Regulation of MHC class II gene expression is an essential aspect of the control of the immune response. Primary MHC class II deficiency is a genetically heterogeneous disease of gene regulation that offers the unique opportunity of a genetic approach for the identification of the functionally relevant regulatory genes and factors. Most patients exhibit a characteristic defect in the binding of a nuclear complex, RFX, to the X box motif of MHC class II promoters. Genetic complementation of a B-lymphocyte cell line from such a patient with a cDNA expression library has allowed us to isolate RFX5, the regulatory gene responsible for the MHC class II deficiency. This gene encodes a novel DNA-binding protein that is indeed a subunit of the RFX complex. Mutations in the RFX5 gene have been characterized in two patients. Transfection of the patient's cells with the RFX5 cDNA repairs the binding defect and fully restores expression of all the endogenous MHC class II genes in vivo.

There are five members of the RFX family of transcription factors in mammals. While RFX5 plays a well-defined role in the immune system, the functions of RFX1 to RFX4 remain largely unknown. We have generated mice with a deletion of the Rfx3 gene. RFX3-deficient mice exhibit frequent left-right (LR) asymmetry defects leading to a high rate of embryonic lethality and situs inversus in surviving adults. In vertebrates, specification of the LR body axis is controlled by monocilia in the embryonic node, and defects in nodal cilia consequently result in abnormal LR patterning. Consistent with this, Rfx3 is expressed in ciliated cells of the node and RFX3-deficient mice exhibit a pronounced defect in nodal cilia. In contrast to the case for wild-type embryos, for which we document for the first time a twofold increase in the length of nodal cilia during development, the cilia are present but remain markedly stunted in mutant embryos. Finally, we show that RFX3 regulates the expression of D2lic, the mouse orthologue of a Caenorhabditis elegans gene that is implicated in intraflagellar transport, a process required for the assembly and maintenance of cilia. In conclusion, RFX3 is essential for the differentiation of nodal monocilia and hence for LR body axis determination.

Determination of left–right asymmetry in mouse embryos is achieved by a leftward fluid flow (nodal flow) in the node cavity that is generated by clockwise rotational movement of 200–300 cilia in the node. The precise action of nodal flow and how much flow input is required for the robust read-out of left–right determination remains unknown. Here we show that a local leftward flow generated by as few as two rotating cilia is sufficient to break left–right symmetry. Quantitative analysis of fluid flow and ciliary rotation in the node of mouse embryos shows that left–right asymmetry is already established within a few hours after the onset of rotation by a subset of nodal cilia. Examination of various ciliary mutant mice shows that two rotating cilia are sufficient to initiate left–right asymmetric gene expression. Our results suggest the existence of a highly sensitive system in the node that is able to sense an extremely weak unidirectional flow, and may favour a model in which the flow is sensed as a mechanical force.

Major Histocompatibility Complex class II (MHC‐II) deficiency is a disease of gene regulation that provides a unique opportunity for the genetic dissection of the molecular mechanisms controlling transcription of MHC‐II genes. Cell lines from MHC‐II deficiency patients have been assigned to three complementation groups (A, B and C) believed to reflect the existence of distinct essential MHC‐II regulatory genes. Groups B and C, as well as an in vitro generated regulatory mutant representing a fourth group (D), are characterized by a specific defect in the binding activity of RFX, a multimeric DNA binding complex that is essential for activation of MHC‐II promoters. RFX5, a subunit of RFX, was recently shown to be mutated in group C. We have now isolated a novel gene, RFXAP (RFX Associated Protein), that encodes a second subunit of the RFX complex. RFXAP is mutated in the 6.1.6 cell line (group D), as well as in an MHC‐II deficiency patient (DA). This establishes that group D is indeed a fourth MHC‐II deficiency complementation group. Complementation of the 6.1.6 and DA cell lines by transfection with RFXAP fully restores expression of all endogenous MHC‐II genes in vivo , demonstrating that RFXAP is a novel essential MHC‐II regulatory gene.

Until recently, the RFX family of DNA binding proteins consisted exclusively of four mammalian members (RFX1–RFX4) characterized by a novel highly conserved DNA binding domain. Strong conservation of this DNA binding domain precluded a precise definition of the motif required for DNA binding. In addition, the biological systems in which these RFX proteins are implicated remained obscure. The recent identification of four new RFX genes has now shed light on the evolutionary conservation of the RFX family, contributed greatly to a detailed characterization of the RFX DNA binding motif, and provided clear evidence for the function of some of the RFX proteins. RFX proteins have been conserved throughout evolution in a wide variety of species, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, mouse and man. The characteristic RFX DNA binding motif has been recruited into otherwise very divergent regulatory factors functioning in a diverse spectrum of unrelated systems, including regulation of the mitotic cell cycle in fission yeast, the control of the immune response in mammals, and infection by human hepatitis B virus.