Nucleic Acids Research, 1995, Vol. 23, No. 12 2328-2336
© 1995
MOLECULAR BIOLOGY |
Structural and functional characterization of the promoter regions of the NFKB2 gene
Laboratories di Ematologia Sperimentale e Genetica Molecolare, Servizio di Ematologia, Istituto.di Scienze Mediche, Università di Milano Ospedale Maggiore IRCCS, Milano 20122, Italy 1Dipartimento di Genetica e Biologia dei Microorganismi, Universita di Milano Milano 20122, Italy
* To whom correspondence should be addressed
Received December 20, 1994. Revised April 12, 1995. Accepted May 2, 1995.
In order to clarify the transcriptional regulation of the NFKB2 gene (lyt-10, NF-
Bp100), we have characterized the structure and function of its promoter regions. Based on the nucleotide sequence of cDNA clones and the 5' flanking genomlc region of the NFKB2 gene, RT-PCR analysis in a number of human cell lines demonstrated the presence of two alternative non-coding first exons (la and Ib). 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 la and high G-C content regions within both PI and P2. Consensus binding sites for transcription factors, Including SP1, API and putative NF-B (B sites), were found upstream of each exon. In particular, six B sites were identified, all but one of them capable of binding NF-B complexes In vitro. Transfectlon in HeLa cells of plasmlds containing PI and P2 sequences linked to a chloramphenicol acetyl-transferase reporter gene indicated that both PI and P2 can act independently as promoters. Co-transfection of NF-B effector plasmids (NF-Bd52 and RelA) with a reporter gene linked to PI and P2 showed that the NFKB2 promoter regions are regulated by NF-B factors. RelA transactlvates the NFKB2 promoter in a dose-dependent manner, whereas NF-kBp52 acts as a repressor, indicating that the NFKB2 gene may be under the control of a negative feedback regulatory circuit
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