Importance of Sp1 consensus motifs in the MYCN promoter.
BACKGROUND
MYCN (N-myc) amplification in neuroblastoma is associated with poor clinical outcome. Factors that regulate MYCN expression have not been elucidated. MYCN is considered a TATA-less promoter, whereas significant promoter activity resides within 160 bp 5' of the major transcription start site. This region contains two GC-rich motifs and a CT box, regions for potential transcription factor interaction.


METHODS
To characterize DNA-protein interactions in this region of the MYCN promoter, electrophoretic mobility shift assays, and promoter-reporter were used.


RESULTS
A MYCN promoter fragment was incubated with HeLa nuclear extract, with or without competitors. Three major protein/DNA complexes were formed. Formation of 2 complexes could be inhibited by unlabeled Sp1 consensus duplex and by the Sp1 site-specific drug WP631. Purified Sp1 protein produced a complex similar to that formed with HeLa extract. To determine whether these DNA/protein interactions could be blocked in a sequence-specific fashion, a triplex forming oligonucleotide (TFO) was used. This TFO was designed to bind in the major groove of the promoter, covering the CT-box (putative Sp1 binding) motif. When triplex formation was followed by addition of nuclear extract, protein binding was indeed inhibited. Functional significance of this inhibition was tested with pE/Bnmyc-luc, a promoter-reporter plasmid containing the human MYCN promoter driving luciferase expression. Incubation with TFO, but not control oligodeoxynucleotides, completely inhibited luciferase activity.


CONCLUSIONS
These data suggest that protein binding does occur in regions of the MYCN promoter containing GC and CT box elements and that this interaction is important for MYCN promoter activity. By inference, these data also suggest that the proteins that bind in this region are Sp1 family members.
Inge
Thomas H
TH
Children's Hospital Research Foundation and Department of Pediatric Surgery, Cincinnati Children's Hospital Medical Center, Ohio 45229,USA.
Casson
Lavona K
LK
Priebe
Waldemar
W
Trent
John O
JO
Georgeson
Keith E
KE
Miller
Donald M
DM
Bates
Paula J
PJ
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D024202:Electrophoretic Mobility Shift Assay; D020862:GC Rich Sequence / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins / Q000235:genetics*; D015513:Oncogene Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000235:genetics* / Q000378:metabolism
C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D016329:Sp1 Transcription Factor