Atrial selectivity from a complex nuclear receptor driven by androgen receptor: a novel gene regulatory network

• Grant number: 18/09839-7
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Effective date (Start): October 01, 2018
• Effective date (End): September 30, 2019
• Field of knowledge: Biological Sciences - Biology
• Principal Investigator: José Xavier Neto
• Grantee: Luana Nunes Santos
• Supervisor: Mirana Soa Manarivo Ramialison
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Research place: Monash University, Australia
• Associated to the scholarship: 15/12549-2 - Identification of genetic networks interacting with COUP-TF II to determinate the atrial cardiac cells identity by the SMyHC III promoter, BP.DD

Abstract

Cardiac development is a complex process that requires various morphogenetic movements and multiple gene regulatory networks. Numerous molecular processes organize the embryonic heart tube into functional units of inflow (atria) and outflow (ventricles) but the mechanisms of chamber expression are still incompletely understood. Only a handful of cardiac genes are known to be expressed in an actual chamber-specific fashion. Rather, genes such as the transcription factors Gata-4, Tbx-5, Tbx-20 and COUP-TFII (chicken ovalbumin promoter-transcription factor II) are more prominently expressed in the cardiac influx, while IRX-4, MLC2-V and HRT-2 are mainly expressed in the ventricles. The Slow Myosin Heavy Chain III gene (SMyHC III) is selectively expressed in atria of quail embryonic hearts and is a useful paradigm to understand the mechanisms of chamber expression. In mice, the SMyHCIII-HAP transgene displays a strong of alkaline phosphatase reporter expression in the heart region occupied by cardiac inflow precursors from 8.25 dpc mouse embryos to adulthood. This atrial preference exhibited by the SMyHC III promoter crosses species barriers and is thus manifested in avians, mammals, even in teleost fishes. Deletion of a distal 72 bp within the 840 bp promoter region of SMyHC III resulted in ventricular expression and reduction of atrial expression of the reporter gene, showing that this 72 bp fragment is essential for atrial expression. Therefore, given the importance of the 72 bp fragment in atrial-specific activation and ventricular repression of SMyHC III expression, we performed bioinformatics analyses, in vitro and in vivo experiments to identify new players of the SMyHC III regulation. We found multiple binding sites for transcription factors of the nuclear receptor family encompassing a 32bp region dubbed complex Nuclear Receptor Element (cNRE). This 32bp region has been showing the presence of ventricular repressors and atrial activators by a host of transcription factors from the nuclear receptor superfamily. Our transactivation assays in Hek293T cells have shown a novel gene regulatory network in cardiac chamber specification, the Androgen Receptor (AR) pathway. In this context, here we propose to investigate protein-chromatin interactions controlling the promoter of the atrial-specific SMyHC III using primary cell culture from quail heart with Androgen Receptor (AR) fused to DamID followed by high throughput DNA sequencing. DamID technology is an alternative approach to detect protein-chromatin interaction at a genomic scale to chromatin immunoprecipitation (ChIP). We will fuse the Androgen Receptor (AR) with Dam in quail primary cell culture, both in normal cells and cNRE deleted by CRISPR to detecting and localizing protein-DNA interactions of SMHCIII regulation and to better understand the molecular mechanism underpinning atrial programming. (AU)