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.
Burd
Amy L
AL
Department of Immunology and Inflammation, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, Post Office Box 368, Ridgefield, Connecticut 06877-0368, USA.
Ingraham
Richard H
RH
Goldrick
Susan E
SE
Kroe
Rachel R
RR
Crute
James J
JJ
Grygon
Christine A
CA
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