A Negative Cis-element Regulates the Level of Enhancement by Hypersensitive Site 2 of the β-Globin Locus Control Region

The core of DNase hypersensitive site (HS) 2 from the β-globin locus control region is a potent enhancer of globin gene expression. Although it has been considered to contain only positive cis-regulatory sequences, our study of the enhancement conferred by segments of HS2 in erythroid cells reveals a novel negative element. Individual cis-regulatory elements from HS2 such as E boxes or Maf-response elements produced as great or greater enhancement than the intact core in mouse erythroleukemia (MEL) cells, indicating the presence of negative elements within HS2. A deletion series through HS2 revealed negative elements at the 5′ and 3′ ends of the core. Analysis of constructs with and without the 5′ negative element showed that the effect is exerted on the promoters of globin genes expressed at embryonic, fetal, or adult stages. The negative effect was observed in bipotential human cells (K562 and human erythroleukemia (HEL) cells), proerythroblastic mouse (MEL) cells, and normal adult human erythroid cells. The novel negative element also functions after stable integration into MEL chromosomes. Smaller deletions at the 5′ end of the HS2 core map the negative element within a 20-base pair region containing two conserved sequences.