Cirhin up-regulates a canonical NF-κB element through strong interaction with Cirip/HIVEP1

Abstract

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-κB 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-κB 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-κB sequence. Our hypothesis is that Cirhin is a transcriptional regulatory factor of this NF-κB sequence and could be a participant in the regulation of other genes with NF-κB responsive elements. Since the activities of genes regulated through NF-κB responsive elements are especially important during development, this interaction may be a key to explain the perinatal appearance of NAIC.