Four P-like elements are required for optimal transcription of the mouse IL-4 gene: involvement of a distinct set of nuclear factor of activated T cells and activator protein-1 family proteins.
We previously identified the P sequence as a critical regulatory element of the human IL-4 promoter. In the mouse IL-4 promoter, there are five elements homologous to the human P sequence designated conserved lymphokine element 0 (CLE0), P, P2, P3 and P4. To characterize the role of these P-like elements and their binding factors in the native promoter, we did transient transfection and electrophoretic mobility shift assays (EMSA). Transfection of EL-4 cells with the IL-4 promoter-reporter constructs carrying mutated P-like elements showed that four P-like elements, CLE0, P, P2 and P4, but not P3, were required for optimal activation of the IL-4 promoter. EMSA showed that both constitutive and inducible complexes bound to CLE0, P, P2 and P4, whereas only a constitutive complex bound to P3. In competition and antibody supershift assays in EMSA, complexes formed with P or P2 proved to contain nuclear factor of activated T cells (NFAT) family proteins as major components. Activator protein (AP)-1 family proteins interacted with CLE0, P, P2 and P4. NFAT/AP-1 complex formed only with P and P2. Cross-competition assays among the P-like elements revealed element-specific and common complexes. Six tandem repeats of the P element linked to the SV40 promoter responded to phorbol 12-myristate 13-acetate, while that of other elements did not. It would thus appear that components of each P-like element-binding complexes are not identical and may coordinately contribute to transcriptional activity.
Takemoto
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N
Department of Stem Cell Regulation, Institute of Medical Science, University of Tokyo, Japan.
Koyano-Nakagawa
N
N
Arai
N
N
Arai
K
K
Yokota
T
T
D000818:Animals; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D018398:Homeodomain Proteins; D006801:Humans; D015847:Interleukin-4 / Q000235:genetics*; D008213:Lymphocyte Activation / Q000235:genetics*; D051379:Mice; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000276:immunology; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000276:immunology; D019253:Proto-Oncogene Proteins c-bcl-2; D012045:Regulatory Sequences, Nucleic Acid / Q000276:immunology*; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D016176:T-Lymphocyte Subsets / Q000276:immunology*; D013945:Thymoma; D018808:Transcription Factor AP-1 / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic / Q000276:immunology*; D014407:Tumor Cells, Cultured
C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D015847:Interleukin-4; D051818:Replication Protein C