Proc. Natl. Acad. Sci. USA Vol 86, pp. 4200-4204, June 1989 Immunology Cloning of the major histocompatibility complex class II promoter binding protein affected in a hereditary defect in class II gene regulation (primary immunodeficiency/DNA-binding proteins/major histocompatibility complex class H X box/Agtll expression library/ DNA-binding site screening) W. REITH, E. BARRAS, S. SATOLA, M. KOBR, D. REINHART, C. HERRERO SANCHEZ, AND B. MACH Department of Microbiology, University of Geneva Medical School, Geneva, Switzerland Communicated by Cesar Milstein, February 6, 1989 (received for review November 7, 1988)

ABSTRACT The regulation of major histocompatibility promoter of the HLA-DR a-chain gene (ref. 14; M.K., W.R., complex class II gene expression is directly involved in the C.H.S., and B.M., unpublished results). One ofthese factors, control of normal and abnormal immune responses. In hu- RF-X, binds to a sequence called the X box, which is present mans, HLA-DR, -DQ, and -DP class II heterodimers are in all human and mouse class II promoters (15, 16). Inter- encoded by a family of a- and fl-chain genes clustered in the estingly, we have shown that RF-X is defective in class major histocompatibility complex. Their expression is devel- II-deficient combined immunodeficiency (CID), a hereditary opmentally controlled and normally restricted to certain cell disease characterized by a regulatory defect leading to an types. This control is mediated by cis-acting sequences in class absence of class II gene expression in all tissues (14, 17, 18). II promoters and by trans-acting regulatory factors. Several It is therefore likely that RF-X is an essential regulatory nuclear proteins bind to class II promoter sequences. In a form factor for the control of normal class II gene expression. In of hereditary immunodeficiency characterized by a defect in a this study, we have used an expression screening procedure trans-acting regulatory factor controlling class II gene tran- to isolate an RF-X cDNA clone. The recombinant protein scription, we have observed that one of these nuclear factors expressed in Escherichia coli has the DNA-binding proper- (RF-X) does not bind to its target sequence (the class H X box). ties ofnatural RF-X and is distinct from other proteins known A cDNA encoding RF-X was isolated by screening a phage to bind in the vicinity of the class II X box. expression library with an X-box binding-site probe. The recombinant protein has the binding specificity of RF-X, including a characteristic gradient of affinity for the X boxes of MATERIALS AND METHODS HLA-DR, -DP, and -DQ promoters. RF-X mRNA is present in Cell Lines and DNAs. Origin and maintenance of cell lines the regulatory mutants, indicating a defect in the synthesis of and preparation of double-stranded oligonucleotides as well a functional form of the RF-X protein. as plasmids used to prepare the BstNI-Hinfl restriction fragment ofthe DRA promoter have been described (14). The Class II major histocompatibility complex antigens are het- X1 and X3 oligonucleotides contain sequences -144 to -70 erodimeric transmembrane glycoproteins. Their expression and -124 to -70, respectively, ofthe DRA promoter. The Xr at the surface of antigen-presenting cells is essential for the oligonucleotide is identical to X3 except that the X box recognition of foreign antigen by the T-cell receptor (1, 2). (CCCCTAGCAACAGATG) is replaced with a random se- T-cell activation and antigen presentation depend not only on quence (ATGTGTCTCGAACGCA). The HLA-DQA and the structural specificity of the highly polymorphic class II HLA-DPA X box oligonucleotides are identical to X3 with molecules (1, 2), but also on the level of expression of class respect to length and position of the X box, but cover II antigens on individual cells (3). Regulation ofexpression of sequences -173 to -119 and -197 to -143 of the HLA-DQA class II genes is therefore an important aspect of the control and HLA-DPA promoters, respectively. of the immune response. Construction and Screening of the cDNA Library. Total In humans, the genes encoding the a and f3 chains of the RNA was extracted from frozen cell pellets by using guanid- HLA-DP, HLA-DQ, and HLA-DR class II molecules are ium isothiocyanate (19), and poly(A)+ RNA was purified by clustered in the D region of the major histocompatibility oligo(dT)-cellulose chromatography. Double-stranded complex on chromosome 6 (4). These genes are subjected to cDNA was synthesized (20) and cloned in Agtll (21) by using tight and complex regulatory controls (4-7). Their expression EcoRI linkers. Plating, induction, transfer to nitrocellulose is generally coordinated and restricted primarily to cells of filters, and screening with 32P-labeled probes were done as the immune system such as B lymphocytes, activated T described (22). To prepare the X1 probe used during the lymphocytes, macrophages, and dendritic cells. Within the primary screening, the X1 oligonucleotide was catenated by B-cell lineage, class II expression is developmentally con- ligation to an average size of 10 and labeled by nick- trolled. Finally, in certain class TI-negative cells, expression translation. To prepare the nonspecific pBR322 probe, a can be induced by stimulation with lymphokines such as 75-base-pair HinfI fragment of pBR322 was treated with interferon y or interleukin 4. Expression appears to be Klenow polymerase to generate blunt ends and then was controlled primarily at the level of transcription. catenated and labeled as described for the X1 probe. X3 and Progress has recently been made in the identification of Xr probes were prepared by labeling the respective oligonu- cis-acting sequences in class II promoters (8-11) and of cleotides with [y-32P]ATP and T4 polynucleotide kinase and nuclear factors interacting with these sequences (8, 11-14). subsequent catenation by ligation to an average size of 10. We have identified five nuclear factors that bind to the Protein Extracts. Preparation of nuclear extracts (23), isolation and induction of Agtll and A9 lysogens (21), and preparation of bacterial extracts (24) were done as described. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: CID, combined immunodeficiency.

Downloaded by guest on September 24, 2021 4200 Immunology: Reith et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4201 Gel Retardation Assays. Probes used for binding were a labeled with [y-32P]ATP and T4 polynucleotide kinase. Bind- ing of 0.2-0.5 ng of end-labeled DNA with 10 of protein 1 pAg m EExON extract was performed for 30 min at 0C in 20 p.1 of 12% Z glycerol/12 mM Hepes, pH 7.9/60 mM KCI/5 mM MgCl2/ -10, T 0.12 mM EDTA/0.3 mM phenylmethylsulfonyl fluoride/0.3 - 108 -9 5 - 74 -6 3 - 52 -4 5 mM dithiothreitol/50 tkg of poly(dI-dC)-poly(dI-dC) (Phar- Xi macia) per ml/25 pAg of sonicated denatured E. coli DNA per ------i-70 ml. After binding, gel electrophoresis was done as described X3 (14). -124: .-70 Methylation Interference Assay. The X1 oligonucleotide was subcloned into the Sma I site of pUC12, cut out with EcoRI (Bst N I) (H inf I) and HindIII, and labeled at the EcoRI site with [y-32P]ATP and T4 polynucleotide kinase for the coding strand or [a-32P]dATP and Klenow polymerase for the noncoding strand. Methyla- b c tion of probe, binding with a protein extract from clone A9, purification of free and bound DNA, and PAGE were per- H 0 R N Ra Ro N Ra Ro formed as described (14). Filter Hybridization and RNase Protection. Analysis of poly(A)+ RNA by filter hybridization was done as described (24). Probes used for filter hybridizations were labeled with RF-Xw- O U OBP4 [a-32P]dCTP by random-primed synthesis. For RNase pro- RF-Y, tection experiments, the cDNA insert ofA9 was subcloned in a Bluescript vector (Stratagene). The plasmid was digested with Fnu4HI and transcribed with T3 RNA polymerase in the presence of [a-32P]UTP. This generates a 139-nucleotide probe homologous to 71 nucleotides located at the 3' end of the mRNA corresponding to A9. Hybridization of the probe with poly(A)+ RNA, digestion with RNase A and T1, and i. PAGE were done as described (25). lot 11 F mm-- Imi F M- RESULTS FIG. 1. CID B-cell lines are deficient in RF-X. (a) Nuclear CID Cells Are Defective in RF-X. The promoter of the factors binding to the DRA promoter in B lymphocytes include HLA-DRA gene contains several conserved sequence motifs; factors specific to the X box (RF-X), Y box (RF-Y), and octamer a TATA box at position -28 to -24, an octamer motif at motif (OBP). DNA fragments used in this study are indicated below position -52 to -45, and two sequence elements called the the map. T, TATA box. (b) Binding of RF-X to oligonucleotide X1 was analyzed by a gel retardation assay using nuclear extracts from class II X and Y boxes at positions -108 to -95 and -74 to normal B-cell lines HHK (H), QBL (Q), and Raji (R) and CID B-cell -63, respectively (Fig. la). The X and Y boxes are conserved lines N, Ra, and Ro. (c) Gel retardation assay showing normal in all murine and human class II promoters (15, 16) and have binding ofRF-Y and OBP to the BstNI-HinfI fragment in CID B-cell been shown to be cis-acting elements involved in the regu- extracts. Positions of free (F) and bound DNA are indicated. lation of class II gene expression (8-11). We have recently identified in B-cell lines five distinct nuclear factors that bind other class II promoters, they can be shown to bind normally to the DRA promoter. These include a factor binding to the in the CID extracts (data not shown). Thus, extracts from X box (RF-X), a factor binding to the Y box (RF-Y), and a CID B lymphocytes are specifically deficient in RF-X. factor binding to the octamer sequence (OBP) (Fig. la; ref. Cloning of A9, a cDNA Encoding RF-X. A technique 14). In addition, we have detected factors that bind weakly to allowing the direct cloning of cDNA clones encoding se- the spacer region between the X and Y boxes (NF-S) and to quence-specific DNA-binding proteins has recently been the W box (NF-W) (13), which is situated 5' of the X box developed (22, 24). The method involves screening a Agtll (M.K., W.R., C.H.S., and B.M., unpublished results). expression library with a 32P-labeled double-stranded DNA RF-X is of particular interest for the regulation of class II probe containing the binding site for the protein of interest. gene transcription because it is absent or defective in inher- The sensitivity ofthe original method (24) has been improved ited CID (14), a disease characterized by a defect in a by two modifications, catenation of the probe to increase trans-acting regulatory factor controlling class II gene binding stability and processing the filters through a guani- expression (17, 18). An oligonucleotide containing the X box dine hydrochloride denaturation/renaturation step prior to of the DRA promoter (Xl, Fig. la) was incubated with screening (22). Treatment of a B-cell nuclear extract with normal or CID B-lymphocyte extracts, and protein-DNA guanidine hydrochloride has shown that RF-X is resistant to complexes were analyzed by nondenaturing PAGE. This gel this denaturation/renaturation cycle (data not shown), a retardation assay shows that, in extracts from normal B-cell property shared by a number of known eukaryotic DNA- lines, RF-X is the only factor that forms a stable complex with binding proteins (22). the X1 oligonucleotide (Fig. lb). In contrast, binding ofRF-X A cDNA library containing 4 x 106 clones was constructed is not detectable in extracts from B-cell lines established from in Agtll with mRNA from a human B-cell line. Seven three unrelated CID patients. In fact, no stable protein-DNA hundred fifty thousand recombinants were screened with a complexes are formed with the X1 oligonucleotide in these probe consisting of catenated and nick-translated X1 oligo- mutants (Fig. lb), although in the same CID extracts both nucleotide. Of 20 phages selected for a second round of RF-Y and OBP bind normally to the BstNI-HinfI fragment screening, only 4 were positive with the X1 probe and containing the Y box and octamer motif (Fig. lc). NF-S and negative with a nonspecific pBR322 probe. This type of NF-W have only low affinity for the DRA promoter and do screening procedure is prone to artifacts such as the binding not bind stably to the Xl oligonucleotide or BstNI-HinfI of proteins to single-stranded DNA. In addition, we know fragment under the conditions used here. However, by using that at least one protein distinct from RF-X (NF-S) binds Downloaded by guest on September 24, 2021 4202 Immunology: Reith et al. Proc. Natl. Acad. Sci. USA 86 (1989) weakly to a sequence immediately 3' of the DRA X box. To methylation of four guanosine residues within and just eliminate such artifacts, a third round of screening was upstream of the X box interferes with binding of the A9- performed with two probes, a specific 54-base-pair-long encoded protein. Methylation of one guanosine residue oligonucleotide centered on the X box (X3, Fig. la) and a appears to enhance binding. This methylation interference nonspecific oligonucleotide (Xr) identical to X3 except that a pattern is strikingly similar to the one observed with native random 16-base-pair sequence replaces the X box. One ofthe RF-X (Fig. 3b) (14). All five contact points, including the four recombinant phages, A9, was strongly positive with X3 guanosine residue at which methylation appears to enhance and totally negative with Xr (Fig. 2a, filters 1 and 2). Clone binding, are shared by both RF-X and the recombinant A9 therefore encodes a protein that binds specifically to the protein. In the case of RF-X, methylation of two additional X box. Interestingly, the guanidine hydrochloride dena- guanosine residues interferes weakly with binding (Fig. 3b) turation/renaturation step used during the screening proce- (14). This minor difference may result from the fact that the dure proved to be essential for identification of A9 because recombinant protein is incomplete and synthesized as a when it was omitted the clone was no longer detectable with fusion protein (data not shown). the X3 probe (Fig. 2a, filter 3). Native RF-X can bind to the X boxes of all class II genes Characterization of the Recombinant Protein Encoded by that have been analyzed. These include the human HLA- A9. Lysogens of Agtll and A9 were isolated and induced to DRA, -DRBI, -DRB3, -DPA, and -DQA genes, the mouse Ea express their respective f-galactosidase genes. Protein ex- and Ej3 genes, and the human class II-associated invariant tracts were prepared from the lysogens and from uninfected chain gene (ref. 14; M.K., W.R., C.H.S., and B.M., unpub- host bacteria. The extracts were then analyzed for X box lished results). However, RF-X has strikingly different af- binding activity by means of a gel retardation assay (Fig. 2b). finities for the X boxes of HLA-DRA, -DPA, and -DQA With the A9 extract, one major protein-DNA complex was genes: it binds most efficiently to the X box of DRA, only formed with oligonucleotide Xl (Fig. 2b, lane 4). In addition very poorly to that of DQA, and with an intermediate affinity to this major band, three weaker bands were detected. All of to that ofDPA (M.K., W.R., C.H.S., and B.M., unpublished these bands are specific to the A9 extract since they were not results). We therefore analyzed the relative binding affinity of observed with extracts from either a Agtll lysogen or the host the recombinant protein for the X boxes of these three genes bacterial strain (Fig. 2b, lanes 2 and 3). As expected, the by competition experiments. The A9 extract and a B-cell A9-encoded protein also binds to the smaller X3 oligonucle- nuclear extract were incubated with the 32P-labeled X1 otide but not to the control Xr probe (Fig. 2b, lanes 5 and 6), oligonucleotide in the presence of different amounts of confirming that A9 encodes a true X box binding protein. The unlabeled DRA, DQA, or DPA competitor oligonucleotides, major protein-DNA complex has a slightly greater electro- and the extent of competition was analyzed by PAGE. RF-X phoretic mobility than the one formed with natural RF-X and the protein encoded by A9 have the same relative binding (Fig. 2b, lane 1). The presence of several bands may be due affinities for the three X boxes (Fig. 2c). Binding of both is to different conformations, modifications, or degradation competed out efficiently by DRA even at a low molar excess products of the same protein. of competitor, very poorly by DQA even at a high molar The binding site and the contact points of the recombinant excess of competitor, and at an intermediate efficiency by protein were further analyzed by methylation interference DPA. Competition with Xr has no effect on binding of either analysis. The Xl oligonucleotide was end-labeled on the RF-X or the recombinant protein. coding or noncoding strand, partially methylated with di- RF-X mRNA Is Rare and Is Expressed Normally in the methyl sulfate, and incubated with the A9 lysogen extract. Regulatory Mutants. Since no binding of RF-X is observed in After gel purification offree and complexed fragments, DNA CID B cells, we investigated the presence of RF-X mRNA in was cleaved at methylated guanosine residues and analyzed these mutants. Poly(A)+ RNA from a normal and two CID by sequencing PAGE (Fig. 3a). Comparison of the cleavage B-cell lines was hybridized with the 3.8-kilobase insert of A9 profiles obtained with free and bound DNA reveals that in a filter hybridization assay (Fig. 4a). A 4.1-kilobase mRNA a b 1 I3 1 2 3 4 5 6

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FIG. 2. Cloning and characterization ofA9, a cDNA clone encoding RF-X. (a) Third round of screening for clone A9. A9 phages were plated, induced to express the P-galactosidase fusion protein, and transferred to nitrocellulose filters. Filters were screened with 32P-labeled and catenated X3 (filters 1 and 3) or Xr oligonucleotides (filter 2). The guanidine-HCI denaturation/renaturation step was omitted for filter 3. (b) Gel retardation assay using oligonucleotides X1 (lanes 1-4), X3 (lane 6), or Xr (lane 5) and extracts from the B-cell line Mann (lane 1), Y1089 host bacteria (lane 2), a Agtll lysogen (lane 3), or a lysogen from clone A9 (lanes 4-6). Positions offree (F) and bound DNA are indicated. (c) Relative affinities of RF-X and the A9-encoded protein for the X boxes of DRA, DQA, and DPA promoters. Binding to the 32P-labeled X1 oligonucleotide was done in the absence of competitor (lane 1) or in the presence of a 20-, 100-, or 200-fold molar excess of unlabeled oligonucleotides DRA-X3 (lanes 2-4), DRA-Xr (lanes 5-7), DQA-X (lanes 8-10), or DPA-X (lanes 11-13). Downloaded by guest on September 24, 2021 Immunology: Reith et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4203 a a b C NC M 1 2 3 4 5 M IG -- G 1 2 3 + + A F B FA a B Z- U ;R. ;I'- -we - 4mm I. 28S 184- 30

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FIG. 4. RF-X mRNA is normal in CID B cells. (a) RNA filter hybridization. Five micrograms of poly(A)+ RNA from the normal b B-cell line Mann (lane 1) and CID B-cell lines N (lane 2) and Ro (lane 3) was fractionated by gel electrophoresis, transferred to a nitrocel- RF-X lulose filter, and hybridized with the 3.8-kilobase cDNA insert of GAAGG IGGATCITIcACI GACr clone A9. A 4.1-kilobase mRNA is detected in all three cell lines 11 t I I following a 10-day exposure. The probe cross-hybridizes weakly with 28S and 18S ribosomal RNA. (b) RNase protection experiment. The A9 cDNA insert was subcloned in a Bluescript vector (Strata- gene) and a 139-nucleotide transcript containing 71 nucleotides SAGGtG9SrCAC IGCAr homologous to the 3' end of RF-X mRNA was synthesized. Protec- tion of this probe against RNase A and T1 was analyzed by denaturing PAGE. Lanes: M, size markers (pBR322 digested with FIG. 3. Analysis of the contact points between the X box and the Hae III); 1, undigested probe; 2, probe digested in the absence of A9-encoded protein. (a) Methylation interference experiment done RNA; 3, probe protected by hybridization with 2.5 Ag of poly(A)+ with the X1 oligonucleotide end-labeled on the coding (C) or non- RNA from the normal B-cell line Mann; 4, CID B-cell line N; 5, CID coding (NC) strand and a A9 lysogen extract. Lanes show G+A B-cell line Ro. Sizes of selected size markers, undigested probe, and sequence ladders (G+A) and the cleavage profiles offree (F) or bound protected probe are indicated. Exposure time was 10 days. (B) DNA. Sequence of the X box region and contact points are indicated along the sides. (b) Comparison of the methylation interfer- fibroblasts contain RF-X with normal binding properties (I. ence profiles obtained for RF-X (14) and the A9-encoded protein. Amaldi, W.R., and B.M., unpublished results). RF-X may Guanosine residues at which methylation interferes strongly or weakly thus require activation or additional cofactors when cells are with binding are indicated by large or small arrow heads, respectively. induced to express class II genes. The arrow indicates a guanosine residue at which methylation en- hances binding. Four lines of evidence indicate that clone A9 encodes RF-X. (i) The recombinant protein binds specifically to the X was detected in both the normal and mutant cells. The box of class II promoters and not to the regions flanking the significance of the shorter transcript detected in all three cell X box. (ii) Its contact points within the X box are practically lines below 18S ribosomal RNA is intriguing because it is identical to those of RF-X. (iii) It shares with RF-X the smaller than the cDNA insert and because Southern blot characteristic and unusual gradient of affinity for the HLA- experiments indicate that A9 hybridizes to a single gene (data DRA, -DPA, and -DQA promoters. (iv) No other protein with not shown). The presence of RF-X mRNA was further the binding characteristics of RF-X is detected in B-cell analyzed by RNase protection experiments. A riboprobe extracts. It is remarkable that, despite the fact that the derived from the 3' end was of the A9 cDNA insert protected recombinant protein is a fusion protein made in its to the same extent by hybridization with RNA from bacteria, poly(A)+ RF-X. normal and CID B cells (Fig. 4b). Thus, RF-X mRNA appears binding characteristics are identical to those of natural to be normal in both size and abundance in the mutant cells. Furthermore, the fact that recombinant RF-X can bind the The exposure times required indicate that RF-X mRNA is DRA promoter sequence indicates that this binding does not present at a very low steady-state level. depend on additional cofactors and reinforces the argument that the CID defect concerns the RF-X protein itself. The procedure described for the cloning of DNA-binding DISCUSSION proteins by screening an expression library with a probe HLA class II-deficient CID probably represents the first containing the target DNA sequence (22, 24) has thus been inherited disease in which a defect in a DNA-binding factor effective in this system. It is of interest that binding of the has been implicated (14). Since the lack of HLA class II recombinant protein during the screening procedure was only expression in these patients is known to result from a defect observed following denaturation and renaturation of the filter- in a trans-acting regulatory factor (17), we have concluded bound protein. This requirement might reflect that the recom- that RF-X, the class II promoter binding protein defective in binant protein is deposited on the filters as an insoluble the mutant cells, is an essential factor for HLA class II gene precipitate or that it is not appropriately folded during its regulation (14). Among the factors that can be shown to bind synthesis (22). It has been pointed out that renaturability is a to class II promoters, RF-X is thus of special interest. RF-X common property of many eukaryotic DNA-binding proteins is likely to be necessary but not sufficient for class II gene (22), and both natural and recombinant RF-X exhibit this expression since nuclear extracts from class II-negative behavior. Downloaded by guest on September 24, 2021 4204 Immunology: Reith et al. Proc. Natl. Acad. Sci. USA 86 (1989) One of the interesting properties of RF-X is the character- We thank S. L. McKnight for advice on the screening procedure; istic gradient of binding affinity for the promoters of HLA- B. Lisowska-Grospierre, C. Griscelli, and M. R. Hadam for CID cell lines; and M. Zufferey for assistance in cell cultures. This work was DRA, -DPA, and -DQA genes (Fig. 2; M.K., W.R., C.H.S., supported by the Swiss National Research Foundation and by the and B.M., unpublished data). The very low affinity of RF-X Armangd Fund. for the DQA promoter may explain the failure to detect the characteristic CID defect in RF-X binding with the use ofDQ 1. Benaceraff, B. (1981) Science 212, 1229-1238. promoter fragments. As expected, RF-X also binds well to 2. Schwartz, R. (1985) Annu. Rev. Immunol. 3, 237-261. the Ea promoter (the murine equivalent of DRA) and very 3. Matis, L. A., Glimcher, L. H., Paul, W. E. & Schwartz, R. H. (1983) Proc. Natl. Acad. Sci. USA 80, 6019-6023. poorly to the Aa promoter (the murine equivalent of DQA) 4. Mach, B., Gorski, J., Rollini, P., Berte, C., Amaldi, I., Berdoz, (M.K., W.R., C.H.S., and B.M., unpublished data). Two J. & Ucla, C. (1986) Cold Spring Harbor Symp. Quant. Biol. 51, distinct DNA-binding proteins recently cloned in the mouse 67-74. system have opposite binding characteristics-i.e., they bind 5. Collins, T., Korman, A. J., Wake, C. T., Boss, J. M., Kappes, well in the vicinity of the X box of Aa but not of Ea (26). D. J., Fiers, W., Ault, K. A., Gimbrone, M. A., Jr., Stromin- ger, J. L. & Pober, J. S. (1984) Proc. Natl. Acad. Sci. USA 81, Moreover, the Southern blot pattern obtained with these 4917-4921. mouse clones is clearly different from the one obtained for the 6. Polla, B. S., Poljak, A., Geier, S. G., Nathenson, S. G., Ohara, mouse homologue of RF-X (data not shown). Consequently, J., Paul, W. E. & Glimcher, L. H. (1986) Proc. Natl. Acad. Sci. these murine proteins (26) do not correspond to the mouse USA 83, 4878-4882. counterparts of RF-X. In humans, we have identified a 7. Blanar, M. A., Boettger, E. C. & Flavell, R. A. (1988) Proc. Natl. Acad. Sci. USA 85, 4672-4676. protein (NF-S) showing binding characteristics that are the 8. Dom, A., Durand, B., Marfing, C., LeMeur, M., Benoist, C. & reverse (DQA > DRA and Aa > Ea) of that exhibited by Mathis, D. (1987) Proc. Natl. Acad. Sci. USA 84, 6249-6253. RF-X (M.K., W.R., C.H.S., and B.M., unpublished data). It 9. Koch, W., Candeias, S., Guardiola, J., Accolla, R., Benoist, C. is possible therefore that NF-S is the human equivalent ofone & Mathis, D. (1988) J. Exp. Med. 167, 1781-1790. of the described murine proteins (26). NF-S does not bind to 10. Sakurai, M. & Strominger, J. L. (1988) Proc. Natl. Acad. Sci. USA 85, 6909-6913. the X box itself but to a sequence located immediately 3' of 11. Sherman, P. A., Basta, P. V. & Ting, J. P.-Y. (1987) Proc. the X box. Natl. Acad. Sci. USA 84, 4254-4258. The lack of RF-X binding in the CID regulatory mutants 12. Dorn, A., Bollekens, J., Staub, A., Benoist, C. & Mathis, D. could result either from a defect in the expression ofthe RF-X (1987) Cell 50, 863-872. gene or from the synthesis of a defective RF-X protein. The 13. Miwa, K., Doyle, C. & Strominger, J. L. (1987) Proc. Nat!. Acad. Sci. USA 84, 4939-4943. presence of RF-X mRNA, of normal size and abundance, 14. Reith, W., Satola, S., Herrero Sanchez, C., Amaldi, I., rules out a deletion ofthe RF-X gene or a mutation abolishing Lisowska-Grospierre, B., Griscelli, C., Hadam, M. R. & its expression. This suggests that the mutation(s) in CID Mach, B. (1988) Cell 53, 897-906. patients affects synthesis ofa functional RF-X protein, either 15. Saito, H., Maki, R. A., Clayton, L. K. & Tonegawa, S. (1983) by causing a premature termination or frameshift or by Proc. Natl. Acad. Sci. USA 80, 5520-5524. 16. Kelly, A. & Trowsdale, J. (1985) Nucleic Acids Res. 13, 1607- leading to amino acid substitutions altering the structural 1619. integrity of RF-X, presumably in its DNA-binding domain. 17. de Preval, C., Lisowska-Grospierre, B., Loche, M., Griscelli, The latter may account for the observation ofoccasional CID C. & Mach, B. (1985) Nature (London) 318, 291-293. mutants that are slightly "leaky" and express trace amounts 18. Griscelli, C., Lisowska-Grospierre, B. & Mach, B. (1989) of class II mRNA (18, 27). Such mutants could reflect Immunodeficiency Rev., in press. structural alterations of RF-X that permit weak residual 19. Chirgwin, J. M., Przbyla, A. E., MacDonald, R. J. & Rutter, J. (1979) Biochemistry 18, 5294-5299. binding activity. 20. Gubler, U. & Hoffman, B. J. (1983) Gene 25, 263-270. In addition to the remarkable polymorphism of major 21. Huynh, T. V., Young, R. A. & Davies, R. W. (1985) in DNA histocompatibility complex class II molecules, responsible Cloning: A Practical Approach, ed. Glover, D. M. (IRL, for the phenomenon of restriction in antigen presentation to Oxford), Vol. 1, pp. 49-77. T lymphocytes, the control ofthe level of expression of class 22. Vinson, C. R., LaMarco, K. L., Johnson, P. F., Landschulz, II affects the extent of T-cell activation W. H. & McKnight, S. L. (1988) Genes Dev. 1, 801-806. molecules directly 23. Dignam, J. D., Leboviz, R. M. & Roeder, R. G. (1983) Nucleic and of the immune response. Furthermore, abnormal control Acids Res. 11, 1475-1489. in the level of expression of class II genes and aberrant 24. Singh, H., LeBowitz, J. H., Baldwin, A. S., Jr., & Sharp, P. A. expression in tissues normally class II-negative (28, 29) are (1988) Cell 52, 415-423. very likely to be implicated in autoimmune phenomena. The 25. Bellocq, C. & Kolakofsky, D. (1987) J. Virol. 61, 3960-3967. cloning of an HLA class II regulatory factor will contribute 26. Liou, H., Boothby, M. R. & Glimcher, L. H. (1988) Science to a better understanding of normal and abnormal class II 242, 69-71. 27. de Preval, C., Hadam, M. R. & Mach, B. (1988) N. Engl. J. gene regulation and opens the way to the possibility of Med. 318, 1295-1300. manipulating the level of class II gene expression. Finally, 28. Massa, P. T., ter Meulen, V. & Fontana, A. (1987) Proc. Natl. the cloning of RF-X may allow us to elucidate the exact Acad. Sci. 84, 4219-4223. molecular basis ofthe CID defect and prepare the appropriate 29. Bottazzo, G. F., Todd, I., Mirakian, R., Belfiore, A. & Pujol- tools for gene therapy in these patients. Borrell, R. (1986) Immunol. Rev. 94, 137-169. 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