Assembly of Major Histocompatability Complex (MHC) Class II Transcription Factors: Association and Promoter Recognition of RFX Proteins

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• 作者：Amy L. Burd ; Richard H. Ingraham ; Susan E. Goldrick ; Rachel R. Kroe ; James J. Crute ; and Christine A. Grygon
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：October 12, 2004
• 年：2004
• 卷：43
• 期：40
• 页码：12750 - 12760
• 全文大小：161K
• 年卷期：v.43,no.40(October 12, 2004)
• ISSN：1520-4995

文摘

Major histocompatibility complex (MHC) class II genes are regulated at the transcriptionallevel by coordinate action of a limited number of transcription factors that include regulatory factor X(RFX), class II transcriptional activator (CIITA), nuclear factor Y (NF-Y), and cyclic AMP-responseelement binding protein (CREB). Here, the MHC class-II-specific transcription factors and CREB wereexpressed in insect cells with recombinant baculoviruses, isolated, and characterized by biochemical andbiophysical methods. Analytical ultracentrifugation (AUC) has demonstrated that RFX is a heterotrimer.A heterodimer of RFX5 and RFX-AP was also observed. A high-affinity interaction (K_d = 25 nM) betweenRFX5 and RFX-AP was measured by isothermal titration calorimetry (ITC), while the interaction betweenRFX-AP and RFX-ANK is at least an order of magnitude weaker. The biophysical data show that theinteraction between RFX-AP and RFX5 is a key event in the assembly of the heterotrimer. Fluorescenceanisotropy was used to determine protein-nucleic acid binding affinities for the RFX subunits andcomplexes binding to duplex DNA. The RFX5 subunit was found to drive recognition of the promoter,while the auxiliary RFX-AP and RFX-ANK subunits were shown to contribute to the specificity of bindingfor the overall complex. AUC experiments demonstrate that in the absence of additional subunits,monomeric RFX5 binds to X-box DNA with a 1:1 stoichiometry. Interactions between CREB, CIITA,and RFX in the absence of DNA were demonstrated using bead-based immunoprecipitation assays,confirming that preassociation with DNA is not required for forming the macromolecular assemblies thatdrive MHC class II gene expression.