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CELL GROWTH AND DEVELOPMENT

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

Jung-whan Kim, Karen I. Zeller, Yunyue Wang, Anil G. Jegga, Bruce J. Aronow, Kathryn A. O'Donnell, Chi V. Dang
Jung-whan Kim
1Graduate Program of Pathobiology
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Karen I. Zeller
2Department of Medicine
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Yunyue Wang
3Graduate Program in Cellular and Molecular Medicine
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Anil G. Jegga
4Division of Pediatric Informatics
5Division of Molecular Developmental Biology, Children's Hospital Research Foundation, Children's Hospital Medical Center
6Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio 45229
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Bruce J. Aronow
4Division of Pediatric Informatics
5Division of Molecular Developmental Biology, Children's Hospital Research Foundation, Children's Hospital Medical Center
6Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio 45229
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Kathryn A. O'Donnell
7Program in Human Genetics and Molecular Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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Chi V. Dang
1Graduate Program of Pathobiology
2Department of Medicine
3Graduate Program in Cellular and Molecular Medicine
7Program in Human Genetics and Molecular Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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  • For correspondence: cvdang@jhmi.edu
DOI: 10.1128/MCB.24.13.5923-5936.2004
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ABSTRACT

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.

  • Copyright © 2004 American Society for Microbiology
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Evaluation of Myc E-Box Phylogenetic Footprints in Glycolytic Genes by Chromatin Immunoprecipitation Assays
Jung-whan Kim, Karen I. Zeller, Yunyue Wang, Anil G. Jegga, Bruce J. Aronow, Kathryn A. O'Donnell, Chi V. Dang
Molecular and Cellular Biology Jun 2004, 24 (13) 5923-5936; DOI: 10.1128/MCB.24.13.5923-5936.2004

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Evaluation of Myc E-Box Phylogenetic Footprints in Glycolytic Genes by Chromatin Immunoprecipitation Assays
Jung-whan Kim, Karen I. Zeller, Yunyue Wang, Anil G. Jegga, Bruce J. Aronow, Kathryn A. O'Donnell, Chi V. Dang
Molecular and Cellular Biology Jun 2004, 24 (13) 5923-5936; DOI: 10.1128/MCB.24.13.5923-5936.2004
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KEYWORDS

DNA Footprinting
E-Box Elements
glycolysis
Phylogeny
Proto-Oncogene Proteins c-myc

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