Regulation Mechanism Of Human SIRT1 Gene Expression

To elucidate the mechanism of lifespan regulation keeps as an intriguing research topic. Recent studies of the silencing information regulator 2 (Sir2), which may closely link the metabolic rate of cells to oxidative stress and aging, cast a new light on the research of longevity.Sir2 functions as nicotinamide-adenine dinucleotide (NAD⁺)-dependent histone deacetylase, which has recently been discovered and shown to be broadly conserved. Sir2 gene homologs have been found in a very wide range of organisms from bacteria to human. The regulation mechanism of Sir2 functioning in lifespan extension made great achievement in low organisms. In the budding yeast, loss of Sir2 shortened lifespan while higher dosage of Sir2 extended lifespan dramatically. It's suggested that nematode Sir2.1 may couple nutrient availability to lifespan through the insulin-like signaling pathway. Recently, the role of mammalian Sir2 playing in longevity regulation arouses much attention.It is suggested that Sir2 is required for epigenetic gene silencing, DNA repair and recombination. Meanwhile, Sir2 protein may also involved in the control of mitosis process and the modulation of lifespan. SIRT1, the closest human homolog of ySir2, acts as a deacetylase that promotes cell survival by negatively regulating the p53 tumor suppressor. By modulating p53 acetylation level, SIRT1 reduces p53 activity and targets gene expression, allowing cells to continue growing. Till now, little is known about the regulation mechanism of SIRT1 gene expression.Caloric restriction (CR) promotes longevity in a wide range of organisms from yeast to human and is the most robust aging intervention studied to date. At present, the exact mechanism of CR benefit for lifespan extension has not been clearly elucidated. Recent studies have revealed that Sir2 is necessary in lifespan extension governed by caloric restriction.Since eating less for the sake of longevity is not likely to gain widespread compliance, any Sir2 agonist that would mimic the effect of CR on Sir2, might offer the promise of long-term intervention to slow the aging process. As definite antioxidants and the sirtuins activators which were newly identified, the polyphenols intrigue great interests in the therapeutic studies of a wide range of age-related diseases and lifespan extension.It is suggested that resversatrol works as an activator of Sir2, which promotes longevity through the same mechanism as CR does. It is thought that the function of Res in promoting longevity to 70% in yeast mother cell appears to be related to the silencing in the rDNA through activating Sir2. Moreover, Res modulated the deacetylating effect of SIRT1 on p53 with a consequent enhanced negative regulation of the p53 tumor suppressor activity. The beneficial effect of Res on lifespan extension was further discovered in nematode and fruitfly. It was reported that polyphenols have many pharmacological effects such as inhibition to carcinoma, aging, oxidative stress etc. Using immature primary culture of embryonic rat cortical cells as a model, we previously found that plant extracts gypenosides significantly protected cells from glutamate-induced cell death. The novel role of gypenosides implies their remarkable preventative and therapeutic potential in treatment of aging-related diseases. What we found further confirmed the strong anti-aging effects of plant extracts.Since Sir2 is suggested as a key factor in anti-damage, anti-cancer and determining longevity, it is very important to explore the regulation mechanism of Sir2 expression. The expression regulation elements and mechanism of human SIRT1 gene is unknown currently. In this study, we extensively studied the regulation mechanism of human SIRT1 gene expression.PART ONE CR and Res Up-regulate Human SIRT1 Gene ExpressionIt is suggested that CR promotes lifespan through activating Sir2, and Res has been proved as a Sirtuin activating compound. We analyzed the effect of both CR and Res on human SIRT1 expression in different cell lines as HEK293, U2OS, HeLa and HepG2 by using RT-PCR and Real-time RT-PCR:1. CR significantly increased SIRT1 mRNA level in HEK293 and HeLa cells, with a three fold enhancement.2. Res promoted SIRT1 expression to nearly three fold in HepG2 and HeLa cells at the concentration of 20μM3. Res induced SIRT1 expression in a dosage-dependent way.It is revealed that the significant promotive effects of CR and Res on SIRT1 expression without cell specificity, since the cell lines tested are of different origins. What we found provided evidence for CR and Res acting in lifespan regulation through the similar mechanism.In order to elucidate the regulation mechanism of SIRT1 gene expression, we studied human SIRT1 promoter .PART TWO Identification and Characterization of human SIRT1 PromoterAt present, the transcription of human SIRT1 gene has not been examined in detail. The aim of this section is to examine human SIRT1 promoter.Firstly, we performed a computer-based searching for the putative transcription factor binding sites within about 1Kb fragment upstream the transcription start site (TSS) of human SIRT1 gene using MatInspector software. Using dual luciferase reporter assay system, we generated defferent luciferase expression constructs with the series 5' deletion of SIRT1 promoter region, including ten 5' deletion constructs as P-883, P-672, P-373, P-243, P-182, P-158, P-111, P-86, P-60, P-42 with respect to the 5' position related to the transcription start site; and four mutant constructs as Sp1-M1, Sp1-M2, Sp1-M3 and P-158-p53(mut). pRL-TK vector was used as an internal control to normalize transfection efficiencies. All the constructs and vehicle control pGL3-basic were objectively transfected into U2OS and HEK293 cells with transfection reagent Lipofectamine^TM2000. 24 h after transfection, cells were harvested and assayed for the luciferase activity. The expression of the target transcription factor was knocked down by siRNA technique and gel retardation analysis was performed to further confirm the identifed functional binding site.1. Binding sites for transcription factors including NF-κB, Sp1, AP2 and CREB were predicted by software analysis and no typical TATA box, and CAAT box were predicted within about 1kb region upstream the transcription start site of SIRT1 gene. A 300bp region upstream the SIRT1 translational start site is GC-rich region (GC 70%)2. The longest luciferase expression construct P-883 showed strong luciferase activity, indicating that this region contains functional elements for the transcripition of human SIRT1 gene.3. Further analysis with increasing 5' deletion constructs revealed that no functional element located within the region between -883bp to -243bp. However, construct P-60 resulted in a～8-fold reduction in luciferase activity compared with construct P-243, indicating this region was involved in controlling basal SIRT1 promoter activity. Similar tendency was observed in the two different cell lines tested. Further analysis confined the key regulation elements within two regions , -86bp to -60bp and -182bp to -111bp.4. It's revealed that the region between -86bp and -60bp is the minimal promoter to control the transcription of the SIRT1 gene. Mutation analysis identified that the 5' Spl binding site of the three tandem ones within this region was the functional element. Sp1 knockdown by RNAi technology reduced SIRT1 expression and gel shift assays further confirmed this response element.5. Furthermore, we identified a p53 responsive element within the SIRT1 promoter upstream of the functional Sp1 binding site. Mutation detection and DNA-protein interaction analysis conferred that p53 is essential to sustain the SIRT1 promoter activity, implying that SIRT1 is a target gene of p53.These results suggest that SIRT1 gene expression is tightly regulated at the transcriptional level and that the transcription factor Sp1 and p53 play important roles in the regulation of SIRT1 gene expression.PART THREE Regulation Mechanism of CR on Human SIRT1 Gene ExpressionWe have found the strong inducing effect of CR and Res on SIRT1 mRNA level and identified a functional p53 element within SIRT1 promoter region. Previous study showed that in the mouse, CR stimulates Sirt1 transcription through two tandem p53 binding half sites in its promoter. However, only one half site for p53 binding in the corresponding region of human SIRT1 gene. It is unknown whether this one half site is functional in regulating SIRT expression. Here, we evaluated the role of the p53 binding site in human SIRT1 promoter.We examined the effects of calorie restriction on the human SIRT1 expression in two sets of cell lines with different origin and p53 status: the osteosarcoma cell lines U2OS (p53⁺) and Saos-2 (p53^-), as well as the hepatoma cell lines HepG2 (p53⁺) and Hep3B (p53^-) For RT-PCR analysis, cells were treated with CR for 12h and the amount of SIRT1 mRNA was measured. We further analysis the effect of CR on SIRT1 promoter activity using constructs P-158, which containing the p53 element, and the construct P-111 without the p53 element as well as construct P-158-p53 (mut) with the p53 binding site mutated. All constructs were objectively transfected into cells with pRL-TK vector served as an internal control to normalize differences in transfection efficiencies. 24 h after transfection, cells were harvested and assayed for the luciferase activity. In addition, to examine whether the transactivation of SIRT1 promoter by p53 depends on p53 binding site, we performed co-transfection experiments using p53 cDNA expression vector in both Saos-2 and Hep3B cells.1. We found that CR significantly increased SIRT1 mRNA level in a p53-dependent way. No significant induction was observed in starved cells that are p53-deficient.2. Using dual luciferase reporter assay system, it is conferred that CR promoted SIRT expression at transcriptional level. Deletion and mutation study further identified a p53 binding site in human SIRT1 promoter between -158bp to -111bp as the target sequence mediating the effect of CR.3. No significant increasing in luciferase activity was observed in Saos-2 and Hep3B (both are p53 null) cells when treated by CR, indicating that p53 and its responsive binding site were both necessary for mediating the effect of CR.4. The increase of promoter activity induced by CR was recapitulated in p53-null cells with the ectopic expression of wild type p53 protein, which further confirmed that SIRT1 expression was mediated by p53 protein expression.We demonstrate that the induction of human SIRT1 by CR is p53-dependent. The single p53 half site is functional and responsible for the CR-induced regulation of SIRT1 transcription. What we found provided a new insight into the transcriptional regulation effect of p53.PART FOUR Effect of FOXO3a on CR-induced SIRT1 expressionIn this part, we further studied the potential signaling pathway involved into CR-induced human SIRT1 expression. It is suggested that forkhead transcription family and Sir2 work in the regulation of lifespan cooperatively. FOXO transcription factors have important roles in metabolism, cellular proliferation, stress tolerance, and aging, which are negatively regulated by protein kinase B/c-Akt-mediated phosphorylation. In C. elegans, the ability of Sirt1 to extend lifespan depends on the presence of Daf-16, a member of the FOXO family of Forkhead transcription factors. It has been revealed that in higher organisms, FOXO transcription factors also are direct substrates of SIRT1.It has been shown that in mammalian cells, acute nutritional stress results in a FOXO3a-dependent increase in SIRT1 levels. FOXO3a regulation of SIRT1 expression occurs through an interaction with p53, which suggests a complicated but undoubtedly important homeostatic regulatory network involving p53, FQXO3a, and SIRT1. Since there is distinct difference in the promoters between the human SIRT1 and mouse Sirt1 gene, we further analyzed the effect of FOXO3a on CR-induced human SIRT1 expression.A nutrient-sensitive physical interaction was observed between FOXO3a and the one p53 responsive element with human SIRT1 promoter. We found that the promotive effect of CR on SIRT1 expression is FOXO3a-dependent. When endogenous FOXO3a expression was inhibited with a specific FOXO3a-targeted siRNA the SIRT1 expression was consequently decreased dramatically in FOXO3a-siRNA cells as demonstrated by RT-PCR. Luciferase reporter construct transfection further confirmed the FOXO3a-siRNA influence on SIRT promoter activity.These results may help us to understand how adaptation to certain cellular stresses, including nutrient availability, may modulate mammalian life-span.