An interferon-sensitive response element is involved in constitutive caspase-8 gene expression in neuroblastoma cells

Abstract

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, hut not in caspase-8-negative neuroblastoma (NB) cells. The P-1161/+16 DNA element regulates both constitutive and interferon IFN-γ-inducihle 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 hp 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-γ-inducible caspase-8 expression. IRF-1 and IRF-2 transcription factors hind 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-γ 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. © 2006 Wiley-Liss, Inc.