Identification of esrp1 as a novel IRF6 Target Gene and Determining its Role in Palate Development
Dawn Truong, BA Candidate, Edward Li, BA, Shawn Hallett, BA, Eric C. Liao, MD-PhD.
Massachusetts General Hospital, Cambridge, MA, USA.
IRF6 transcription factor is one of the most important genetic regulators of palate development, mutations of which result in cleft lip and palate (CLP). IRF6 directly regulates another transcription factor GRHL3 which is also a key regulator of palatogenesis and implicated in CLP pathogenesis. Therefore we hypothesized that systematic identification of IRF6 target genes will further elucidate the molecular regulation of palate development, and discover additional genetic determinants of CLP pathogenesis.
A irf6 zebrafish mutant was generated and used in a subtractive ChIP-seq and RNA-seq strategy. These datasets were overlapped to show genes directly under IRF6 transcriptional regulation and also differentially expressed between wild type and irf6 mutants. Integration of ChIP-seq and mRNA-seq dataset enriched for a subset of irf6 target candidate genes of interest. We further refined this candidate gene list based on high-throughput gene expression studies and quantitative PCR of the relative expression levels of these genes in irf6-/- tissues compared to wild type controls.
Several irf6 target genes were identified, shown to be down-regulated in irf6 mutants, and were previously not recognized to be regulated by Irf6. For example, epithelial splicing regulatory protein 1 (esrp1), is a RNA-binding protein that regulates alternative splicing in epithelial cells and has been shown to have a role in the alternative splicing of Fibroblast Growth Factor Receptors (FGFR), epithelial-to-mesenchymal transition and cell polarity. Esrp1 -/- mouse models have the presence of an anterior-to-poster fully penetrant cleft palate and microcephaly. Interestingly, irf6 -/- mutant mice also have cleft palate. Esrp1 knockout mice also have shortened limbs, suggesting a functional role for the protein in FGFR-regulated limb induction due to its role in FGFR alternative splicing. Furthermore, in irf6 -/- tissues, esrp1 expression levels were significantly reduced compared to wild type tissues.
This work revealed esrp1 to be a direct downstream target of irf6. The craniofacial phenotype in an esrp1-/- mouse model suggests that esrp1 may be necessary for palate morphogenesis, acting under Irf6 regulation. Due to its function as a RNA-binding protein and role in FGFR slicing, studying the process by which esrp1 modulates craniofacial development and neural crest cell migration is ongoing. Current work also focuses on examining whole genome sequencing data from CLP trios to uncover ESRP1 gene variants to examine its role in cleft pathogenesis.
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