Identification of a Novel Enhancer/Chromatin Opening Element Associated with High-Level gamma-Globin Gene Expression

The organization of the five beta-type globin genes on chromosome 11 reflects the timing of expression during erythroid cell development, with the embryonic epsilon-globin gene being located at the 5' end, followed by the two fetal gamma-globin genes, and with the adult beta- and delta-globin genes being located at the 3' end. Here, we functionally characterized a DNase I-hypersensitive site (HS) located 4 kb upstream of the G gamma-globin gene (HBG-4kb HS). This site is occupied by transcription factors USF1, USF2, EGR1, MafK, and NF-E2 in the human erythroleukemia cell line K562 and exhibits histone modifications typical for enhancers. We generated a synthetic zinc finger (ZF) DNA-binding domain targeting the HBG-4kb HS (HBG-4kb ZF). The HBG-4kb ZF interacted with the target site in vitro and in the context of cells with a high affinity and specificity. Direct delivery of the HBG-4kb ZF to K562 and primary human erythroid cells caused a reduction in gamma-globin gene expression which was associated with decreased binding of transcription factors and active histone marks at and downstream of the HS. The data demonstrate that the HBG-4kb HS is important for fetal globin production and suggest that it may act by opening chromatin in a directional manner.