The -175T----C mutation increases promoter strength in erythroid cells: correlation with evolutionary conservation of binding sites for two trans-acting factors.
A point mutation at position -175 has been detected in Agamma as well as Ggamma globin genes in individuals with hereditary persistence of fetal hemoglobin (HPFH). To prove that this single point mutation results in increased promoter strength, we transfected erythroid and nonerythroid cell lines with constructs containing normal and mutant promoters linked to the bacterial chloramphenicol acetyl transferase (CAT) gene. Differences in transfection efficiency were controlled by cotransfection of pRSVgpt. In K562 erythroleukemia cells, the -175 HPFH promoter directed three- to fourfold more CAT activity than its wild type counterpart. However, in HeLa cells the two promoters were similar in strength. The -195 to -165 region of the gamma-globin promoter contains binding sites for two proteins: a ubiquitously distributed octamer binding protein, OBP, and the erythroid-specific protein, GF-1. We find that while the GF-1 binding site is highly conserved among related primate gamma-globin genes, the octamer binding site is not. The evolutionary conservation of GF-1 as well as its erythroid-specific distribution suggest that this protein is important in gamma-globin gene expression. A role for OBP in the regulation of gamma-globin, if any, must have arisen recently in primate evolution.
Department of Internal Medicine, University of Michigan, Ann Arbor 48109-0650.
D L
DL
Gumucio
W K
WK
Lockwood
J L
JL
Weber
A M
AM
Saulino
K
K
Delgrosso
S
S
Surrey
E
E
Schwartz
M
M
Goodman
F S
FS
Collins
D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D004268:DNA-Binding Proteins / Q000235:genetics; D005319:Fetal Hemoglobin / Q000235:genetics*; D005914:Globins / Q000235:genetics*; D006453:Hemoglobinopathies / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011323:Primates / Q000235:genetics; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D014157:Transcription Factors; D005914:Globins; D005319:Fetal Hemoglobin