Major histocompatibility complex class II transcriptional platform: assembly of nuclear factor Y and regulatory factor X (RFX) on DNA requires RFX5 dimers.
Major histocompatibility complex class II (MHC-II) genes are regulated in a B-cell-specific and gamma interferon-inducible manner. Conserved upstream sequences (CUS) in their compact promoters bind nuclear factor Y (NFY) and regulatory factor X (RFX) complexes. These DNA-bound proteins form a platform that attracts the class II transactivator, which initiates and elongates MHC-II transcription. In this report, we analyzed the complex assembly of these DNA-bound proteins. First, we found that NFY can interact with RFX in cells. In particular, NFYA and NFYC bound RFXANK/B in vitro. Next, RFX5 formed dimers in vivo and in vitro. Within a leucine-rich stretch N-terminal to the DNA-binding domain in RFX5, the leucine at position 66 was found to be critical for this self-association. Mutant RFX5 proteins that could not form dimers also did not support the formation of higher-order DNA-protein complexes on CUS in vitro or MHC-II transcription in vivo. We conclude that the MHC-II transcriptional platform begins to assemble off CUS and then binds DNA via multiple, spatially constrained interactions. These findings offer one explanation of why in the Bare Lymphocyte Syndrome, which is a congenital severe combined immunodeficiency, MHC-II promoters are bare when any subunit of RFX is mutated or missing.
Jabrane-Ferrat
Nabila
N
Department of Surgery, University of California-San Francisco, San Francisco, CA 94115-0703, USA.
Nekrep
Nada
N
Tosi
Giovanna
G
Esserman
Laura J
LJ
Peterlin
B Matija
BM
D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D019556:COS Cells; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005786:Gene Expression Regulation / Q000502:physiology; D005802:Genes, MHC Class II / Q000502:physiology*; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000235:genetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000502:physiology; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection; D020798:Two-Hybrid System Techniques
D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D000949:Histocompatibility Antigens Class II; D046911:Macromolecular Substances; D021122:Protein Subunits; C093524:RFX5 protein, human; D011993:Recombinant Fusion Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; C402244:nuclear factor Y; D004247:DNA