title: Inhibition of CD28 expression by oligonucleotide decoys to the regulatory element in exon 1 of the CD28 gene.
abstract: 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.
authors.affiliation: Immunology Laboratory, ICN Research Center, Costa Mesa, CA 92626, USA. rctam@icnpharm.com
authors.forename: R C
authors.initials: RC
authors.lastname: Tam
authors.forename: C J
authors.initials: CJ
authors.lastname: Lin
authors.forename: C
authors.initials: C
authors.lastname: Lim
authors.forename: B
authors.initials: B
authors.lastname: Pai
authors.forename: V
authors.initials: V
authors.lastname: Stoisavljevic
mesh_terms: D015703:Antigens, CD / Q000096:biosynthesis; D018106:CD28 Antigens / Q000096:biosynthesis* / Q000235:genetics*; D015815:Cell Adhesion Molecules / Q000096:biosynthesis; D004247:DNA / Q000378:metabolism; D005091:Exons / Q000276:immunology*; D006151:Guanosine / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D019169:Jurkat Cells; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000276:immunology*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000276:immunology; D019014:fas Receptor / Q000096:biosynthesis
chemical_list: D015703:Antigens, CD; D018106:CD28 Antigens; D015815:Cell Adhesion Molecules; D018960:Hyaluronan Receptors; C065289:ICAM2 protein, human; D009838:Oligodeoxyribonucleotides; D016271:Proto-Oncogene Proteins c-myc; D016329:Sp1 Transcription Factor; D013873:Thionucleotides; D019014:fas Receptor; D006151:Guanosine; D004247:DNA