Loading of DNA-binding factors to an erythroid enhancer

The HS-40 enhancer is the major cis-acting regulatory element responsible f or the developmental stage- and erythroid lineage-specific expression of th e human alpha-like globin genes, the embryonic zeta and the adult alpha 2/a lpha/1. A model has been proposed in which competitive factor binding at on e of the HS-40 motifs, 3'-NA, modulates the capability of HS-40 to activate the embryonic zeta-globin promoter. Furthermore, this modulation was thoug ht to be mediated through configurational changes of the HS-40 enhanceosome during development. In this study, we have further investigated the molecu lar basis of this model. First, human erythroid K562 cells stably integrate d with various HS-40 mutants cis linked to a human ar-globin promoter-growt h hormone hybrid gene were analyzed by genomic footprinting and expression analysis. By the assay, we demonstrate that factors bound at different moti fs of HS-40 indeed act in concert to build a fully functional enhanceosome. Thus, modification of factor binding at a single motif could drastically c hange the configuration and function of the HS-40 enhanceosome. Second, a s pecific 1-bp, GC-->TA mutation in the 3'-NA motif of HS-40, 3'-NA(II), has been shown previously to cause significant derepression of the embryonic ze ta-globin promoter activity in erythroid cells. This derepression was hypot hesized to be regulated through competitive binding of different nuclear fa ctors, in particular AP1 and NF-E2, to the 3'-NA motif. By gel mobility shi ft and transient cotransfection assays, we now show that 3'-NA(II) mutation completely abolishes the binding of small MafK homodimer. Surprisingly, NF -E2 as well as AP1 can still bind to the 3'-NA(II) sequence. The associatio n constants of both NF-E2 and AP1 are similar to their interactions with th e wild-type 3'-NA motif. However, the 3'-NA(LI) mutation causes an approxim ately twofold reduction of the binding affinity of NF-E2 factor to the 3'-N A motif. This reduction of affinity could be accounted for by a twofold-hig her rate of dissociation of the NF-E2-3'-NA(II) complex. Finally, we show b y chromatin immunoprecipitation experiments that only binding of NF-E2, not AP1, could be detected in vivo in K562 cells around the HS-40 region. Thes e data exclude a role for AP1 in the developmental regulation of the human or-globin locus via the 3'-NA motif of HS-40 in embryonic/fetal erythroid c ells. Furthermore, extrapolation of the in vitro binding studies suggests t hat factors other than NF-E2, such as the small Maf homodimers, are likely involved in the regulation of the HS-40 function in vivo.