The regulation of MHC class II gene expression controls T-cell activation and, hence, the immune response. Among the nuclear factors observed to bind to conserved DNA sequences in human leukocyte antigen (HLA) class II gene promoters, RFX is of special interest: Its binding is defective in congenital HLA class II deficiency, a disease of class II gene regulation. The cloning of an RFX cDNA has allowed us to show by transfection of a plasmid directing the synthesis of antisense RFX RNA that RFX is a class II gene regulatory factor. RFX is a novel 979-amino-acid DNA-binding protein that contains three structurally and functionally separate domains. The 91-amino-acid DNA-binding domain is distinct from other known DNA-binding motifs but may be distantly related to the helix-loop-helix motif. The most striking property of RFX is that it can bind stably to the class II X box as either a monomer or a homodimer and that the domain responsible for dimerization is distant from and functionally independent of the DNA-binding domain. This distinguishes RFX from other known dimeric DNA-binding proteins. It also implies that an RFX homodimer has two potential DNA-binding sites. We therefore speculate that RFX could form a DNA loop by cross-linking the two X-box sequences found far apart upstream of MHC class II genes.

RFX1 is a transactivator of human hepatitis B virus enhancer I. We show here that RFX1 belongs to a previously unidentified family of DNA-binding proteins ofwhich we have cloned three members, RFX1, RFX2, and RFX3, from humans and mice. Members of the RFX family constitute the nuclear complexes that have been referred to previously as enhancer factor C, EP, methylation-dependent DNA-binding protein, or rpL30a. RFX proteins share five strongly conserved regions which include the two domains required for DNA binding and dimerization. RFX1 is a transcription factor that was initially cloned by virtue of its affinity for the X-box motif (31, 32), a conserved cis-acting regulatory element present in the promoters of all major histocompatibility complex (MHC) class II genes from all species examined (for reviews, see references 3, 13, and 41). RFX1 was subsequently also found to bind with high affinity to inverted repeats known as enhanced factor C (EF-C) sites (also called EP or methylation-dependent DNAbinding protein [MDBP] sites), which are cis-acting regulatory elements present in the enhancers of several unrelated viruses, including hepatitis B virus (HBV), polyomavirus, and cytomegalovirus (CMV) (7,10,15,28,29,42,(50)(51)(52). Among these EF-C sites, the functional importance of the site in the HBV enhancer (EnhI) has been most clearly demonstrated (7,11,15,29,37,42 . Second, there is a C-terminal dimerization domain which does not display obvious sequence similarity to known dimerization motifs (32). Moreover, in contrast to the majority of dimeric DNAbinding proteins, the DNA-binding and dimerization domains of RFX1 are not closely linked in the protein and are functionally independent, such that RFX1 can bind in vitro as either a monomer or a dimer (32, 37). Finally, identity between RFX1 and EF-C (EP or MDBP) (37) has allowed us to document a unique feature not previously described for other cloned eukaryotic transcription factors. Namely, in addition to its classical site-specific DNA-binding activity, RFX1 (MDBP or EF-C) also shows a peculiar site-specific affinity for certain methylated sequences (10,22,37,45,50,52). These methylation-dependent RFX1 (MDBP or EF-C) binding sites are recognized specifically only when they contain 5-methylcytosine at CpG dinucleotides.Depending on the cell line examined, EF-C (EP or MDBP) sites are bound by one to three different nuclear complexes having identical target site specificity (10,29,37,50), and only one of these complexes represents an RFX1 homodimer (37). The rpL30(x and MHC class II X-box sites are also bound by several closely related complexes (17,24,35). This suggested the existence of a family of RFX proteins. Considering the novel features of RFX1, its crucial role in activating the enhancer of HBV, and its suspected role for transcription of the MHC class II and rpL30 genes, it was of interest to identify these additional RFX factors. We have now cloned and characterized two other highly conserved RFX genes, RFX2 and RFX3, from both humans and mice, the...

The regulation of major histocompatiblity complex (MHC) class H gene expression is a key feature of the control of normal and abnormal immune responses. In humans, class II a-and ,8-chain genes are organized in a multigene family with three distinct subregions, HLA-DR, -DQ, and -DP. The regulation of these genes is generally coordinated, and their promoters contain highly conserved motifs, in particular the X and Y boxes. We have identified five distinct proteins that bind to specific DNA sequences within the first 145 base pairs of the HLA-DRA promoter, a segment known to be functionally essential for class H gene regulation. Among these, RF-X is of special interest, since mutants affected in the regulation of MHC class II gene expression have a specific defect in RF-X binding. Unexpectedly, RF-X displays a characteristic gradient of binding affinities for the X boxes of three a-chain genes (DRA > DPA >> DQA). The same observation was made with recombinant RF-X. We also describe a novel factor, NF-S, which bound to the spacer region between the X and Y boxes of class II promoters. NF-S exhibited a reverse gradient of affinity compared with RF-X (DQA > DPA >> DRA).As expected, RF-X bound well to the mouse IEa promoter, while NF-S bound well to IAa. The drastic differences in the binding of RF-X and NF-S to different MHC class II promoters contrasts with the coordinate regulation of HLA-DR, -DQ, and -DP genes.Major histocompatibility complex (MHC) class II genes, or immune response genes, encode highly polymorphic transmembrane glycoproteins that are directly responsible for the recognition of antigens by the receptors of T lymphocytes. The immune response is controlled on one hand by the extensive structural diversity of MHC molecules, which is responsible for allelic differences in the efficiency of T-cell stimulation by a given antigen and for the existence of highand low-responder phenotypes (4, 5). In addition, normal and abnormal immune responses are controlled by the quantitative regulation of MHC class II gene expression. Since expression of class II molecules at the cell surface confers the ability to stimulate T cells, the tissue specificity and level of MHC class II expression control T-cell activation (6, 21).Because of this biological relevance, there is great interest in the mechanisms responsible for the regulation, developmental control, and tissue specificity of MHC class II gene expression. Finally, since MHC class II genes form a multigene family, the mechanisms responsible for the global regulation observed for the entire family of HLA-DR, -DQ, and -DP a-and a-chain genes (8) are of interest. Rare cases of dissociated expression of HLA-DR versus -DQ have also been described elsewhere (13).In transfection experiments, a DNA sequence containing less than 160 base pairs upstream of MHC class II genes is sufficient to confer both B-cell-specific expression (23, 30) and inducibility by gamma interferon (3,28,30). Within this region, three highly conserved sequences are observed in human and mouse c...

RFX1 is a transactivator of human hepatitis B virus enhancer I. We show here that RFX1 belongs to a previously unidentified family of DNA-binding proteins of which we have cloned three members, RFX1, RFX2, and RFX3, from humans and mice. Members of the RFX family constitute the nuclear complexes that have been referred to previously as enhancer factor C, EP, methylation-dependent DNA-binding protein, or rpL30 alpha. RFX proteins share five strongly conserved regions which include the two domains required for DNA binding and dimerization. They have very similar DNA-binding specificities and heterodimerize both in vitro and in vivo. mRNA levels for all three genes, particularly RFX2, are elevated in testis. In other cell lines and tissues, RFX mRNA levels are variable, particularly for RFX2 and RFX3. RFX proteins share several novel features, including new DNA-binding and dimerization motifs and a peculiar dependence on methylated CpG dinucleotides at certain sites.

The regulation of major histocompatibility complex (MHC) class II gene expression is a key feature of the control of normal and abnormal immune responses. In humans, class II alpha - and beta-chain genes are organized in a multigene family with three distinct subregions, HLA-DR, -DQ, and -DP. The regulation of these genes is generally coordinated, and their promoters contain highly conserved motifs, in particular the X and Y boxes. We have identified five distinct proteins that bind to specific DNA sequences within the first 145 base pairs of the HLA-DR promoter, a segment known to be functionally essential for class II gene regulation. Among these, RF-X is of special interest, since mutants affected in the regulation of MHC class II gene expression have a specific defect in RF-X binding. Unexpectedly, RF-X displays a characteristic gradient of binding affinities for the X boxes of three alpha-chain genes (DRA greater than DPA much greater than DQA). The same observation was made with recombinant RF-X. We also describe a novel factor, NF-S, which bound to the spacer region between the X and Y boxes of class II promoters. NF-S exhibited a reverse gradient of affinity compared with RF-X (DQA greater than DPA much greater than DRA). As expected, RF-X bound well to the mouse IE alpha promoter, while NF-S bound well to IA alpha. The drastic differences in the binding of RF-X and NF-S to different MHC class II promoters contrasts with the coordinate regulation of HLA-DR, -DQ, and -DP genes.