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A Critical E-box in Barhl1 3′ Enhancer Is Essential for Auditory Hair Cell Differentiation

1
Institute of Cell and Developmental Biology, College of Life Sciences, Zhejiang University, Hangzhou 310058, China
2
Department of Biotechnology and Genetic Engineering, Faculty of Biological Sciences, Islamic University, Kushtia 7003, Bangladesh
3
Institute of Genetics, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310058, China
4
Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Hangzhou 310058, China
*
Author to whom correspondence should be addressed.
Cells 2019, 8(5), 458; https://doi.org/10.3390/cells8050458
Received: 10 April 2019 / Revised: 6 May 2019 / Accepted: 7 May 2019 / Published: 15 May 2019
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Abstract

Barhl1, a mouse homologous gene of Drosophila BarH class homeobox genes, is highly expressed within the inner ear and crucial for the long-term maintenance of auditory hair cells that mediate hearing and balance, yet little is known about the molecular events underlying Barhl1 regulation and function in hair cells. In this study, through data mining and in vitro report assay, we firstly identified Barhl1 as a direct target gene of Atoh1 and one E-box (E3) in Barhl1 3’ enhancer is crucial for Atoh1-mediated Barhl1 activation. Then we generated a mouse embryonic stem cell (mESC) line carrying disruptions on this E3 site E-box (CAGCTG) using CRISPR/Cas9 technology and this E3 mutated mESC line is further subjected to an efficient stepwise hair cell differentiation strategy in vitro. Disruptions on this E3 site caused dramatic loss of Barhl1 expression and significantly reduced the number of induced hair cell-like cells, while no affections on the differentiation toward early primitive ectoderm-like cells and otic progenitors. Finally, through RNA-seq profiling and gene ontology (GO) enrichment analysis, we found that this E3 box was indispensable for Barhl1 expression to maintain hair cell development and normal functions. We also compared the transcriptional profiles of induced cells from CDS mutated and E3 mutated mESCs, respectively, and got very consistent results except the Barhl1 transcript itself. These observations indicated that Atoh1-mediated Barhl1 expression could have important roles during auditory hair cell development. In brief, our findings delineate the detail molecular mechanism of Barhl1 expression regulation in auditory hair cell differentiation. View Full-Text
Keywords: Barhl1; Atoh1; E-box; mESCs; auditory hair cells Barhl1; Atoh1; E-box; mESCs; auditory hair cells
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Hou, K.; Jiang, H.; Karim, M.R.; Zhong, C.; Xu, Z.; Liu, L.; Guan, M.; Shao, J.; Huang, X. A Critical E-box in Barhl1 3′ Enhancer Is Essential for Auditory Hair Cell Differentiation. Cells 2019, 8, 458.

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