Myc Stimulates Nuclearly Encoded Mitochondrial Genes and Mitochondrial Biogenesis<sup><a href="#fn1" rid="fn1" class=" fn">†</a></sup>

Feng Li

Yunyue Wang

Karen Zeller

James Potter

Divisions of Hematology,^{Gastroenterology, Department of Medicine,}^{Department of Cell Biology,}^{Graduate Programs in Human Genetics and Molecular Biology,}^{Pathobiology,}^{Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland}⁵

^{Corresponding author. Mailing address: Ross Building, Room 1032, 720 Rutland Avenue, Baltimore, MD 21205. Phone: (410) 955-2773. Fax: (410) 955-0185. E-mail for Chi V. Dang:}ude.imhj@gnadvc. E-mail for Feng Li: ude.imhj@3ilf.

^{Present address: 200 Cambridge Park Drive, Cambridge, MA 02140.}

Received 2005 Jan 12; Revised 2005 Feb 22; Accepted 2005 Apr 11.

Abstract

Although several genes involved in mitochondrial function are direct Myc targets, the role of Myc in mitochondrial biogenesis has not been directly established. We determined the effects of ectopic Myc expression or the loss of Myc on mitochondrial biogenesis. Induction of Myc in P493-6 cells resulted in increased oxygen consumption and mitochondrial mass and function. Conversely, compared to wild-type Myc fibroblasts, Myc null rat fibroblasts have diminished mitochondrial mass and decreased number of normal mitochondria. Reconstitution of Myc expression in Myc null fibroblasts partially restored mitochondrial mass and function and normal-appearing mitochondria. Concordantly, we also observed in primary hepatocytes that acute deletion of floxed murine Myc by Cre recombinase resulted in diminished mitochondrial mass in primary hepatocytes. Our microarray analysis of genes responsive to Myc in human P493-6 B lymphocytes supports a role for Myc in mitochondrial biogenesis, since genes involved in mitochondrial structure and function are overrepresented among the Myc-induced genes. In addition to the known direct binding of Myc to many genes involved in mitochondrial structure and function, we found that Myc binds the TFAM gene, which encodes a key transcriptional regulator and mitochondrial DNA replication factor, both in P493-6 lymphocytes with high ectopic MYC expression and in serum-stimulated primary human 2091 fibroblasts with induced endogenous MYC. These observations support a pivotal role for Myc in regulating mitochondrial biogenesis.

Abstract

MYC, which encodes a transcription factor, c-Myc (herein termed Myc), plays a central role in the regulation of cell size, cell proliferation, and apoptosis (6, 11, 28, 40, 41) through its regulation of RNA polymerase I-, II-, and III-dependent genes (2, 17, 18, 39). Deregulated expression of MYC contributes to at least 100,000 U.S. cancer deaths annually, but 20 years after its discovery as a proto-oncogene, its full spectrum of regulatory functions remains incompletely understood (8, 34). Myc dimerizes with Max and binds E boxes (5′-CACGTG-3′) to activate transcription (16). Myc also represses transcription by interfering with the function of other transcriptional activators, such as Miz-1, NF-Y, or Sp1 (23, 45). Myc regulates several major cellular functions, such as cell proliferation, cell adhesion, and cell size, which explains the diversity of Myc target genes found in a variety of metabolic pathways, including amino acid, nucleotide, lipid, and glucose metabolism (www.myccancergene.org) (48). While Myc has been linked to energy metabolism through its regulation of glycolysis (26), its role in mitochondrial biogenesis is less well understood. It is noteworthy that mitochondria are required for a number of important biochemical pathways, including heme synthesis, fatty acid oxidation, and amino acid and steroid metabolism, among many others. Hence, it stands to reason that in driving cellular replication, Myc could also directly or indirectly affect the generation of mitochondria to maintain a steady-state respiratory and metabolic capacity as cells traverse through the cell cycle (1).

Large-scale gene expression analysis in rat or human systems suggests that overexpression of Myc induces nuclearly encoded mitochondrial genes (7, 20, 31, 37, 38, 46). Genes that bind to Myc in Drosophila, rat, or human systems include those encoding mitochondrial proteins or those involved in mitochondrial biogenesis (15, 31, 33, 36, 38, 47). Collectively, these studies suggest that Myc could affect mitochondrial protein expression. Here, we sought to determine the effects of Myc on mitochondrial biogenesis in an inducible Myc-dependent human B-cell model of cell proliferation, a well-defined Rat1 fibroblast system that has been rendered Myc null through homologous recombination, and a model of conditional Myc knockout in primary murine hepatocytes (10, 30, 44). Not only did we observe that mitochondrial biogenesis depends on Myc, but we also found that among the Myc target genes most highly induced in the human B-cell system are those involved in mitochondrial biogenesis. Furthermore, we found that Myc directly regulates TFAM, which encodes a key factor involved in mitochondrial transcription and mitochondrial DNA (mtDNA) replication. These observations support a pivotal role for Myc in regulating mitochondrial biogenesis.

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Acknowledgments

We thank D. Eick for P493-6 cells, J. Sedivy for TGR and HO15 cells, and P. Puigserver for comments on the manuscript. We thank D. Murphy for kind instruction on sample preparation and data analysis for confocal microscopy and electron microscopy, Y. Ko for technical assistance with the stimulated cellular oxygen consumption assay, and L. Blosser for the flow cytometry assay. We thank Francisco Martinez Murillo and Chunfa Jie for microarray hybridization and data analysis.

This work was supported by NIH/NCI grants CA52497, CA57341, and CA09159 and the Training Program in Human Genetics and Molecular Biology. J. Kim is a Howard Hughes Medical Institute predoctoral fellow. C. Dang is the Johns Hopkins Family Professor in Oncology Research.

Acknowledgments

Footnotes

^{Supplemental material for this article may be found at}http://mcb.asm.org/.

Footnotes

REFERENCES

References

1. Amati, B., K. Alevizopoulos, and J. Vlach. 1998. Myc and the cell cycle. Front Biosci.3:d250-d268. [[PubMed]
2. Arabi, A., S. Wu, K. Ridderstrale, H. Bierhoff, C. Shiue, K. Fatyol, S. Fahlen, P. Hydbring, O. Soderberg, I. Grummt, L. G. Larsson, and A. P. Wright. 2005. c-Myc associates with ribosomal DNA and activates RNA polymerase I transcription. Nat. Cell. Biol.7:303-310. [[PubMed]
3. Blackwell, T. K., J. Huang, A. Ma, L. Kretzner, F. W. Alt, R. N. Eisenman, and H. Weintraub. 1993. Binding of myc proteins to canonical and noncanonical DNA sequences. Mol. Cell. Biol.13:5216-5224.
4. Boyd, K. E., J. Wells, J. Gutman, S. M. Bartley, and P. J. Farnham. 1998. c-Myc target gene specificity is determined by a post-DNA binding mechanism. Proc. Natl. Acad. Sci. USA95:13887-13892.
5. Cam, H., E. Balciunaite, A. Blais, A. Spektor, R. C. Scarpulla, R. Young, Y. Kluger, and B. D. Dynlacht. 2004. A common set of gene regulatory networks links metabolism and growth inhibition. Mol. Cell16:399-411. [[PubMed]
6. Cole, M. D., and S. B. McMahon. 1999. The Myc oncoprotein: a critical evaluation of transactivation and target gene regulation. Oncogene18:2916-2924. [[PubMed]
7. Coller, H. A., C. Grandori, P. Tamayo, T. Colbert, E. S. Lander, R. N. Eisenman, and T. R. Golub. 2000. Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. Proc. Natl. Acad. Sci. USA97:3260-3265.
8. Dang, CV. 1999. c-Myc targets genes involved in cell growth, apoptosis, and metabolism. Mol. Cell. Biol.19:1-11. [Google Scholar]
9. de Alboran, I. M., E. Baena, and A. C. Martinez. 2004. c-Myc-deficient B lymphocytes are resistant to spontaneous and induced cell death. Cell Death Differ.11:61-68. [[PubMed]
10. de Alboran, I. M., R. C. O'Hagan, F. Gartner, B. Malynn, L. Davidson, R. Rickert, K. Rajewsky, R. A. DePinho, and F. W. Alt. 2001. Analysis of C-MYC function in normal cells via conditional gene-targeted mutation. Immunity14:45-55. [[PubMed]
11. Eisenman, RN. 2001. Deconstructing myc. Genes Dev.15:2023-2030. [[PubMed][Google Scholar]
12. Ekstrand, M. I., M. Falkenberg, A. Rantanen, C. B. Park, M. Gaspari, K. Hultenby, P. Rustin, C. M. Gustafsson, and N. G. Larsson. 2004. Mitochondrial transcription factor A regulates mtDNA copy number in mammals. Hum. Mol. Genet.13:935-944. [[PubMed]
13. Elkon, R., K. I. Zeller, C. Linhart, C. V. Dang, R. Shamir, and Y. Shiloh. 2004. In silico identification of transcriptional regulators associated with c-Myc. Nucleic Acids Res.32:4955-4961.
14. Elmore, S. P., Y. Nishimura, T. Qian, B. Herman, and J. J. Lemasters. 2004. Discrimination of depolarized from polarized mitochondria by confocal fluorescence resonance energy transfer. Arch. Biochem. Biophys.422:145-152. [[PubMed]
15. Fernandez, P. C., S. R. Frank, L. Wang, M. Schroeder, S. Liu, J. Greene, A. Cocito, and B. Amati. 2003. Genomic targets of the human c-Myc protein. Genes Dev.17:1115-1129.
16. Grandori, C., S. M. Cowley, L. P. James, and R. N. Eisenman. 2000. The Myc/Max/Mad network and the transcriptional control of cell behavior. Annu. Rev. Cell. Dev. Biol.16:653-699. [[PubMed]
17. Grandori, C., N. Gomez-Roman, Z. A. Felton-Edkins, C. Ngouenet, D. A. Galloway, R. N. Eisenman, and R. J. White. 2005. c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I. Nat. Cell. Biol.7:311-318. [[PubMed]
18. Grewal, S. S., L. Li, A. Orian, R. N. Eisenman, and B. A. Edgar. 2005. Myc-dependent regulation of ribosomal RNA synthesis during Drosophila development. Nat. Cell. Biol.7:295-302. [[PubMed]
19. Guidot, DM. 1998. Endotoxin pretreatment in vivo increases the mitochondrial respiratory capacity in rat hepatocytes. Arch. Biochem. Biophys.354:9-17. [[PubMed][Google Scholar]
20. Guo, Q. M., R. L. Malek, S. Kim, C. Chiao, M. He, M. Ruffy, K. Sanka, N. H. Lee, C. V. Dang, and E. T. Liu. 2000. Identification of c-myc responsive genes using rat cDNA microarray. Cancer Res.60:5922-5928. [[PubMed]
21. Hosack, D. A., G. Dennis, Jr., B. T. Sherman, H. C. Lane, and R. A. Lempicki. 2003. Identifying biological themes within lists of genes with EASE. Genome Biol.4:R70.
22. Irizarry, R. A., B. Hobbs, F. Collin, Y. D. Beazer-Barclay, K. J. Antonellis, U. Scherf, and T. P. Speed. 2003. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics4:249-264. [[PubMed]
23. Izumi, H., C. Molander, L. Z. Penn, A. Ishisaki, K. Kohno, and K. Funa. 2001. Mechanism for the transcriptional repression by c-Myc on PDGF beta-receptor. J. Cell Sci.114:1533-1544. [[PubMed]
24. Kanki, T., K. Ohgaki, M. Gaspari, C. M. Gustafsson, A. Fukuoh, N. Sasaki, N. Hamasaki, and D. Kang. 2004. Architectural role of mitochondrial transcription factor A in maintenance of human mitochondrial DNA. Mol. Cell. Biol.24:9823-9834.
25. Kelly, D. P., and R. C. Scarpulla. 2004. Transcriptional regulatory circuits controlling mitochondrial biogenesis and function. Genes Dev.18:357-368. [[PubMed]
26. Kim, J. W., K. I. Zeller, Y. Wang, A. G. Jegga, B. J. Aronow, K. A. O'Donnell, and C. V. Dang. 2004. Evaluation of myc E-box phylogenetic footprints in glycolytic genes by chromatin immunoprecipitation assays. Mol. Cell. Biol.24:5923-5936.
27. Kim, S., Q. Li, C. V. Dang, and L. A. Lee. 2000. Induction of ribosomal genes and hepatocyte hypertrophy by adenovirus-mediated expression of c-Myc in vivo. Proc. Natl. Acad. Sci. USA97:11198-11202.
28. Lutz, W., J. Leon, and M. Eilers. 2002. Contributions of Myc to tumorigenesis. Biochim. Biophys. Acta1602:61-71. [[PubMed]
29. Maniura-Weber, K., S. Goffart, H. L. Garstka, J. Montoya, and R. J. Wiesner. 2004. Transient overexpression of mitochondrial transcription factor A (TFAM) is sufficient to stimulate mitochondrial DNA transcription, but not sufficient to increase mtDNA copy number in cultured cells. Nucleic Acids Res.32:6015-6027.
30. Mateyak, M. K., A. J. Obaya, S. Adachi, and J. M. Sedivy. 1997. Phenotypes of c-Myc-deficient rat fibroblasts isolated by targeted homologous recombination. Cell Growth Differ.8:1039-1048. [[PubMed]
31. Menssen, A., and HHermeking. 2002. Characterization of the c-MYC-regulated transcriptome by SAGE: identification and analysis of c-MYC target genes. Proc Natl. Acad. Sci. USA99:6274-6279. [Google Scholar]
32. Mezey, E., L. Rennie-Tankersley, and J. J. Potter. 2001. Liver alcohol dehydrogenase is degraded by the ubiquitin-proteasome pathway. Biochem. Biophys. Res. Commun.285:644-648. [[PubMed]
33. Morrish, F., C. Giedt, and D. Hockenbery. 2003. c-MYC apoptotic function is mediated by NRF-1 target genes. Genes Dev.17:240-255.
34. Nesbit, C. E., J. M. Tersak, and E. V. Prochownik. 1999. MYC oncogenes and human neoplastic disease. Oncogene18:3004-3016. [[PubMed]
35. Newton, M. A., C. M. Kendziorski, C. S. Richmond, F. R. Blattner, and K. W. Tsui. 2001. On differential variability of expression ratios: improving statistical inference about gene expression changes from microarray data. J. Comput. Biol.8:37-52. [[PubMed]
36. Nikiforov, M. A., S. Chandriani, B. O'Connell, O. Petrenko, I. Kotenko, A. Beavis, J. M. Sedivy, and M. D. Cole. 2002. A functional screen for Myc-responsive genes reveals serine hydroxymethyltransferase, a major source of the one-carbon unit for cell metabolism. Mol. Cell. Biol.22:5793-5800.
37. O'Connell, B. C., A. F. Cheung, C. P. Simkevich, W. Tam, X. Ren, M. K. Mateyak, and J. M. Sedivy. 2003. A large scale genetic analysis of c-Myc-regulated gene expression patterns. J. Biol. Chem.278:12563-12573. [[PubMed]
38. Orian, A., B. van Steensel, J. Delrow, H. J. Bussemaker, L. Li, T. Sawado, E. Williams, L. W. Loo, S. M. Cowley, C. Yost, S. Pierce, B. A. Edgar, S. M. Parkhurst, and R. N. Eisenman. 2003. Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. Genes Dev.17:1101-1114.
39. Oskarsson, T., and ATrumpp. 2005. The Myc trilogy: lord of RNA polymerases. Nat. Cell. Biol.7:215-217. [[PubMed][Google Scholar]
40. Oster, S. K., C. S. Ho, E. L. Soucie, and L. Z. Penn. 2002. The myc oncogene: MarvelouslY Complex. Adv. Cancer Res.84:81-154. [[PubMed]
41. Pelengaris, S., M. Khan, and G. Evan. 2002. c-MYC: more than just a matter of life and death. Nat. Rev. Cancer2:764-776. [[PubMed]
42. Rajagopalan, H., P. V. Jallepalli, C. Rago, V. E. Velculescu, K. W. Kinzler, B. Vogelstein, and C. Lengauer. 2004. Inactivation of hCDC4 can cause chromosomal instability. Nature428:77-81. [[PubMed]
43. Schuhmacher, M., F. Kohlhuber, M. Holzel, C. Kaiser, H. Burtscher, M. Jarsch, G. W. Bornkamm, G. Laux, A. Polack, U. H. Weidle, and D. Eick. 2001. The transcriptional program of a human B cell line in response to Myc. Nucleic Acids Res.29:397-406.
44. Schuhmacher, M., M. S. Staege, A. Pajic, A. Polack, U. H. Weidle, G. W. Bornkamm, D. Eick, and F. Kohlhuber. 1999. Control of cell growth by c-Myc in the absence of cell division. Curr. Biol.9:1255-1258. [[PubMed]
45. Wanzel, M., S. Herold, and M. Eilers. 2003. Transcriptional repression by Myc. Trends Cell Biol.13:146-150. [[PubMed]
46. Watson, J. D., S. K. Oster, M. Shago, F. Khosravi, and L. Z. Penn. 2002. Identifying genes regulated in a Myc-dependent manner. J. Biol. Chem.277:36921-36930. [[PubMed]
47. Wonsey, D. R., K. I. Zeller, and C. V. Dang. 2002. The c-Myc target gene PRDX3 is required for mitochondrial homeostasis and neoplastic transformation. Proc. Natl. Acad. Sci. USA99:6649-6654.
48. Zeller, K. I., A. G. Jegga, B. J. Aronow, K. A. O'Donnell, and C. V. Dang. 2003. An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets. Genome Biol.4:R69.