LCR-regulated transgene expression levels depend on the Oct-1 site in the AT-rich region of β-globin intron-2

Abstract

Human β-globin transgenes regulated by the locus control region (LCR) express at all integration sites in transgenic mice. For such LCR activity at ectopic sites, the 5′HS3 element requires the presence of the AT-rich region (ATR) in β-globin intron-2. Here, we examine the dependence of 5′HS3 LCR activity on transcription factor binding sites in the ATR. In vitro DNasel footprint analysis and electrophoretic mobility shift assays of the ATR identified an inverted double Gata-1 site composed of 2 noncanonical sequences (GATT and GATG) and an Oct-1 consensus site. Mutant Oct-1, Gata-1, or double mutant sites were created in the ATR of the BGT50 construct composed of a 5′HS3 β/γ-globin hybrid transgene. Transgenes with double mutant sites expressed at all sites of integration, but mean expression levels in transgenic mice were reduced from 64% per copy (BGT50) to 37% (P < .05). Mutation of the inverted double Gata-1 site had no effect at 61% per copy expression levels. In contrast, mutation of the Oct-1 site alone reduced per-copy expression levels to 31% (P