Promoter identification of CIKS, a novel NF-kappaB activating gene, and regulation of its expression.
We have recently identified a novel gene, named CIKS (Connection to IKK-complex and SAPK), able to activate the transcription factor NF-kappaB, after interaction with the regulatory subunit NEMO/IKKgamma of IKK complex, and the stress-activated protein kinase (SAPK)/JNK. CIKS mRNA is ubiquitously expressed, although its levels differ greatly among different tissues. The aim of this study is to identify and characterize the promoter region of CIKS gene and to analyse the regulation of its expression by different cytokines. The transcription start site of CIKS mRNA was mapped both by primer extension and by a polymerase chain reaction (PCR)-based strategy. The proximal 5'-flanking region of CIKS gene was 'TATA-less', but contained other consensus promoter elements including an initiator (Inr), 'GC' and 'CAAT' boxes. Transfection of luciferase reporter plasmids containing 1.8 kb of the 5'-flanking region increased luciferase activity in epithelial MDCK cells, but not in endothelial HUVEC cells. Deletion analysis identified a sequence from -464 to -220 bp of the 5'-flanking region of CIKS gene essential for basal promoter activity in MDCK cells. Competitive reverse transcriptase-PCR, Northern and Western blot assays showed that different cytokines, such as tumor necrosis factor (TNF)-alpha, Interleukin (IL)-1beta and transforming growth factor (TGF)-beta, dramatically increased CIKS mRNA expression in HeLa cells. We conclude that the proximal 5'-flanking region of CIKS gene contains a functional promoter and binding sites for nuclear proteins leading to its basal transcription. Moreover, we demonstrate that the expression of CIKS is up-regulated by different cytokines.
Pacifico
Francesco
F
Istituto di Endocrinologia ed Oncologia Sperimentale, CNR, Federico II University of Naples, Naples, Italy.
Barone
Ciro
C
Mellone
Stefano
S
Di Jeso
Bruno
B
Consiglio
Eduardo
E
Formisano
Silvestro
S
Vito
Pasquale
P
Leonardi
Antonio
A
D024506:5' Flanking Region / Q000235:genetics; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics*; D002460:Cell Line; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D024363:Transcription Initiation Site; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D007375:Interleukin-1; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C414202:TRAF3IP2 protein, human; D016212:Transforming Growth Factor beta; D047988:Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases