ABSTRACT
The major histocompatibility complex (MHC) class II transactivator (CIITA) regulates the expression of genes involved in the immune response, including MHC class II genes and the interleukin-4 gene. Interactions between CIITA and sequence-specific, DNA-binding proteins are required for CIITA to function as an activator of MHC class II genes. CIITA also interacts with the coactivators CBP (also called p300), and this interaction leads to synergistic activation of MHC class II promoters. Here, we report that CIITA forms complexes with itself and that a central region, including the GTP-binding domain is sufficient for self-association. Additionally, this central region interacts with the C-terminal leucine-rich repeat as well as the N-terminal acidic domain. LXXLL motifs residing in the GTP-binding domain are essential for self-association. Finally, distinct differences exist among various CIITA mutant proteins with regard to activation function, subcellular localization, and association with wild-type protein and dominant-negative potential.
Subject(s)
Trans-Activators/metabolism , Transcriptional Activation , Amino Acid Motifs , Amino Acids/chemistry , Blotting, Western , Cell Line , Dose-Response Relationship, Drug , Flow Cytometry , Genes, Dominant , Genes, MHC Class II , Guanosine Triphosphate/metabolism , Humans , Interleukin-4/metabolism , Luciferases/metabolism , Mutation , Nuclear Proteins/metabolism , Plasmids/metabolism , Precipitin Tests , Promoter Regions, Genetic , Protein Binding , Protein Biosynthesis , Protein Structure, Tertiary , Transcription, Genetic , TransfectionABSTRACT
The MHC class II transactivator (CIITA) is a critical transcription factor that regulates genes involved in antigen presentation function. At least three functional forms of CIITA gene products are transcribed from three different promoters. The CIITA gene expressed in dendritic cells (DC-CIITA) has a unique first exon encoding an extended N-terminal region of CIITA. Here, we show that the N terminus of DC-CIITA has high homology to a caspase recruitment domain (CARD) found in components of apoptosis and nuclear factor-kappaB signaling pathways. However, DC-CIITA does not regulate cell death, nor does it induce nuclear factor-kappaB activity. Instead, DC-CIITA is transcriptionally a more potent activator of the MHC class II gene than the form expressed in B cells. A single amino acid substitution in the CARD of DC-CIITA, predicted to disrupt CARD-CARD interactions, diminished the transactivation potential of DC-CIITA. These results indicate that the CARD in the context of CIITA serves as a regulatory domain for transcriptional activity and may function to selectively enhance MHC class II gene expression in dendritic cells.
Subject(s)
Caspases/chemistry , Dendrites/metabolism , Nuclear Proteins , Trans-Activators/chemistry , Amino Acid Sequence , Apoptosis , Blotting, Western , Cell Line , Cell Nucleus/metabolism , DNA/metabolism , Dendritic Cells/metabolism , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Exons , Flow Cytometry , Humans , Luciferases/metabolism , Models, Genetic , Molecular Sequence Data , NF-kappa B/metabolism , Plasmids/metabolism , Promoter Regions, Genetic , Protein Isoforms , Protein Structure, Tertiary , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Signal Transduction , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription, Genetic , Transcriptional Activation , TransfectionABSTRACT
The MHC class II transactivator (CIITA) activates the expression of multiple genes involved in Ag presentation, but inhibits Th2-type cytokine production, including IL-4, during Th1 cell differentiation. Th1 cells derived from CIITA-deficient mice produce both Th1- and Th2-type cytokines, and the introduction of CIITA to Th2 cells down-regulates Th2-type cytokine gene transcription. Here we show that the IL-4 promoter is regulated by multiple protein-protein interactions among CIITA, NF-AT, and coactivator CBP/p300. The introduction of CBP/p300 and NF-AT enhances the IL-4 promoter activity, and this activation was repressed by CIITA. Furthermore, our data show that CIITA competes with NF-AT to bind CBP/p300 and that this competition dramatically influences transcriptional activation of the IL-4 promoter. We identified two domains of CIITA that interact with two distinct domains of CBP/p300 that are also recognized by NF-AT. CIITA mutants that retain the ability to interact with CBP/p300 are sufficient to inhibit NF-AT-mediated IL-4 gene expression.
