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[Functional analysis of human A gamma-globin gene promoter with-173 T-->C mutation]
1National Laboratory of Medical Molecular Biology, Institute of Basic of Medical Sciences, CAMS and PUMC, Beijing 100005.
Objective:
To investigate the effect of the human A gamma-globin gene-173 T-->C mutation on the binding of transacting factors to the promoter and the activity of the promoter.
Methods:
Electrophoretic mobility shift assay and transient transfection assay were used in this study.
Results:
The A gamma-globin gene-173 T-->C mutation decreased the affinity of GATA-1 to the mutant promoter fragment (-201(-)-158) 96% (P < 0.01) and the binding of Oct-1 to the same DNA fragment 55% (P < 0.05). The activity of the mutant promoter was 2-fold (P < 0.05) as strong as that of the normal one in MELGM979 cells. The mutant and the normal promoters exhibited basically same activities in K562 and Hela cells.
Conclusions:
The -173 T-->C mutation decreased dramatically the binding of GATA-1, implicating that GA-TA-1 may act as a negative regulator of A gamma-globin gene in adults. The -173 T-->C mutation may enhance the activity of A gamma-globin gene promoter in the adult erythroid cell environment.