In vivo transcriptional regulation of N-Myc target genes is controlled by E-box methylation.

N-Myc is a transcription factor that forms heterodimers with the protein Max and binds gene promoters by recognizing a DNA sequence, CACGTG, called E-box. The identification of N-myc target genes is an important step for understanding N-Myc biological functions in both physiological and pathological contexts. In this study, we describe the identification of N-Myc-responsive genes through chromatin immunoprecipitation and methylation-sensitive restriction analysis. Results show that N-Myc is a direct regulator of several identified genes, and that methylation of the CpG dinucleotide within the E-box prevents the access of N-Myc to gene promoters in vivo. Furthermore, methylation profile of the E-box within the promoters of EGFR and CASP8, two genes directly controlled by Myc, is cell type-specific, suggesting that differential E-box methylation may contribute to generating unique patterns of Myc-dependent transcription. This study illuminates a central role of DNA methylation in controlling N-Myc occupancy at gene promoters and modulating its transcriptional activity in cancer cells.

[1]  G. Prendergast,et al.  Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region. , 1991, Science.

[2]  F. Behm,et al.  Caspase 8 is deleted or silenced preferentially in childhood neuroblastomas with amplification of MYCN , 2000, Nature Medicine.

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

[4]  A. Janulaitis,et al.  The eco72IC gene specifies a trans-acting factor which influences expression of both DNA methyltransferase and endonuclease from the Eco72I restriction-modification system. , 1995, Gene.

[5]  H. Kondoh,et al.  Defects of embryonic organogenesis resulting from targeted disruption of the N-myc gene in the mouse. , 1993, Development.

[6]  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.

[7]  Stephen K. Burley,et al.  X-Ray Structures of Myc-Max and Mad-Max Recognizing DNA Molecular Bases of Regulation by Proto-Oncogenic Transcription Factors , 2003, Cell.

[8]  R. Eisenman,et al.  N-myc is essential during neurogenesis for the rapid expansion of progenitor cell populations and the inhibition of neuronal differentiation. , 2002, Genes & development.

[9]  P. Farnham,et al.  Direct Examination of Histone Acetylation on Myc Target Genes Using Chromatin Immunoprecipitation* , 2000, The Journal of Biological Chemistry.

[10]  M. Schwab,et al.  The N‐Myc oncoprotein is associated in vivo with the phosphoprotein Max(p20/22) in human neuroblastoma cells. , 1991, The EMBO journal.

[11]  L. Tonon,et al.  Retinoic acid-induced growth arrest and differentiation of neuroblastoma cells are counteracted by N-myc and enhanced by max overexpressions. , 1996, Oncogene.

[12]  P. Fernandez,et al.  Binding of c-Myc to chromatin mediates mitogen-induced acetylation of histone H4 and gene activation. , 2001, Genes & development.

[13]  S. Cawley,et al.  Unbiased Mapping of Transcription Factor Binding Sites along Human Chromosomes 21 and 22 Points to Widespread Regulation of Noncoding RNAs , 2004, Cell.

[14]  R. Roeder,et al.  Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. , 1983, Nucleic acids research.

[15]  H. Sather,et al.  Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. , 1985, The New England journal of medicine.

[16]  F. Alt,et al.  N-myc can functionally replace c-myc in murine development, cellular growth, and differentiation. , 2000, Genes & development.

[17]  Peter A. Jones,et al.  Identification of DNA methylation differences during tumorigenesis by methylation-sensitive arbitrarily primed polymerase chain reaction. , 2002, Methods.

[18]  M. Cole,et al.  The Essential Cofactor TRRAP Recruits the Histone Acetyltransferase hGCN5 to c-Myc , 2000, Molecular and Cellular Biology.

[19]  G. Church,et al.  Genomic sequencing. , 1993, Methods in molecular biology.

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

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

[22]  M. Schwab,et al.  Conditional expression of N-myc in human neuroblastoma cells increases expression of alpha-prothymosin and ornithine decarboxylase and accelerates progression into S-phase early after mitogenic stimulation of quiescent cells. , 1996, Oncogene.

[23]  V. Stewart,et al.  Embryonic lethality in mice homozygous for a targeted disruption of the N-myc gene. , 1992, Genes & development.

[24]  R. Weinberg.  Oncogenes and the molecular origins of cancer , 1989 .

[25]  J. Rossant,et al.  Dissecting the role of N-myc in development using a single targeting vector to generate a series of alleles , 1998, Current Biology.

[26]  G. Evan,et al.  Transcriptional activation by the human c-Myc oncoprotein in yeast requires interaction with Max , 1992, Nature.

[27]  Peter A. Jones,et al.  The fundamental role of epigenetic events in cancer , 2002, 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. Amati,et al.  Myc-Max-Mad: a transcription factor network controlling cell cycle progression, differentiation and death. , 1994, Current opinion in genetics & development.

[30]  J. Trent,et al.  Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour , 1983, Nature.

[31]  R. Jaenisch,et al.  Induction of Tumors in Mice by Genomic Hypomethylation , 2003, Science.

[32]  N. Benvenisty,et al.  A DNA microarray screen for genes involved in c-MYC and N-MYC oncogenesis in human tumors , 2001, Oncogene.

[33]  P. Farnham,et al.  Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. , 2002, Methods.

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

[35]  S. McMahon,et al.  Analysis of genomic targets reveals complex functions of MYC , 2004, Nature Reviews Cancer.

[36]  A. Bird.  CpG islands as gene markers in the vertebrate nucleus , 1987 .