Identification of two novel regulatory elements within the 5'-untranslated region of the human A gamma-globin gene.
Interaction between the stage selector element (SSE) in the proximal gamma-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the beta-globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5'-untranslated region of the gamma-gene also contributes to this competitive silencing of the beta-gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal gamma-promoter in the absence of the SSE, completely silencing a linked beta-promoter. Mutation of a 20-base pair sequence of the gamma-gene 5'-untranslated region (UTR) led to derepression of beta-promoter activity. A marked activation of gamma-promoter activity was also observed with this mutation, suggesting the presence of a repressor. Fine mutagenesis dissected these activities to different regions of the 5'-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5'-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor GATA-1. These results indicate that the 5'-UTR of the gamma-gene contains sequences that may be important for its transcriptional and developmental regulation.
Amrolia
P J
PJ
Division of Experimental Hematology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101, USA.
Cunningham
J M
JM
Ney
P
P
Nienhuis
A W
AW
Jane
S M
SM
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004274:DNA, Recombinant; D004742:Enhancer Elements, Genetic / Q000235:genetics; D015672:Erythroid Precursor Cells / Q000378:metabolism*; D005333:Fetus / Q000166:cytology; D005786:Gene Expression Regulation; D005914:Globins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D004274:DNA, Recombinant; D012097:Repressor Proteins; D005914:Globins