Mutations in the bare lymphocyte syndrome define critical steps in the assembly of the regulatory factor X complex

The regulatory factor X (RFX) complex, which contains RFXANK(B), RFXAP, and RFX5, binds to X and S boxes in major histocompatibility complex class II (MHC II) promoters. In the bare lymphocyte syndrome (BLS), which is a human severe combined immunodeficiency, MHC II promoters are neither occupied nor transcribed. Thus, the absence of any one subunit prevents the formation of the RFX complex. Nevertheless, except for a weak binding between RFX5 and RFXAP, no other interactions between RFX proteins have been described. In this study, we demonstrate that RFXANK(B) binds to RFXAP to form a scaffold for the assembly of the RFX complex, which then binds to DNA. Moreover, mutant RFXANK(B) and RFXAP proteins from complementation groups B and D of BLS, respectively, cannot support this interaction. Our data elucidate an intriguing medical situation, where a genetic disease targets two different surfaces that are required for the nucleation of a multisubunit DNA-protein complex.     

Assembly of the RFX complex on the MHCII promoter: role of RFXAP and RFXB in relieving autoinhibition of RFX5

The RFX complex is key component of a multi-protein complex that regulates the expression of the Major Histocompatibility Class II (MHCII) genes, whose products are essential for the initiation and development of the adaptive immune response. The RFX complex is comprised of three proteins--RFX5, RFXAP, and RFXB--all of which are required for expression of MHCII genes. We have used electrophoretic mobility shift assays to characterize the DNA binding of RFX5 and the complexes it forms with RFXB and RFXAP, to the proximal regulatory region of the MHCII promoter. DNA binding of RFX5 is inhibited by domains flanking its DNA binding domain, and both RFXAP and RFXB are required to overcome the inhibition of both domains. We provide evidence that a single RFX complex binds to the proximal regulatory region of the MHCII promoter and identify regions of the DNA that are important for high affinity binding of the RFX complex. Together, our results provide the most detailed view to date of the assembly of the RFX complex on the MHCII promoter and how its DNA binding is regulated.     

Analysis of ankyrin repeats reveals how a single point mutation in RFXANK results in bare lymphocyte syndrome.

Ankyrin repeats are well-known structural modules that mediate interactions between a wide spectrum of proteins. The regulatory factor X with ankyrin repeats (RFXANK) is a subunit of a tripartite RFX complex that assembles on promoters of major histocompatibility complex class II (MHC II) genes. Although it is known that RFXANK plays a central role in the nucleation of RFX, it was not clear how its ankyrin repeats mediate the interactions within the complex and with other proteins. To answer this question, we modeled the RFXANK protein and determined the variable residues of the ankyrin repeats that should contact other proteins. Site-directed alanine mutagenesis of these residues together with in vitro and in vivo binding studies elucidated how RFXAP and CIITA, which simultaneously interact with RFXANK in vivo, bind to two opposite faces of its ankyrin repeats. Moreover, the binding of RFXAP requires two separate surfaces on RFXANK. One of them, which is located in the ankyrin groove, is severely affected in the FZA patient with the bare lymphocyte syndrome. This genetic disease blocks the expression of MHC II molecules on the surface of B cells. By pinpointing the interacting residues of the ankyrin repeats of RFXANK, the mechanism of this subtype of severe combined immunodeficiency was revealed.