Assembly of major histocompatibility complex (MHC) class II transcription factors: association and promoter recognition of RFX proteins.
Major histocompatibility complex (MHC) class II genes are regulated at the transcriptional level 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-response element binding protein (CREB). Here, the MHC class-II-specific transcription factors and CREB were expressed in insect cells with recombinant baculoviruses, isolated, and characterized by biochemical and biophysical 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) between RFX5 and RFX-AP was measured by isothermal titration calorimetry (ITC), while the interaction between RFX-AP and RFX-ANK is at least an order of magnitude weaker. The biophysical data show that the interaction between RFX-AP and RFX5 is a key event in the assembly of the heterotrimer. Fluorescence anisotropy was used to determine protein-nucleic acid binding affinities for the RFX subunits and complexes 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 binding for 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 that drive MHC class II gene expression.
Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Post Office Box 368, Ridgefield, Connecticut 06877-0368, USA.
Amy L
AL
Burd
Richard H
RH
Ingraham
Susan E
SE
Goldrick
Rachel R
RR
Kroe
James J
JJ
Crute
Christine A
CA
Grygon
D000818:Animals; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005786:Gene Expression Regulation; D000949:Histocompatibility Antigens Class II / Q000235:genetics*; D046911:Macromolecular Substances; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020836:Protein Structure, Quaternary; D000071701:Regulatory Factor X Transcription Factors; D018411:Spodoptera; D013379:Substrate Specificity; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D000949:Histocompatibility Antigens Class II; C083728:MHC class II transactivator protein; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D000071701:Regulatory Factor X Transcription Factors; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA