Genetic network interacting with COUPTF-II to determine the atrial identity through the complex nuclear receptor element (cNRE).

Author(s)

Luana Nunes Santos

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2020-01-22

Examining board members

José Xavier Neto; Henrique Marques Barbosa de Souza; Tatiana Teixeira Torres; Chao Yun Irene Yan

• Advisor: José Xavier Neto

Abstract

The interplay between morphogenetic movements and gene regulatory networks taking place during embryonic development is responsible for proper heart development. In this process, spatial gene expression patterns lead to the development of the primary heart tube into a complex four-chamber organ. However, little is known about how these events are regulated at the molecular level. For instance, it has been evidenced that the SMyHC III gene has a dual pattern of expression during heart development. In the early stages, it is expressed throughout the entire heart tube and then, upon chamber specification, its expression is restricted to atria. It has been postulated that a Vitamin D/Retinoic Acid Responsive Element (VDRE/RARE), present at the SMyHC III promoter is in charge of its ventricular repression. However, the element driving its atrial expression remains elusive. As a means to fill this gap, our research group previously established that the SMyHC III gene promoter carries the complex Nuclear Receptor Element (cNRE), a new 32-bp regulatory element that is composed of three tandem-arrayed hexads, harboring binding sites for multiple transcription factors. Here, we hypothesized that cNRE is responsible for the atria-specific activation of the SMyHC III promoter, and the molecular mechanism regulating this characteristic. Transgenesis assays performed in zebrafish with the SMyHC III promoter driving GFP expression revealed that cNRE is necessary for atrial GFP expression and sufficient to switch the ventricular specific promoter (vmhc) towards an atrial direction. Thus, not only cNRE drives atrial specification of gene expression, it crosses the species barrier. Next, we tested whether the master regulator of atrial identity COUPTF-II (Chicken Ovalbumin Upstream Promoter Transcription Factor II), would activate the SMyHC III promoter via cNRE. We found that COUPTF-II binds a promoter fragment that harbors the cNRE. Unexpectedly, transactivation assays showed that COUPTF-II represses SMyHC III promoter activation. We then employed a Mass Spectrometry-based strategy and identified the Androgen Receptor (AR) as a new COUPTF-II ligand. AR was also found to be a repressor of the SMyHC III promoter, but in synergic interaction with COUTF-II, it activated the promoter in a cNRE-dependent manner. In synthesis, in this thesis, we validated a new gene regulatory sequence (cNRE) that is responsible for the atrial activation of the SMyHC III promoter, as well as for the repression of the same promoter in the ventricular context. We also showed that this element has a binding site for COUTF-II that, synergically with AR activates SMyHC III promoter via cNRE. Taken together, these findings point out to a new complex nuclear receptor element (cNRE) carrying species barrier-crossing information to provide chamber-specific gene expression and unveil new roles for known transcription factors in cell fate and cardiac chamber specification. (AU)

• FAPESP's process: 15/12549-2 - Identification of genetic networks interacting with COUP-TF II to determinate the atrial cardiac cells identity by the SMyHC III promoter
• Grantee: Luana Nunes Santos
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)