Project Grant F31DE036114

REGULATION OF TGF-BETA SUPERFAMILY SIGNALING IN THE NEURAL CREST LINEAGE AND CRANIOFACIAL PATHOLOGY - PROJECT SUMMARY THE NEURAL CREST (NC) IS AN EARLY EMBRYONIC CELL POPULATION THAT DIFFERENTIATES INTO MULTIPLE TERMINAL DERIVATIVES AT ALL AXIAL LEVELS. IMPROPER FORMATION OF NC CELLS RESULTS IN A VARIETY OF COMPLEX PATHOLOGICAL SYNDROMES AND PEDIATRIC CANCER. A KEY ASPECT OF NC FORMATION IS SIGNALING INPUT FROM THE TGF-B SUPERFAMILY (BMP, TGF-B, ACTIVIN, NODAL). PRIOR WORK HAS ELABORATED ON THE CONTRIBUTIONS OF BMP SIGNALING TO NC FORMATION AND ITS TEMPORAL REQUIREMENT. TGF-B SIGNALING IS ALSO KNOWN TO BE REQUIRED FOR PROPER NC FORMATION, BUT THE TEMPORAL DYNAMICS OF ITS ACTIVITY ARE NOT WELL UNDERSTOOD. FURTHERMORE, THE DIRECT TARGETS OF THESE SIGNALING PATHWAYS IN THE HUMAN NC (HNC) GENE REGULATORY NETWORK (GRN) ARE NOT KNOWN. CONTINUED CONTRIBUTIONS OF TGF-B SUPERFAMILY SIGNALING ARE ALSO REQUIRED FOR THE DIFFERENTIATION OF CRANIOFACIAL NC LINEAGES, INCLUDING TEETH, BONE, AND CARTILAGE. THEREFORE, THE OBJECTIVE OF THIS PROPOSAL IS TO STUDY THE MECHANISMS REGULATING TGF-B SUPERFAMILY TARGET SPECIFICITY IN THE HNC GRN. THE IDENTIFICATION OF SMAD-MEDIATED CHROMATIN INTERACTIONS (CIS- REGULATORY MODULES; CRMS) IN HNC CELLS WILL ENABLE THE IDENTIFICATION OF DIRECT TGF-B SUPERFAMILY TARGETS IN THE HNC GRN. IN OUR STUDIES OF ONE NC PATHOLOGY, MOWAT-WILSON SYNDROME (MWS), WE OBSERVED DYSREGULATION OF BMP SIGNALING IN HNC GENERATED FROM PATIENT-DERIVED INDUCED PLURIPOTENT STEM CELLS. THROUGH A LOSS-OF-FUNCTION APPROACH, WE ALSO IDENTIFIED DYSREGULATED FACTORS KNOWN TO INTERACT WITH SMADS-THE DOWNSTREAM EFFECTORS OF TGF-B SUPERFAMILY PATHWAYS. SOX5, DACH1, AND HIVEP2 ARE KNOWN SMAD CO-FACTORS IN OTHER CONTEXTS, BUT ARE SIGNIFICANTLY UNDERSTUDIED IN NC, PARTICULARLY IN HUMAN NC. CONSIDERING THEY ARE DYSREGULATED IN MWS HNC, IT MAY BE SUGGESTED THESE SMAD CO-FACTORS CONTRIBUTE TO THE DYSREGULATED TGF-B SUPERFAMILY STATE IN MWS. THUS, MY OVERALL HYPOTHESIS IS THAT SMAD-MEDIATED CHROMATIN INTERACTIONS UNDERLIE NC DEFECTS IN THE MWS PHENOTYPE, WHILE THE SMAD CO-TRANSCRIPTION FACTORS SOX5, DACH1, AND HIVEP2 REGULATE SMAD TARGET SPECIFICITY IN THE HNC GRN. THIS WILL BE ADDRESSED IN THE FOLLOWING SPECIFIC AIMS. IN AIM 1, SMAD-MEDIATED CRMS WILL BE INTERROGATED USING HI-CHIP WITH BIOINFORMATIC INTEGRATION OF SMAD CUT & RUN TO IDENTIFY CRMS ASSOCIATED WITH SMAD OCCUPANCY. IN AIM 2, REGULATORY ROLES OF SOX5, DACH1, AND HIVEP2 WILL BE INTERROGATED BY LOSS-OF-FUNCTION APPROACHES. THEN, CUT & RUN IN WT AND MWS HNC WILL EXAMINE THE ASSOCIATION OF THESE FACTORS WITH SMAD OCCUPANCY. FINALLY, SOX5 WILL BE PROBED IN GREATER DETAIL IN HUMAN TOOTH ORGANOIDS DUE TO THE PREVALENCE OF DENTAL ANOMALIES OBSERVED IN NEUROCRISTOPATHIES. OVERALL, THESE STUDIES WILL DEFINE SMAD- MEDIATED CRMS AND REVEAL DIRECT TARGETS OF TGF-B SUPERFAMILY SIGNALING PATHWAYS IN THE HNC GRN. THEY WILL ALSO DEFINE SOX5, DACH1, AND HIVEP2 AS MODULATORS OF TGF-B SUPERFAMILY INPUTS THROUGH A SMAD CO-FACTOR MECHANISM. THE PROPOSED WORK HAS BROAD IMPLICATIONS FOR THE DEVELOPMENT OF DIAGNOSTIC AND THERAPEUTIC STRATEGIES IN THE TREATMENT OF CRANIOFACIAL PATHOLOGIES.