© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Transcriptional regulation of the murine Connexin40 promoter by cardiac factors Nkx2-5, GATA4 and Tbx5
aLaboratório de Cardiologia Celular e Molecular-Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 20941-900, Brazil
bDevelopmental Biology Unit, Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia
cSection of Pediatric Hematology/Oncology, Department of Pediatrics, University of Chicago, Chicago, IL 60637-1470, USA
* Corresponding author. Developmental Biology Unit, Victor Chang Cardiac Research Institute, 384 Victoria St, Darlinghurst, NSW, 2010, Australia. Tel.: +61 2 92958522; fax: +61 2 92958501. Email address: m.costa{at}victorchang.unsw.edu.au
Objective: Connexin40 (Cx40) is a gap junction protein expressed specifically in developing and mature atrial myocytes and cells of the conduction system. In this report, we identify cis-acting elements within the mouse Cx40 promoter and unravel part of the complex pathways involved in the cardiac expression of this gene.
Methods: To identify the factors involved in the cardiac expression of Cx40, we used transient transfections in mammalian cells coupled with electrophoretic mobility shift assays (EMSA) and RT-PCR.
Results: Within the promoter region, we identified the minimal elements required for transcriptional activity within 150 base pairs (bp) upstream of the transcriptional start site. Several putative regulatory sites for transcription factors were predicted within this region by computer analysis, and we demonstrated that the nuclear factors Sp1, Nkx2-5, GATA4 and Tbx5 could interact specifically with elements present in the minimal promoter region of the Cx40. Furthermore, co-transfection experiments showed the ability of Nkx2-5 and GATA4 to transactivate the minimal Cx40 promoter while Tbx5 repressed Nkx2-5/GATA4-mediated activation. Mutagenesis of the Nkx2-5 core site in the Cx40 promoter led to significantly decreased activity in rat smooth muscle cell line A7r5. Consistent with this, mouse embryos lacking Nkx2-5 showed a marked decrease in Cx40 expression.
Conclusion: In this work, we cloned the promoter region of the Cx40 and demonstrated that the core promoter was modulated by cardiac transcriptional factors Nkx2-5, Tbx5 and GATA4 acting together with ubiquitous Sp1.
KEYWORDS Gap junctions; Cx40; Nkx2-5; GATA4; Tbx5; Muscle gene regulation
Abbreviations: Cx40, connexin40 • bp, base pairs • kb, kilobase pairs • PCR, polymerase chain reaction • EMSA, electrophoretic mobility shift assay • dpc, days post coitum • AVC, atrioventricular canal • OFT, outflow tract
1 These authors contributed equally to this work.
2 Current address: The Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Rd., Cambridge, CB2 1QR, UK.
Time for primary review 25 days
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