Alternative titles; symbols
HGNC Approved Gene Symbol: RFXAP
Cytogenetic location: 13q13.3 Genomic coordinates (GRCh38): 13:36,819,222-36,829,104 (from NCBI)
| Location | Phenotype |
Phenotype MIM number |
Inheritance |
Phenotype mapping key |
|---|---|---|---|---|
| 13q13.3 | Bare lymphocyte syndrome, type II, complementation group D | 209920 | Autosomal recessive | 3 |
Major histocompatibility complex (MHC) class II molecules are heterodimeric transmembrane glycoproteins expressed on specialized cells of the immune system. There are 3 MHC class II isotypes, HLA-DR (see 142860), HLA-DP (see 142880), and HLA-DQ (see 146880), encoded by distinct pairs of alpha- and beta-chain genes. RFXAP, RFX5 (601863), and RFXANK (603200) are subunits of the trimeric RFX DNA-binding complex that binds specifically to the X box present in all MHC class II promoters to regulate transcription (summary by Peretti et al., 2001).
Durand et al. (1997) cloned RFXAP, which encodes the 36-kD subunit of RFX, from a human B-cell cDNA library. The deduced protein contains 272 amino acids and has a calculated molecular mass of 30 kD. In vitro translation experiments confirmed that RFXAP had an apparent molecular mass of 36 kD by SDS-PAGE. RFXAP lacks an RFX DNA-binding motif, but it contains an acidic residue-rich domain and a glutamine-rich domain, which are reminiscent of transcriptional activation domains, and a basic residue-rich domain containing a bipartite nuclear localization signal (NLS).
Peretti et al. (2001) cloned mouse Rfxap, which encodes a 231-amino acid protein that shares 65% overall amino acid identity with the human protein. Conservation is highest in the C-terminal region containing the acidic segment, the glutamine-rich segment, and the basic segment with the NLS.
Mach (1998) stated that the RFXAP gene maps to chromosome 13q13-q14.
Gross (2020) mapped the RFXAP gene to chromosome 13q13.3 based on an alignment of the RFXAP sequence (GenBank BC026088) with the genomic sequence (GRCh38).
Using EMSA, Durand et al. (1997) confirmed that RFXAP was a subunit of the human RFX complex. Yeast 2-hybrid experiments showed that RFXAP interacted with RFX5. In transfection experiments, RFXAP restored expression of all MHC class II genes to normal levels in the 6.1.6 cell line, representing MHC class II deficiency complementation group D (see 209920).
Villard et al. (1997) reported that in all the patients with bona fide MHC class II deficiency that they had studied, the mutations responsible belonged to 1 of the 4 known complementation groups: A, B, C, or D. A diagram (their Figure 4) illustrated their concept of the relationship between the MHC class II promoter and the transcription factors affected in complementation groups A, C, and D. The 3 regulatory genes affected in MHC class II deficiency, CIITA (MHC2TA; 600005), RFX5, and RFXAP, are not only absolutely essential, with no bypass or alternative pathways, but are also highly specific for MHC class II genes. These properties are unusual for transcriptional regulatory factors, which generally control multiple genes and thus have pleiotropic effects.
Nekrep et al. (2000) demonstrated a direct interaction between the C terminus of RFXAP and RFXANK; mutant RFXAP or RFXANK proteins failed to bind. The authors found that RFX5 bound only to the RFXANK-RFXAP scaffold and not to either protein alone. However, neither the scaffold nor RFX5 alone could bind DNA. Nekrep et al. (2000) concluded that the binding of the RFXANK-RFXAP scaffold to RFX5 leads to a conformational change in the latter that exposes the DNA-binding domain of RFX5. The DNA-binding domain of RFX5 anchors the RFX complex to MHC class II X and S promoter boxes. Another part of the RFX5 protein interacts with MHC2TA (RFX1). The authors pointed out that mutation of either protein in complementation group B or group D of BLS patients prevents its binding to the other protein, explaining why MHC class II promoters are bare in the bare lymphocyte syndrome.
By expression of mouse Rfxap in human 6.1.6 cells lacking functional RFXAP, Peretti et al. (2001) found that mouse Rfxap could substitute functionally for the human protein and associate with the other human RFX subunits to form a complex capable of binding to the X box. The authors found that over 80% of mouse Rfxap was dispensable for activation of HLA-DR expression, and that only a short C-terminal segment of Rfxap was sufficient for binding of the RFX complex and activation of HLA-DR expression in 6.1.6 cells and RFXAP-deficient fibroblasts. Deletion analyses showed that the putative NLS of Rfxap was not essential for Rfxap function, whereas the C-terminal glutamine-rich region was essential. Optimal expression of HLA-DQ and HLA-DP required a larger C-terminal segment of Rfxap compared with HLA-DR. The differential Rfxap C-terminal domain requirement for expression of the 3 MHC II isotypes was due to differential dependence on this domain for promoter occupation and recruitment of the transcription factor CIITA.
Masternak et al. (2003) pointed out that the promoter-proximal S-Y module can direct cell-type-specific and gamma-interferon (IFNG; 147570)-induced expression of transiently expressed transfected genes, but it is not sufficient to reproduce the normal pattern of MHC class II expression in the context of chromatin. There is, in addition, a distal region that also functions as a regulatory element known as the locus control region (LCR), which contains clusters of Dnase I hypersensitive sites in MHC class II-positive cells and is required to express class II genes in a cell-type-specific pattern. Masternak et al. (2003) showed that RFXAP and CIITA bound to the LCR and induced long-range histone acetylation from the promoter to as far as 16 kb upstream, RNA polymerase II recruitment, and the synthesis of extragenic transcripts within the LCR.
Using RFX-deficient human 5637 cells, Niesen et al. (2005) found that DNA methylation repressed transcriptional activity of the HLA-DRA promoter. Exogenous expression analysis showed that RFX increased transcriptional activity by binding to the methylated HLA-DRA promoter and enhancing recruitment of CIITA. RFX did not facilitate activation of nonmethylated HLA-DRA, nor did it mediate demethylation. In RFX-deficient cells, the authors identified a methylated CpG immediately adjacent to the canonical RFX binding site of the HLA-DRA promoter sequence. In vivo analysis of 5637 cells confirmed that RFX bound preferentially to methylated HLA-DRA promoter sequences.
Long et al. (2006) found a high degree of conservation in the RFXAP C terminus extending from human to fish. Mutation analysis revealed that the glutamine-rich region of RFXAP was required for its function, and RFXAP displayed differential MHC II isotype sensitivity, as a longer C-terminal fragment was necessary for HLA-DP and HLA-DQ expression compared with HLA-DR. In addition, the glutamine residues could be replaced with alanine or glutamic acid without affecting RFXAP activity. Mutation of 2 hydrophobic residues in the region caused reduced activity, indicating their importance for RFXAP function. Moreover, alanine substitution at the conserved putative phosphorylation sites N-terminal to the glutamine-rich region resulted in RFXAP proteins with reduced or absent activity. Quantitative RT-PCR on transfected 6.1.6 cells revealed different transcription efficiencies among the 3 MHC-II isotypes, and these differences were exacerbated by RFXAP mutants. RFXAP mutations affected multiple activities, including the ability of RFXAP to associate with the other RFX subunits, bind DNA, translocate to the nucleus, and recruit CIITA to MHC II promoters in vivo.
By analyzing HeLa cell nuclear extracts, Zhang et al. (2008) identified RFX as a 5-prime-directed mismatch excision stimulatory factor. Both purified native RFX and recombinant RFX strongly stimulated the excision reaction, whereas minimal excision occurred in the absence of native or recombinant RFX. Knockdown of RFX inhibited mismatch-provoked excision in HeLa cells, and addition of recombinant RFX to RFX5-depleted extract reversed the effect. Further examination of the subclones of the knockdown cells revealed DNA instability, as expected in cells with defects in mismatch repair.
In the 6.1.6 cell line, representing MHC class II deficiency complementation group D (see 209920), Durand et al. (1997) identified a frameshift mutation in each allele of the RFXAP gene (see 601861.0001). The authors also found that RFXAP fully complemented a cell line from an MHC class II deficiency patient of complementation group D, and they found that the patient had a homozygous frameshift mutation within the RFXAP gene (601861.0003). These results provided unequivocal evidence that MHC class II deficiency complementation group D is caused by mutations in RFXAP.
The identification of transacting factors controlling MHC class II gene transcription via the proximal enhancer of the promoter was greatly facilitated by a genetic approach, namely, the analysis of cell lines characterized by regulatory defects abolishing transcription of these genes (Mach et al., 1996). Most of these MHC class II regulatory mutants were cell lines derived from patients suffering from MHC class II deficiency, also referred to as bare lymphocyte syndrome type II (BLS; 209920), a rare autosomal recessive disorder characterized by lack of constitutive and inducible MHC class II expression in all cell types and tissues. The genetic lesions responsible for this lack of expression occurred not in MHC class II genes themselves, but in transacting regulatory genes required for their transcription (Mach et al., 1996). Durand et al. (1997) noted that different complementation groups had been identified by means of somatic cell fusion experiments, reflecting the existence of different essential MHC class II regulatory genes. The molecular defect in complementation group A lies in the gene encoding CIITA (RFX1; 600005). CIITA is a non-DNA-binding transactivator that functions as a molecular switch controlling both cell-type specific and inducible MHC class II gene transcription. In contrast, the defects in complementation groups B, C, and D all lead to a deficiency in RFX, a nuclear protein complex that binds to the X box of MHC class II promoters. The lack of RFX binding activity in complementation group C was shown by Steimle et al. (1995) to result from mutations in the gene encoding the 75-kD subunit of RFX, which they called RFX5 because it was the fifth member of the RFX family of DNA-binding proteins. These proteins share a novel and highly characteristic DNA-binding domain called the RFX motif.
Schwartz (1997) pointed out that bare lymphocyte syndrome was the first genetic disease in which the mutation was traced to a transcription factor. Other examples now include combined pituitary hormone deficiency, caused by mutation of the PIT1 gene (173110), deafness due to mutation in the X-linked POU3F4 gene (300039), and cleidocranial dysplasia due to a mutant form of the transcription factor CBFA1 (600211). In transcription-mutant forms of beta-thalassemia, the fault lies in the promoter, not the transcription factors. Somatic mutations of transcription factor genes are major players in carcinogenesis. Somatic mutations of p53 (191170) are the most frequent genetic abnormality in human cancer.
In cell line 6.1.6, representing bare lymphocyte syndrome type II, complementation group D (209920), Durand et al. (1997) found a frameshift mutation in each allele of the RFXAP gene. One allele contained an additional G inserted into a run of 6 G residues (nucleotides 413 to 418). The other allele contained an additional G inserted into a run of 4 G residues (nucleotides 505 to 508; 601861.0002). The resulting frameshifts led to the use of premature out-of-frame stop codons situated at nucleotide 433 in the first allele and nucleotide 638 in the second. The cell line tested was generated by mutagenesis with ICR-191, a frameshift mutagen that leads preferentially to the introduction of an additional G:C basepair in runs of G:C basepairs.
See 601861.0001 and Durand et al. (1997).
Durand et al. (1997) studied cell lines from several bare lymphocyte syndrome type II (209920) patients that had not yet been classified in the known complementation groups A, B, and C. In an EBV-transformed B-cell line established from one of the first MHC class II deficiency patients described (Touraine et al., 1978; Touraine and Betuel, 1981), Durand et al. (1997) found that transfection with RFXAP cDNA restored a normal MHC class II positive phenotype. Cell surface expression of the 3 MHC class II isotypes was restored to wildtype levels, indicating that the alpha and beta chain genes for HLA-DR, -DP, and -DQ were all reactivated coordinately. Mutation analysis showed that the cell line, from a patient designated DA, was homozygous for a deletion of a G residue at nucleotide 484. The resulting frameshift led to the use of a premature out-of-frame stop codon at nucleotide 525, and thus to the synthesis of a severely truncated and inactive protein of only 136 amino acids. The parents of DA were not available for study, but they were stated to have been first cousins.
Villard et al. (1997) found this same mutation in a patient of complementation group D.
In a Turkish patient with bare lymphocyte syndrome type II (209920), Villard et al. (1997) demonstrated a C-to-T transition at nucleotide 279 that converted a glutamine codon (CAG) to a premature stop codon (TAG). This mutation led to a severely truncated protein of only 52 amino acids. Direct sequencing of a genomic PCR fragment demonstrated that the patient was homozygous for the mutated allele.
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Gross, M. B. Personal Communication. Baltimore, Md. 3/30/2020.
Long, A. B., Ferguson, A. M., Majumder, P., Nagarajan, U. M., Boss, J. M. Conserved residues of the bare lymphocyte syndrome transcription factor RFXAP determine coordinate MHC class II expression. Molec. Immun. 43: 395-409, 2006. [PubMed: 16337482] [Full Text: https://doi.org/10.1016/j.molimm.2005.03.008]
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Mach, B. Personal Communication. Geneva, Switzerland 10/28/1998.
Masternak, K., Peyraud, N., Krawczyk, M., Barras, E., Reith, W. Chromatin remodeling and extragenic transcription at the MHC class II locus control region. Nature Immun. 4: 132-137, 2003. [PubMed: 12524537] [Full Text: https://doi.org/10.1038/ni883]
Nekrep, N., Jabrane-Ferrat, N., Peterlin, B. M. Mutations in the bare lymphocyte syndrome define critical steps in the assembly of the regulatory factor X complex. Molec. Cell Biol. 20: 4455-4461, 2000. [PubMed: 10825209] [Full Text: https://doi.org/10.1128/MCB.20.12.4455-4461.2000]
Niesen, M. I., Osborne, A. R., Yang, H., Rastogi, S., Chellappan, S., Cheng, J. Q., Boss, J. M., Blanck, G. Activation of a methylated promoter mediated by a sequence-specific DNA-binding protein, RFX. J. Biol. Chem. 280: 38914-38922, 2005. [PubMed: 16166088] [Full Text: https://doi.org/10.1074/jbc.M504633200]
Peretti, M., Villard, J., Barras, E., Zufferey, M., Reith, W. Expression of the three human major histocompatibility complex class II isotypes exhibits a differential dependence on the transcription factor RFXAP. Molec. Cell. Biol. 21: 5699-5709, 2001. [PubMed: 11486010] [Full Text: https://doi.org/10.1128/MCB.21.17.5699-5709.2001]
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Zhang, Y., Yuan, F., Wang, D., Gu, L., Li, G.-M. Identification of regulatory factor X as a novel mismatch repair stimulatory factor. J. Biol. Chem. 283: 12730-12735, 2008. [PubMed: 18319249] [Full Text: https://doi.org/10.1074/jbc.M800460200]