Unraveling the Regulation of Transcription and Activity of Calcium Transporter SLC24A4 in Enamel Formation - Project Summary/Abstract The exceptional strength of enamel can be attributed to its high mineral content within the matured matrix. This biomineralization process relies heavily on maturation ameloblasts (MABs), responsible for transporting approximately 86% of the required minerals. Among the key players in this intricate process is the K+- dependent Ca2+/Na+-exchanger SLC24A4. Disruptions of SLC24A4 contribute to the development of an enamel disorder called amelogenesis imperfecta. SLC24A4 is highly upregulated in those post-secretory stages of ameloblasts, where it shuttles between the apical plasma membrane (PM) of the ruffle-ended and the cytosol of the smooth-ended MAB subpopulations. The mechanisms governing SLC24A4’s temporal and spatial expression in MABs remain largely unexplored. During development, secretory ameloblasts (SABs) are succeeded by the transition stage of ameloblasts (TABs). TABs play a crucial role in modifying the protein composition and mechanical stress within the underlying enamel matrix by introducing the potent proteinase KLK4. Our preliminary data indicated that factors present in the transition stage of enamel, including amelogenin hydrolysis peptides (Amelx20, C-domain, and C-P1), along with gradually intensified mechanical stiffness, enhance the transcription of Slc24a4. Additional data indicated a correlation between the transcription of Slc24a4 and heightened levels of H3K4 methylation. Simultaneously, we discovered that exposure to BSA-saturated palmitic acid (PA) elevated the levels of protein palmitoylation and enriched SLC24A4 along the PM of ameloblast lineage cells (ALCs). Furthermore, our findings revealed a significant concentration of palmitoyltransferase ZDHHC21 within PM of the ruffle-ended MABs, implying a potential role of protein palmitoylation in the cellular trafficking of SLC24A4. We hypothesize that the combined influence of developmental cues and protein palmitoylation regulates the calcium transport capacity of SLC24A4 at both the transcriptional and activity levels. To investigate this hypothesis, we have formulated two specific aims. Specific aim 1: To determine the molecular mechanisms underlying the upregulation of SLC24A4 transcription in MABs. To assess the impact of development cues, we will conduct a comparative analysis of H3K4me3 marks in ALCs treated with and without these cues using ChIP-seq. To validate the intensity of H3K4me3 marks in the regulatory region of the SLC24A4 gene within rare cell populations of wt mouse SABs and MABs, we will employ the recently developed CUT&Run-seq analysis. Specific aim 2: To define the significance of protein palmitoylation in governing the activity of SLC24A4. We propose to quantitate the protein palmitoylation levels of SLC24A-transduced ALCs treated with or without BSA-PA using click chemistry and proximity ligation assay. Next, we aim to determine the palmitoylation sites of SLC24A4 using immunoprecipitation and mass spectrometry. Understanding the factors and mechanisms that regulate SLC24A4 transcription and activity is important for both enamel biology and regenerating ameloblast/enamel.
