Functional importance of the cyclic AMP response element-like decamer motif in the CD8 alpha promoter.
Expression of the CD8 gene is highly regulated during lymphocyte differentiation and in a tissue-specific manner. We characterized the human CD8 alpha promoter region to determine whether tissue specificity resides within the promoter and to define important regulatory elements. A set of six fragments 5' of the CD8 alpha gene were linked to a luciferase reporter gene. The luciferase activity was then measured in a transient transfection assay. We found that CD8 alpha promoter activity can be detected from a 146-bp fragment upstream of the translation start site, but not from a 133-bp fragment. The cyclic AMP response element (CRE)-like site within the 10 bp from -143 to -133 is critical for promoter activity. Mutation of the CRE/decamer in the context of a 429-bp fragment causes loss of activity. Tissue specificity does not reside in the 146-bp fragment because this fragment directs expression in both T and non-T cell lines. Fragments longer than 146 bp are generally expressed less well in the cell lines suggesting the potential existence of negative regulatory elements upstream of -146. Using a CRE/decamer-containing oligomer as a probe in an electrophoresis mobility shift assay, three retarded bands formed by proteins binding to the DNA were detected using nuclear extracts from two T cell lines. Two of the three bands contain proteins of the CRE-binding protein (CREB)/activating transcription factor (ATF) family. Because the CRE-binding protein/activating transcription factor proteins play a role in the expression of many other T cell-specific genes, our work strengthens the hypothesis that the CRE motif is important for regulating the expression of T cell-specific genes.
Gao
M H
MH
Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06510.
Kavathas
P B
PB
D001483:Base Sequence; D001665:Binding Sites; D016827:CD8 Antigens / Q000235:genetics*; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D013601:T-Lymphocytes / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D016827:CD8 Antigens; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D000242:Cyclic AMP