Chromatin Immunoprecipitation Assays Footprints in Glycolytic Genes by Evaluation of Myc E-box Phylogenetic Supplemental Material

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.

[1]  G. Semenza,et al.  Oncogenic alterations of metabolism. , 1999, Trends in biochemical sciences.

[2]  A. Menssen,et al.  Characterization of the c-MYC-regulated transcriptome by SAGE: Identification and analysis of c-MYC target genes , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Didier Picard,et al.  Chimaeras of Myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells , 1989, Nature.

[4]  L. Pachter,et al.  rVista for comparative sequence-based discovery of functional transcription factor binding sites. , 2002, Genome research.

[5]  J. Hoek,et al.  Mitochondrial Binding of Hexokinase II Inhibits Bax-induced Cytochrome c Release and Apoptosis* , 2002, The Journal of Biological Chemistry.

[6]  G. Rubin,et al.  Exploiting transcription factor binding site clustering to identify cis-regulatory modules involved in pattern formation in the Drosophila genome , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[7]  P. Farnham,et al.  c-Myc target gene specificity is determined by a post-DNAbinding mechanism. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[8]  R. Sharan,et al.  Genome-wide in silico identification of transcriptional regulators controlling the cell cycle in human cells. , 2003, Genome research.

[9]  P. Farnham,et al.  c-Myc Mediates Activation of the cad Promoter via a Post-RNA Polymerase II Recruitment Mechanism* , 2001, The Journal of Biological Chemistry.

[10]  R. Eisenman,et al.  Myc and Max proteins possess distinct transcriptional activities , 1992, Nature.

[11]  U. Weidle,et al.  Control of cell growth by c-Myc in the absence of cell division , 1999, Current Biology.

[12]  Chi V. Dang,et al.  A strategy for identifying transcription factor binding sites reveals two classes of genomic c-Myc target sites , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[13]  James W Carman,et al.  Detection and visualization of compositionally similar cis-regulatory element clusters in orthologous and coordinately controlled genes. , 2002, Genome research.

[14]  R. Passantino,et al.  ENO1 gene product binds to the c‐myc promoter and acts as a transcriptional repressor: relationship with Myc promoter‐binding protein 1 (MBP‐1) , 2000, FEBS letters.

[15]  R. Eisenman,et al.  The Myc/Max/Mad network and the transcriptional control of cell behavior. , 2000, Annual review of cell and developmental biology.

[16]  P. Neiman,et al.  Analysis of gene expression during myc oncogene-induced lymphomagenesis in the bursa of Fabricius , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[17]  U. Weidle,et al.  The transcriptional program of a human B cell line in response to Myc. , 2001, Nucleic acids research.

[18]  Peter W. Markstein,et al.  Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[19]  Kathryn A. O’Donnell,et al.  An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets , 2003, Genome Biology.

[20]  Peggy J. Farnham,et al.  Analysis of Myc Bound Loci Identified by CpG Island Arrays Shows that Max Is Essential for Myc-Dependent Repression , 2003, Current Biology.

[21]  Mouse Genome Sequencing Consortium.  Initial sequencing and comparative analysis of the mouse genome , 2002, Nature.

[22]  Mathieu Blanchette,et al.  FootPrinter: a program designed for phylogenetic footprinting , 2003, Nucleic Acids Res..

[23]  M. Cole,et al.  The Myc oncoprotein: a critical evaluation of transactivation and target gene regulation , 1999, Oncogene.

[24]  R A Jungmann,et al.  c-Myc transactivation of LDH-A: implications for tumor metabolism and growth. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[25]  S. R. Datta,et al.  BAD and glucokinase reside in a mitochondrial complex that integrates glycolysis and apoptosis , 2003, Nature.

[26]  O. Warburg.  On the origin of cancer cells. , 1956, Science.

[27]  L. Pennacchio,et al.  Genomic strategies to identify mammalian regulatory sequences , 2001, Nature Reviews Genetics.

[28]  E. Lander,et al.  Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[29]  B. Howard,et al.  Phosphotyrosine-containing lactate dehydrogenase is restricted to the nuclei of PC12 pheochromocytoma cells , 1990, Molecular and cellular biology.

[30]  C. Dang,et al.  c-Myc Overexpression Uncouples DNA Replication from Mitosis , 1999, Molecular and Cellular Biology.

[31]  K. Zeller,et al.  Characterization of Nucleophosmin (B23) as a Myc Target by Scanning Chromatin Immunoprecipitation* , 2001, The Journal of Biological Chemistry.

[32]  Colin N. Dewey,et al.  Initial sequencing and comparative analysis of the mouse genome. , 2002 .

[33]  L. Penn,et al.  The myc oncogene: MarvelouslY Complex. , 2002, Advances in cancer research.

[34]  A. Sandelin,et al.  Identification of conserved regulatory elements by comparative genome analysis , 2003, Journal of biology.

[35]  John D. Watson,et al.  Identifying Genes Regulated in a Myc-dependent Manner* , 2002, The Journal of Biological Chemistry.

[36]  E. Kandel,et al.  Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. , 2001, Genes & development.

[37]  J L Cleveland,et al.  The ornithine decarboxylase gene is a transcriptional target of c-Myc. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[38]  John M Sedivy,et al.  A Large Scale Genetic Analysis of c-Myc-regulated Gene Expression Patterns* 210 , 2003, The Journal of Biological Chemistry.

[39]  C. Dang,et al.  Deregulation of Glucose Transporter 1 and Glycolytic Gene Expression by c-Myc* , 2000, The Journal of Biological Chemistry.

[40]  Andrea Cocito,et al.  Genomic targets of the human c-Myc protein. , 2003, Genes & development.

[41]  Michael Q. Zhang,et al.  A global transcriptional regulatory role for c-Myc in Burkitt's lymphoma cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[42]  B. Edgar,et al.  Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network. , 2003, Genes & development.

[43]  R. Roeder,et al.  S Phase Activation of the Histone H2B Promoter by OCA-S, a Coactivator Complex that Contains GAPDH as a Key Component , 2003, Cell.

[44]  R. Eisenman,et al.  Myc target genes. , 1997, Trends in biochemical sciences.

[45]  A. Subramanian,et al.  Structural analysis of alpha-enolase. Mapping the functional domains involved in down-regulation of the c-myc protooncogene. , 2000, The Journal of biological chemistry.