CD28 provides an essential costimulatory signal required for Ag-mediated T cell activation via the TCR. Although accumulating evidence exists for the signaling properties of CD28, less is known regarding the regulation of CD28 expression. In this study, we have identified a novel promoter element of CD28, CD28GR (GGGGAGGAGGGG), which is located between +181 and +192 in exon 1 of the CD28 gene. Mutations within the 12-bp CD28GR sequence abolished its transcriptional activity. CD28GR contains a Sp1/EGR-1 binding site, which was found to act as the predominant functional element for regulating CD28 gene expression in Jurkat cells. Exon 1/CD28GR-driven transcription in Jurkat cells was augmented by cotransfection with Sp1 or EGR-1 expression plasmid. Similar augmentation was also shown with pharmacologic activation. This study is the first to identify a regulatory element that is critical for conferring constitutive and activation-induced transcriptional activation of the CD28 gene. Furthermore, our results proposed potential involvement of Sp1 in regulating CD28 expression. The linkage between Sp1 and the expression of CD28 has important implications in how viral infections, such as HIV, can induce immunosuppression.

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Ligation of CD28 provides a costimulatory signal essential for Ag-mediated T cell activation via the TCR. Previously we demonstrated that inhibition of human and murine CD28 expression by a guanosine (G)-rich oligonucleotide (ODN), GR1, led to immunosuppression in vitro and in vivo. The bioactivity of GR1 was dependent on a G-rich DNA sequence motif consisting of two G tetrads separated by four nucleotides, (G4N4G4). We have shown recently that a G-rich region, designated CD28GR, in exon 1 of the CD28 gene is such a motif and is a positive regulatory element that binds the transcription factors Sp1 and EGR-1. Here we showed that the bioactivity of GR1 and the related GR2 correlated with the sequence-specific formation of distinct nuclear protein complexes and a high degree of ODN secondary structure. In addition, these ODN blocked transcription factor binding to CD28GR (also in a sequence-specific manner) and prevented CD28GR from driving transcription of a reporter gene. Interestingly, GR1 potently inhibited CD28, but not the expression of other Sp1- and EGR-1-regulated genes, an effect associated with lower Sp1 protein binding affinity of GR1 and GR2 compared with that of canonical Sp1 sites. These data show that DNA sequences that contain the G-rich sequence motif, G4N4G4, such as GR1 and GR2, can functionally mimic the regulatory protein binding ability of CD28GR. Thus, GR1 and GR2 act as molecular decoys to selectively interfere with transcriptional regulation of the CD28 gene.