Loading of DNA-Binding Factors to an Erythroid Enhancer

Abstract

The HS-40 enhancer is the major cis-acting regulatory element responsible for the developmental stage- and erythroid lineage-specific expression of the human α-like globin genes, the embryonic ζ and the adult α2/α/1. A model has been proposed in which competitive factor binding at one of the HS-40 motifs, 3′-NA, modulates the capability of HS-40 to activate the embryonic ζ-globin promoter. Furthermore, this modulation was thought to be mediated through configurational changes of the HS-40 enhanceosome during development. In this study, we have further investigated the molecular basis of this model. First, human erythroid K562 cells stably integrated with various HS-40 mutants cis linked to a human α-globin promoter-growth hormone hybrid gene were analyzed by genomic footprinting and expression analysis. By the assay, we demonstrate that factors bound at different motifs of HS-40 indeed act in concert to build a fully functional enhanceosome. Thus, modification of factor binding at a single motif could drastically change the configuration and function of the HS-40 enhanceosome. Second, a specific 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 ζ-globin promoter activity in erythroid cells. This derepression was hypothesized to be regulated through competitive binding of different nuclear factors, in particular AP1 and NF-E2, to the 3′-NA motif. By gel mobility shift 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 association constants of both NF-E2 and AP1 are similar to their interactions with the wild-type 3′-NA motif. However, the 3′-NA(II) mutation causes an approximately twofold reduction of the binding affinity of NF-E2 factor to the 3′-NA motif. This reduction of affinity could be accounted for by a twofold-higher rate of dissociation of the NF-E2–3′-NA(II) complex. Finally, we show by chromatin immunoprecipitation experiments that only binding of NF-E2, not AP1, could be detected in vivo in K562 cells around the HS-40 region. These data exclude a role for AP1 in the developmental regulation of the human α-globin locus via the 3′-NA motif of HS-40 in embryonic/fetal erythroid cells. Furthermore, extrapolation of the in vitro binding studies suggests that factors other than NF-E2, such as the small Maf homodimers, are likely involved in the regulation of the HS-40 function in vivo.

ACKNOWLEDGMENTS

The first three authors contributed equally to this work.

We thank Qingyi Zhang for HS-40 mutants used in the study. We also appreciate Volker Blank and Nancy Andrews' generosity in providing pMT2-p18.

K.R. was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. This research has been supported by grants from the Academia Sinica, the National Science Council, and National Health Research Institute of Taiwan, Republic of China, and by Public Health Service grant NIH DK 29800 to C.-K. J. S.