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Molecular and Cellular Biology, July 2004, p. 5923-5936, Vol. 24, No. 13
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.13.5923-5936.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Evaluation of Myc E-Box Phylogenetic Footprints in Glycolytic Genes by Chromatin Immunoprecipitation Assays{dagger}

Jung-whan Kim,1 Karen I. Zeller,2 Yunyue Wang,3 Anil G. Jegga,4,5,6 Bruce J. Aronow,4,5,6 Kathryn A. O'Donnell,7 and Chi V. Dang1,2,3,7*

Graduate Program of Pathobiology,1 Department of Medicine,2 Graduate Program in Cellular and Molecular Medicine,3 Program in Human Genetics and Molecular Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,7 Division of Pediatric Informatics,4 Division of Molecular Developmental Biology, Children's Hospital Research Foundation, Children's Hospital Medical Center,5 Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio 452296

Received 23 January 2004/ Returned for modification 15 March 2004/ Accepted 8 April 2004

Prediction of gene regulatory sequences using phylogenetic footprinting has advanced considerably but lacks experimental validation. Here, we report whether transcription factor binding sites predicted by dot plotting or web-based Trafac analysis could be validated by chromatin immunoprecipitation assays. MYC overexpression enhances glycolysis without hypoxia and hence may contribute to altered tumor metabolism. Because the full spectrum of glycolytic genes directly regulated by Myc is not known, we chose Myc as a model transcription factor to determine whether it binds target glycolytic genes that have conserved canonical Myc binding sites or E boxes (5'-CACGTG-3'). Conserved canonical E boxes in ENO1, HK2, and LDHA occur in 31- to 111-bp islands with high interspecies sequence identity (>65%). Trafac analysis revealed another region in ENO1 that corresponds to a murine region with a noncanonical E box. Myc bound all these conserved regions well in the human P493-6 B lymphocytes. We also determined whether Myc could bind nonconserved canonical E boxes found in the remaining human glycolytic genes. Myc bound PFKM, but it did not significantly bind GPI, PGK1, and PKM2. Binding to BPGM, PGAM2, and PKLR was not detected. Both GAPD and TPI1 do not have conserved E boxes but are induced and bound by Myc through regions with noncanonical E boxes. Our results indicate that Myc binds well to conserved canonical E boxes, but not nonconserved E boxes. However, the binding of Myc to unpredicted genomic regions with noncanonical E boxes reveals a limitation of phylogenetic footprinting. In aggregate, these observations indicate that Myc is an important regulator of glycolytic genes, suggesting that MYC plays a key role in a switch to glycolytic metabolism during cell proliferation or tumorigenesis.

* Corresponding author. Mailing address: Ross Research Building, Room 1032, The Johns Hopkins University School of Medicine, 720 Rutland Ave., Baltimore, MD 21205. Phone: (410) 955-2773. Fax: (410) 955-0185. E-mail: cvdang{at}jhmi.edu.

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

Molecular and Cellular Biology, July 2004, p. 5923-5936, Vol. 24, No. 13
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.13.5923-5936.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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