Long-Range Enhancement of β-Globin Gene Expression Is Dependent on Gt Motifs Residing in the HS3 Core Element of the Locus Control Region.

Normal expression of the human β-globin genes is dependent on a powerful regulatory element residing upstream of the globin gene cluster referred as the locus control region (LCR), physically characterized by five DNAseI-hypersensitive sites (HS1 to HS5). Of particular interest is HS3, which is characterized by a 225 bp core element region containing seven GT motifs (GT1 to GT7) that alternate with four GATA-1 binding sites. The GT motifs are bound by a family of Kruppel-like factors and are important for proper expression of many housekeeping and tissue-specific genes. We previously demonstrated in transgenic mice carrying a 248 kb human β-globin yeast artificial chromosome in which the GT6 motif (GT6^m β-YAC) was mutated resulted in a decrease in ε- and γ-globin gene expression during embryonic erythropoiesis and a decrease in γ-globin expression during definitive erythropoiesis, thus, providing evidence that a single transcriptional motif distantly located can have profound effects on gene expression. We have used the same β-YAC transgenic mouse model system to analyze the function of the remaining six GT motifs of the HS3 core element. Currently we have produced transgenic mice carrying either a mutation of GT1/2 (GT1 and GT2 motifs overlap) or GT3. Total RNA was isolated from yolk sac, liver and blood samples of transgenic F₂ embryos at 12- and 14-day postconception and adult blood, and subjected to RNAse protection analysis. The GT1/2^m β-YAC transgenic mice exhibited normal ε- and γ-globin gene expression during embryonic erythropoiesis, but during definitive erythropoiesis γ-globin gene expression was reduced 4 to 5 fold in day 12- and day 14- fetal livers. In the two GT1/2^m β-YAC transgenic lines produced, β-globin gene expression was reduced and levels varied from 12.3% ± 2.9% to 63.2% ± 5.8% of endogenouse murine α-globin indicating that β-globin expression is strongly influenced by the position of integration of the transgene. In contrast, the GT3^m β-YAC transgenic mice the mutation to GT3 had no effect on globin gene expression during development. Our results suggest that multiple GT motifs within the same highly complex regulatory element contribute differently to the enhancement of the downstream genes.