We previously established that the expression of numerous nuclear encoded mitochondrial genes was up-regulated in Drosophila
tissue culture cells following the loss of the corepressor SIN3 [5
]. In research described in this current work, we first determined that the changes in gene expression following loss of SIN3 are unlikely due to off target effects of SIN3 RNAi. Targeting two different regions of the SIN3 mRNA results in similar changes in gene expression, indicating that the changes are likely due to loss of SIN3 and not to an effect of another gene that contains a short similar sequence. In addition to the changes in expression of the nuclear encoded genes, loss of SIN3 results in an up-regulation of mitochondrial encoded genes. Although we observe changes in gene expression, we find that mitochondrial genome copy number is unaltered. The changes in gene expression prompted us to examine mitochondrial function in SIN3-deficient cells. SIN3-deficient cells show changes in ATP levels and respiration rates, suggesting that the increases in gene expression result in altered oxidative phosphorylation capacity. A necessary role of SIN3 for normal mitochondrial function is supported by the finding that yeast sin3
mutants are unable to grow on non-fermentable carbon sources, conditions which require active mitochondria for ATP production.
Consistent with numerous published reports, we have determined that loss of ySin3 is dispensable for fermentative growth [9
]. In contrast, ySin3 is critical for growth when respiration is required (Figure , Additional files 2
). Genome wide studies have been conducted to identify yeast mutant strains that have the ability to grow on YPD and exhibit reduced growth on non-fermentable carbon sources but failed to identify SIN3
]. Several possible explanations may account for false negatives in global screens, three of which are discussed by the authors of one of the global screen studies [40
]. First, methods of analysis for growth and scoring differ. Second, there may be phenotypic plasticity between mutants in different strain backgrounds. Third, the yeast deletion collections are known to accumulate errors, and it is well-established that deletion strains must first be tested to ensure that the deletion is correct. Obviously, this cannot be done when carrying out global screens. In addition, in the global study of Steinmetz et al.
], the sin3
mutant did not grow well when cultured in any of the different carbon sources under the assay conditions tested, including YPD. Pools of mutants were cultured in various media and growth of individual mutant strains of the pool was compared to average growth in the culture. It is possible that the sin3
strain was not able to grow in any of the assay conditions in such pools of mutants. All of the above are potential reasons as to why SIN3
was not identified in a global screen for genes required for respiratory activity. In this study we demonstrate that, when assayed as an individual strain, the sin3
mutant grows very poorly in non-fermentable media, indicating that ySin3 is required when cells are forced to respire. Consistent with this data, sin3
mutants have lower levels of ATP and decreased respiration rates.
Work from multiple laboratories has indicated that there are many similarities between yeast and metazoan SIN3, including the fact that SIN3 acts as a global regulator of transcription and that it functions in the context of the SIN3 histone deactylase complex [9
]. One major reported difference between yeast and metazoan SIN3 is the requirement of this gene for viability. ySin3 is dispensable for growth while metazoan SIN3 is an essential gene [9
]. Analysis of conditional knockdown of Drosophila
SIN3 mutants suggests that SIN3-deficiency leads to death due to defects in cell cycle progression rather than due to alteration of a specific developmental pathway [8
]. In this work, we have found that ySin3 is required for cell growth when mitochondrial function is necessary for ATP production. It is thus possible that, rather than being essential for some specific process of development, the metazoan requirement for SIN3 is due to its requirement for normal mitochondrial activity. We hypothesize that loss of SIN3 leads to a defect in mitochondrial function that in turn leads to a defect in cell cycle progression. Links between cell cycle control and mitochondrial activity are well established. Myc: Max heterodimers positively regulate the cell cycle while Rb-E2F complexes repress genes necessary for cell cycle progression [46
]. Both sets of factors have been linked to regulation by NRF-1, a key transcription activator of nuclear encoded mitochondrial genes [48
]. SIN3 has been linked to both the Max transcription network and to regulation by Rb, supporting the hypothesis that SIN3 regulation of mitochondrial biogenesis and activity is linked to cell cycle control [18
]. It was recently shown in mammalian cells that changes in mitochondrial morphology are crucial for cell cycle control. At the G1
-S checkpoint mitochondria are organized as a large fused network with higher mitochondrial membrane potentials, increased ATP levels and increased respiration rates, whereas in other stages of the cell cycle, mitochondria are predominantly in the fission state [51
]. Lower ATP levels and respiration rates caused by SIN3 knockdown (Figure , depleted condition) may thus interfere with cell cycle progression at the G1
In addition to its role in gene regulation, SIN3 might directly regulate energy metabolism as a participant in cell signaling. There is clear evidence that signaling pathways regulate mitochondrial respiration and energy levels, e.g., the cAMP and inflammatory pathways lead to phosphorylation of cytochrome c
oxidase, the terminal enzyme of the electron transport chain, followed by changes in respiration and ATP levels [52
]. SIN3 might exert a stimulatory signal required for normal mitochondrial function. The observation that reduction in SIN3 results in decreased ATP levels and respiration is perhaps surprising in light of the finding that expression of many genes encoding proteins with mitochondrial function are up-regulated. Up-regulation of nuclear encoded mitochondrial genes upon SIN3 knockdown may therefore be a compensatory response to an increase in the mitochondrial component due to dysfunctional energy metabolism, as is seen when yeast must adapt to mitochondrial respiration in full dependence on glycolysis (Figure , YPD). Alternatively, mis-coordination of mitochondrial gene expression in the SIN3-deficient cells may result in the observed altered mitochondrial function because loss of SIN3 leads to an increase in expression of many but not all genes involved in mitochondrial processes [5
]. As has been previously suggested, an imbalance in the level of the proteins present in complexes important for energy production may result in mitochondrial malfunction [48
]. Finally, key links between regulation of acetylation and metabolism are highlighted in recently published data indicating that ATP-citrate lyase is important for maintenance of histone acetylation levels in growing cells [55
]. It is possible that SIN3 knockdown affects acetylation resulting not only in changes in gene expression but also altered pools of key metabolites and an inappropriate response of the mitochondria to these abnormal signals. It will be of interest to further investigate the relationship between histone acetylation and mitochondrial function to better understand control of cellular metabolism.