title: Novel mutations in the RFXANK gene: RFX complex containing in-vitro-generated RFXANK mutant binds the promoter without transactivating MHC II.
abstract: MHC class II deficiency is a combined immunodeficiency caused by defects in the four regulatory factors, CIITA, RFXANK, RFX5 and RFXAP, that control MHC II expression at the transcriptional level. The RFXANK gene encodes one subunit of the heterotrimeric RFX complex that is involved in the assembly of several transcription factors on MHC II promoters. Seven different RFXANK mutations have previously been reported in 26 unrelated patients. The most frequent mutation, a 26-bp deletion (752delG-25), has been identified in 21 patients. The other mutations are all nonsense or splice-site mutations, leading to proteins lacking all or part of the RFXANK ankyrin repeat region. We report two novel missense mutations, D121V and R212X, resulting in loss of function of the gene. We investigated the in vivo effects of these mutations and of three other point mutations on the expression of the RFXANK RNA and protein. The number of RFXANK transcripts was severely reduced in all patients except one. The RFXANK protein was barely detected in two cases. In addition, guided by a structural model of RFXANK, we investigated experimental mutants of the C-terminal tyrosine 224. Substitution Y224A, but not Y224F, led to the loss of function of RFXANK. Two null mutants, D121V and Y224A, were tested in protein interaction and DNA binding assays. The D121V mutant was unable to form the RFX complex, indicating that D121 is required for RFXAP binding. The Y224A mutant formed an RFX complex that bound normally to the MHC II promoter, but did not lead to MHC class II expression, whereas Y224F RFXANK retained the wild-type function. This indicates that an aromatic ring, but not the phenyl chain of tyrosine, is necessary at position 224 for normal RFXANK function. Studies on the Y224A mutant suggest that, in addition to the RFX subunits and CIITA, another protein is essential for MHC class II expression. This protein appears to interact with the fourth ankyrin repeat of RFXANK.
authors.affiliation: INSERM U 429, Hopital Necker-Enfants Malades, Paris, France. wojtekw@imid.med.pl
authors.forename: Wojciech
authors.initials: W
authors.lastname: Wiszniewski
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Fondaneche
authors.forename: Pascale
authors.initials: P
authors.lastname: Louise-Plence
authors.forename: Ada
authors.initials: A
authors.lastname: Prochnicka-Chalufour
authors.forename: Françoise
authors.initials: F
authors.lastname: Selz
authors.forename: Cappucine
authors.initials: C
authors.lastname: Picard
authors.forename: Françoise
authors.initials: F
authors.lastname: Le Deist
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Eliaou
authors.forename: Alain
authors.initials: A
authors.lastname: Fischer
authors.forename: Barbara
authors.initials: B
authors.lastname: Lisowska-Grospierre
mesh_terms: D019943:Amino Acid Substitution; D017089:Ankyrin Repeat; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002461:Cell Line, Transformed; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005802:Genes, MHC Class II; D006801:Humans; D008958:Models, Molecular; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D017384:Sequence Deletion; D016511:Severe Combined Immunodeficiency / Q000235:genetics / Q000276:immunology; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation
chemical_list: D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D021122:Protein Subunits; C117892:RFXANK protein, human; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors