Phosphorylation of class II transactivator regulates its interaction ability and transactivation function

The MHC class II transactivator (CIITA) plays a central role in adaptive immune responses by controlling the expression of MHC class II genes. CIITA binds DNA-binding proteins and co-activator proteins to form an enhanceosome complex necessary for MHC class II gene expression. Here we demonstrate that CIITA interactions depend upon the phosphorylation status of CIITA. Hyper-phosphorylated CIITA interacts with co-activator p300, RFX5 and CIITA itself, which in turn results in induction of MHC class II promoter activity. Moreover, the C-terminal region of CIITA containing leucine-rich repeats (LRR) is a regulatory domain for CIITA self-association and LRR binding to CIITA is negatively regulated by phosphorylation. cAMP-dependent protein kinase (PKA) phosphorylates CIITA, and serine residues residing in a region between the proline/serine/threonine-rich domain and the GTP-binding domain are phosphorylated by PKA in vitro. The maximum transactivation potential of CIITA requires PKA phosphorylation as demonstrated by reduced transactivation activities of the mutant bearing substitutions of serine residues at the PKA site.     

Structural and functional characteristics of a dominant‐negative isoform of porcine MHC class II transactivator

The MHC class II transactivator, CIITA, is critical for MHC class II gene expression in all species studied to date. We isolated an interferon (IFN)‐γ‐inducible isoform of porcine CIITA (pCIITA′) encoding a protein of 566 amino acids (aa) with significant homology to human CIITA (hCIITA). Analysis indicated that pCIITA′ lacks the entire GTP‐binding domain that is important for nuclear translocation and activation of target genes by hCIITA. In pCIITA′ this region is replaced by a 14‐aa motif with homology to several signalling peptide sequences. Expression of pCIITA′ in porcine (ST‐IOWA) and human (HeLa) cell lines resulted in suppression of IFN‐γ‐stimulated MHC class II gene expression, at the protein and mRNA levels. We also identified two IFN‐γ‐inducible variants of hCIITA, hCIITAlo and hCIITA′ from Hela cells, both exhibiting dominant‐negative suppression of MHC class II gene expression. Interestingly, hCIITA′ encodes a predicted protein of 546 aa with a strikingly similar organization to pCIITA′ including the 14‐aa GTP‐binding domain‐replacement motif in which 10 out of 14 amino acids are identical to the pig sequence. Expression of hCIITA′ and hCIITAlo sequences in Hela cells suppressed IFN‐γ‐induced MHC class II gene expression. hCIITAlo, a predicted 303‐aa protein with deleted GTP‐binding and carboxy‐terminal domain, displayed a more subtle suppression of IFN‐γ‐induced MHC class II expression. These in vitro data indicate that there may be a role in vivo for isoforms of CIITA that can suppress full‐length CIITA‐mediated MHC class II gene expression. Both humans and now, potentially, pigs are candidate donors for organ and tissue allografts and xenografts, respectively. Regulation of MHC class II gene expression by manipulation of CIITA isoform expression in humans and pigs may provide a useful strategy for attenuation of T‐cell‐mediated cellular rejection.     

MHC class II transactivator (CIITA) expression is upregulated in multiple myeloma cells by IFN-gamma

The MHC class II transactivator (CIITA) acts in the cell nucleus as the master regulator of MHC class II (MHC II) gene expression. It is important to study CIITA regulation in multiple myeloma since MHC expression is central to ability of myeloma cells to present antigen and to the ability of the immune system to recognize and destroy this malignancy. Regulation of CIITA by IFN-gamma in B lymphocytes occurs through the CIITA type IV promoter (pIV), one of the four potential promoters (pI-pIV) of this gene. To investigate regulation of CIITA by IFN-gamma in multiple myeloma cells, first the ability of these cells to respond to IFN-gamma was examined. RT-PCR analyses show that IFN-gammaR1, the IFN-gamma-binding chain of the IFN-gamma receptor, is expressed in myeloma cells and IRF-1 expression increases in response to IFN-gamma treatment. Western blotting demonstrates that STAT1 is activated by phosphorylation in response to IFN-gamma. RT-PCR and functional promoter analyses show that IFN-gamma upregulates the activity of CIITA pIV, as does ectopic expression of IRF-1 or IRF-2. In vivo protein/DNA binding studies demonstrate protein binding at the GAS, E box and IRF-E sites. In vitro studies confirm the binding of IRF-1 and IRF-2 to CIITA pIV. Although multiple myeloma cells express PRDI-BF1/Blimp-1, a factor that represses both the CIITA type III and IV promoters, they retain the capability to upregulate CIITA pIV and MHC II expression in response to IFN-gamma treatment. These findings are the first to demonstrate that although PRDI-BF1/Blimp-1 diminishes the constitutive ability of these cells to present antigen by limiting CIITA and MHC II expression, it is possible to enhance this expression through the use of cytokines, like IFN-gamma.     

Dendritic cells and differential usage of the MHC class II transactivator promoters in the central nervous system in experimental autoimmune encephalitis

In the normal central nervous system (CNS) expression of MHC class II is minimal, but has been found to be highly up-regulated on microglia cells in experimental autoimmune encephalitis (EAE). Here we used the EAE model to examine the regulation of expression of the class II transactivator (CIITA), which is required for activation of MHC class II genes. EAE was induced in C57BL/6 mice by immunization with myelin oligodendrocyte glycoprotein peptide 35-55. CIITA mRNA form I (specific for dendritic cells) and form IV (IFN-gamma inducible) but not form III (B cell specific) were detected in brain and spinal cord of mice with acute EAE. In unimmunized or mock-immunized mice, none of the three CIITA forms was found to be induced. Dendritic cells (DC) were identified by immunostainings for CD11c in perivascular and meningeal cell infiltrates in EAE spinal cord and brain. Time-course analysis showed (1) the appearance of DC in the CNS shortly before onset of disease, (2) the recruitment of CD11b cells occuring much earlier and (3) the absence of CIITA and MHC class II expression in these CD11b+ cells at preclinical stages.