Role of the glucocorticoid-induced leucine zipper transcription factor TSC22D3 in the molecular circadian clock

• Grant number: 19/04451-3
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: July 01, 2019
• End date: June 30, 2020
• Field of knowledge: Biological Sciences - Physiology
• Principal Investigator: Gisele Akemi Oda
• Grantee: Danilo Eugênio de França Laurindo Flôres
• Supervisor: John B Hogenesch
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Institution abroad: Cincinnati Children's Hospital Medical Center, United States
• Associated to the scholarship: 17/16242-4 - Computational studies on photoperiodism in light and food synchronization of multi-oscillatory circadian systems, BP.PD

Abstract

In mammals, daily rhythms in physiology and behavior are regulated by a circadian system, composed of a central oscillator in the suprachiasmatic nucleus (SCN) and peripheral oscillators in all body tissues. In central and peripheral oscillators, a self-sustained rhythm is generated by an intracellular mechanism composed of interlocked molecular transcriptional/translational feedback loops. The circadian system interacts with the hypothalamus-pituitary-adrenal axis at different regulatory points, with a resulting daily rhythm in glucocorticoid (GC) release into the bloodstream. Within target tissues, GC activates the transcription of GC-regulated genes. One of these is the glucocorticoid-induced leucine zipper transcription factor Tsc22d3. This gene is rhythmically expressed in different tissues of mice, rats and humans. Biochemical evidence indicates that the Tsc22dD3 gene may work as an input, an output, or a component of the circadian clock. In this BEPE project, we will investigate the role played by the gene in regulating circadian oscillations, by genetically perturbing Tsc22d3 and then evaluating the effects on clock gene expression. In our first aim, Tsc22d3 function will be studied in mouse cell lines in vitro, by transcriptional knockdown using RNAi in fibroblasts, adipocytes and hepatocytes. A more profound inhibition will be later performed with complete gene knockout using the CRISPR/Cas9 system to inactivate the gene. Genetic disturbances can have different effects on cells and complex tissues, like the SCN. Therefore, as a second aim, we will observe clock gene expression in SCN explants subjected to Tsc22d3 knockdown or over-expression. As a third aim, we will observe whether other biochemical pathways are affected by the inhibition, since the transcription factor may work as an output. To that end, we will profile, with RNA-seq, genome-wide transcriptional changes in cell lines upon Tsc22d3 knockout. Finally, in the last aim, we will start the process of generating a Tsc22d3 knockout mouse line with the CRISPR/Cas9 system for future evaluation of behavior and tissue rhythms, under complete inhibition of the gene. This project on the molecular mechanisms of circadian oscillators will be a conceptual and methodological complement to the current postdoctoral project with generic oscillator models.

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)

FLORES, DANILO E. F. L.; ODA, GISELE A.. Quantitative Study of Dual Circadian Oscillator Models under Different Skeleton Photoperiods. JOURNAL OF BIOLOGICAL RHYTHMS, v. 35, n. 3, FEB 2020. (19/04451-3, 17/16242-4, 17/19680-2)

STOKES, KYLE; NUNES, MALIKA; TROMBLEY, CHANTELLE; FLORES, DANILO E. F. L.; WU, GANG; TALEB, ZAINAB; ALKHATEEB, ABEDALRHMAN; BANSKOTA, SUHRID; HARRIS, CHRIS; LOVE, OLIVER P.; et al. The Circadian Clock Gene, Bmal1, Regulates Intestinal Stem Cell Signaling and Represses Tumor Initiation. CELLULAR AND MOLECULAR GASTROENTEROLOGY AND HEPATOLOGY, v. 12, n. 5, p. 1847-1872, 2021. (19/04451-3)

ROSSELOT, ANDREW E.; PARK, MIRI; KIM, MARI; MATSU-URA, TORU; WU, GANG; FLORES, DANILO E.; SUBRAMANIAN, KRITHIKA R.; LEE, SUENGWON; SUNDARAM, NAMBIRAJAN; BRODA, TAYLOR R.; et al. Ontogeny and function of the circadian clock in intestinal organoids. EMBO Journal, v. 41, n. 2, OCT 2021. (19/04451-3, 17/16242-4)