TY - JOUR
T1 - Cloning and functional characterization of the 5'-regulatory region of the human CD86 gene
AU - Li, Jianfeng
AU - Colovai, Adriana I.
AU - Cortesini, Raffaello
AU - Suciu-Foca, Nicole
PY - 2000/5
Y1 - 2000/5
N2 - The induction of CD86 expression by IFN-γ on the surface of various antigen presenting cells has been previously reported. In order to understand the mechanisms by which the expression of the CD86 gene is regulated by IFN- γ at the transcriptional level, we have cloned and characterized the 5'- flanking region of the human CD86 gene. To functionally analyze the upstream regulatory region of the CD86 gene, a series of luciferase reporter gene constructs were prepared and used for transfection of cells from the monocytic line U937 and Raji B cell line. Under basal conditions, functional activity of these constructs was detected in Raji cells, which show high constitutive expression of the CD86 molecule, but not in U937 cells, which show low expression of CD86 in non-activated state. Induction of CD86 expression by stimulation of U937 cells with IFN-γ revealed the presence of two functional GAS (gamma-interferon activation site) elements. Gel mobility shift assays showed that these two GAS elements specifically bind an IFN-γ- induced transcriptional complex. The DNA-protein complex was supershifted by antibody to Stat1 α (signal transducer and activator of transcription), but not by antibodies to Stat 2, Stat 3 and Sp1, indicating that GAS elements interact with Stat1 α. Point mutations in the GAS elements prevented the formation of DNA-protein complex and significantly reduced the responsiveness of the reporter gene to IFN-γ. These findings suggest that two functional GAS elements within the human CD86 promoter play an important role in the induction of CD86 gene by binding to IFN-γ-induced Stat1 α. (C) 2000 American Society for Histocompatibility and Immunogenetics.
AB - The induction of CD86 expression by IFN-γ on the surface of various antigen presenting cells has been previously reported. In order to understand the mechanisms by which the expression of the CD86 gene is regulated by IFN- γ at the transcriptional level, we have cloned and characterized the 5'- flanking region of the human CD86 gene. To functionally analyze the upstream regulatory region of the CD86 gene, a series of luciferase reporter gene constructs were prepared and used for transfection of cells from the monocytic line U937 and Raji B cell line. Under basal conditions, functional activity of these constructs was detected in Raji cells, which show high constitutive expression of the CD86 molecule, but not in U937 cells, which show low expression of CD86 in non-activated state. Induction of CD86 expression by stimulation of U937 cells with IFN-γ revealed the presence of two functional GAS (gamma-interferon activation site) elements. Gel mobility shift assays showed that these two GAS elements specifically bind an IFN-γ- induced transcriptional complex. The DNA-protein complex was supershifted by antibody to Stat1 α (signal transducer and activator of transcription), but not by antibodies to Stat 2, Stat 3 and Sp1, indicating that GAS elements interact with Stat1 α. Point mutations in the GAS elements prevented the formation of DNA-protein complex and significantly reduced the responsiveness of the reporter gene to IFN-γ. These findings suggest that two functional GAS elements within the human CD86 promoter play an important role in the induction of CD86 gene by binding to IFN-γ-induced Stat1 α. (C) 2000 American Society for Histocompatibility and Immunogenetics.
KW - CD86 promoter
KW - Costimulatory molecules
KW - Cytokines
KW - Transcription factors
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U2 - 10.1016/S0198-8859(00)00099-9
DO - 10.1016/S0198-8859(00)00099-9
M3 - Article
C2 - 10773351
AN - SCOPUS:0034005749
SN - 0198-8859
VL - 61
SP - 486
EP - 498
JO - Human Immunology
JF - Human Immunology
IS - 5
ER -