PERFORMED IN MY INDEPENDENT LAB (R00) TO UTILIZE THE NETWORK MODELS FOR ENGINEERING PRECISE MODIFICATIONS IN VISUAL CIRCUITS. I WILL SEEK TO SELECTIVELY UNCOUPLE THE CIRCUIT THAT COMPUTES WIDE-FIELD MOTION FROM THE ONE THAT DETECTS SMALL MOVING OBJECTS. I WILL USE SYNAPTIC TRACING METHODS AS WELL AS INTRAVITAL CALCIUM IMAGING TO DEMONSTRATE THE FUNCTIONAL CONSEQUENCES OF DEVELOPMENTAL PERTURBATIONS. ALTOGETHER, THIS PROJECT WILL ESTABLISH DIRECT MECHANISTIC LINKS BETWEEN THE ENCODING OF NEURONAL IDENTITY AND THE MOLECULES THAT MEDIATE INTERCELLULAR INTERACTIONS DURING SYNAPTIC PARTNER SELECTION, WHICH ARE COMMONLY AFFECTED IN NEURODEVELOPMENTAL DISORDERS. THE MENTORSHIP I WILL RECEIVE FROM DR. DESPLAN AND DR. BONNEAU, COMBINED WITH THE IMPRESSIVE RESOURCES OF NEW YORK UNIVERSITY PROVIDE THE IDEAL ENVIRONMENT FOR PREPARING ME TO BUILD A SUCCESSFUL INDEPENDENT RESEARCH PROGRAM THAT LINK GENE REGULATION TO BRAIN WIRING.

INVESTIGATION OF NICHE CONTROL OF GERMLINE STEM CELL LINEAGE DIFFERENTIATION

CRISPR-CAS13D: TRANSGENIC ZEBRAFISH LINES TOKNOCKDOWN MRNA - PROJECT SUMMARY DETERMINING THE FUNCTION OF GENES IS FUNDAMENTAL FOR UNDERSTANDING VERTEBRATE DEVELOPMENT, REGULATORY MECHANISMS AND HUMAN DISEASES. GENOME EDITING TECHNOLOGIES, SUCH CRISPR-CAS9, HAVE ALLOWED ASSOCIATING SPECIFIC PHENOTYPES TO PERMANENT GENE ALTERATION. HOWEVER, SOME KEY TECHNICAL AND CONCEPTUAL ISSUES REMAIN PROBLEMATIC IN VERTEBRATES, PARTICULARLY IN AQUATIC MODEL ORGANISMS SUCH ZEBRAFISH. FOR EXAMPLE, THE MATERNALLY PROVIDED MRNA CAN RESCUE THE PHENOTYPE OF HOMOZYGOUS MUTANTS; GENOTYPING STEPS ARE TEDIOUS; AND LONG NON- CODING RNA, LETHAL OR TISSUE/TEMPORAL GENES ARE DIFFICULT TO STUDY USING DNA MANIPULATION AS WELL AS COMPLEX GENOMIC LOCI. COMPLEMENTARY ‘KNOCK-DOWN’ APPROACHES ARE INVALUABLE TOOLS TO CIRCUMVENT SOME OF THESE PROBLEMS, HOWEVER, THERE WAS NO SYSTEMATIC TOOL TO KNOCKDOWN MRNAS IN ZEBRAFISH OR OTHER TELEOST FISH. OUR LONG-TERM GOAL IS TO UNRAVEL THE FUNCTION OF GENES RELATED TO REGULATORY MECHANISMS, DEVELOPMENT, AND HUMAN DISEASES. OUR RECENT PUBLICATION DEMONSTRATES THAT INJECTION OF THE CRISPR-RFXCAS13D SYSTEM INTO VERTEBRATE EMBRYOS PROVIDES A ROBUST AND COST-EFFECTIVE TECHNOLOGY TO SYSTEMATICALLY DISRUPT GENE FUNCTION. HOWEVER, THE INJECTION OF THIS SYSTEM ONLY PROVIDES TRANSIENT KNOCKDOWN FOR ~3 DAYS. THEREFORE, THE CENTRAL GOAL OF THE PROPOSAL IS TO TRANSFER THE CRISPR-RFXCAS13D TECHNOLOGY FROM BEING INJECTED INTO VERTEBRATE EMBRYOS, TO BEING ENDOGENOUSLY EXPRESSED THROUGH TRANSGENESIS. WE PROPOSE THAT THE DEVELOPMENT OF A REPERTOIRE TRANSGENIC ZEBRAFISH EXPRESSING THE RFXCAS13D ENZYME (UBIQUITOUS AND TISSUES-SPECIFIC) AND GUIDERNA WOULD FACILITATE RAPID AND VIGOROUS INVESTIGATION INTO GENE FUNCTIONS. OUR PRELIMINARY DATA INDICATE THAT TRANSGENIC EXPRESSION OF RFXCAS13D IN ZEBRAFISH IS FUNCTIONAL AND NOT TOXIC. THE OBJECTIVES ARE: 1) DEFINE THE OPTIMAL SYSTEM TO KNOCKDOWN MRNA EXPRESSION IN CRISPR-RFXCAS13D TRANSGENIC ZEBRAFISH. 2) KNOCKDOWN MRNA EXPRESSION IN A TISSUE-SPECIFIC MANNER USING SPECIFIC TRANSGENIC LINES. THIS PROPOSAL IS CONCEPTUALLY INNOVATIVE AS IT IS BASED ON THE EXPLORATION OF A NOVEL TECHNIQUE, CRISPR-RFXCAS13D, TO KNOCKDOWN MRNA IN A TISSUE-SPECIFIC MANNER IN ZEBRAFISH. THIS APPROACH HAS NEVER BEEN DONE IN VERTEBRATE MODEL SYSTEMS WHERE RNAI DOES NOT WORK. THE OUTCOMES OF THIS PROJECT WILL BE THE FIRST TRANSGENIC SYSTEM TO DISSECT AND STUDY GENE FUNCTION BY KNOCKING DOWN MRNA EXPRESSION IN ZEBRAFISH. OUR APPROACH WILL HELP THE SCIENTIFIC COMMUNITY INVESTIGATE GENE FUNCTION IN A FASTER AND TISSUE-TEMPORAL SPECIFIC MANNER, AS WELL AS ANSWERING QUESTIONS THAT ARE VERY CHALLENGING TO ADDRESS BY CURRENT METHODOLOGIES, SUCH AS THE FUNCTION OF NON-CODING RNAS OR PHENOTYPES CAUSED BY MULTIPLE GENES. MOREOVER, BY FOLLOWING THE GUIDELINES WE WILL OPTIMIZE THROUGH THIS PROJECT, RESEARCHERS MAY PRODUCE A WHOLE GUIDERNA COLLECTION, TARGETING ALL GENES, WHICH SHOULD BE AVAILABLE FOR ANYONE TO ORDER TO ADDRESS THEIR INDIVIDUAL RESEARCH QUESTIONS. FINALLY, AS WE HAVE SUCCESSFULLY IMPLEMENTED THE CRISPR-CAS13D SYSTEM BY INJECTION IN OTHER ORGANISMS SUCH AS MEDAKA, KILLIFISH, AND MOUSE EMBRYOS, OUR WORK DESCRIBED IN THIS PROPOSAL MAY SERVE AS THE FOUNDATION FOR TRANSFERRING THIS EFFICIENT KNOCKDOWN TECHNOLOGY INTO A RANGE OF OTHER SPECIES.

IN VIVO ANALYSIS OF TRKB SIGNALING DURING SYMPATHETIC NERVOUS SYSTEM DEVELOPMENT AND NEUROBLASTOMA PATHOGENESIS

FUNCTION OF MATERNAL MRNA DURING ZEBRAFISHEMBRYOGENESIS - PROJECT SUMMARY OUR RESEARCH FOCUSES ON UNRAVELING THE FUNDAMENTAL PROCESSES OF EARLY VERTEBRATE EMBRYOGENESIS, PARTICULARLY THE DYNAMIC MOLECULAR MECHANISMS THAT REGULATE MATERNAL AND ZYGOTIC RNAS DURING THE MATERNAL-TO-ZYGOTIC TRANSITION (MZT). THIS CRUCIAL PERIOD INVOLVES THE DEGRADATION OF MATERNAL RNAS AND ACTIVATION OF THE ZYGOTIC GENOME, PROCESSES ESSENTIAL FOR PROPER EMBRYONIC DEVELOPMENT. OUR GOAL IS TO DISSECT THE SPATIAL AND TEMPORAL REGULATION OF MATERNAL AND ZYGOTIC TRANSCRIPTS, IDENTIFY THE FUNCTIONS OF INDIVIDUAL MATERNAL RNAS, AND UNDERSTAND THEIR ROLES IN GENE EXPRESSION, INCLUDING MRNA STABILITY AND TRANSLATIONAL REGULATION. OUR RECENT WORK HAS ESTABLISHED TRANSFORMATIVE TOOLS AND FRAMEWORKS FOR INVESTIGATING EMBRYOGENESIS, INCLUDING THE DEVELOPMENT OF THE CRISPR-CAS13D SYSTEM FOR EFFICIENT MATERNAL RNA KNOCKDOWN IN ZEBRAFISH EMBRYOS. THIS SYSTEM HAS OVERCOME LIMITATIONS IN TRADITIONAL GENETIC APPROACHES, INCLUDING LETHAL AND/OR MASKED PHENOTYPES DUE TO MATERNAL CONTRIBUTION, ENABLING US TO SYSTEMATICALLY TARGET MATERNAL RNAS AND INVESTIGATE THEIR FUNCTIONS WITH UNPRECEDENTED PRECISION. THROUGH INTEGRATIVE MULTI-OMICS APPROACHES—SUCH AS RNA-SEQ, SLAM-SEQ, RIBOSOME PROFILING, AND QUANTITATIVE PROTEOMICS—WE HAVE MADE SIGNIFICANT PROGRESS IN UNDERSTANDING CODON-DEPENDENT MRNA STABILITY, MATERNAL-ZYGOTIC GENE EXPRESSION, AND THE INTERPLAY OF TRANSCRIPTION, TRANSLATION, AND PROTEIN ACCUMULATION DURING EARLY EMBRYOGENESIS. OVER THE NEXT FIVE YEARS, WE AIM TO EXPAND THESE EFFORTS BY ADDRESSING KEY QUESTIONS SURROUNDING THE SPATIAL AND TEMPORAL REGULATION OF MATERNAL RNAS AND THEIR INFLUENCE ON EARLY DEVELOPMENT. OUR STUDIES WILL FOCUS ON: MATERNAL RNA DECAY, LOCALIZATION AND FUNCTION DURING MZT: INVESTIGATING HOW MATERNAL RNAS, SUCH AS CTH1, ARE REGULATED BY SPATIAL AND CELL-SPECIFIC DECAY MECHANISMS. THIS WILL INCLUDE IDENTIFYING CIS-REGULATORY ELEMENTS IN THEIR UNTRANSLATED REGIONS AND CHARACTERIZING THE MOLECULAR FACTORS THAT MEDIATE THEIR LOCALIZATION AND STABILITY. LEVERAGING CRISPR-CAS13D AND MULTI-OMICS TECHNIQUES TO UNCOVER THE ROLES OF MATERNAL RNAS IN ZYGOTIC GENOME ACTIVATION AND EARLY CELLULAR PROCESSES. BROADER EXPLORATION OF MATERNAL RNAS: SYSTEMATICALLY INVESTIGATING ADDITIONAL MATERNAL GENES, INCLUDING GENES ENCODING SMALL TRANSLATED OPEN READING FRAMES, GENES WITH SPECIFIC TEMPORAL EXPRESSION AND SPATIALLY LOCALIZED TRANSCRIPTS WITH CRITICAL DEVELOPMENTAL ROLES. THE OVERARCHING VISION OF OUR RESEARCH IS TO ESTABLISH A COMPREHENSIVE UNDERSTANDING OF HOW GENE REGULATION IS ORCHESTRATED AT MULTIPLE LEVELS—SPATIAL, TEMPORAL, AND MOLECULAR—DURING THE EARLIEST STAGES OF VERTEBRATE DEVELOPMENT. OUR WORK HAS BROAD IMPLICATIONS FOR ADVANCING KNOWLEDGE IN DEVELOPMENTAL BIOLOGY, REPRODUCTIVE HEALTH, AND GENE REGULATION, WITH POTENTIAL APPLICATIONS IN AREAS SUCH AS FERTILITY, REGENERATIVE MEDICINE, AND MRNA-BASED THERAPEUTICS. BY INTEGRATING CUTTING-EDGE TECHNOLOGIES WITH INNOVATIVE APPROACHES, OUR RESEARCH PROGRAM IS POISED TO UNCOVER FUNDAMENTAL PRINCIPLES OF GENE REGULATION AND MRNA STABILITY, CONTRIBUTING TO A DEEPER UNDERSTANDING OF THE MOLECULAR PROCESSES THAT DRIVE THE TRANSFORMATION OF A SINGLE CELL INTO A MULTICELLULAR ORGANISM.

MULTILINEAGE DAMFRET TO INVESTIGATE AD/ADRD PROTEIN PHASE BEHAVIOR IN NEURAL TISSUE MODELS - PROJECT SUMMARY/ABSTRACT ALZHEIMER'S DISEASE AND RELATED DEMENTIAS (AD/ADRD) ARE THE MOST COMMON FORMS OF DEMENTIA. NO TREATMENTS EXIST TO STOP OR PREVENT THEM. AD/ADRD ARE CAUSED IN PART BY THE AGGREGATION OF ONE, OR MORE OFTEN A COMBINATION OF, SPECIFIC PROTEINS. TDP-43 IS ONE OF A HANDFUL OF SUCH PROTEINS. IT CHARACTERISTICALLY TRANSITIONS FROM A DYNAMIC LIQUID PHASE OF ASSEMBLY TO A RIGID AND PATHOGENIC PHASE IN A LARGE FRACTION OF DEMENTIA CASES. THIS TRANSITION OCCURS AGAINST A BACKDROP OF PROGRESSIVE CHANGES IN THE INTERACTIONS BETWEEN DIFFERENT CELL TYPES IN THE BRAIN. AN INTERDEPENDENCE OF THESE MOLECULAR AND CELLULAR CHANGES LIKELY DETERMINES THE CLINICAL COURSES OF AD/ADRD, BUT TO UNDERSTAND THAT INTERDEPENDENCE, WE NEED NEW TOOLS WITH WHICH TO COMPARE PROTEIN PHASE TRANSITIONS AND THEIR CORRESPONDING PHENOTYPE EFFECTS IN THE CONTEXT OF INTERACTING BRAIN CELL TYPES. WE PROPOSE HERE TO DEVELOP SUCH A TOOL BY MODIFYING A CELL-BASED BIOPHYSICAL METHOD, DISTRIBUTED AMPHIFLUORIC FRET (DAMFRET) FOR USE IN HUMAN INDUCED PLURIPOTENT STEM CELL (HIPSC)-DERIVED BRAIN TISSUE MODELS (AIM 1). WE WILL SIMULTANEOUSLY USE DAMFRET TO DEVELOP TDP-43 AS A REPORTER OF CELL TYPE-SPECIFIC DIFFERENCES IN THE HOMEOSTASIS OF PROTEIN PHASE TRANSITIONS (AIM 2). BY COMPLETING THESE AIMS, WE WILL HAVE CREATED AND VALIDATED A UNIQUELY POWERFUL TOOL FOR BETTER UNDERSTANDING THE COMPLEX MOLECULAR AND CELLULAR CAUSES OF AD/ADRD.

PATHOGENESIS, PHENOTYPIC VARIATION, AND PREVENTION OF CRANIOFACIAL ANOMALIES - PROJECT SUMMARY CRANIOFACIAL ANOMALIES ACCOUNT FOR ABOUT ONE-THIRD OF ALL BIRTH DEFECTS AND CONSEQUENTLY ARE A MAJOR CAUSE OF INFANT MORTALITY. TO DATE OVER 700 DISTINCT CRANIOFACIAL SYNDROMES HAVING BEEN DESCRIBED, AND ANOMALIES SUCH AS CLEFT LIP AND PALATE, MICROGNATHIA AND CRANIOSYNOSTOSIS HAVE SERIOUS LIFETIME FUNCTIONAL, AESTHETIC AND SOCIAL CONSEQUENCES THAT ARE DEVASTATING TO BOTH CHILDREN AND PARENTS ALIKE. COMPREHENSIVE SURGERY TOGETHER WITH WELL-COORDINATED AND INTEGRATED DENTAL CARE, PSYCHOLOGICAL COUNSELLING AND REHABILITATION CAN HELP AMELIORATE AND MANAGE EACH CONDITION. HOWEVER, THE RESULTS ARE OFTEN VARIABLE AND RARELY FULLY CORRECTIVE, HENCE CONSIDERABLE EFFORT NEEDS TO BE INVESTED IN DEVELOPING PREVENTATIVE THERAPIES. THIS CAN ONLY COME FROM A THOROUGH APPRECIATION OF THE ETIOLOGY AND PATHOGENESIS OF INDIVIDUAL CRANIOFACIAL MALFORMATION SYNDROMES, WHICH IS BUILT UPON A DEEP FOUNDATIONAL KNOWLEDGE AND UNDERSTANDING OF NORMAL CRANIOFACIAL DEVELOPMENT. ADVANCES IN GENOMICS CONTINUES TO ELUCIDATE THE COMPLEX ETIOLOGY UNDERLYING BIRTH DEFECTS, BUT KNOWING THE MOLECULAR GENOTYPE OF A SINGLE LOCUS IS OFTEN INSUFFICIENT FOR PREDICTING THE PHENOTYPE OF MANY MALFORMATION SYNDROMES. FURTHERMORE, BIRTH DEFECT DISORDERS ARE TYPICALLY CHARACTERIZED BY CONSIDERABLE PHENOTYPIC VARIANCE, OFTEN DUE TO GENE-ENVIRONMENT INTERACTIONS, BUT OUR UNDERSTANDING OF ENVIRONMENTAL RISK FACTORS IS POOR, AS BIOLOGICAL AND TECHNICAL CONSTRAINTS HAVE MADE DEFINING GENE-ENVIRONMENT INTERACTIONS IN BIRTH DEFECT ETIOLOGY CHALLENGING. CRANIOFACIAL MORPHOGENESIS IS ONE EXAMPLE OF A COMPLEX TRAIT IN WHICH EPISTATIC AND GENE-ENVIRONMENT INTERACTIONS LIKELY CONTRIBUTE EXTENSIVELY TO PHENOTYPIC VARIATION DURING NORMAL DEVELOPMENT AND IN THE PATHOGENESIS OF CRANIOFACIAL DYSMORPHOLOGY. HOWEVER, WHILE IT HAS BEEN POSITED THAT MOST CRANIOFACIAL VARIATION IS GENETICALLY DETERMINED, A LARGE GAP IN KNOWLEDGE EXISTS WITH RESPECT TO THE CONTRIBUTIONS OF ENVIRONMENTAL FACTORS AND GENETIC BACKGROUND IN THE PATHOGENESIS OF CRANIOFACIAL ANOMALIES. FOCUSING ON THE CRANIOFACIAL DISORDER, TREACHER COLLINS SYNDROME, THIS RESEARCH PROPOSAL: (I) UNCOVERS AND VALIDATES SPECIFIC GENE-ENVIRONMENT INTERACTIONS WHICH UNDERLIE THE ETIOLOGY AND PATHOGENESIS OF ANOMALIES OF THE HEAD AND FACE; (II) IDENTIFIES AND VALIDATES GENETIC MODIFIERS THAT CONTRIBUTE TO INTER-FAMILIAL AND INTRA-FAMILIAL VARIABILITY CHARACTERISTIC OF CRANIOFACIAL SYNDROMES; AND (III) DEFINES AND VALIDATES IN UTERO THERAPEUTIC APPROACHES TO PREVENT CRANIOFACIAL ANOMALIES.

THE ROLE OF RRNA TRANSCRIPTION AND RIBOSOME BIOGENESIS IN NEURAL CREST PROGENITORS AND STEM CELLS DURING EMBRYONIC AND POSTNATAL CRANIOFACIAL DEVELOPMENT - ABSTRACT CRANIOFACIAL ANOMALIES ARE THE MOST COMMON MALFORMATIONS PRESENT AT BIRTH AND COMPRISE UP TO 1/3 OF ALL CONGENITAL DEFECTS. MOST OF THE CRANIOFACIAL CARTILAGE, BONE AND CONNECTIVE TISSUE ARE DERIVED FROM NEURAL CREST CELLS (NCC), A MIGRATORY STEM AND PROGENITOR CELL POPULATION BORN DURING EARLY EMBRYOGENESIS. THE MAJORITY OF CRANIOFACIAL DISORDERS ARE THEREFORE THOUGHT TO BE A RESULT OF DISRUPTIONS IN NCC DEVELOPMENT3. IN ORDER TO IMPROVE THE PROGNOSIS FOR INDIVIDUALS AFFECTED WITH CRANIOFACIAL ANOMALIES, AND TO DEVELOP POTENTIAL PREVENTATIVE THERAPIES, THERE IS A CRITICAL NEED FOR A DEEPER UNDERSTANDING OF THE FUNDAMENTAL MECHANISMS THAT GOVERN THE FORMATION, MIGRATION AND DIFFERENTIATION OF NCCS DURING CRANIOFACIAL MORPHOGENESIS4. RECENT WORK IN OUR LAB DEMONSTRATED THAT RNA POLYMERASE I (POL I)-MEDIATED RIBOSOMAL RNA (RRNA) TRANSCRIPTION, WHICH IS THE RATE LIMITING STEP IN RIBOSOME BIOGENESIS, IS ELEVATED IN NCCS COMPARED TO OTHER CELL TYPES5, 6. THIS IS NECESSARY TO MEET NCC’S HIGHER PROTEIN TRANSLATIONAL REQUIREMENTS AND UNDERPINS THEIR HIGH RATE OF PROLIFERATION, EPITHELIAL TO MESENCHYMAL TRANSFORMATION, AND METABOLICALLY EXPENSIVE MIGRATION PROPERTIES5. IN SUPPORT OF THIS IDEA, NCC- SPECIFIC DISRUPTION OF POL I FUNCTION LEADS TO INCREASED NCC DEATH AND CRANIOFACIAL ANOMALIES CHARACTERISTIC OF CONGENITAL CRANIOFACIAL DISORDERS SUCH AS TREACHER COLLINS SYNDROME AND ACROFACIAL DYSOSTOSIS-CINCINNATI TYPE5, 7, 8. ADDITIONALLY, EVIDENCE FROM OTHER STUDIES SUPPORTS ELEVATED REQUIREMENTS FOR RIBOSOMES AND TRANSLATIONAL CAPACITY IN ORDER FOR STEM CELLS TO ACQUIRE NEW FATES, SUGGESTING THAT RIBOSOME BIOGENESIS ALSO PLAYS A CRITICAL ROLE DURING THE DIFFERENTIATION STAGE OF NCC DEVELOPMENT. BASED ON OUR PREVIOUS FINDINGS I HYPOTHESIZE THAT INCREASED RRNA TRANSCRIPTION AND RIBOSOME BIOGENESIS ARE ESSENTIAL FOR BOTH NCC-DERIVED PROGENITOR AND STEM CELL MAINTENANCE AND DIFFERENTIATION DURING EMBRYONIC AND ALSO JUVENILE PHASES OF CRANIOFACIAL DEVELOPMENT. TO TEST THIS HYPOTHESIS, I WILL GENETICALLY DELETE POLR1A, THE CATALYTIC CORE OF POL I, IN TAMOXIFEN-INDUCIBLE GLOBAL AND TISSUE-SPECIFIC MOUSE LINES. THEN THROUGH PHENOTYPIC AND CELLULAR ANALYSES, IN COMBINATION WITH TRANSCRIPTOMIC AND PROTEOMIC APPROACHES, I WILL DEFINE THE MECHANISMS DRIVING THE ANATOMICAL ANOMALIES THAT RESULT FROM DISRUPTIONS IN POL I FUNCTION. MY RESULTS WILL PROVIDE NEW KNOWLEDGE ON THE ROLES OF RRNA TRANSCRIPTION AND RIBOSOME BIOGENESIS IN CRANIOFACIAL PROGENITOR AND STEM CELL POPULATIONS DURING EMBRYONIC AND POSTNATAL DEVELOPMENT. THE FINDINGS CAN POTENTIALLY UNCOVER NEW THERAPIES FOR PREVENTING OR AMELIORATING RIBOSOMOPATHIES, AND IMPROVE CLINICAL OUTCOMES FOR CRANIOFACIAL PATIENTS, ESPECIALLY WHEN IT COMES TO CORRECTIVE SURGERIES, THROUGH IMPROVED POSTNATAL GROWTH PREDICTIONS.

INVESTIGATING PROTEIN SUPERSATURATION AS A DRIVER OF AGING - PROJECT SUMMARY PROTEIN AGGREGATION IS A HALLMARK OF AGING AND AGE-ASSOCIATED DISEASE, HOWEVER A CAUSAL RELATIONSHIP HAS NOT BEEN DEMONSTRATED. ELUCIDATING WHETHER THERE IS A PREDESTINED, IRREVERSIBLE DRIVING FORCE IN CELL AGING WOULD ENABLE THE DEVELOPMENT OF NOVEL THERAPIES TO DECELERATE AGING. THEREFORE, MY LONG-TERM GOAL IS TO UNDERSTAND THIS RELATIONSHIP AT A FUNDAMENTAL LEVEL. MY CENTRAL HYPOTHESIS IS THAT PROBABILISTIC, IRREVERSIBLE CONFORMATIONAL TRANSITIONS IN PHYSIOLOGICALLY SUPERSATURATED PROTEINS NOT ONLY INITIATE THE PROCESS OF CELLULAR AGING, BUT DRIVE IT!. THE ACCUMULATION OF AMYLOIDS FOLLOWING SUCH TRANSITIONS WILL COMPROMISE KINETIC PROTEOSTASIS, OR KINETIC BARRIERS FOR SUPERSATURATED PROTEINS TO REMAIN SOLUBLE, AND THIS ULTIMATELY COMPROMISES THERMODYNAMIC PROTEOSTASIS -- OR THE PROCESSES THAT MAINTAIN THE CONCENTRATIONS AND STABILITIES OF SOLUBLE PROTEINS. I WILL UTILIZE THE FOLLOWING SPECIFIC AIMS AND SYNERGISTIC APPROACHES, DISTRIBUTED AMPHIFLUORIC FRET (DAMFRET), EPIGENETIC CLOCKS, AND RNA-SEQ, TO DISTINGUISH THE KINETIC FROM THERMODYNAMIC DETERMINANTS OF PROTEIN SOLUBILITY AS A FUNCTION OF CELL AGE. IN AIM 1, I WILL COMPARE THERMODYNAMIC AND KINETIC PROTEOSTASIS AS A FUNCTION OF BIOLOGICAL AGE. TO DO SO, I WILL FIRST OBTAIN PRIMARY HUMAN FIBROBLASTS (PHFS) FROM DIFFERENTIALLY AGED DONORS, AND VALIDATE THEIR EPIGENETIC AGE USING DNA METHYLATION SIGNATURES (DNAM) REFERENCED AGAINST PREVIOUSLY DEVELOPED DNAM AGE PREDICTION ALGORITHMS, AS WELL AS RNA-SEQ. I WILL THEN PERFORM DAMFRET EXPERIMENTS IN EACH OF THE PHFS USING A PANEL OF INDUCIBLE CONSTRUCTS THAT RELIABLY AGGREGATE IN A NUCLEATION- AND/OR CONCENTRATION-LIMITED MANNER. THESE DATA WILL REVEAL THE DEGREE TO WHICH KINETIC PROTEOSTASIS AND/OR THERMODYNAMIC PROTEOSTASIS ARE IMPACTED BY BIOLOGICAL AGE. IN AIM 2, I WILL TEST IF CONFORMATIONAL NUCLEI ACCELERATE THE AGING OF PHFS. I WILL GENERATE GENERIC LIGHT-ACTIVATED OPTOSEEDS FROM OUR REPORTER LIBRARY IN AIM 1 TO ELICIT A CONFORMATIONAL TRANSITION, OR CROSS-SEEDING EVENT, IN PHFS OF YOUNG AGE. I WILL THEN USE MULTIPLE MASS SPECTROMETRY APPROACHES TO EVALUATE WHETHER THE NUCLEATION EVENT PRECIPITATED ENDOGENOUS PROTEINS, AND DETERMINE THEIR IDENTITIES. I WILL THEN DETERMINE IF THE TREATMENT ACCELERATES THE PROGRESSION OF CELL AGE VIA DNAMAGE AND RNA-SEQ. IN AIM 3, I WILL TEST IF PERTURBING KINETIC PROTEOSTASIS IN THE NUCLEUS ENHANCES THE RATE OF AGING AS COMPARED TO THE CYTOSOL. USING OUR OPTOSEEDS, I WILL ELICIT A CONFORMATIONAL TRANSITION IN THE NUCLEAR AND CYTOPLASMIC COMPARTMENTS. I WILL AGAIN USE DNAM AGE PREDICTION AND RNA-SEQ TO DETERMINE WHETHER AGE IS ACCELERATED VIA CONFORMATIONAL TRANSITIONING IN THE NUCLEUS VERSUS THE CYTOSOL. COMPLETION OF THESE AIMS WILL PROVIDE FUNDAMENTAL INSIGHTS INTO THE THERMODYNAMIC REASONS FOR WHY WE AGE. IN ADDITION, COMPLETION OF THE PROPOSED STUDIES WILL PROVIDE ME WITH A STRONG FOUNDATION TO CONTINUE MY RESEARCH AS AN INDEPENDENT INVESTIGATOR.

A GENETIC PROGRAM FOR ORGAN REGENERATION IN ZEBRAFISH

ZEBRAFISH DEVELOPMENT & GENETICS CONFERENCE

EVOLUTIONARY MECHANISMS OF GASTRULATION AND LEFT-RIGHT PATTERNING IN AMNIOTES. - PROJECT SUMMARY ALTHOUGH AS HUMANS WE APPEAR SYMMETRICAL ON THE OUTSIDE, OUR INTERNAL ORGANS ARE ASYMMETRICALLY POSITIONED ALONG THE LEFT AND RIGHT SIDES OF OUR BODY. LEFT-RIGHT (L-R) PATTERNING IS A FUNDAMENTAL BIOLOGICAL PROCESS THAT HELPS TO ENSURE THE CORRECT POSITIONING OF OUR ORGANS, AND ITS PERTURBATION IS TYPICALLY ASSOCIATED WITH CONGENITAL HEART MALFORMATIONS AND HIGH MORTALITY. IN A TYPICAL DEUTEROSTOME, INCLUDING HUMANS, PROPER L-R PATTERNING INVOLVES MOTILE CILIA IN THE L-R ORGANIZING (LRO) REGION, WHICH TRIGGER A CA2+ WAVE ON THE LEFT SIDE OF THE EMBRYO. THIS RESULTS IN ASYMMETRIC GENE EXPRESSION AND ULTIMATELY ASYMMETRIC ORGANOGENESIS. HOWEVER, MORE THAN 65% OF ALL TETRAPODS, INCLUDING REPTILES AND EVEN-TOED UNGULATES, DO NOT USE MOTILE CILIA FOR L-R PATTERNING. INSTEAD, TILTING OF THE LRO AND ASYMMETRIC CELLULAR MOVEMENTS SOMEHOW LEAD TO MOLECULAR ASYMMETRY. HOWEVER, THE MECHA- NISMS UNDERPINNING L-R ASYMMETRY IN THESE ORGANISMS ARE POORLY UNDERSTOOD. IT IS UNCLEAR HOW ASYMMETRIC CELL MOVEMENTS ORIGINATE, WHETHER THEY TRIGGER THE ASYMMETRICAL CA2+ WAVE, OR IF CILIA ARE INVOLVED IN ANY ASPECT OF L-R PATTERNING. CURRENTLY, THE CHICKEN EMBRYO IS USED TO REPRESENT THE DIVERSITY OF ALL 25,000 SPECIES OF REPTILES, AND NEW MODELS ARE REQUIRED FOR A DEEPER EVOLUTIONARY UNDERSTANDING OF FUNDAMENTAL DEVELOPMENTAL EVENTS. VEILED CHAMELEONS (CHAMAELEO CALYPTRATUS) ARE PERFECT FOR THE STUDY OF EARLY DEVELOPMENT AND EVOLUTION IN NON- AVIAN REPTILES, SINCE THEY LAY LARGE CLUTCHES OF EGGS AT PRE-GASTRULATION STAGES. THEIR LRO LACKS MOTILE CILIA, AND INSTEAD MOLECULAR ASYMMETRY IS ESTABLISHED THROUGH LARGE-SCALE MORPHOLOGICAL CHANGES. VEILED CHAMELEONS HAVE A SEQUENCED AND ANNOTATED GENOME, AND ARE AMENABLE TO CELL AND EMBRYO CULTURE, AS WELL AS LIVE IMAGING. THE OBJECTIVE OF THIS APPLICATION THEREFORE IS TO DEFINE THE MECHANISMS GOVERNING L-R PATTERNING IN CHAMELEONS AND THUS EXPAND OUR UNDERSTANDING OF AMNIOTE DEVELOPMENT AND EVOLUTION. THE CENTRAL HYPOTHESIS IS THAT CELLULAR FLOW AND LARGE-SCALE MORPHOLOGICAL CHANGES TRIGGER AN EVOLUTIONARILY CONSERVED ASYMMETRIC CA2+ WAVE, LEADING TO MOLECULAR L-R ASYMMETRY, WHICH HAS UNDERGONE EVOLUTIONARY CHANGE ACROSS AMNIOTES. THIS HYPOTHESIS WILL BE ADDRESSED IN THE FOLLOWING AIMS: (AIM 1) DETERMINE THE MECHANICS OF ESTABLISHING L-R PATTERNING IN VEILED CHAMELEON. (AIM 2) EVALUATE GENETIC CHANGES AND CONSERVATION OF THE L-R PATTERNING PATHWAY ACROSS AMNIOTES. THE PATTERNS OF CELL MIGRATION AND THE DYNAMICS OF CA2+ SIGNALING WILL BE EVALUATED THROUGH LIVE IMAGING, PROVIDING TRAINING IN ADVANCED MICROSCOPY. CRISPR/CAS9 GENE EDITING WILL BE ADAPTED FOR USE IN CHAMELEONS AND WILL INCLUDE TRAINING IN SURGERY AND VIRUS PRODUCTION. THIS STUDY WILL RESULT IN THE FIRST SCRNA-SEQ AND SCATAC-SEQ LIBRARIES FOR ASYMMETRIC GENE EXPRESSION BETWEEN THE LEFT AND RIGHT SIDES OF CHICKEN, CHAMELEON, AND MOUSE EMBRYOS, AND WILL INVOLVE COMPUTATIONAL BIOLOGY TRAINING. SUCCESSFUL COMPLETION OF THIS PROJECT WILL BE SIGNIFICANT IN THE FIELDS OF L-R PATTERNING AND EVO-DEVO, PROVIDING THE FIRST DETAILED STUDY OF THE EARLY STEPS OF L-R PATTERNING IN A NON-AVIAN, NON-MAMMALIAN AMNIOTE, WHICH MAY REVOLUTIONIZE OUR CURRENT THINKING ABOUT ROLES FOR CILIA AND CA2+ SIGNALING IN L-R PATTERNING. IT WILL ALSO LAY THE FOUNDATION FOR A SUCCESSFUL INDEPENDENT CAREER.

UNCOVERING MECHANISMS CONTROLLING CHROMOSOME-SPECIFIC BEHAVIORS DURING MEIOSIS

UNDERSTANDING RNA POLYMERASE III TRANSCRIPTION IN NEURAL CREST CELL AND CRANIOFACIAL DEVELOPMENT - PROJECT SUMMARY CRANIOFACIAL ANOMALIES ACCOUNT FOR ONE THIRD OF ALL BIRTH DEFECTS AND ARE A SIGNIFICANT CAUSE OF INFANT MORTALITY. NEURAL CREST CELLS (NCC) GIVE RISE TO THE MAJORITY OF CRANIOFACIAL BONE, CARTILAGE, AND CONNECTIVE TISSUE AND UN- DERSTANDING THEIR DEVELOPMENT IS CRUCIAL FOR ADVANCING THE PREVENTION OF CRANIOFACIAL BIRTH DEFECTS. DISRUPTIONS IN NCC DEVELOPMENT ARE KNOWN TO UNDERLIE SEVERAL CRANIOFACIAL DISORDERS INCLUDING TREACHER COLLINS SYNDROME, WHICH IS CAUSED BY MUTATIONS IN TCOF1, POLR1B, POLR1C, AND POLR1D. POLR1C AND POLR1D ARE SUBU- NITS OF BOTH RNA POLYMERASES (POL) I AND III AND ARE IMPORTANT FOR TRANSCRIPTION OF RIBOSOMAL RNA. I PREVIOUSLY DEMONSTRATED IN POLR1C AND POLR1D ZEBRAFISH MODELS THAT RIBOSOMAL RNA TRANSCRIPTION IS REDUCED LEADING TO TP53-DEPENDENT CELL DEATH OF NCC PROGENITORS WHICH RESULTS IN CRANIOFACIAL ANOMALIES. HOWEVER, HOW GLOBAL DISRUPTIONS IN POLR1C AND POLR1D SPECIFICALLY AFFECT NCC DEVELOPMENT REMAINS UNRESOLVED AND THE CONTRIBUTION OF POL III, WHICH TRANSCRIBES NON-CODING RNAS INCLUDING 5S RIBOSOMAL RNA AND TRANSFER RNAS, TO CRANIOFACIAL DEVELOPMENT IS NOT KNOWN. I HYPOTHESIZE THAT IN ADDITION TO DISRUPTION OF POL I TRANSCRIPTION IN THE PATHOGENESIS OF TREACHER COLLINS SYNDROME, POL III TRANSCRIPTION IS ALSO DISRUPTED AND CONTRIBUTES TO THE TISSUE-SPECIFIC PHE- NOTYPES OBSERVED. TRANSCRIPTS PRODUCED BY POL III, INCLUDING TRNAS, HAVE BEEN SHOWN IN MULTIPLE SYSTEMS TO BE TISSUE-SPECIFICALLY EXPRESSED. TO GENERATE A NEW UNDERSTANDING OF THE ROLE OF POL III TRANSCRIPTION SPECIFICALLY IN NCC, I WILL RECEIVE TRAINING IN PROFILING NCC FOR CHANGES IN POL III TRANSCRIPTS AND IN EVALUATING THE EFFECT OF THESE CHANGES ON TRANSLATION. IT HAS BEEN POSTULATED THAT DISTINCT PATHOGENIC VARIANTS IN POLR3A, THE LARGEST SUBUNIT OF POL III, LEAD TO DIFFERENTIAL EFFECTS ON POL III TRANSCRIPTION. IN ORDER TO TEST THIS HYPOTHESIS IN A NCC- SPECIFIC MANNER, I WILL USE HIPSCS DERIVED FROM PATIENT FIBROBLASTS WITH PATHOGENIC VARIANTS IN POLR3A AND ANALYZE THEM FOR PROLIFERATION, TRANSLATION, DIFFERENTIATION, AND POL I AND III TRANSCRIPTION. GIVEN THE PREVALENCE OF DENTAL ANOMALIES IN INDIVIDUALS WITH MUTATIONS IN POLR3A, I EXPECT TO IDENTIFY POL III-SPECIFIC EFFECTS IN A SUBSET OF NCC DERIVATIVES. IN THE INDEPENDENT PHASE OF THIS AWARD, I WILL GENERATE NEW ZEBRAFISH MODELS TO UNDER- STAND THE ROLE OF SPECIFIC VARIANTS IN POL III IN A DEVELOPMENTAL CONTEXT AND ASSESS NCC FORMATION, MIGRATION, DIFFERENTIATION, AND PROLIFERATION IN COMBINATION WITH THE EFFECT ON POL I AND III TRANSCRIPTION. THESE MODELS WILL PROVIDE NEW RESOURCES TO THE RESEARCH COMMUNITY FOR THE UNDERSTANDING OF POL III TRANSCRIPTION. ALTOGETHER, I WILL RECEIVE THE TRAINING NECESSARY TO ANALYZE POL III TRANSCRIPTION AND TRANSLATION AND MODEL PATIENT-SPECIFIC VAR- IANTS IN NCC WHICH WILL FORM THE FOUNDATION OF MY INDEPENDENT RESEARCH PROGRAM AND FURTHER MY GOAL OF UNDER- STANDING AND PREVENTING CRANIOFACIAL BIRTH DEFECTS.

A NEW SYSTEM FOR MODELING SUPERNUMERARY CHROMOSOME DYNAMICS AND FORMATION DURING MEIOSIS.

EXAMINING SIGNALS THAT SCULPT CRANIAL NEURAL CREST MIGRATION

PROFILING EARLY METASTATIC MELANOMA IN VIVO USING THE CHICK EMBRYO MODEL

EXTRINSIC SIGNALING IN NEURAL CREST AND CRANIOFACIAL DEVELOPMENT

FUNCTIONAL CHARACTERIZATION OF MEDIATOR COMPLEX PROTEINS IN NEURAL CREST AND CRANIOFACIAL DEVELOPMENT - PROJECT SUMMARY CRANIOFACIAL DEVELOPMENTAL DISORDERS SUCH AS CLEFT PALATE AND RETROGNATHIA (SMALLER LOWER JAW) OFTEN ARISE DUE TO DEFECTS IN NEURAL CREST CELL DEVELOPMENT AND AFFECT 1 IN 700 AND 1 IN 1,500 LIVE HUMAN BIRTHS, RESPECTIVELY. CLEFT PALATE AND RETROGNATHIA OFTEN PRESENT WITH OTHER CRANIOFACIAL ANOMALIES IN CONDITIONS SUCH AS TREACHER COLLINS SYNDROME AND DIGEORGE SYNDROME. ALTHOUGH GENETIC ANALYSES HAVE IDENTIFIED THE GENES RESPONSIBLE FOR SOME CRANIOFACIAL ANOMALIES, THE VAST MAJORITY HAVE AN UNDIAGNOSED MOLECULAR ETIOLOGY AND CELLULAR PATHOGENESIS. IN AN ENU MUTAGENESIS SCREEN, THE MED23 GENE WAS IDENTIFIED TO BE CRITICAL FOR CRANIOFACIAL DEVELOPMENT. A POINT MUTATION IN MED23 RESULTS IN CRANIOFACIAL AND VASCULAR DEFECTS, LEADING TO EMBRYONIC LETHALITY AT MID-GESTATION. MED23 BELONGS TO THE TAIL MODULE OF THE MEDIATOR COMPLEX, WHICH IS A GLOBAL TRANSCRIPTION COREGULATOR FOR GENES TRANSCRIBED BY RNA POLYMERASE II. MED23 IS UBIQUITOUSLY EXPRESSED IN MOUSE EMBRYOS, THEREBY, RAISING AN IMPORTANT QUESTION OF HOW A UBIQUITOUSLY EXPRESSED PROTEIN REGULATES TISSUE SPECIFIC DEVELOPMENT DURING EMBRYOGENESIS, PARTICULARLY IN CRANIOFACIAL DEVELOPMENT. TO UNDERSTAND THE FUNCTION OF MED23 IN CRANIOFACIAL AND NEURAL CREST CELL DEVELOPMENT, I GENERATED NEURAL CREST CELL SPECIFIC CONDITIONAL KNOCKOUTS OF MED23 THAT EXHIBIT CLEFT PALATE, RETROGNATHIA, GLOSSOPTOSIS AND CLEIDOCRANIAL DYSPLASIA. INTERESTINGLY, ENDOTHELIAL CELL SPECIFIC CONDITIONAL KNOCKOUTS OF MED23 ALSO RESULT IN CRANIOFACIAL DEFECTS TOGETHER WITH VASCULAR DEFECTS. IN THIS PROPOSAL, I WILL TEST THE OVERARCHING HYPOTHESIS THAT MED23 AND OTHER TAIL MODULE SUBUNITS OF MEDIATOR HAVE IMPORTANT AND DISTINCT TRANSCRIPTIONAL REGULATORY FUNCTIONS IN CRANIOFACIAL DEVELOPMENT VIA THE CONTROL OF NEURAL CREST CELL AND ENDOTHELIAL CELL TRANSCRIPTOMES. SPECIFICALLY, I WILL ADDRESS THE FOLLOWING AIMS. (AIM 1) IDENTIFY THE MOLECULAR MECHANISM UNDERLYING THE CRANIOFACIAL DEFECT IN NEURAL CREST CELL SPECIFIC MUTANTS OF MED23 BY ANALYZING TRANSCRIPTOMIC CHANGES IN THESE MUTANTS AS WELL AS BY IDENTIFYING MED23 DNA AND PROTEIN BINDING PARTNERS. (AIM 2) CHARACTERIZE THE PATHOGENESIS OF CRANIOFACIAL DEFECTS IN ENDOTHELIAL CELL SPECIFIC MUTANTS OF MED23 AND THEIR UNDERLYING MOLECULAR MECHANISMS BY ANALYSIS OF THE CRANIOFACIAL TRANSCRIPTOME. FURTHERMORE, I WILL TEST THE MECHANISM OF MED23-MEDIATED CONTROL OF KEY REGULATORY GENES THAT FUNCTION IN CRANIOFACIAL DEVELOPMENT AND ARE LINKED TO CRANIOFACIAL DISORDERS. SPECIFICALLY, I WILL INVESTIGATE THE MOLECULAR BASIS OF MED23 FUNCTION VIA ITS CONTROL OF RUNX2 AND SS-CATENIN IN THE MANDIBULAR MESENCHYME. (AIM 3) GENERATE AND CHARACTERIZE LOSS OF FUNCTION MUTANTS OF THE MEDIATOR TAIL SUBMODULE PROTEIN, MED24. THE BROAD IMPACT OF THIS INNOVATIVE PROPOSAL WILL ADVANCE OUR MECHANISTIC UNDERSTANDING OF THE GENE AND PROTEIN NETWORKS UNDERLYING THE FUNCTION OF GLOBAL TRANSCRIPTION CO-FACTOR MEDIATOR COMPLEX PROTEINS IN FUNDAMENTAL CELLULAR PROCESSES AND DEVELOPMENT.

DEFINING MECHANISTIC DIFFERENCES BETWEEN EMBRYONIC AND REGENERATIVE ORGANOGENESIS

DECIPHERING GENOME INTEGRITY MAINTENANCE USING CYTOGENOMICS - PROJECT SUMMARY/ABSTRACT CHROMOSOME MISSEGREGATION LEADS TO ANEUPLOIDY AND GENOMIC INSTABILITY—HALLMARKS OF CANCER AND CONTRIBUTORS TO REPRODUCTIVE AGING. UNDERSTANDING HOW CENTROMERE SEQUENCE COMPOSITION, STRUCTURE, AND EPIGENETIC BACKGROUND INFLUENCE CHROMOSOME SEGREGATION IS IMPORTANT FOR UNRAVELING THE MOLECULAR ORIGINS OF GENOME INSTABILITY THAT COULD PROMOTE CANCER EVOLUTION. CENTROMERES ARE SPECIALIZED CHROMOSOMAL REGIONS THAT SERVE AS SITES OF KINETOCHORE FORMATION AND MICROTUBULE BINDING THAT PARTITION CHROMOSOMES INTO DAUGHTER CELLS DURING CELL DIVISION. THE GERTON LAB AT THE STOWERS INSTITUTE FOR MEDICAL RESEARCH, SUPPORTED BY NCI FUNDING (R01CA266339, MAINTAINING THE INTEGRITY OF A GENOME), INVESTIGATES CENTROMERE BIOLOGY AND KINETOCHORE FUNCTION TO UNCOVER THE MECHANISMS DRIVING CHROMOSOME MISSEGREGATION AND ANEUPLOIDY. AS A RESEARCH SPECIALIST II IN THE GERTON LAB, I DESIGN AND IMPLEMENT EXPERIMENTAL STRATEGIES ADDRESSING CENTROMERE COMPOSITION, FUNCTIONAL ACTIVITY, AND CHROMOSOME DYNAMICS. OUR CURRENT RESEARCH INVESTIGATES HOW NATURAL VARIATION IN HUMAN CENTROMERIC ARRAY SIZE AND ACTIVITY IMPACTS THE ACCURACY OF CHROMOSOME SEGREGATION. MULTIPLE COMPLETE TELOMERE- TO-TELOMERE HUMAN GENOME ASSEMBLIES EXPOSED CENTROMERES AS SOME OF THE MOST VARIABLE REGIONS IN THE HUMAN GENOME. CENTROMERES OF THE SAME CHROMOSOMES CAN DIFFER BETWEEN INDIVIDUALS IN BOTH SIZE AND THE EPIGENETIC DETERMINANTS OF KINETOCHORE POSITIONING. WE HYPOTHESIZE THAT INDIVIDUAL SEQUENCE AND EPIGENETIC PATTERN VARIATIONS IN HUMAN CENTROMERES CAN ALTER THEIR INTERACTIONS WITH CELL DIVISION MACHINERY, PREDISPOSING INDIVIDUALS TO DISTINCT CHROMOSOME MISSEGREGATION EVENTS. TO TEST THIS HYPOTHESIS, WE ARE WORKING ON MAPPING THE CENTROMERIC SEQUENCE LANDSCAPE AND ASSOCIATED KINETOCHORE PROTEINS IN INDIVIDUAL HUMAN CENTROMERES, AND INVESTIGATING HOW THESE VARIATIONS IMPACT CHROMOSOME SEGREGATION IN CULTURED CELLS AND TUMOR TISSUES. I UTILIZE CUTTING-EDGE GENOMIC AND CYTOGENETIC TECHNIQUES, ALONG WITH HIGH-RESOLUTION MICROSCOPY, TO MEASURE CENTROMERE SIZES AND THE DEPOSITION OF INNER KINETOCHORE PROTEINS. THESE DATA ARE BEING INTEGRATED WITH GENETIC AND EPIGENETIC INFORMATION FROM THE TELOMERE-TO-TELOMERE (T2T) CONSORTIUM, A GLOBAL MULTI-LAB INITIATIVE FOCUSED ON GENERATING COMPLETE GENOME ASSEMBLIES. THIS COLLABORATION ALLOWS OUR GROUPS TO INTEGRATE GENOMIC DATA WITH CYTOGENETICS, A METHOD WE CALL “CYTOGENOMICS”. THIS INFORMATION WILL BE USED TO FIND THE CENTROMERE-SPECIFIC GENETIC AND EPIGENETIC SIGNATURES INFLUENCING THE ACCURACY OF CHROMOSOME SEGREGATION IN BOTH CULTURED CELLS AND XENOGRAFT TUMOR TISSUES. OUR LONG-TERM GOAL IS TO DEVELOP COMPREHENSIVE MODELS EXPLAINING HOW CENTROMERE SEQUENCES AND ACTIVITIES CONTRIBUTE TO THE RISE OF ANEUPLOIDY AND TO TRANSLATE THESE FINDINGS INTO INSIGHTS RELEVANT TO CANCER BIOLOGY. THIS RESEARCH IS INNOVATIVE BECAUSE IT LEVERAGES THE LATEST INFORMATION ON HUMAN CENTROMERIC DNA ARRAYS, INTRODUCES NEW MODELS FOR CENTROMERE ORGANIZATION, AND EMPLOYS CUTTING-EDGE MOLECULAR, GENOMIC, AND IMAGING TOOLS. THE PROPOSED WORK IS SIGNIFICANT BECAUSE IT ADDRESSES FUNDAMENTAL QUESTIONS ABOUT THE ROLE OF INNATE CENTROMERE VARIATION IN GENOMIC ALTERATIONS, PROVIDING INSIGHTS INTO CANCER BIOLOGY AND AGING.

CHARACTERIZATION OF THE SIN3A AND SIN3B HDAC COMPLEXES

INTRINSIC VS EXTRINSIC DEFECTS IN NEURAL CREST CELL PATTERNING

CHARACTERIZATION OF NOD, A DROSOPHILA PROTEIN REQUIRED FOR CHROMOSOME SEGREGATION

OPENING SMALL PACKAGES: UNRAVELING ROLES FOR MICROPROTEINS DURING EARLY VERTEBRATE DEVELOPMENT - PROJECT SUMMARY HUMAN DEVELOPMENT RELIES ON HIGHLY COORDINATED CELL DIVISION, SIGNALING, MIGRATION, AND DIFFERENTIATION. ANY PERTURBATIONS OF THE PROTEIN EFFECTORS THAT ORCHESTRATE THESE CRITICAL PROCESSES CAN LEAD TO HUMAN BIRTH DEFECTS AND/OR DISEASES1-3. WHILE OUR CLASSICAL CATALOG CONTAINS AROUND 20,000 PROTEINS, EVIDENCE FROM ‘OMICS-BASED TECHNIQUES HAS GENERATED A RAPID PARADIGM SHIFT IN RNA BIOLOGY4-12. NOTABLY, RNA SEQUENCES DEFINED AS NON- CODING IN FACT PRODUCE SHORT PROTEINS (£ 100 AMINO ACIDS) CALLED MICROPROTEINS THAT MODULATE DIVERSE PROCESSES13- 40 INCLUDING HEART FUNCTION26, IMMUNITY22,27,41, AND CELL GROWTH29,30. FOR EXAMPLE, A MICROPROTEIN CALLED APELA MAINTAINS PLURIPOTENCY IN HUMAN EMBRYONIC STEM CELLS20 AND IS CRITICAL FOR ZEBRAFISH HEART DEVELOPMENT16,19. HOWEVER, ADDITIONAL MICROPROTEIN FUNCTION(S) DURING VERTEBRATE DEVELOPMENT REMAIN LARGELY UNKNOWN. ZEBRAFISH IS AN OUTSTANDING MODEL FOR INTERROGATING VERTEBRATE GENE FUNCTION. THEIR GENETIC TRACTABILITY COUPLED WITH EXTERNAL, SYNCHRONOUS DEVELOPMENT IS WELL-SUITED FOR DEVELOPMENTAL ANALYSES. FURTHER, HUNDREDS OF MICROPROTEINS HAVE BEEN IDENTIFIED ACROSS ZEBRAFISH DEVELOPMENT USING RIBOSOME PROFILING, MASS- SPECTROMETRY, AND CONSERVATION ANALYSES16,42. REMARKABLY, APELA IS THE ONLY MICROPROTEIN OUT OF THESE 400 THAT IS CURRENTLY CHARACTERIZED. A KEY BARRIER TO FURTHER MICROPROTEIN INVESTIGATION IS THAT A MAJORITY OF MESSENGER RNAS (MRNA) DURING EARLY DEVELOPMENT ARE MATERNALLY PROVIDED AND CAN MASK THE EFFECTS OF A TARGETED GENE DISRUPTION. OUR NOVEL CRISPR/CAS13D SYSTEM43,44 OVERCOMES THIS BARRIER BECAUSE IT ACTIVELY DEGRADES ITS TARGET MRNA AND THEREFORE ENABLES SELECTIVE KNOCKDOWN OF MATERNALLY PROVIDED MRNAS IN ZEBRAFISH. MY PRELIMINARY EXPERIMENTS WITH CRISPR/CAS13D HAVE REVEALED THAT KNOCKDOWN OF ONE MICROPROTEIN MRNA INHIBITS ZYGOTIC GENOME ACTIVATION AND DISRUPTS POSTERIOR PATTERNING. THIS STUDY WILL COMBINE CRISPR/CAS13D AND ‘OMICS-BASED TECHNIQUES TO INTERROGATE MICROPROTEIN FUNCTION DURING ZEBRAFISH DEVELOPMENT. AIM 1 WILL LEVERAGE CRISPR/CAS13D TO ELUCIDATE MICROPROTEINS IMPORTANT FOR EARLY DEVELOPMENT. THEN, AIM 2 WILL DETERMINE THE CELL AND MOLECULAR PROCESSES THAT RELY ON DEVELOPMENTAL MICROPROTEINS. TOGETHER, THESE AIMS WILL DEFINE AND CHARACTERIZE A POPULATION OF MICROPROTEINS INVOLVED IN VERTEBRATE DEVELOPMENT. EXPERIMENTAL APPROACHES WILL DEVELOP MY SKILLS IN BIOINFORMATICS, MOLECULAR GENETICS, DEVELOPMENTAL BIOLOGY, AND PROTEIN BIOCHEMISTRY. MICROPROTEINS CRITICAL FOR ZEBRAFISH DEVELOPMENT WILL BE INFORMATIVE FOR EXPANDING THE CATALOG OF HUMAN PROTEINS THROUGH COMPARATIVE ANALYSES. FURTHER, MICROPROTEINS WITH FUNCTIONS DURING DEVELOPMENT REPRESENT UNCHARTED THERAPEUTIC AND/OR DIAGNOSTIC OPPORTUNITIES FOR HUMAN BIRTH DEFECTS AND/OR HUMAN DISEASES WITH DEVELOPMENTAL ORIGINS.

TRITHORAX-RELATED, MLL3 AND MLL4 IN ENHANCER-MEDIATED CANCER PATHOGENESIS

RIBOSOMAL RNA TRANSCRIPTION IS SPATIOTEMPORALLY REGULATED AND FUNCTIONALLY REQUIRED DURING NEURAL CREST,BONE AND CARTILAGE DEVELOPMENT

INVESTIGATING HOW TRANSCRIPTION FACTORS COOPERATE AND OVERCOME THE ENHANCER NUCLEOSOME BARRIER DURING EMBRYONIC PATTERNING - PROJECT SUMMARY THIS MISREGULATION OF GENE EXPRESSION UNDERLIES SEVERAL HUMAN DISEASES, INCLUDING MANY CANCERS, DIABETES, OBESITY, AND MULTIPLE DEVELOPMENTAL DISORDERS. GENOME-WIDE STUDIES AND NEXT-GENERATION SEQUENCING HAVE REVEALED THAT SEQUENCE VARIANTS IN ENHANCERS, CIS-REGULATORY DNA SEQUENCES THAT CONTROL SPACIOTEMPORAL GENE EXPRESSION PROGRAMS, CONTRIBUTE TO THE DEVELOPMENT OF THESE DISEASES. THESE MUTATIONS OFTEN AFFECT ENHANCER ACTIVITY, WHICH MUST BE TIGHTLY CONTROLLED SINCE ENHANCERS DRIVE TISSUE AND CELL-TYPE SPECIFIC GENE EXPRESSION PATTERNS. ONE WAY THAT ENHANCER ACTIVITY IS CONTROLLED IS THROUGH THE REGULATION OF ENHANCER ACCESSIBILITY BY THE NUCLEOSOME: THE STRUCTURAL UNIT OF CHROMATIN COMPRISED OF 147 BP OF DNA AND A HISTONE OCTAMER. ENHANCERS ARE CHARACTERIZED BY AN INTRINSICALLY STRONG NUCLEOSOME BARRIER THAT PREVENTS THE BINDING OF TRANSCRIPTION FACTORS (TFS), THE PROTEINS THAT ACTIVATE ENHANCERS, UNTIL THE PROPER CONTEXT FOR ACTIVATION IS REACHED, AT WHICH POINT TFS MUST OVERCOME THE NUCLEOSOME BARRIER AND BIND TO THE DNA. WHILE NUCLEOSOME DEPLETION IS A KEY EARLY STEP IN ENHANCER ACTIVATION, WE DO NOT YET UNDERSTAND HOW THE NUCLEOSOME BARRIER IS OVERCOME AND HOW ENHANCERS ARE MADE ACCESSIBLE FOR GENE ACTIVATION, DESPITE ACCESSIBILITY BEING A MAJOR REGULATOR OF ENHANCER ACTIVITY. CURRENT MODELS SUGGEST THAT SPECIALIZED TFS CALLED PIONEER FACTORS CAN ACCESS THEIR MOTIFS IN THE PRESENCE OF NUCLEOSOMES AND FOMENT NUCLEOSOME DEPLETION THROUGH COOPERATIVITY WITH ADDITIONAL TFS. EVEN STILL, HOW PIONEER AND NON-PIONEER TFS COOPERATE TO GENERATE CHROMATIN ACCESSIBILITY AT ENHANCERS IS NOT YET KNOWN. FURTHERMORE, HOW PIONEER FACTORS PERTURB THE NUCLEOSOMAL LANDSCAPE TO FACILITATE CHROMATIN ACCESSIBILITY AND COOPERATIVE TF BINDING IS UNCLEAR. THIS STUDY SEEKS TO IDENTIFY HOW TFS OVERCOME THE NUCLEOSOME BARRIER AT ENHANCERS USING HIGH-RESOLUTION EXPERIMENTAL AND COMPUTATIONAL GENOMICS TECHNIQUES TO MAP TF BINDING, CHROMATIN ACCESSIBILITY, AND NUCLEOSOME POSITIONING. AIM 1 WILL CHARACTERIZE HOW PIONEER AND NON-PIONEER TFS COOPERATE FOR BINDING TO THE DNA AND FOR ESTABLISHING CHROMATIN ACCESSIBILITY. THIS AIM WILL COMBINE HIGH-RESOLUTION TF BINDING (CHIP- NEXUS) AND TEMPORALLY RESOLVED CHROMATIN ACCESSIBILITY (TIME-COURSE ATAC-SEQ) INFORMATION WITH DEEP LEARNING MODELS (BPNET) THAT WILL REVEAL THE SEQUENCES AND SEQUENCE CONSTRAINTS THAT ARE IMPORTANT FOR AND PREDICTIVE OF TF COOPERATIVITY. AIM 2 WILL PROFILE GENOME-WIDE NUCLEOSOME POSITIONAL CHANGES OVER DEVELOPMENTAL TIME AT UNPRECEDENTED RESOLUTION, USING A CHEMICAL MAPPING OF NUCLEOSOME CENTERS APPROACH. THIS AIM WILL UNCOVER HOW THE NUCLEOSOME STATE AT ENHANCERS IS ALTERED OVER TIME TO GENERATE ACCESSIBILITY AND HOW NUCLEOSOMES ARE POSITIONED WITH RESPECT TO THE UNDERLYING REGULATORY DNA SEQUENCES. TAKEN TOGETHER, THESE AIMS WILL ILLUMINATE HOW TFS PIONEER THE CHROMATIN LANDSCAPE FOR ENHANCER ACTIVATION, THEREBY DEEPENING THE FIELD’S UNDERSTANDING OF THE MECHANISMS OF GENE REGULATION AND HOW MISREGULATION CONTRIBUTES TO HUMAN DISEASE.

DEFINING THE MECHANISMS OF NUCLEAR PORE COMPLEX ASSEMBLY IN FISSION YEAST

THE REGULATION AND CELLULAR DYNAMICS OF NEURAL CREST CELL DELAMINATION IN MAMMALIAN CRANIOFACIAL DEVELOPMENT - PROJECT SUMMARY CRANIOFACIAL ANOMALIES ACCOUNT FOR A THIRD OF ALL HUMAN CONGENITAL BIRTH DEFECTS AND SIGNIFICANTLY IMPACT NATIONAL HEALTH CARE BUDGETS. AFFECTED INDIVIDUALS TYPICALLY UNDERGO MULTIPLE SURGERIES THROUGHOUT THEIR LIFETIME, WHICH ARE RARELY FULLY CORRECTIVE. THEREFORE, IT IS CRITICAL TO DEVELOP THERAPIES FOR IMPROVED PROGNOSIS AND PREVENTION, BUT THIS CAN ONLY COME FROM A BETTER UNDERSTANDING OF THE GENETIC AND CELLULAR MECHANISMS GOVERNING CRANIOFACIAL DEVELOPMENT AND THE ETIOLOGY AND PATHOGENESIS OF INDIVIDUAL DISORDERS. DISRUPTIONS IN NEURAL CREST CELL (NCC) DEVELOPMENT ARE CONSIDERED THE UNDERLYING CAUSE OF MANY CRANIOFACIAL BIRTH DEFECTS. THEREFORE, UNDERSTANDING THE GENETIC AND CELLULAR MECHANISMS THAT REGULATE NCC DEVELOPMENT AND THEIR ULTIMATE GENERATION OF CRANIOFACIAL TISSUE IS CRUCIAL FOR DEVELOPING PREVENTATIVE THERAPIES AND IMPROVED SURGICAL PROGNOSIS. DELAMINATION FROM THE NEUROEPITHELIUM IS A CRITICAL STEP IN THE FORMATION OF MIGRATING NCC. HOWEVER, THE MOLECULAR AND CELLULAR MECHANISMS GOVERNING NCC DELAMINATION IN MAMMALIAN EMBRYOS ARE POORLY UNDERSTOOD. DELAMINATION IS A BIOPHYSICAL PROCESS BY WHICH A CELL DEPARTS ITS TISSUE ENVIRONMENT, AND MY EXTENSIVE PRELIMINARY DATA INDICATES THAT CELL EXTRUSION MAY BE A NOVEL MECHANISM FACILITATING CRANIAL NCC DELAMINATION. THIS PROPOSAL FOCUSES ON CELL EXTRUSION AND WILL BROADEN OUR UNDERSTANDING OF NCC DELAMINATION BY ELUCIDATING THE CELLULAR AND GENETIC SYSTEMS REGULATING NCC DELAMINATION VIA CELL EXTRUSION. AIM1 WILL VISUALIZE AND CAPTURE THE DYNAMIC CYTOARCHITECTURAL AND MORPHOLOGICAL CHANGES THAT DRIVE CRANIAL NCC DELAMINATION IN MOUSE EMBRYOS. AIM2 WILL INVESTIGATE THE ROLE OF PIEZO1, A MECHANOSENSITIVE ION CHANNEL IN CRANIAL NCC DELAMINATION AND CRANIOFACIAL DEVELOPMENT. THE IMPACT OF PIEZO1 LOSS-OF-FUNCTION ON NCC DELAMINATION AND CRANIOFACIAL DEVELOPMENT WILL BE ANALYZED THROUGH NULL AND CONDITIONAL GENETIC KNOCK OUT OF PIEZO1 MECHANOSENSITIVE ION CHANNELS IN MOUSE EMBRYOS. THE DOWNSTREAM REGULATORY NETWORK AND SIGNALING PATHWAYS WILL THEN BE ELUCIDATED THROUGH TRANSCRIPTOMIC COMPARISONS MEASURING THE DIFFERENCES IN CANDIDATE DOWNSTREAM REGULATORS BETWEEN WILD TYPE AND KNOCK OUT EMBRYOS. COMPLETION OF THE STUDY WILL ADVANCE FUNDAMENTAL KNOWLEDGE AND FURTHER OUR UNDERSTANDING OF NEURAL CREST CELL AND CRANIOFACIAL DEVELOPMENT AND OF CONGENITAL BIRTH DEFECTS.

ESTABLISHING THE AXIAL-SPECIFIC REGULATORY ROLE OF HOXA2 IN MOUSE CRANIAL NEURAL CREST CELL DEVELOPMENT

INVESTIGATING RIBOSOME BIOGENESIS AND FUNCTION IN THE AGING MAMMALIAN OOCYTE. - PROJECT SUMMARY THE FEMALE REPRODUCTIVE SYSTEM IS THE FIRST SYSTEM TO EXPERIENCE AGE-DEPENDENT DECLINE IN MAMMALS. IN HUMANS, THIS DECLINE BEGINS IN ONE’S MID-THIRTIES AND CONTINUES TO PROGRESS UNTIL MENOPAUSE IS REACHED AT APPROXIMATELY 50 YEARS OF AGE. THIS AGE-DEPENDENT DECLINE IS CHARACTERIZED BY MANY FERTILITY COMPLICATIONS THAT LEAD TO NEGATIVE OUTCOMES FOR MOTHER AND FETUS, SUCH AS PROLONGED TIME UNTIL CONCEPTION, SPONTANEOUS ABORTION, CHROMOSOME ABNORMALITIES, PREECLAMPSIA, PRETERM BIRTH, AND BIRTH DEFECTS. THIS PHENOMENON HAS BECOME A SIGNIFICANT HEALTH CARE CONCERN, AS AN ESCALATING NUMBER OF WOMEN ARE POSTPONING CHILDBEARING UNTIL ADVANCED REPRODUCTIVE AGE, LEADING TO INCREASED RELIANCE ON ARTIFICIAL REPRODUCTIVE TECHNOLOGIES (ART). UNFORTUNATELY, ART HAS YET TO OVERCOME THE PRIMARY SOURCE OF AGE-DEPENDENT FERTILITY DECLINE: OOCYTE QUALITY. DESPITE THE SIGNIFICANT ROLE OOCYTE QUALITY PLAYS IN FEMALE REPRODUCTIVE AGING, THE MOLECULAR COMPONENTS THAT LEAD TO ITS DECLINE REMAIN ELUSIVE. THE LONG-TERM GOAL OF THIS PROJECT IS TO IDENTIFY NOVEL MOLECULAR MECHANISMS THAT CONTRIBUTE TO OOCYTE QUALITY DECLINE. THE MAMMALIAN OOCYTE IS AN EXTREMELY LONG-LIVED CELL THAT ORIGINATES DURING EARLY EMBRYONIC DEVELOPMENT. NOT ONLY DOES IT CONTRIBUTE HALF OF AN EMBRYO’S GENOME, BUT IT ALSO PROVIDES MOST OF THE PROTEINS, LIPIDS, MRNA, AND NUTRIENTS REQUIRED FOR DEVELOPMENTAL COMPETENCE. TRANSLATION IS A VITAL MOLECULAR PLAYER THAT SUPPORTS DEVELOPMENTAL COMPETENCE, YET IT REMAINS UNCLEAR IF AN OOCYTE’S TRANSLATION ABILITIES CHANGE WITH AGE. I HAVE GENERATED SUBSTANTIAL PRELIMINARY EVIDENCE THAT SUGGEST TWO KEY ASPECTS OF TRANSLATION, (1) RIBOSOME BIOGENESIS AND (2) PROTEIN SYNTHESIS, BECOME PERTURBED WITH AGE IN OOCYTES. MY PROPOSAL WILL ELUCIDATE HOW TRANSLATION BECOMES PERTURBED WITH AGE THROUGHOUT OOCYTE DEVELOPMENT AND MATURATION USING A MOUSE MODEL. AIM 1 WILL CHARACTERIZE HOW RIBOSOME BIOGENESIS CHANGES WITH AGE BY TAKING A RIBOSOMAL RNA (RRNA)-CENTRIC APPROACH, AS RRNA IS THE RATE LIMITING FACTOR OF RIBOSOME BIOGENESIS. THIS AIM WILL BE ADDRESSED BY EXAMINING RRNA PRODUCTION AND THE THREE KEY COMPONENTS OF RRNA TRANSCRIPTION REGULATION: RDNA METHYLATION, RDNA TRANSCRIPTION FACTOR BINDING, AND RNA POLYMERASE 1, IN OOCYTES FROM REPRODUCTIVELY YOUNG AND OLD MICE. AIM 2 WILL DETERMINE HOW TRANSLATION ACTIVITY AND ACCURACY CHANGES WITH AGE, WITH A COMBINED IN VIVO AND IN VITRO APPROACH. GLOBAL TRANSLATION ACTIVITY, POLYSOME AND MONOSOME DISTRIBUTIONS, AND THE OCCURRENCE OF TRANSLATION ERRORS WILL BE MEASURED IN OOCYTES FROM REPRODUCTIVELY YOUNG AND OLD MICE. THE COMPLETION OF THE PROPOSED STUDY WILL PROVIDE NOVEL INSIGHT TO AGE-DEPENDENT CHANGES IN A CRITICAL COMPONENT OF OOCYTE QUALITY AND COULD CONTRIBUTE TO NEW THERAPEUTIC TARGETS TO IMPROVE REPRODUCTIVE LONGEVITY, AND ULTIMATELY MATERNAL AND FETAL HEALTH.

IN VIVO FUNCTIONAL CHARACTERIZATION OF MYELODYSPLASIA/MYELOID LEUKEMIA FACTOR

DISSECTING THE GENETIC BASIS OF SELFISH SEX CHROMOSOMES

POLR1C AND POLR1D MUTANT ZEBRAFISH AS NEW MODELS FOR TREACHER COLLINS SYNDROME

EVALUATING THE ROLE OF PROTEIN SELF-ASSEMBLY IN THE INNATE IMMUNE SYSTEM

TRANSLATION OF SMALL OPEN READING FRAMES IN 3'UTR ENHANCES TRANSLATION OF CANONICAL OPEN READING FRAMES

CONFLICT-DRIVEN ANEUPLOIDY AND GENOMIC INSTABILITY CAUSED BY MEIOTIC PROTEINS

ADVANCED IMAGING TOOLS TO EXAMINE INTERACTIONS, DYNAMICS OF SECRETORY COMPONENETS

DECIPHERING THE PARALOGUE SPECIFIC INTERACTIONS WITHIN THE SIN3 PROTEIN INTERACTION NETWORK

INVESTIGATING THE MECHANISM OF WTF KILLER MEIOTIC DRIVERS

NOVEL HDAC-INTERACTING PROTEINS THAT REGULATE BREAST CANCER CELL GROWTH

ANNUAL MEETING OF THE SOCIETY FOR CRANIOFACIAL GENETICS AND DEVELOPMENTAL BIOLOGY

THE 2016 SANTA CRUZ DEVELOPMENTAL BIOLOGY MEETING