Identification of a novel enhancer/chromatin opening element associated with high-level -globin gene expression

The organization of the five -type globin genes on chromosome 11 reflects the timing of expression during erythroid cell development, with the embryonic -globin gene being located at the 5= end, followed by the two fetal -globin genes, and with the adult - and -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-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 -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.