Cirhin up-regulates a canonical NF-kappaB element through strong interaction with Cirip/HIVEP1.
North American Indian childhood cirrhosis (NAIC/CIRH1A) is a severe autosomal recessive intrahepatic cholestasis. All NAIC patients have a homozygous mutation in CIRH1A that changes conserved Arg565 to Trp (R565W) in Cirhin, a nucleolar protein of unknown function. Subcellular localization is unaffected by the mutation. Yeast two-hybrid screening identified Cirip (Cirhin interaction protein) and found that interaction between Cirip and R565W-Cirhin was weakened. Co-immunoprecipitation of the two proteins from nuclear extracts of HeLa cells strongly supports the yeast two hybrid results. Cirip has essentially the same sequence as the C-terminal of HIVEP1, a regulator of a canonical NF-kappaB sequence. Since Cirip has the zinc fingers required for this interaction, we developed an in vitro assay based on this element in mammalian cells to demonstrate functional Cirhin-Cirip interaction. The strong positive effect of Cirip on the NF-kappaB sequence was further increased by both Cirhin and R565W-Cirhin. Importantly, the effect of R565W-Cirhin was weaker than that of the wild type protein. We observed increased levels of Cirhin-Cirip complex in nuclear extracts in the presence of this NF-kappaB sequence. Our hypothesis is that Cirhin is a transcriptional regulatory factor of this NF-kappaB sequence and could be a participant in the regulation of other genes with NF-kappaB responsive elements. Since the activities of genes regulated through NF-kappaB responsive elements are especially important during development, this interaction may be a key to explain the perinatal appearance of NAIC.
Yu
Bin
B
Génétique Médicale, Centre de Recherche CHU Sainte-Justine, Département de Pédiatrie, Université de Montréal, Montréal, QC, Canada.
Mitchell
Grant A
GA
Richter
Andrea
A
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D012261:Ribonucleoproteins; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation / Q000235:genetics / Q000502:physiology
D004268:DNA-Binding Proteins; C063232:HIVEP1 protein, human; D016328:NF-kappa B; D011506:Proteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D014157:Transcription Factors; C470475:UTP4 protein, human