Gail A. M. Breen
Educational and Professional Affiliations
B.Sc., Pharmacy, University of Toronto
Ph.D., Neuroscience, University of California, Los Angeles
Postdoctoral Fellow, Roswell Park Memorial Institute
Research Associate, University of California, San Diego
Member, American Society for Cell Biology
Member, American Association for the Advancement of Science
Most cellular energy in the form of ATP in synthesized in the mitochondria by the oxidative phosphorylation system. The activities of the enzymes of the mammalian oxidative phosphorylation system vary in response to a number of physiological conditions, including hormonal stimulation, development, differentiation, cellular proliferation, and oxygen tension. Current evidence indicates that the levels of the enzymes of the oxidative phosphorylation system are controlled to a large extent at the level of gene transcription. Dysfunction of the mitochondrial energy production system appears to play an important role in a number of diseases, including Alzheimer's disease, Parkinson's, Freiderich's ataxia, Huntington's, diabetes mellitus, malignant tumors and cardiovascular disease.
1. Transcriptional Regulation of the Mammalian Mitochondrial ATP Synthase Complex
The ATP synthase complex is the central enzyme of the oxidative phosphorylation system synthesizing ATP from ADP and Pi. The ATP synthase complex is a multisubunit enzyme made up of 14 different subunits that are encoded by two distinct genetic systems. Our laboratory has been begun analyzing the expression and regulation of the nuclear gene (ATPA) that encodes the alpha-subunit of the mammalian ATP synthase complex. We have determined that the ATPA gene is expressed at high levels in tissues with high oxidative energy needs, such as heart and skeletal muscle, and at lower levels in tissues such as liver, intestine and stomach (Pierce et al., 1992).
In order to examine how the expression of the ATPA gene is regulated, we fused the 5'-flanking region of the ATPA gene to a reporter gene and transfected this construct into human cells. These experiments have identified several positive cis-acting regulatory elements that are important for expression of the ATPA gene (Vander Zee et al., 1994). These include cis-acting regulatory regions 1 and 2 in the upstream promoter and a regulatory region surrounding the major sites of transcriptional initiation. We have determined that the transcription factor, upstream stimulatory factor 2 (USF2), binds to and activates transcription of the ATPA gene through an E-box element (CANNTG) located in the cis-acting regulatory region 1 (Breen and Jordan, 1997). Furthermore, we have identified two trans-acting regulatory factors that bind to the initiator element- the multifunctional regulatory factor, YY1 (Breen et al., 1996) and USF2 (Breen and Jordan, 1998). Interestingly, expression of YY1 together with USF2 dramatically reduces the level of activation of the ATPA initiator element relative to transfection of USF2 alone (Breen and Jordan, 1998). We have also determined that the transactivation of the ATPA initiator element by USF2 involves the recruitment of the coactivator, p300 (Breen and Jordan, 1999). We have used the yeast one-hybrid screening method to identify another factor, COUP-TFII/ARP-1, which also binds to the ATPA cis-acting regulatory element 1 (Jordan et al., 2003). Functional assays in HeLa cells showed that COUP-TFII/ARP-1 represses the ATPA promoter activity in a dose- and sequence-dependent manner. Furthermore, cotransfection assays demonstrated that COUP-TFII/ARP-1 inhibits the USF2-mediated activation of the wild-type ATPA gene promoter but not a mutant promoter that is defective in COUP-TFII/ARP-1-binding. Overexpression of USF2 reversed the COUP-TFII/ARP-1-mediated repression of the ATPA promoter. Electrophoretic mobility shift assays revealed that COUP-TFII/ARP-1 and USF2 compete for an overlapping binding site in the ATPA regulatory element 1.
We are currently examining the effects of the transcriptional coativator, PGC-1, on the expression of the ATPA gene. We have determined that ectopic expression of PGC-1 stimulates the activity of the ATPA gene promoter in a dose-dependent manner. Interestingly, expression of PGC-1 together with USF2 blunts the USF2-mediated activation of the ATPA gene promoter. It is likely that the net expression of the ATPA gene in a given cell will result from the relative concentration and affinity of a number of transcription factors and cofactors, including USF2, YY1, COUP-TFII/ARP-1, p300 and PGC-1.
2. Comparative Profiling of the Mitochondrial Proteome in Alzheimer's Disease
Alzheimer's disease (AD) is an age-dependent irreversible neurodegenerative disorder that causes a progressive deterioration of cognitive functions, including a profound loss of memory (www.alzheimers.org). Reduced brain metabolism is a prominent and early feature of Alzheimer's disease. One of the mechanisms reducing brain metabolism in AD appears to be damage to or reduction of key mitochondrial components, including enzymes of the Krebs cycle and the oxidative phosphorylation system. The goal of our research is to use a quantitative proteomics approach to compare the levels of the mitochondrial proteins in the hippocampus and cortex of AD brain versus normal brain during the course of the disease (Breen et al., 2006). A triple transgenic mouse model of AD (3xTg-AD; PS1M146V, APPSwe and TauP301L) that exhibits both amyloid and tau pathologies in a region-dependent manner, as well as cognitive defects, is being used in these experiments.
Pierce, D. J., Jordan, E. M. and. Breen, G. A. M. (1992) Structural Organization of a Nuclear Gene for the α-Subunit of the Bovine Mitochondrial ATP Synthase Complex, Biochim. Biophys. Acta 1132: 265-275.
Jordan, E. M. and. Breen, G. A. M. (1993) Upstream Region of a Nuclear Gene Encoding the α-Subunit of the Human Mitochondrial F0F1 ATP Synthase, Biochim. Biophys. Acta 1173: 115-117.
Vander Zee, C. A., Jordan, E. M. and Breen, G. A. M. (1994) ATPF1 Binding Site, a Positive Cis-Acting Regulatory Element of the Mammalian ATP Synthase α-Subunit Gene, J. Biol Chem. 269: 6972-6977.
Breen, G. A. M., Vander Zee, C. A. and Jordan, E. M (1996) Nuclear Factor YY1 Activates the Mammalian F0F1 ATP Synthase α-Subunit Gene, Gene Expression 5: 181-191.
Breen, G. A. M. and Jordan, E. M. (1997) Regulation of the Nuclear Gene that Encodes the α-Subunit of the Mitochondrial F1F0-ATP Synthase Complex: Activation by Upstream Stimulatory Factor 2, J. Biol. Chem. 272: 10538-10564.
Breen, G. A. M. and Jordan, E. M. (1998) Upstream Stimulatory Factor 2 Activates the Mammalian F1F0 ATP Synthase α-Subunit Gene Through an Initiator Element, Gene Expression 7: 163-170.
Breen, G. A. M. and Jordan, E. M. (1999) Transcriptional Activation of the F1F0 ATP Synthase α-Subunit Initiator Element by USF2 is Mediated by p300, Biochim. Biophys. Acta 1428: 169-176.
Breen, G. A. M. and Jordan, E. M. (2000) Upstream Stimulatory Factor 2 Stimulates Transcription through an Initiator Element in the Mouse Cytochrome c Oxidase Subunit Vb Promoter, Biochim. Biophys. Acta 1517: 119-127.
Jordan, E. M., Worley, T. and Breen, G. A. M. (2003) Transcriptional Regulation of the Nuclear Gene that Encodes the α-Subunit of the Mammalian Mitochondrial ATP Synthase Complex: Role for the Orphan Nuclear Receptor, COUP-TFII/ARP-1, Biochemistry 42: 2656-2663.
Breen, G. A., Chou, J. and Goodman, S. R. (2006) Proteomics of Mitochondria in Alzheimer’s Disease, Proceedings of the 10th International Conference on Alzheimer’s Disease and Related Disorders, in press
- Updated: February 6, 2006