The stage-specific regulation of ameloblastin and enamelin by the distinct nuclear factors

University Of California, San Francisco, San Francisco CA

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Abstract

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. The initial phase of ameloblast differentiation becomes apparent with the transition from low columnar epithelial cells, known as pre-ameloblasts (PABs), to tall columnar secretory ameloblasts (SABs). It is the SABs that synthesize and deposit unique enamel matrix proteins (EMPs), which scaffold the formation of the organic enamel matrix. These EMPs template the assembly of hydroxyapatites, ultimately occupying the matrix and creating the hardest tissue in the human body. To bioengineer enamel, a tissue that lacks regenerative capacity, a thorough comprehension of the transcriptional regulation of ameloblasts is essential. Specifically, we need to grasp how cells construct their transcriptional programs to facilitate the transition from PABs to SABs. Building upon this foundation, we identified the distinctive temporal expression profile of chromatin organizer, special AT-rich binding protein 1 (SATB1), and have been exploring its indispensable role in orchestrating the transition from pre-ameloblasts (PABs) to secretory ameloblasts (SABs). SATB1 governs the establishment of polarity in secretory ameloblasts (SABs), the secretion of enamel matrix (ECM) proteins, and the structural integrity of the enamel matrix. We also found that ablation of SATB1, highly expressed in PABs, repressed H3K27ac level and Ambn & Enam transcription in SABs. Ameloblastin (Ambn) and enamelin (Enam) encode the major EMPs co-upregulated in SABs and then co-downregulated MABs advance. Our organ culture showed that elevated histone acetylation upregulated Ambn & Enam. An enhancer and base unpairing region (BUR, selective SATB1 DNA binding site) have been predicted in the vicinity of Ambn & Enam gene loci. To further understand the roles of SATB1 in establishing SAB gene transcriptional program and to overcome the low survival rate observed in global SATB1 knock-out mouse lines, we generated additional mouse lines: Satb1 cKO, achieved by crossing K14 Cre with Satb1fl/fl, and Satb1BUR-/-, accomplished by targeted deletion of the SATB1 DNA binding site within Ambn & Enam gene cluster. To address the overarching hypothesis stated in the parental R01, which suggests SATB1 organizes chromatin conformation and poises a transcriptional complex to upregulate Ambn & Enam to advance PABs to SABs, we propose additional experiments in this diversity supplement to complement the original specific aim 1. In alignment with the parental grant, we're conducting chromatin openness profiling, initially targeting the gene loci housing ameloblastin and enamelin. Our investigation spans conditions with or without SATB1, or where SATB1 cannot bind to the BUR site near the ameloblastin/enamelin gene cluster. We're utilizing enamel organ cells and ATAC-seq to achieve this. In further pursuit, we will explore the prospect of employing single cells as input materials for ATAC-seq analyses.