An interferon-sensitive response element is involved in constitutive caspase-8 gene expression in neuroblastoma cells.
We previously identified a 1.2 Kb DNA element (P-1161/+16), 5' to caspase-8 exon-1, that acts as promoter in caspase-8-positive, but not in caspase-8-negative neuroblastoma (NB) cells. The P-1161/+16 DNA element regulates both constitutive and interferon IFN-gamma-inducible caspase-8 expression. Two GAS (IFN-activated sequence, STAT-1 binding site) and two ISRE (interferon sensitive response element, IRF binding site) were present in P-1161/+16. Deletion studies indicated that elements essential for promoter activity in NB cells were present in a 167 bp region 5' flanking exon-1 (P-151/+16), which contains an ISRE at position -32. The transcription initiation site was mapped by 5' rapid amplification of cDNA end (RACE) at position -20 from caspase-8 cDNA reference sequence. Disruption of the ISRE-32 indicated that it is required for both constitutive and IFN-gamma-inducible caspase-8 expression. IRF-1 and IRF-2 transcription factors bind to the (-151/+16) DNA fragment in vitro. Chromatin immunoprecipitation (ChIP) assays showed that IRF-1 and IRF-2 bind to the DNA region at the 5' of caspase-8 gene in NB cells, which show constitutive expression but not in caspase-8 negative cells. In these last cells, up-regulation of caspase-8 by IFN-gamma was associated to induction of IRF-1 and IRF-2 binding to caspase-8 promoter and increased histone acetylation. Moreover, RNA interference experiments also supported the involvement of IRF-1 and IRF-2 in constitutive caspase-8 expression in NB cells.
De Ambrosis
Alessandro
A
Laboratory of Immunological Therapy, Istituto Nazionale per la Ricerca sul Cancro (IST-Genova) Largo Rosanna Benzi 10, 16132 Genova, Italy.
Casciano
Ida
I
Croce
Michela
M
Pagnan
Gabriella
G
Radic
Luana
L
Banelli
Barbara
B
Di Vinci
Angela
A
Allemanni
Giorgio
G
Tonini
Gian Paolo
GP
Ponzoni
Mirco
M
Romani
Massimo
M
Ferrini
Silvano
S
D000998:Antiviral Agents / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D053181:Caspase 8 / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D017353:Gene Deletion; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D050836:Interferon Regulatory Factor-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020218:Response Elements / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D015854:Up-Regulation
D000998:Antiviral Agents; C494257:IRF1 protein, human; C494229:IRF2 protein, human; D050856:Interferon Regulatory Factor-1; D050836:Interferon Regulatory Factor-2; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D007371:Interferon-gamma; C505530:CASP8 protein, human; D053181:Caspase 8