Bioinformatic and molecular study of the regulation of SIRT3 expression

Abstract

Calorie restriction (CR) is a dietary intervention that extends lifespan, delays the onset of age-related diseases, and induces a wide-ranging metabolic adaptation in multiple model organisms. One of its primary effectors is the mitochondrial NAD+-dependent deacetylase sirtuin 3 (SIRT3). SIRT3 expression is upregulated by CR in multiple tissues, yet the mechanism of this induction is unclear. We therefore pursued multiple avenues in the study of the regulation of SIRT3 expression. To study SIRT3 transcriptional activity, we developed a plasmid with the SIRT3 promoter driving expression of the reporter gene luciferase, and we used it to demonstrate that SIRT3 expression in human 293T cells is upregulated by rapamycin, an inhibitor of the nutrient-sensing Target of Rapamycin pathway. Because SIRT3 expression level is a predictor of clinical outcome in breast cancer, this construct could be applied as a diagnostic and prognostic tool. We next conducted a bioinformatic analysis to identify transcription factors that may induce SIRT3 expression and identified nuclear respiratory factor 2 (NRF-2) as a top candidate. We showed that SIRT3 levels respond to NRF-2 overexpression or knockdown and that NRF-2 binds the SIRT3 promoter. Notably, NRF-2 and estrogen-related receptor α – the only other transcription factor previously identified as binding the SIRT3 promoter directly – are both co-activated by peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α), a major regulator of the expression of mitochondrial and metabolic genes. Future study will be necessary to determine whether this pathway underlies the upregulation of SIRT3 expression in CR. Finally, we also used high-throughput RNA sequencing to suggest that calorie restriction was capable of reversing not just age-related changes in gene expression, but also age-related changes in the usage of different isoforms of the same gene. This may be a new mechanism by which CR controls the biological activity of certain genes. Together, these studies provide novel tools and insights in the study of the regulation of SIRT3 expression and the effects of CR.