Saturday, April 20, 2024
4/20/2024
DESCRIPTION (provided by applicant): Mammals born with mutations in the ptfla gene, a basic helix-loop-helix transcription factor, suffer severe disease manifested by pancreatic and cerebellar agenesis. However, transcriptional targets of PTF1a during embryogenesis are unknown. Similar to mammals, mutations in zebrafish ptfla cause exocrine pancreatic agenesis and embryonic lethality. The zebrafish presents a unique opportunity to study ptf1a- mediated pathology in a genetically tractable model system. The goal of this proposal is unbiased genome- wide evaluation of chromosomal occupancy by PTF1a during zebrafish embryogenesis at two important developmental time points, 24 and 48 hpf. The approach identifies novel PTF1a-DNA interactions in two steps. First chromatin immunoprecipitation (ChIP) isolates chromosomal DNA physically bound to PTF1a. This is followed by a modified longSAGE technique involving paired-end ditag (PET) serial analysis of chromosomal occupancy (SACO) to map PTFla-associated DNA to a specific chromosomal location within the zebrafish genome. Identifying dynamic temporal interactions of PTF1a and DNA during development will be invaluable for determining novel PTF1 target genes and their role in ptfl a-dependent organogenesis of exocrine pancreas and hindbrain in CNS. To perform ChIP and PET-SACO to identify PTF1a chromosomal occupancy, we will require a transgenic zebrafish expressing epitope tagged PTF1 a within endogenous ptf1a expressing tissues using the GAL4- UAS expression system. Specific aim one involves creation of ptf1a:gal4; UAS:flag-ptf1 a transgenic fish and demonstrates the feasibility of in vivo ChIP analysis of PTF1a-DNA interactions using known PTF1 a target genes. Specific aim two applies ptf1a:gal4; UAS:flag-ptf1 a transgenic fish to in vivo analysis of PTF1a chromosomal occupancy during zebrafish embryogenesis using ChIP and PET-SACO methodology. Specific aim three validates putative PTF1a target genes and explores alternate approaches for identifying PTF1a target genes during development. Data from these specific aims will mechanistically identify and define PTF1a target genes and their signaling networks during embryogenesis. The ptf1a gene is required for proper pancreas and brain development. Disruptions of ptf1a result in life- long disease. We aim to discover novel roles for ptf1a during development. These discoveries may result in translation to therapeutics for individuals suffering neurological and pancreatic disease.
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