1: J Biol Chem. 2004 Feb 13;279(7):5110-7. Epub 2003 Nov 26. 

E2F and Sp1/Sp3 Synergize but are not sufficient to activate the MYCN gene in
neuroblastomas.

Kramps C, Strieder V, Sapetschnig A, Suske G, Lutz W.

Institute of Molecular Biology and Tumor Research, 35033 Marburg, Germany.

Amplification of the MYCN gene, resulting in overexpression of MYCN,
distinguishes a subset of neuroblastomas with poor prognosis. We recently
identified MYCN as a target gene of the E2F transcription factors. Here we show
that Sp1 and Sp3 cooperate with E2F-1 to activate the MYCN promoter. However, in
a neuroblastoma cell line that does not express MYCN, overexpression of E2F-1
was not sufficient to activate the MYCN promoter even in the presence of
trichostatin A and 5-aza-cytidine. This was because of a failure of E2F-1 to
bind to the MYCN promoter in these cells, although access of E2F-1 to the
inactive MYCN promoter was not blocked by a nucleosome. Differences in
nucleosomal organization of the MYCN promoter in different cell lines did not
correlate with gene activation per se but with the switch from basal to
activated transcription. Binding of E2F and Sp1/Sp3 to the MYCN promoter in vivo
correlated with acetylation of histones H3 and H4 and recruitment of RNA
polymerase II and the protein acetyltransferase Tip60 but not with nucleosome
remodeling. Our results define distinct chromatin states of the MYCN promoter,
indicate that factors in addition to E2F and Sp1/Sp3 are required to activate
MYCN in neuroblastomas, and provide evidence for a novel mechanism of
controlling access of E2F to selected target genes.

PMID: 14645238 [PubMed - indexed for MEDLINE]


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2: Surgery. 2002 Aug;132(2):232-8. 

Importance of Sp1 consensus motifs in the MYCN promoter.

Inge TH, Casson LK, Priebe W, Trent JO, Georgeson KE, Miller DM, Bates PJ.

Children's Hospital Research Foundation and Department of Pediatric Surgery,
Cincinnati Children's Hospital Medical Center, Ohio 45229,USA.

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.

PMID: 12219017 [PubMed - indexed for MEDLINE]


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