Project Summary/abstract Cell identity is largely determined by specific epigenetic landscapes and transcriptional networks. Ameloblast is the only epithelial cell that can generate calcified tissue during development. Classified into two major stages, the secretory stage of ameloblasts (SABs) synthesize and deposit enamel matrix proteins (EMPs) to scaffold organic matrix, and the maturation stage of ameloblasts (MABs) hydrolyze, endocytose EMPs, and transport ions to mineralize enamel. To bioengineer enamel, a nonregenerative tissue, we must understand the transcriptional regulation of ameloblasts. To date, understanding of the unique transcriptional regulation of ameloblasts has been limited due to a loss of ameloblasts after the tooth eruption and a lack of cell line fully recapitulating the characteristics of ameloblasts. Previous funding allows us to establish a novel and comprehensive list of genes significant to each developmental stage of ameloblasts across species and to explore the functions of chromatin organizer SATB1, peptidase KLK4, and the major calcium transporter NCKX4 in the context of ameloblast differentiation. These efforts resulted in a discovery that all SATB1, KLK4, and NCKX4 contribute to the transcriptional regulation of ameloblastin (Ambn) and enamelin (Enam), encoding the major EMPs co-upregulated in SABs and then co-downregulated in MABs. In SABs, we found that ablation of SATB1, highly expressed in preameloblasts (PABs), greatly repressed Ambn & Enam transcription and H3K27ac level. Our organ culture showed that elevated histone acetylation upregulated Ambn & Enam. An enhancer and base unpairing region (BUR, preferentially selected by SATB1) have been predicted in the vicinity of Ambn & Enam. These data suggest that SATB1 plays an important role in establishing enhancer for the upregulation of Ambn & Enam in SABs. In the case of mice lacking Klk4 and Nckx4—the causative genes for amelogenesis imperfecta—we found a retention of proline-rich EMPs resulting from defective hydrolysis. Responding to the reduced proline retake from enamel, the Nckx4-/- and Klk4-/- MABs upregulated Ambn & Enam and downregulated Hif1a. In vitro studies showed that proline downregulated Ambn & Enam and upregulated Hif1a. HIF1A, a transcription factor regulating cell responses to oxidative stress, had a 6-fold upregulation in MABs vs SABs in our RNA-seq analyses, reflecting MAB’s robust anti-oxidative capacity to continuously provide energy for ion transport and protein degradation. These data suggest that retake of proline upregulates Hif1a, which in turn downregulats Ambn & Enam. Our in vivo and in vitro studies allow us to hypothesize that the dynamic expression of Ambn & Enam in the two major functional stages of ameloblasts is coordinately regulated by distinct factors chromatin organizer SATB1 and transcription factor HIF1A. This hypothesis will be addressed by specific aim 1: To determine the roles of SATB1 as a pioneer factor in PABs t...