Saturday, May 4, 2024
5/4/2024
PROJECT SUMMARY/ABSTRACT ALX4 (Aristaless-like homeobox 4) is a conserved homeodomain transcription factor important for bone, skull, and limb development. Mutations in ALX4 cause the autosomal dominant disease parietal foramina 2 (PFM2) and the autosomal recessive disease frontonasal dysplasia 2 (FND2), and autosomal dominant variants in ALX4 are associated with an increased risk for craniosynostosis 5 (CRS5). Despite its important role in development and association with rare human diseases, ALX4 is an understudied protein with limited biochemical/cellular, biophysical, and structural characterization, which limits our understanding of ALX4 function under normal physiological and human disease contexts, such as PFM2, FND2, and CRS5. The overall goal of this proposal is to purify and characterize WT and disease associated ALX4 variants using (1) X-ray crystallography to solve its high-resolution structure, (2) biochemical/biophysical approaches, such as electrophoretic mobility shift assays (EMSAs) and isothermal titration calorimetry (ITC), to analyze its binding interactions with DNA, and (3) luciferase assays to assess their transcriptional regulatory properties. Our preliminary data supports the hypothesis that these variants negatively affect ALX4 interactions with DNA and/or cooperative binding, which underlies the pathophysiologic basis of these variants in PFM2, FND2, and CSR5. To test our hypothesis and achieve our goal, we will pursue the following two experimental aims – Aim1: Purify and determine high-resolution structures of ALX4 and Aim2: Characterize the DNA binding and regulatory properties of WT ALX4 and disease variants. Completion of this proposal will provide a deeper understanding of ALX4 structure and function for both WT and disease associated variants that map to its HD. Moreover, completion of this pilot project grant will lay the groundwork for subsequent collaborative studies that characterize ALX4 function in human development and disease using a multidisciplinary approach, combining structural and in vitro biophysical and biochemical/cellular studies with organoid and mouse models of ALX4.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
