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 e xpression in transfected cells. In this study, electrophoretic mobilit y shift assays revealed that a battery of nuclear proteins from both e rythroid and nonerythroid cells bind specifically to these regulatory regions. Assays based on exonuclease III digestion, methylation interf erence, and DNase I footprinting identified sequences bound by protein s in crude nuclear extracts and by purified transcription factor Spl. 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 extr acts, as are three extended upstream regions. Two recognition sites fo r Sp1 in intron 1 are also bound both by proteins in crude nuclear ext racts and by purified Sp1. The sequences CCAC in intron 2 and C-5 in t he 3'-untranslated region also bind proteins. A major binding site fou nd in exon 1, TATGGCGC, matches in sequence and methylation interferen ce pattern the binding site for nuclear protein YY1, and binding is in hibited through competition by YY1-specific oligonucleotides. The prot ein-binding sites flanking and internal to the rabbit alpha-globin gen e may form an extended promoter.