Generation of a knockout mouse for the orphan nuclear receptor Coup-TFII: investigation of the molecular mechanisms underlying the atrial-specific expression of the SMyHC3 gene

• Grant number: 15/10166-9
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Effective date (Start): September 01, 2015
• Effective date (End): June 30, 2017
• Field of knowledge: Biological Sciences - Morphology - Embryology
• Principal Investigator: José Xavier Neto
• Grantee: Ângela Saito
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil

Abstract

The CRISPR/Cas9 technology has recently emerged as an effective alternative for the introduction of site-specific modifications into the genome. Compared to the previously techniques, CRISPR/Cas9 has lower cost, greater efficiency and speed to edit the genome in multiple cell types and organisms. The CRISPR/Cas9 system is based on the use of a small RNA which is able to pair with target DNA in the genome and to direct the endonuclease spCas9 for DNA modification. Because of the simplicity and speed on generation, the technique has become a viable resource in our environment. Our goal is to use the technology CRISPR/Cas9 as the best possible approach to answer an old question from our group: the identification of the atrial-specific regulator of the Slow Myosin Heavy Chain gene (SMyHC3) expression, a transgene indicator of the atrial differentiation program in mice. Many results obtained with various techniques over the past 15 years indicate the nuclear receptor COUPTF-II, implicated in the development of atrial and venous system as the best candidate for atrial SMyHC3 activator gene. Our group has identified a region of 33 base pairs (bp) in SMyHC3 promoter, called the Complex Nuclear Receptor Response Element (ECRRN) and determined the nucleotides into ECRRN responsible for the atrial activation and ventricular suppression in transgenic mouse embryos. Transactivation assays using the luciferase reporter gene under the control of the promoter SMyHC3 demonstrated that the activation of this promoter by COUPTF-II requires the presence of ECRRN. Also, biophysical studies support the physical interaction between COUP-TFII and ECRRN. A proteomic approach for the characterization of COUPTF-II interactors through COUPTF-FLAG immunoprecipitation revealed proteins in the androgen receptor (AR) and glucocorticoid receptors (GR) signal transduction pathways. Consistently, transactivation assays showed that SMyHC3 promoter is synergistically activated in the presence of both, COUP-TFII and androgen receptor. An even more restrictive test for ECRRN fused to a basal promoter also showed the synergic activation by COUP-TFII and AR. To physiological and definitively establish whether COUP-TFII is an activator or not of the atrial-specific expression of SMyHC3 in vivo, we will use the technology CRISPR/Cas9 to generate a knockout mouse line for the nuclear receptor COUP-TFII. The knockout embryos for COUP-TFII will be characterized mainly as the atrial and ventricular phenotype as a source of transcripts for differential expression analysis against wild-type embryos. Essential for our analysis it will also be the generation of a transgenic animal SMyHC3-eGFP, which expresses the eGFP reporter gene under the control of SMyHC3 promoter. The ultimate test for our hypothesis will be the evaluation of the SMyHC3-eGFP expression in COUPTF-II knockout mice. Therefore, the line containing the heterozygous knockout allele for COUP-TFII will be mated with the SMyHC3-eGFP transgene. That knockout at the SMyHC3-eGFP mice will also be used to investigate the role of COUP-TFII in the androgen receptor binding capacity to ECRRN. For that, this receptor will be immunoprecipitated from the sino-atrial structures of SMyHC3-eGFP transgene containing the wild-type and knockout COUP-TFII allele and the detection of the immunoprecipitated SMyHC3 promoter at the receptor will be performed by quantitative real-time PCR (ChIP- qPCR). Therefore, the characterization of the line generated from the crossing of SMyHC3-eGFP transgene with a knockout strain for COUP-TFII will be an important approach for the definitive demonstration that COUP-TFII regulates the atrial-specific expression of SMyHC3 and it will contribute to the elucidation of the COUP-TFII regulating mechanisms, in concert with other nuclear receptors, for the atrial differentiation program in mice. (AU)