Anti-HLA-DRA monoclonal antibody, clone IM-49 [FITC]

Major histocompatibility class 11 (MHC-II) genes are coordinately regulated by conserved, upstream promoter elements that are bound cooperatively by cyclic AMP response element binding protein (CREB), regulatory factor X (RFX), and nuclear factor Y (NF-Y). These DNA-binding proteins serve as a scaffold for the transcriptional coactivator class 11 transactivator (CIITA). To determine how CREB interacts with RFX and CIITA, co-immunoprecipitations and reporter assays were performed using a variety of CREB mutants. These assays demonstrated that CREB interacted with CIITA and the RFX5 subunit of RFX through the C-terminal portion of CREB. This C-terminal portion of CREB was fully functional in MHC-II promoter reporter assays. Phosphorylation of CREB enhanced transcription from the reporter, but was not required for transcription. Phospho-CREB was found at the HLA-DRA promoter by chromatin immunoprecipitation, providing evidence for its role. Together, these data provide genetic and biochemical evidence of the specific associations between CREB and two elements of the MHC-II regulatory complex and of the role played by phosphorylated CREB at MHC-II promoters. (c) 2006 Elsevier Ltd. All rights reserved.

The class II major histocompatibility complex gene HLA-DRA is expressed in B cells, activated T lymphocytes, and in antigen-presenting cells. In addition, HLA-DRA gene expression is inducible in a variety of cell types by interferon-gamma (IFN-gamma). Here we show that the lymphoid-specific transcription factor Oct-2A plays a critical role in HLA-DRA gene expression in class II-positive B cell lines, and that the high mobility group protein (HMG) I/Y binds to multiple sites within the DRA promoter, including the Oct-2A binding site. Coexpression of HMG I/Y and Oct-2 in cell lines lacking Oct-2 results in high levels of HLA-DRA gene expression, and in vitro DNA-binding studies reveal that HMG I/Y stimulates Oct-2A binding to the HLA-DRA promoter. Thus, Oct-2A and HMG I/Y may synergize to activate HLA-DRA expression in B cells. By contrast, Oct-2A is not involved in the IFN-gamma induction of the HLA-DRA gene in HeLa cells, but antisense HMG I/Y dramatically decreases the level of induction. We conclude that distinct sets of transcription factors are involved in the two modes of HLA-DRA expression, and that HMG I/Y may be important for B cell-specific expression, and is essential for IFN-gamma induction.