Expression-dependent perturbation of nucleosomal phases at HS2 of the human β-LCR: possible correlation with periodic bent DNA

Abstract

DNA bend sites appear periodically at average intervals of 680 bp, corresponding to a length of four nucleosomes, in the human ϵ-, β- and Gγ- Aγ-ψβ-globin gene regions. We found that the HS2 region flanked by two DNA bend sites accommodated five nucleosomes and they were regularly phased throughout the region with the exception of that located in the middle, which corresponded to the precise location of HS2 and included the binding site for NF-E2. There appeared to be several phases in this region in the reconstituted chromatin and in erythroid K562 cells where the globin genes are expressed, whereas only one phase was adopted in non-erythroid HeLa cells. Meanwhile, almost unique phases were adopted at the flanking bend sites in vitro as well as in vivo. Sequences of 30 bp containing the bend centers cloned into the vector alone showed identical nucleosomal phases to those observed with the in vitro and in vivo experiments and removing the bend sites caused disruption of the phases at the bend sites as well as those in their direct vicinity. Finally, the nucleosome in this HS2 region had an inhibitory effect on NF-E2 binding, although remodeling occurred with the nuclear extract from K562 cells in the presence of ATP. This suggests that HS2 is placed at a region of weak nucleosome phasing activity along with factor binding sites.