Two DNA-binding proteins discriminate between the promoters of different members of the major histocompatibility complex class II multigene family.

Abstract

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...

MHC class II regulatory factor RFX has a novel DNA-binding domain and a functionally independent dimerization domain

“…The protein encoded by the RFX9 cDNA clone binds specifically to the X box, has essentially the same contact points within the X box as RFX, and shares with RFX a characteristic gradient of affinity for the promoters of the DRA, DPA, and DQA genes Kobr et al 1990). Because a given D N A sequence can frequently bind several different nuclear factors, we provide here additional evidence confirming that RFX9 indeed encodes RFX.…”

“…As observed previously for both native and recombinant RFX Kobr et al 1990), binding of the monomeric rRFX36/HaeII polypeptide to 32p-labeled DRA-X1 is competed for completely by DRA-X3, poorly by DQA-X, and with intermediate efficiency by DPA-X (Fig. 6A).…”

“…The more slowly migrating complex that is also inhibited specifically by the antibodies is always associated with RFX and has been shown by competition and methylation interference experiments to contain RFX (data not shown). The inhibition is restricted to RFX because the complex due to NF-S, a protein that binds to a sequence situated next to the 3' end of the X box (Kobr et al 1990) is not affected (Fig. 1A, lane 3).…”

“…These sequences are cis-acting elements playing a central role in class II gene expression (Dom et al 1987b;Sherman et al 1987Sherman et al , 1989Basta et al 1988;Koch et al 1988;Sakurai and Strominger 1988;Tsang et al 1988Tsang et al , 1990Dedrick and Jones 1990). We and others have identified nuclear factors binding specifically to these sequences (Dorn et al 1987a, b;Miwa et al 1987;Reith et al 1988Reith et al , 1989Sherman et al 1989;Dedrick and Jones 1990;Kobr et al 1990;Tsang et al 1990). Among these, the factor we have called RFX is of special interest because there is evidence that it is in-volved in the regulation of class II gene expression.…”

“…Among these, the factor we have called RFX is of special interest because there is evidence that it is in-volved in the regulation of class II gene expression. RFX is a nuclear protein that binds specifically, although with variable affinity, to the X boxes of the h u m a n DRA, DPA, DQA, DRB1, and DRB3 genes, as well as the mouse Ea and h u m a n class II-associated invariant chain gene (Reith et al 1988Kobr et al 1990). Binding of RFX is defective in nuclear extracts from B-cell lines derived from patients suffering from class II-deficient combined immunodeficiency (CID), a disease in which the absence of class II gene expression in all tissues has been shown to be due to a defect in a trans-acting regulatory factor (de Pr6val et al , 1988Lisowska-Grospierre et al 1985;Reith et al 1988Reith et al , 1989Griscelli et al 1989).…”