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

> **NIH NIH R56** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2022 · $323,000

## Abstract

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...

## Key facts

- **NIH application ID:** 10645781
- **Project number:** 2R56DE027076-06
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Yan Zhang
- **Activity code:** R56 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $323,000
- **Award type:** 2
- **Project period:** 2022-09-08 → 2023-09-05

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10645781

## Citation

> US National Institutes of Health, RePORTER application 10645781, The stage-specific regulation of ameloblastin and enamelin by the distinct nuclear factors (2R56DE027076-06). Retrieved via MORVS 2026-10-25 from https://api.morvs.ai/grant/nih/10645781. Licensed CC0.

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