Full Activity From Human β-Globin Locus Control Region Transgenes Requires 5′HS1, Distal β-Globin Promoter, and 3′ β-Globin Sequences

Abstract

AbstractThe locus control region (LCR) activates high-level human β-globin transgene expression. LCR cassettes composed of 5′HS2-4 linked to the 815 bp β-globin proximal promoter do not express fully. Here, we show that LCR (5′HS2-4) β-globin transgenes that also contain either 5′HS1 or the distal promoter fail to express fully in single- and low-copy transgenic mice. In contrast, full expression is obtained in the presence of both 5′HS1 and the distal promoter. Nine factor binding sites were identified in 5′HS1, using in vitro DNaseI footprint and gel retardation assays, and these include a strong Sp1/Sp3 site, four GATA-1 sites, and two sites that encompass an ACTAAC motif. LCR (5′HS1-4) β-globin transgene constructs with the distal promoter deleted or replaced by spacer DNA show that specific distal promoter sequences are required for full expression. An LCR (5′HS1-4) transgene construct with truncated downstream β-globin gene sequences indicates that 3′ sequences also play an important role. These results show that full expression of the β-globin gene directed by the LCR requires 5′HS1, the distal β-globin promoter, and 3′ sequences, and has implications for gene therapy construct design and models of LCR activation.