Early growth response protein 1 regulates promoter activity of α-plasma membrane calcium ATPase 2, a major calcium pump in the brain and auditory system

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Abstract

Background

Along with sodium/calcium (Ca ²⁺) exchangers, plasma membrane Ca ²⁺ ATPases (ATP2Bs) are main regulators of intracellular Ca ²⁺ levels. There are four ATP2B paralogs encoded by four different genes. Atp2b2 encodes the protein pump with the fastest activation, ATP2B2. In mice, the Atp2b2 transcript has several alternate transcriptional start site variants: α, β, µ and δ. These variants are expressed in developmental and tissue specific manners. The α and β Atp2b2 transcripts are equally expressed in the brain. αAtp2b2 is the only transcript found in the outer hair cells of young mice (Silverstein RS, Tempel BL. in Neuroscience 141:245–257, 2006). Mutations in the coding region of the mouse Atp2b2 gene indicate a narrow window for tolerated dysfunction of the ATP2B2 protein, specifically in the auditory system. This highlights the necessity of tight regulation of this gene for normal cell physiology.

Results

Although ATP2Bs are important regulators of Ca ²⁺ in many cell types, little is known about their transcriptional regulation. This study identifies the proximal promoter of the αAtp2b2 transcript. Further investigations indicate that ATOH1 and EGR1 modulate promoter activity. Additionally, we report that EGR1 increases endogenous expression of Atp2b2 transcript in two cell lines. Electrophoretic mobility shift assays (EMSA) indicate that EGR1 binds to a specific site in the CpG island of the αAtp2b2 promoter.

Conclusion

This study furthers our understanding of Atp2b2 regulation by: (I) elucidating transcriptional regulatory mechanisms for Atp2b2, and (II) identifying transcription factors that modulate expression of Atp2b2 in the brain and peripheral auditory system and (III) allows for future studies modulating gene expression of Atp2b2.

Electronic supplementary material

The online version of this article (doi:10.1186/s12867-017-0092-1) contains supplementary material, which is available to authorized users.

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Most cited references 37

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Metazoan promoters: emerging characteristics and insights into transcriptional regulation.

Boris Lenhard, Albin Sandelin, Piero Carninci (2012)

Promoters are crucial for gene regulation. They vary greatly in terms of associated regulatory elements, sequence motifs, the choice of transcription start sites and other features. Several technologies that harness next-generation sequencing have enabled recent advances in identifying promoters and their features, helping researchers who are investigating functional categories of promoters and their modes of regulation. Additional features of promoters that are being characterized include types of histone modifications, nucleosome positioning, RNA polymerase pausing and novel small RNAs. In this Review, we discuss recent findings relating to metazoan promoters and how these findings are leading to a revised picture of what a gene promoter is and how it works.

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Transcription profiling of inner ears from Pou4f3(ddl/ddl) identifies Gfi1 as a target of the Pou4f3 deafness gene.

Ronna Hertzano, Gideon Rechavi, Tarik Möröy … (2004)

Pou4f3 (Brn3.1, Brn3c) is a class IV POU domain transcription factor that has a central function in the development of all hair cells in the human and mouse inner ear sensory epithelia. A mutation of POU4F3 underlies human autosomal dominant non-syndromic progressive hearing loss DFNA15. Through a comparison of inner ear gene expression profiles of E16.5 wild-type and Pou4f3 mutant deaf mice using a high density oligonucleotide microarray, we identified the gene encoding growth factor independence 1 (Gfi1) as a likely in vivo target gene regulated by Pou4f3. To validate this result, we performed semi-quantitative RT-PCR and in situ hybridizations for Gfi1 on wild-type and Pou4f3 mutant mice. Our results demonstrate that a deficiency of Pou4f3 leads to a statistically significant reduction in Gfi1 expression levels and that the dynamics of Gfi1 mRNA abundance closely follow the pattern of expression for Pou4f3. To examine the role of Gfi1 in the pathogenesis of Pou4f3-related deafness, we performed comparative analyses of the embryonic inner ears of Pou4f3 and Gfi1 mouse mutants using immunohistochemistry and scanning electron microscopy. The loss of Gfi1 results in outer hair cell degeneration, which appears comparable to that observed in Pou4f3 mutants. These results identify Gfi1 as the first downstream target of a hair cell specific transcription factor and suggest that outer hair cell degeneration in Pou4f3 mutants is largely or entirely a result of the loss of expression of Gfi1.

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A Novel Atoh1 “Self-Terminating” Mouse Model Reveals the Necessity of Proper Atoh1 Level and Duration for Hair Cell Differentiation and Viability

Ning Pan, Israt Jahan, Jennifer Kersigo … (2012)

Atonal homolog1 (Atoh1) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO) mouse line using Tg(Atoh1-cre), in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to “self-terminate” its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.

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Author and article information

Contributors

Rebecca R. Minich:

ORCID: http://orcid.org/0000-0002-8700-237X

minire@uw.edu

Jin Li: jliyan@uw.edu

Bruce L. Tempel: bltempel@uw.edu

Journal

Journal ID (nlm-ta): BMC Mol Biol

Journal ID (iso-abbrev): BMC Mol. Biol

Title: BMC Molecular Biology

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2199

Publication date (Electronic): 22 May 2017

Publication date PMC-release: 22 May 2017

Publication date Collection: 2017

Volume: 18

Electronic Location Identifier: 14

Affiliations

[1 ]ISNI 0000000122986657, GRID grid.34477.33, Department of Pharmacology, School of Medicine, , University of Washington, ; Seattle, WA 98195 USA

[2 ]ISNI 0000000122986657, GRID grid.34477.33, Department of Otolaryngology-HNS, School of Medicine, , University of Washington, ; Box 357923, Seattle, WA 98195 USA

[3 ]ISNI 0000000122986657, GRID grid.34477.33, Virginia Merrill Bloedel Hearing Research Center, School of Medicine, , University of Washington, ; Seattle, WA 98195 USA

Author information

Rebecca R. Minich http://orcid.org/0000-0002-8700-237X

Article

Publisher ID: 92

DOI: 10.1186/s12867-017-0092-1

PMC ID: 5441030

PubMed ID: 28532435

SO-VID: 874e4933-dabd-4a01-84ed-d3dc33f44a03

License:

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

History

Date received : 17 February 2016

Date accepted : 8 May 2017

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000009, Foundation for the National Institutes of Health;

Award ID: RO1 DC02739

Award ID: R21 DC014864

Award ID: P30 DC04661

Award ID: T32 DC005361

Award Recipient : Rebecca R. Minich Bruce L. Tempel

Custom metadata

ScienceOpen disciplines: Molecular biology

Keywords: atonal bhlh transcription factor 1 (atoh1),plasma membrane calcium atpase 2 (atp2b2),plasma membrane calcium-transporting atpase 4 (atp2b4),early growth response protein 1 (egr1),gene regulation,gene transcription,minimal promoter,promoter

Data availability: