Abstract
The interleukin-2 CD28 response element (CD28RE) acts as a composite enhancer, in conjunction with a 3'-12-O-tetradecanoylphorbol-13-acetate response element (TRE)-like element, to confer CD28 receptor-dependent inducibility to the interleukin-2 promoter in T-cells. When inserted as a single copy upstream of a basal promoter, this composite enhancer, termed the CD28RE-TRE, is both highly active and CD28-inducible in transactivation assays. A multicomponent nuclear protein complex that binds the CD28RE-TRE was isolated by DNA affinity chromatography from nuclear extracts of mitogen- and CD28 receptor-costimulated human T-cells. Immunological and biochemical analyses of this complex reveal the presence of c-Rel, ATF-1, and CREB2 as major DNA-binding components. Coexpression of c-Rel in combination with ATF-1, CREB2, or ATF-1/CREB2 leads to synergistic transactivation of a CD28RE-TRE reporter plasmid in quiescent Jurkat T-cells. Furthermore, CD28-dependent transactivation of the CD28RE-TRE is specifically inhibited by cAMP response element-binding protein (CREB) dominant-negative expression vectors. Moreover, mutant promoter constructs in which the internal 5'-CD28RE and 3'-TRE-like sequences have been topologically positioned 180 degrees out of phase with one another show loss of mitogen- and CD28-dependent inducibility. Finally, the addition of the CREB-binding transcriptional coactivator p300 leads to a dramatic CREB-dependent increase in both mitogen- and CD28-mediated transactivation of the CD28RE-TRE. These findings demonstrate that full physiological responsiveness to CD28 receptor stimulation in T-cells is dependent on topologically linked sequences within the CD28RE-TRE composite enhancer and provide strong support of a direct role for the CREB family of transcription factors and p300/CREB-binding protein coactivator proteins in cytokine gene induction during T-cell activation.
Highlights
A large part of these receptor-mediated responses are governed at the level of transcription and, are mediated by multiple transcription factors that regulate the expression of genes encoding various cytokines responsible for propagating the immune activated state
We propose that the CD28 response element (CD28RE)-TPA response element (TRE) sequence, which spans positions Ϫ174 to Ϫ146 in the interleukin-2 promoter, can act individually as a single enhancer element to promote the transcriptional activation of the IL-2 gene
The activity of the transfected interleukin-2 promoter is closely paralleled by the ionomycin/ PMA and anti-CD28 mAb inducibility of a reporter plasmid containing a single copy of the CD28RE-TRE linked to a basal promoter (Fig. 1B)
Summary
Vol 273, No 1, Issue of January 2, pp. 552–560, 1998 Printed in U.S.A. Coordinate Transactivation of the Interleukin-2 CD28 Response Element by c-Rel and ATF-1/CREB2*. The addition of the CREB-binding transcriptional coactivator p300 leads to a dramatic CREB-dependent increase in both mitogen- and CD28-mediated transactivation of the CD28RE-TRE These findings demonstrate that full physiological responsiveness to CD28 receptor stimulation in T-cells is dependent on topologically linked sequences within the CD28RE-TRE composite enhancer and provide strong support of a direct role for the CREB family of transcription factors and p300/CREB-binding protein coactivator proteins in cytokine gene induction during T-cell activation. We show that a near 100-fold superinduction of the CD28RE-TRE by enforced expression of the CREB-binding coactivator p300 is CREB-dependent These findings establish that the CD28RE-TRE enhancer functions as a composite element in response to CD28 costimulation and provide compelling evidence for a prominent and direct role for the CREB family of proteins and p300/CREB-binding protein in the transcriptional activation of interleukin-2
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