Structural and functional characterization of the promoter regions of the NFKB2 gene

Abstract

In order to clarify the transcriptional regulation of the NFKB2 gene (lyt-10, NF-kappa Bp100), we have characterized the structure and function of its promoter regions. Based on the nucleotide sequence of cDNA clones and the 5' flanking genomic region of the NFKB2 gene, RT-PCR analysis in a number of human cell lines demonstrated the presence of two alternative noncoding first exons (1a and 1b). Two distinct promoter regions, P1 and P2, were identified upstream of each exon, containing multiple sites of transcription initiation, as shown by RNase protection analysis. Sequence analysis of these regions showed a CAAT box upstream of exon 1a and high G-C content regions within both P1 and P2. Consensus binding sites for transcription factors, including SP1, AP1 and putative NF-kappa B (kappa B sites), were found upstream of each exon. In particular, six kappa B sites were identified, all but one of them capable of binding NF-kappa B complexes in vitro. Transfection in HeLa cells of plasmids containing P1 and P2 sequences linked to a chloramphenicol acetyltransferase reporter gene indicated that both P1 and P2 can act independently as promoters. Co-transfection of NF-kappa B effector plasmids (NF-kappa Bp52 and RelA) with a reporter gene linked to P1 and P2 showed that the NFKB2 promoter regions are regulated by NF-kappa B factors. RelA transactivates the NFKB2 promoter in a dose-dependent manner, whereas NF-kappa Bp52 acts as a repressor, indicating that the NFKB2 gene may be under the control of a negative feedback regulatory circuit.

Transcriptional regulatory effects of lymphoma-associated NFKB2/lyt10 protooncogenes

“…Again, p100 was strongly repressive of p65 activation in a dose dependent manner. p52 NF-kB-2 actually exhibited a similar strong inhibitory e ect on P2-directed gene expression, leading to almost complete repression of P2 promoter activity at higher levels of transfection (similar to the previous data from Lombardi et al, 1995). In contrast, p80HT expression resulted in a maximal reduction of RelA activation by only approximately 2 ± 3-fold, leaving strong persistent residual activation of the P2 promoter by p65 even at the highest levels of p80HT expression.…”

“…In order to characterize the speci®c autoregulatory e ects of di erent NFKB2 gene products, we molecularly cloned the NFKB2 promoter region. We con®rmed the published P1 transcription start site by primer extension analysis, used PCR to subclone a 589 bp segment 5' to the P1 transcription start site and demonstrated that this sequence was su cient to confer full regulation of the NFKB2 promoter in response to phorbol esters (data not shown), as previously reported by others (Liptay et al, 1994;Lombardi et al, 1995). A PCR approach was also used to speci®cally subclone a 624 bp segment of the P2 promoter including the published functional NF-kB sites.…”

“…Regulatory effects of wild-type NF-kB-2 gene products and tumor-associated p80HT expression on NFKB2 and IL-6 kB site promoter activity As the NFKB2 promoter was previously shown to contain several NF-kB binding sites (Liptay et al, 1994;Lombardi et al, 1995), we hypothesized that the NFKB2 promoter could itself represent a target promoter whose expression is altered by the loss of p100 NF-kB-2 and/or the new synthesis of p80HT NFkB-2. The genomic structure and sequence of the NFKB2 gene revealed the presence of two distinct promoter regions (P1 and P2) each containing several functional kB motifs (Liptay et al, 1994;Lombardi et al, 1995).…”

“…The genomic structure and sequence of the NFKB2 gene revealed the presence of two distinct promoter regions (P1 and P2) each containing several functional kB motifs (Liptay et al, 1994;Lombardi et al, 1995). Cotransfection of p52 NF-kB-2 with RelA resulted in a dose-dependent activation of the NFKB2 promoters (Liptay et al, 1994) or biphasic response [activation and then repression with increasing amounts of p52 ].…”

“…A 5 kb HindIII fragment was subcloned (pEV1), and DNA sequence analysis was performed by dideoxynucleotide chain termination method (Sanger et al, 1977). A 586 bp segment representing the core elements of the P1 promoter (Liptay et al, 1994;Lombardi et al, 1995) was ampli®ed by PCR using a 5' oligonucleotide primer beginning at 7586 bp with respect to the major P1 start site and containing a HindIII restriction enzyme site, with the sequence: 5'-CCCAAGCTTTCCCGGAGCCTTGAGGCT-GA-3'; and a 3' oligonucleotide primer extending from the +70 bp position of the P1 promoter, containing and XbaI restriction enzyme site, and having the sequence: 5'-CGTCTAGAGCTAAAGGCCCTCCTCCCTC-3'. The ampli®ed DNA was ligated to the HindIII and XbaI sites upstream of the CAT (chloramphenicol acetyl transferase) reporter gene in the pCAT vector (Promega) to construct P1 CAT.…”