Structural analysis and mapping of DNase I hypersensitivity of HS5 of the beta-globin locus control region

The beta-globin locus control region (LCR) is a cis regulatory element that is located in the 5' part of the locus and confers high-level erythroid li neage-specific and position-independent expression of the globin genes. The LCR is composed of five DNase I hypersensitive sites (HSs), four of which are formed in erythroid cells. The function of the 5'-most site, HS5, remai ns unknown. To gain insights into its function, mouse HS5 was cloned and se quenced. Comparison of the HS5 sequences of mouse, human, and galago reveal ed two extensively conserved regions, designated HS5A and HS5B. DNase I hyp ersensitivity mapping revealed that two hypersensitive sites are located wi thin the HS5A region (designated HS5A(major) and HSA(minor)), and two are l ocated within the HS5B region (HS5B(major), HS5B(minor)). The positions of each of these HSs colocalize with either GATA-1 or Ap1/NF-E2 motifs, sugges ting that these protein binding sites are implicated in the formation of HS 5. Gel retardation assays indicated that the Ap1/NF-E2 motifs identified in murine HS5A and HS5B interact with NF-E2 or similar proteins. Studies of p rimary murine cells showed that HS5 is formed in all hemopoietic tissues te sted (fetal liver, adult thymus, and spleen), indicating that this HS is no t erythroid lineage specific. HS5 was detected in murine brain but not in m urine kidney or adult liver, suggesting that this site is not ubiquitous. T he presence of GATA-1 and NF-E2 motifs (which are common features of the DN ase I hypersensitive sites of the LCR) suggests that the HS5 is organized i n a manner similar to that of the other HSs. Taken together, our results su ggest that HS5 is an inherent component of the beta-globin locus control re gion. (C) 1999 Academic Press.