Nuclear protein-binding sites in a transcriptional control region of the rabbit alpha-globin gene.
The 5'-flanking and internal regions of the rabbit alpha-globin gene, which constitute a CpG island, are required for enhancer-independent expression in transfected cells. In this study, electrophoretic mobility shift assays revealed that a battery of nuclear proteins from both erythroid and nonerythroid cells bind specifically to these regulatory regions. Assays based on exonuclease III digestion, methylation interference, and DNase I footprinting identified sequences bound by proteins in crude nuclear extracts and by purified transcription factor Sp1. In the 5' flank, recognition sites for the transcription factors alpha-IRP (positions -53 to -44 relative to the cap site), CP1 (-73 to -69), and Sp1 (-95 to -90) are bound by proteins in K562 cell nuclear extracts, as are three extended upstream regions. Two recognition sites for Sp1 in intron 1 are also bound both by proteins in crude nuclear extracts and by purified Sp1. The sequences CCAC in intron 2 and C5 in the 3'-untranslated region also bind proteins. A major binding site found in exon 1, TATGGCGC, matches in sequence and methylation interference pattern the binding site for nuclear protein YY1, and binding is inhibited through competition by YY1-specific oligonucleotides. The protein-binding sites flanking and internal to the rabbit alpha-globin gene may form an extended promoter.
Yost
S E
SE
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Shewchuk
B
B
Hardison
R
R
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D005786:Gene Expression Regulation; D005796:Genes; D005914:Globins / Q000235:genetics*; D006041:Goats; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011817:Rabbits; D016415:Sequence Alignment; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D016329:Sp1 Transcription Factor; D005914:Globins