TY - JOUR
T1 - Novel mutations in the RFXANK gene
T2 - RFX complex containing in-vitro-generated RFXANK mutant binds the promoter without transactivating MHC II
AU - Wiszniewski, Wojciech
AU - Fondaneche, Marie Claude
AU - Louise-Plence, Pascale
AU - Prochnicka-Chalufour, Ada
AU - Selz, Françoise
AU - Picard, Cappucine
AU - Deist, Françoise
AU - Eliaou, Jean François
AU - Fischer, Alain
AU - Lisowska-Grospierre, Barbara
N1 - Funding Information:
Acknowledgements We wish to thank Fabienne Mazerolles for performing the phosphorylation studies and for expert advice. We thank Viktor Steimle and Walter Reith for the critical reading of the manuscript. This work was supported in part by the Progrès Programme (INSERM) and KBN grant No. 4 PO5E 11819. W.W. is a PhD student at the Postgraduate School of Molecular Medicine in Warsaw Medical University and the recipient of a Young Scientist Award from the Foundation for Polish Science.
PY - 2003/2/1
Y1 - 2003/2/1
N2 - 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.
AB - 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.
KW - Ankyrin repeat
KW - MHC II
KW - RFXANK
KW - Transcription
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U2 - 10.1007/s00251-002-0521-1
DO - 10.1007/s00251-002-0521-1
M3 - Article
C2 - 12618906
AN - SCOPUS:0037304181
SN - 0093-7711
VL - 54
SP - 747
EP - 755
JO - Immunogenetics
JF - Immunogenetics
IS - 11
ER -