Silencing of Agamma-globin gene expression during adult definitive erythropoiesis is mediated by GATA-1 binding

摘要

Autonomous silencing of gene expression is one mechanism operative in the control of human beta-like globin gene switching and is best exemplified by the epsilon-globin gene. Experiments using variously truncated Agamma-globin genes linked to LCR sequences suggested that a region of the Agamma-globin gene between -730 to -378 relative to the mRNA CAP site may function as an adult stage-specific silencer element. A marked copy of the Agamma-globin gene (Agammam-globin) was inserted between LCR 5'HS1 and the epilon-globin gene in a human beta-globin locus yeast artificial chromosome (Agammam 5' epsilon beta-YAC). The Agammam-globin gene was autonomously silenced in Agammam 5' epsilon beta-YAC transgenic mice, even in the absence of an adult beta-globin gene. A -730 to -378 deletion of the Agammam-globin gene was introduced into the Agammam 5' epsilon beta-YAC to produce a DELTA1s Agammam 5' epsilon beta-YAC. Transgenic lines-containing intact beta-globin loci expressed the DELTA1s Agm-globin gene in embryonic yolk sac, fetal liver, and adult blood. To further delineate the function of the DELTA1s fragment, transient transfection assays to test silencer function and protein-DNA interaction assays were performed. The Als fragment was found to act as a repressor of a constitutively active SV40 promoter in K562 cells. Electro-mobility shift assays and DNaseI footprinting analysis demonstrated GATA-1-binding at a site -570 bp upstream of the Agamma-globin CAP site. These data suggest that the -730 to - 378 Agamma-globin gene region contains a silencer element at the - 570 GATA site that binds a GATA-1 repressor complex during the adult stage of definitive erythropoiesis to silence expression of the Agamma-globin gene.