Loss of the SIN3 transcriptional corepressor results in aberrant mitochondrial function.

BACKGROUND: SIN3 is a transcriptional repressor protein known to regulate many genes, including a number of those that encode mitochondrial components. RESULTS: By monitoring RNA levels, we find that loss of SIN3 in Drosophila cultured cells results in up-regulation of not only nuclear encoded mitochondrial genes, but also those encoded by the mitochondrial genome. The up-regulation of gene expression is accompanied by a perturbation in ATP levels in SIN3-deficient cells, suggesting that the changes in mitochondrial gene expression result in altered mitochondrial activity. In support of the hypothesis that SIN3 is necessary for normal mitochondrial function, yeast sin3 null mutants exhibit very poor growth on non-fermentable carbon sources and show lower levels of ATP and reduced respiration rates. CONCLUSIONS: The findings that both yeast and Drosophila SIN3 affect mitochondrial activity suggest an evolutionarily conserved role for SIN3 in the control of cellular energy production.

Systematic Analysis of SIN3 Histone Modifying Complex Components During Development.

Establishment and maintenance of histone acetylation levels are critical for metazoan development and viability. Disruption of the balance between acetylation and deacetylation by treatment with chemical histone deacetylase (HDAC) inhibitors results in loss of cell proliferation, differentiation and/or apoptosis. Histone deacetylation by the SIN3 complex is essential in Drosophila and mice, as loss of the scaffolding factor SIN3 or the associated HDAC results in lethality. The objective of this study is to elucidate contributions of SIN3 complex components to these essential processes. We used the Drosophila model organism to carry out a systematic functional analysis of the SIN3 complex. We find that SIN3 associated proteins are essential for viability and cell proliferation during development. Additionally, tissue specific reduction of SIN3 complex components results in abnormal wing development. Interestingly, while knockdown of each factor resulted in similar phenotypes, their individual effects on recruitment of SIN3 to polytene chromosomes are distinct. Reduction of some factors leads to large changes in the morphology of the chromosome and/or greatly reduced SIN3 binding. These findings suggest that while individual SIN3 complex components work through distinct molecular mechanisms, they each make a substantial contribution to the overall function of this highly conserved histone deacetylase complex.

The histone demethylase dKDM5/LID interacts with the SIN3 histone deacetylase complex and shares functional similarities with SIN3.

BACKGROUND: Regulation of gene expression by histone-modifying enzymes is essential to control cell fate decisions and developmental processes. Two histone-modifying enzymes, RPD3, a deacetylase, and dKDM5/LID, a demethylase, are present in a single complex, coordinated through the SIN3 scaffold protein. While the SIN3 complex has been demonstrated to have functional histone deacetylase activity, the role of the demethylase dKDM5/LID as part of the complex has not been investigated. RESULTS: Here, we analyzed the developmental and transcriptional activities of dKDM5/LID in relation to SIN3. Knockdown of either Sin3A or lid resulted in decreased cell proliferation in S2 cells and wing imaginal discs. Conditional knockdown of either Sin3A or lid resulted in flies that displayed wing developmental defects. Interestingly, overexpression of dKDM5/LID rescued the wing developmental defect due to reduced levels of SIN3 in female flies, indicating a major role for dKDM5/LID in cooperation with SIN3 during development. Together, these observed phenotypes strongly suggest that dKDM5/LID as part of the SIN3 complex can impact previously uncharacterized transcriptional networks. Transcriptome analysis revealed that SIN3 and dKDM5/LID regulate many common genes. While several genes implicated in cell cycle and wing developmental pathways were affected upon altering the level of these chromatin factors, a significant affect was also observed on genes required to mount an effective stress response. Further, under conditions of induced oxidative stress, reduction of SIN3 and/or dKDM5/LID altered the expression of a greater number of genes involved in cell cycle-related processes relative to normal conditions. This highlights an important role for SIN3 and dKDM5/LID proteins to maintain proper progression through the cell cycle in environments of cellular stress. Further, we find that target genes are bound by both SIN3 and dKDM5/LID, however, histone acetylation, not methylation, plays a predominant role in gene regulation by the SIN3 complex. CONCLUSIONS: We have provided genetic evidence to demonstrate functional cooperation between the histone demethylase dKDM5/LID and SIN3. Biochemical and transcriptome data further support functional links between these proteins. Together, the data provide a solid framework for analyzing the gene regulatory pathways through which SIN3 and dKDM5/LID control diverse biological processes in the organism.

Disruption of Methionine Metabolism in Drosophila melanogaster Impacts Histone Methylation and Results in Loss of Viability.

Histone methylation levels, which are determined by the action of both histone demethylases and methyltransferases, impact multiple biological processes by affecting gene expression activity. Methionine metabolism generates the major methyl donor S-adenosylmethionine (SAM) for histone methylation. The functions of methionine metabolic enzymes in regulating biological processes as well as the interaction between the methionine pathway and histone methylation, however, are still not fully understood. Here, we report that reduced levels of some enzymes involved in methionine metabolism and histone demethylases lead to lethality as well as wing development and cell proliferation defects in Drosophila melanogaster. Additionally, disruption of methionine metabolism can directly affect histone methylation levels. Reduction of little imaginal discs (LID) histone demethylase, but not lysine-specific demethylase 2 (KDM2) demethylase, is able to counter the effects on histone methylation due to reduction of SAM synthetase (SAM-S). Taken together, these results reveal an essential role of key enzymes that control methionine metabolism and histone methylation. Additionally, these findings are an indication of a strong connection between metabolism and epigenetics.

SIN3 is critical for stress resistance and modulates adult lifespan.

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.

Identification of genetic suppressors of the Sin3A knockdown wing phenotype.

The role of the Sin3A transcriptional corepressor in regulating the cell cycle is established in various metazoans. Little is known, however, about the signaling pathways that trigger or are triggered by Sin3A function. To discover genes that work in similar or opposing pathways to Sin3A during development, we have performed an unbiased screen of deficiencies of the Drosophila third chromosome. Additionally, we have performed a targeted loss of function screen to identify cell cycle genes that genetically interact with Sin3A. We have identified genes that encode proteins involved in regulation of gene expression, signaling pathways and cell cycle that can suppress the curved wing phenotype caused by the knockdown of Sin3A. These data indicate that Sin3A function is quite diverse and impacts a wide variety of cellular processes.