Regulation of the Human Alpha-Globin Genes by GKLF

EKLF and related Krueppel-like factors (KLFs) are variably implicated in the regulation of the ?- and ?-like globin genes. Prompted by the observation that four KLF sites are distributed in the human -globin promoter, we investigated if any of the ?-globin cluster regulating KLFs could also act to modulate the expression of the -globin genes. We found that, among the globin regulating KLFs (EKLF, LKLF, BKLF, GKLF, KLF6, FKLF and FKLF2), only GKLF and BKLF bound specifically to three out of four KLF sites. In K562 cells, over-expressed GKLF transactivated at high levels a -globin-luciferase reporter and its action was impaired by point mutations of the KLF sites that disrupted GKLFDNA binding. In K562 cells stably transfected with a Tet-off regulated GKLF expression cassette, GKLF induction stimulated the expression of the endogenous -globin genes. In a complementary assay in K562 cells, knocking down GKLF expression with small interfering RNAs caused a parallel decrease in the transcription of the -globin genes. All experiments combined support a main regulatory role of GKLF in the control of -globin gene expression.