SIN3 is critical for stress resistance and modulates adult lifespan

Abstract

Coordinate control of gene activity is critical for fitness and longevity of an organism. The SIN3 histone deacetylase (HDAC) complex functions as a transcriptional repressor of many genes. SIN3-regulated genes include those that encode proteins affecting multiple aspects of mitochondrial function, such as energy production and stress responsiveness, important for health maintenance. Here we used Drosophila melanogaster as a model organism to examine the role of SIN3 in the regulation of fitness and longevity. Adult flies with RNA interference (RNAi) induced knockdown expression of Sin3A have reduced climbing ability; an activity that likely requires fully functional mitochondria. Additionally, compared to wild type, adult Sin3A knockdown flies were more sensitive to oxidative stress. Interestingly, media supplementation with the antioxidant glutathione largely restored fly tolerance to oxidative stress. Although Sin3A knockdown flies exhibited decreased longevity compared to wild type, no significant changes in expression of many well-categorized aging genes were observed. We found, however, that Sin3A knockdown corresponded to a significant reduction in expression of genes encoding proteins involved in the de novo synthesis of glutathione. Taken together, the data support a model whereby SIN3 regulates a gene expression program required for proper mitochondrial function and effective stress response during adulthood.

Figure 1. The Gene-Switch GAL4 driver can be used to effectively knock down expression of Sin3A in adult Drosophila

RT-qPCR analysis was performed using tissue isolated from the indicated fly lines without RU486 (control) or with RU486 (RNAi knockdown). cDNA prepared from 25 day-old flies was used as a template in PCR with primer pairs for Sin3A. Numbers I and II refer to two distinct lines designed to express dsRNA to target different regions of the Sin3A transcript. Relative levels of gene expression are indicated. Error bars represent standard error of the mean. * p<0.01.

Figure 2. Sin3A knockdown (KD) affects locomotor function

Climbing ability of control (-RU486), SIN3 KD or GFP RNAi (+RU486) flies is indicated. The average percentage of flies that crossed the 30 cm mark on a 30 ml graduated cylinder in a 30 second time period, measured on indicated days is plotted. Error bars represent standard error of the mean. A and B. SIN3 KD I, C and D. SIN3 KD II, E and F. GFP RNAi serves as an additional control. p<0.001 for the comparison of control and SIN3 KD for each data set starting at day eight.

Figure 3. Enhanced Sin3A knockdown (KD) paraquat sensitivity is partially suppressed by glutathione

Survival of control (-RU486), SIN3 KD or GFP RNAi (+RU486) flies on 5 mM paraquat is indicated. For glutathione treatment, flies were placed on media containing 0.22mM glutathione for seven days prior to paraquat treament. Error bars represent standard error of the mean. A and B. SIN3 KD. C and D. GFP RNAi serves as an additional control. * p<0.01.

Figure 4. Ubiquitous Sin3A knockdown (KD) causes decreased survival

Mean survival of control (-RU486), SIN3 KD or GFP RNAi (+RU486) flies is indicated. A and B. SIN3 KD I (p<0.0001 using the logrank (Mantel-Cox) test), C and D. SIN3 KD II (p<0.0001), E and F. GFP RNAi serves as an additional control.

Figure 5. Effects of ubiquitous knock down of Sin3A on expression of genes known to impact longevity and glutathione synthesis

RT-qPCR performed using cDNA prepared from RNA isolated from whole flies from control (-RU486), SIN3 KD or GFP RNAi (+RU486). Fold changes in gene expression for GFP RNAi, SIN3 KD I and II are shown for predicted longevity relevant genes in seven day- (A) or 20 day-old flies (B) and genes important for the de novo synthesis of glutathione in seven day-old flies (C). Error bars represent standard error of the mean. * p<0.05.