Variegated Expression from the Murine Band 3 (AE1) Promoter in Transgenic Mice Is Associated with mRNA Transcript Initiation at Upstream Start Sites and Can Be Suppressed by the Addition of the Chicken β-Globin 5′ HS4 Insulator Element

The anion exchanger protein 1 (AE1; band 3) is an abundant erythrocyte transmembrane protein that regulates chloride-bicarbonate exchange and provides an attachment site for the erythrocyte membrane skeleton on the cytoplasmic domain. We analyzed the function of the erythroid AE1 gene promoter by using run-on transcription, RNase protection, transient transfection, and transgenic mouse assays. AE1 mRNA was transcribed at a higher level and maintained at a higher steady-state level than either ankyrin or β-spectrin in mouse fetal liver cells. When linked to a human γ-globin gene, two different AE1 promoters directed erythroid-specific expression of γ-globin mRNA in 18 of 18 lines of transgenic mice. However, variegated expression of γ-globin was observed in 14 of 18 lines. While there was a significant correlation between transgene copy number and the amount of γ-globin mRNA in all 18 lines, the transgene mRNAs initiated upstream of the start site of the endogenous AE1 mRNA. Addition of the insulator element from 5′HS4 of the chicken β-globin cluster to the AE1/γ-globin transgene allowed position-independent, copy-number-dependent expression at levels similar to the AE1 transcription rate in six of six lines of transgenic mice. The mRNA from the insulated AE1/γ-globin transgene mapped to the start site of the endogenous AE1 mRNA, and γ-globin protein was expressed in 100% of erythrocytes in all lines. We conclude that the chicken β-globin 5′HS4 element is necessary for full function of the AE1 promoter and that position effect variegation is associated with RNA transcription from the upstream start sites