Characterization of the RFX complex and the RFX5(L66A) mutant: implications for the regulation of MHC class II gene expression

Major histocompatability complex class II (MHCII) molecules are an essential component of the mammalian adaptive immune response. The expression of MHCII genes is regulated by a cell-specific multiprotein complex, termed the MHCII enhanceosome. The heterotrimeric RFX complex is the key DNA-binding component of the MHCII enhanceosome. The RFX complex is comprised of three proteins, RFXB, RFXAP, and RFX5, all of which are required for DNA binding and activation of MHCII gene expression. Static light scattering and chemical cross-linking of the three RFX proteins show that RFXB and RFXAP are monomers and that RFX5 dimerizes through two separate domains. One of these domains, the oligomerization domain, promotes formation of a dimer of dimers of RFX5. In addition, we show that the RFX complex forms a 2:1:1 complex of RFX5.RFXAP.RFXB, which can associate with a further dimer of RFX5 to form a 4:1:1 complex through the oligomerization domain of RFX5. On the basis of these studies, we propose DNA-binding models for the interaction between the RFX complex and the MHCII promoter including a DNA looping model. We also provide direct evidence that the RFX5(L66A) point mutation prevents dimerization of the RFX complexes and propose a model for how this results in a loss of MHCII gene expression.     

NFY和RFX1对人PNRC启动子的调控作用

探讨核因子Y（nuclear factor Y, NFY）和调节因子X1（regulatory factors that bind to the X box, RFX1）对人PNRC（proline rich nuclear receptor coactivator）基因的调控作用及机制.根据凝胶电泳迁移率变化实验,分析NFY和RFX1与PNRC启动子区域的结合.将含有NFY和RFX1的真核表达质粒(pCMV-NFY, pCMV-RFX1)和含有PNRC启动子的荧光素酶报告基因质粒共转染HepG2细胞,检测转染细胞的荧光素酶活性,并用RT-PCR和Western印迹检测PNR的表达情况.Quick-Change法对PNRC启动子区NFY和RFX1结合位点进行突变,将包含突变点的重组荧光素酶报告质粒与含有NFY和RFX1的真核表达质粒共同转染HepG2细胞,检测各组荧光素酶活性.结果发现,NFY和RFX1能与PNRC启动子区域特异性结合;转染pCMV-NFY和pCMV-RFX1可抑制PNRC启动子活性并下调PNRC在HepG2细胞中的表达;包含NFY和RFX1结合位点的突变质粒与pCMV-NFY和pCMV-RFX1共转染后,NFY和RFX1对PNRC启动子活性的抑制作用消失. 以上结果提示,NFY和RFX1能调控PNRC基因的表达,其机制是与PNRC启动子区域的特异性结合位点相结合,发挥其反式抑制作用.     

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.