Interaction of Sp1 with the human gamma globin promoter: binding and transactivation of normal and mutant promoters.
We have analyzed the binding of Sp1, a ubiquitously expressed transactivator, to the promoter region of the gamma genes. Low-affinity Sp1 sites were found at -50 and -200. A high-affinity site was detected at -140, over the CACCC sequence. To analyze the function of these sites, Drosophila SL-2 cells, which lack Sp1, were cotransfected with an Sp1 expression plasmid and gamma globin promoter-CAT constructs. In these assays, the gamma promoter was significantly stronger in the presence than in the absence of Sp1. Thus, the three Sp1 sites in the gamma promoter allow binding as well as transactivation of the promoter. The majority of this transactivation was due to the strong binding site at -140 because introduction of a point mutation at -144 (CACCC----AACCC) reduced Sp1-dependent promoter strength by 57%. Analysis of the -200 region suggested that in the wild-type promoter, Sp1 binding at this site contributes little to promoter strength. However, a point mutation (-198 T----C) associated with hereditary persistence of fetal hemoglobin (HPFH) dramatically increased the affinity of this site for Sp1 and significantly increased Sp1 dependent promoter strength in SL-2 cells. Three other point mutations associated with HPFH did not significantly affect the interaction of Sp1 with the -200 region.
Gumucio
D L
DL
Department of Internal Medicine, Howard Hughes Medical Institute, University of Michigan, Ann Arbor 48109-0650.
Rood
K L
KL
Blanchard-McQuate
K L
KL
Gray
T A
TA
Saulino
A
A
Collins
F S
FS
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003850:Deoxyribonuclease I; D004330:Drosophila / Q000235:genetics; D005914:Globins / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D015345:Oligonucleotide Probes; D016329:Sp1 Transcription Factor; D005914:Globins; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I