Associations and Interactions between Bare Lymphocyte Syndrome Factors

The bare lymphocyte syndrome, a severe combined immunodeficiency due to loss of major histocompatibility complex (MHC) class II gene expression, is caused by inherited mutations in the genes encoding the heterotrimeric transcription factor RFX (RFX-B, RFX5, and RFXAP) and the class II transactivator CIITA. Mutagenesis of the RFX genes was performed, and the properties of the proteins were analyzed with regard to transactivation, DNA binding, and protein-protein interactions. The results identified specific domains within each of the three RFX subunits that were necessary for RFX complex formation, including the ankyrin repeats of RFX-B. DNA binding was dependent on RFX complex formation, and transactivation was dependent on a region of RFX5. RFX5 was found to interact with CIITA, and this interaction was dependent on a proline-rich domain within RFX5. Thus, these studies have defined the protein domains required for the functional regulation of MHC class II genes. Type II bare lymphocyte syndrome (BLS), an inherited severe combined immunodeficiency in humans, is caused by the inability to transcribe major histocompatibility complex (MHC) class II genes (9, 15, 32). MHC class II genes encode heterodimeric glycoproteins that present antigens to CD4 1 T cells to initiate the acquired arm of the immune response. They are also crucial for determining the repertoire of CD4 1 T cells during positive and negative selection in the thymus. Patients with BLS typically present in the first year of life with recurrent infections and have reduced levels of CD4 1 T cells (9, 11). Their humoral immune response is severely impaired as well, and most patients die before reaching puberty. Patient and experimentally derived cell lines were used to separate the BLS phenotype into four complementation groups: BLS groups A, B, C, and D (3, 46, 54). The genes responsible for each of these groups have been identified and found to encode proteins required for MHC class II gene transcription. MHC class II genes are expressed on the surface of B cells, dendritic cells, macrophages, thymic epithelia, and activated T cells. Additionally, non-antigen-presenting cells can be induced to express MHC class II by exposure to the cytokine gamma interferon (IFN-g) (8). Aberrant expression of MHC class II genes is associated with autoimmunity, tumor growth, and failure to mount an immune response. The three MHC class II isotypes, HLA-DR, HLA-DP, and HLA-DQ, contain conserved cis-acting elements in their promoters (the W, X1, X2, and Y boxes) that allow their coordinate regulation (reviewed in references 4 and 31). Homologous sequence elements are also found in the HLA-DM, invariant chain, and MHC class I genes. These elements allow the coordinate expression of the different isotypes in antigen-presenting cells and the induction of these genes by IFN-g. Regulatory factor X (RFX) and the X2 box-binding protein (X2BP), which was identified as the cyclic AMP response element-binding protein (CREB) (34), bind to the X1 and X2 boxes, respectively. The Y box, an inverted CAAT box, is bound by the heterotrimeric nuclear factor Y (NF-Y) (4). The W box has not been extensively studied, but it was suggested to bind the X1 box factor, RFX (22). While all of these promoter-bound factors are required for MHC class II expression, they are not sufficient. The class II transactivator, CIITA, is also required. CIITA does not bind DNA and is believed to interact with factors on the MHC class II promoter, as well as the general transcriptional machinery, to activate transcription through its acidic activation domain (44, 49, 55). CIITA expression correlates directly with MHC class II expression and is regulated by IFN-g (6, 7, 50). Thus, the presence of CIITA functions as a molecular switch for MHC class II gene regulation.