Study on the molecular mechanisms of the orphan nuclear receptor COUP-TFII action during cardiac chambers development

• Grant number: 12/14859-0
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Effective date (Start): June 01, 2013
• Effective date (End): May 31, 2015
• Field of knowledge: Biological Sciences - Morphology - Embryology
• Principal Investigator: José Xavier Neto
• Grantee: Carlo Donato Simões Caiaffa Feliciano de Carvalho
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil

Abstract

The embryonic development integrates different levels of organization that are activated and distributed during the events of cell specialization in tissues and organs. Over the past 14 years, our group has generated strong evidence that retinoic acid (RA) signaling is essential for the specification of cardiac progenitor cells in anterior and posterior domains, which give rise to ventricular and sino-atrial tissues respectively. During early somitogenesis, RA induces a caudo-rostral wave of RALDH2 expression, which extends from lateral plate mesoderm to a posterior region in the heart field. The SMyHC3 gene (slow myosin heavy chain 3), initially characterized in quails (Coturnix japonica), shows a preference for atrial expression due the presence of inserted elements in the promoter region, that are requested to activate this gene in atrial precursor cells and to repress it in ventricles. To understand this feature, a fragment with 840 base pairs (bp) of the SMyHC3 gene promoter was used to drive human alkaline phosphatase (HAP) expression in transgenic mice. The reporter gene staining indicated that the transgene is induced by RA, and that the promoter activity is localized in the sino-atrial region, showing an expression pattern coincident with the cardiac fields rich in RALDH2 expression and endogenous RA production. Our group has isolated a complex element of nuclear receptor response (CENRR) with 33 bp, which is inserted in an atrial regulatory domain of the SMyHC3 gene promoter. Electrophoretic mobility shift assays using a control probe to compete against the CENRR for binding to RA receptors (RAR/RXR) demonstrate that the RAR/RXR heterodimer does not have significant influence on the SMyHC3 gene promoter, suggesting that the RA role during promoter activation may be indirect. Recent findings from our group demonstrated that the orphan nuclear receptor COUP-TFII (Chicken Ovalbumin Upstream Promoter - Transcription Factor 2) is able to activate the SMyHC3 promoter in transfection assays, while RNA interference against COUP-TFII inhibited the activation. In order to dissect the mechanism of the SMyHC3 promoter activation, we intend to identify COUP-TFII interaction partners using yeast two-hybrid technology. In a parallel approach we will use the yeast one-hybrid system to identify proteins that are able to associate with CENRR. To find the ligand complex that interacts with COUP-TFII downstream of RA action, during cardiac chamber specification, a strategy that combines immunoprecipitation and mass spectrometry will also be used in the course of this project. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

XAVIER-NETO, JOSE; SOUSA COSTA, ANGELA M.; FIGUEIRA, ANA CAROLINA M.; CAIAFFA, CARLO DONATO; DO AMARAL, FABIO NEVES; CERQUEIRA PERES, LARA MALDANIS; PIRES DA SILVA, BARBARA SANTOS; SANTOS, LUANA NUNES; MOISE, ALEXANDER R.; CASTILO, HOZANA ANDRADE. Signaling through retinoic acid receptors in cardiac development: Doing the right things at the right times. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS, v. 1849, n. 2, SI, p. 94-111, FEB 2015. (12/14859-0, 13/12008-6, 11/15273-7)

XAVIER-NETO, JOSE; SOUSA COSTA, ANGELA M.; FIGUEIRA, ANA CAROLINA M.; CAIAFFA, CARLO DONATO; DO AMARAL, FABIO NEVES; CERQUEIRA PERES, LARA MALDANIS; PIRES DA SILVA, BARBARA SANTOS; SANTOS, LUANA NUNES; MOISE, ALEXANDER R.; CASTILO, HOZANA ANDRADE. Signaling through retinoic acid receptors in cardiac development: Doing the right things at the right times. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS, v. 1849, n. 2, p. 18-pg., 2015-02-01. (11/15273-7, 13/12008-6, 12/14859-0)