Sp1 functions in a chromatin-dependent manner to augment human alpha-globin promoter activity.
The 5' flanking region of the human alpha-globin gene is highly G + C rich and contains multiple copies of the consensus sequence for the Sp1 binding site. We investigated the role of this G + C-rich region in augmenting alpha-globin promoter activity in the presence of the far-upstream alpha-globin enhancer, HS-40. We show that in transiently transfected erythroid cells, deletion of the alpha-globin G + C-rich 5' flanking region has no effect on alpha-globin promoter activity. However, upon stable integration into chromatin, deletion of this region causes a nearly 90% decrease in promoter activity compared with expression from an alpha-globin promoter retaining this region. These results suggest that the alpha-globin G + C-rich 5' flanking region augments alpha-globin promoter activity in a chromatin-dependent manner. We further show that this G + C-rich region is required for the activation of alpha-globin gene expression during erythroid differentiation. Finally, we show by both footprint analysis and functional assays that the ability of the G + C-rich region to increase alpha-globin promoter activity from a stably integrated alpha-globin gene is mediated by its multiple binding sites for the transcription factor Sp1.
Pondel
M D
MD
Sir William Dunn School of Pathology, Chemical Pathology Unit, Oxford University, United Kingdom.
Murphy
S
S
Pearson
L
L
Craddock
C
C
Proudfoot
N J
NJ
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D016384:Consensus Sequence; D004742:Enhancer Elements, Genetic; D004912:Erythrocytes / Q000378:metabolism; D004920:Erythropoiesis / Q000235:genetics; D005914:Globins / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D014162:Transfection
D002843:Chromatin; D016329:Sp1 Transcription Factor; D005914:Globins