title: Analysis of the defect in IFN-gamma induction of MHC class II genes in G1B cells: identification of a novel and functionally critical leucine-rich motif (62-LYLYLQL-68) in the regulatory factor X 5 transcription factor.
abstract: MHC class II deficiency found in bare lymphocyte syndrome patients results from the absence or dysfunction of MHC class II transcriptional regulators, such as regulatory factor X (RFX) and class II transactivator (CIITA). Understanding the roles of these factors has been greatly facilitated by the study of genetic defects in cell lines of bare lymphocyte syndrome patients, as well as in cell lines that have been generated by chemical mutagenesis in vitro. The latter group includes MHC class II-deficient lines that are no longer responsive to induction by IFN-gamma. Here, we show that the defect in G1B, one such cell line, is attributed to the lack of functional RFX5, the largest subunit of RFX. The RFX5 gene isolated from G1B cells contains two separate single-base pair mutations. One alteration does not exhibit a phenotype, whereas a leucine-to-histidine mutation eliminates DNA-binding and transactivating functions. This mutation lies outside of previously defined functional domains of RFX5 but within an unusual, leucine-rich region (62-LYLYLQL-68). To further investigate the significance of the leucine-rich region, we targeted all neighboring leucine residues for mutagenesis. These mutants were also unable to transactivate a MHC class II reporter gene, confirming that these leucine residues play an essential role in RFX activity and characterize a novel leucine-rich motif.
authors.affiliation: UNC Lineberger Comprehensive Cancer Center, Department of Immunology and Microbiology, School of Dentistry, University of North Carolina, Chapel Hill 27599, USA.
authors.forename: W J
authors.initials: WJ
authors.lastname: Brickey
authors.forename: K L
authors.initials: KL
authors.lastname: Wright
authors.forename: X S
authors.initials: XS
authors.lastname: Zhu
authors.forename: J P
authors.initials: JP
authors.lastname: Ting
mesh_terms: D020816:Amino Acid Motifs / Q000276:immunology; D000595:Amino Acid Sequence; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000276:immunology*; D005802:Genes, MHC Class II / Q000276:immunology*; D005816:Genetic Complementation Test; D006684:HLA-DR Antigens / Q000096:biosynthesis / Q000378:metabolism; D059805:HLA-DR alpha-Chains; D006801:Humans; D007371:Interferon-gamma / Q000502:physiology*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D010641:Phenotype; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D006684:HLA-DR Antigens; D059805:HLA-DR alpha-Chains; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; C093524:RFX5 protein, human; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D007371:Interferon-gamma; D007930:Leucine; D003545:Cysteine