Lochamy Jonathan, Rogers Edward M, Boss Jeremy M
Department of Microbiology and Immunology, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322, USA.
Mol Immunol. 2007 Feb;44(5):837-47. doi: 10.1016/j.molimm.2006.04.004. Epub 2006 May 24.
Major histocompatibility class II (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 II 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.
主要组织相容性复合体II类（MHC-II）基因由保守的上游启动子元件协同调控，这些元件由环磷酸腺苷反应元件结合蛋白（CREB）、调节因子X（RFX）和核因子Y（NF-Y）协同结合。这些DNA结合蛋白作为转录共激活因子II类反式激活因子（CIITA）的支架。为了确定CREB如何与RFX和CIITA相互作用，使用多种CREB突变体进行了共免疫沉淀和报告基因分析。这些分析表明，CREB通过其C末端部分与CIITA和RFX的RFX5亚基相互作用。CREB的这一C末端部分在MHC-II启动子报告基因分析中具有完全功能。CREB的磷酸化增强了报告基因的转录，但转录并不需要磷酸化。通过染色质免疫沉淀在HLA-DRA启动子处发现了磷酸化CREB，为其作用提供了证据。总之，这些数据提供了遗传和生化证据，证明了CREB与MHC-II调节复合体的两个元件之间的特异性关联以及磷酸化CREB在MHC-II启动子处发挥的作用。
