New pathway to thermogenesis: determining the role of Nr2f6 in regulating the expression of thermogenesis-related genes in skeletal muscle.

• Grant number: 24/22304-6
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: April 01, 2025
• End date: March 31, 2027
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Leonardo dos Reis Silveira
• Grantee: Lucas Paulo Jacinto Saavedra
• Host Institution: Instituto de Biologia (IB). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil
• Associated research grant: 23/08207-5 - New road to thermogenesis: targeting nuclear receptor Nr2f6 in skeletal muscle, AP.TEM

Abstract

Transcription factors (TFs) are proteins that bind specific DNA sequences to control gene expression, impacting cellular metabolism. This regulation occurs at promoters and enhancers, enabling rapid gene expression adjustments in response to cellular needs. When TF-mediated signaling malfunctions, metabolic diseases like obesity, diabetes, and cancer may result. Thus, identifying TFs and their regulatory sequences is vital for understanding gene regulation in various physiological conditions, including metabolic diseases characterized by mitochondrial dysfunction. In preliminary analyzes using large RNAseq and ChIPseq datasets from the National Center for Biotechnology Information (NCBI) to investigate TFs linked to metabolism, supervisors group found the reduced expression of the transcription factor Nr2f6 in the skeletal muscle and adipocytes of mice exposed to cold or physical exercise-conditions known to stimulate an oxidative phenotype in tissues. To further explore Nr2f6's function, the gene was knocked down in C2C12 muscle cells, and RNAseq analysis was performed to identify impacted pathways. Knockdown of Nr2f6 resulted in significant upregulation of genes involved in metabolism, cellular differentiation, muscle contraction, and Ca2+ transport, suggesting that Nr2f6 influences cellular metabolic processes. Notably, the results pointed to Nr2f6's role in muscle thermogenesis. The knockdown increased phosphorylation of AMPK, a marker of thermogenic and metabolic activity, and enhanced insulin responsiveness. Additionally, genes associated with thermogenesis-such as Slc25a4 (ATP/ADP carrier), ATP1A1 (ATPase Na+/K+ transporter), and Ryr1 (ryanodine receptor)-were upregulated in Nr2f6-deficient cells. Further investigation identified Nr2f6 binding sites on DNA regulatory sequences of Ca2+ transport genes, including Sln, ATP2a1 (Serca 1A), ATP2a2 (Serca 2A), and Ryr1. Nr2f6 was also found at the regulatory sequence of PPARGC1A, a key thermogenesis gene in brown adipocytes and skeletal muscle cells. Taken together this finding suggest that the nuclear receptor, Nr2f6, might be an important regulator of mitochondrial function and thermogenic process in skeletal muscle. In this sense the present proposal aims to determine if Nr2f6 physically binds to Sln and Serca DNA sequences (using chromatin immunoprecipitation - ChIP); To determine the repressive role of Nrf26 on Sln and Serca DNA regions by a reporter essay using luciferase/GFP; To seek for other TFs associated with Sln/Serca regulation in C212 cells.