International Immunology, Vol 9, 1329-1338, Copyright © 1997 by Oxford University Press
N Takemoto, N Koyano-Nakagawa, N Arai, K Arai and T Yokota
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.
ARTICLES
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
Department of Stem Cell Regulation, Institute of Medical Science, University of Tokyo, Japan.
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