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

Abstract

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 ␤-spectrin in mouse fetal liver cells. When linked to a human ␥-globin gene, two different AE1 promoters directed erythroidspecific expression of ␥-globin mRNA in 18 of 18 lines of transgenic mice. However, variegated expression of ␥-globin was observed in 14 of 18 lines. While there was a significant correlation between transgene copy number and the amount of ␥-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 5HS4 of the chicken ␤-globin cluster to the AE1/␥-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/␥-globin transgene mapped to the start site of the endogenous AE1 mRNA, and ␥-globin protein was expressed in 100% of erythrocytes in all lines. We conclude that the chicken ␤-globin 5HS4 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.

Mutation of a barrier insulator in the human ankyrin-1 gene is associated with hereditary spherocytosis

“…In the absence of intervening sequences, both HS2 and GATA-1 enhancers promoted the growth of numerous G418-resistant colonies ( Figure 3). As shown previously, the cHS4 insulator suppressed this activity (46). The ankyrin -282 to -101 5′HS region could not block the effects of either the mouse β-globin HS2 or GATA-1 enhancer elements ( Figure 3, A and B).…”

“…and mice have used barrier insulators to flank reporter genes to prevent gene silencing and reduce PEV in vivo (22,23,29,30,32,35,46,(56)(57)(58)(59)(60)(61). Based on the data above, we hypothesized that the -108/-153 spherocytosis-associated mutations disrupt the barrier activity of 5′HS, leading to perturbations in ankyrin gene expression in erythroid cells, and this defect would be corrected by flanking the mutant -108/-153 erythroid promoter/ γ-globin transgene with the well-defined cHS4 insulator ( Figure 8A; cHS4-296 mutant -108/-153/ A γ cHS4).…”

Prospects and implications of using chromatin insulators in gene therapy and transgenesis

“…The impact of insulator use in transgenic animals is to reduce the number of transgenic founders required in order to obtain animals with an appropriate expression level and tissue distribution of the transgene. (47)(48)(49)(50) An alternative use of insulators is related to the improvement of inducible transgene expression, which can be easily explained by the fact that insulator sequences contribute to the generation of an open chromatin context that allows much faster and direct regulatory factor recognition and action, with no necessity for extensive chromatin remodeling. (40,51) This becomes relevant, particularly for inducible systems in which gene expression needs to be highly controlled, but most importantly in the scenario where the gene product is toxic.…”

A 914-bp promoter is sufficient to reproduce the endogenous prolyl oligopeptidase gene localization in the mouse placenta if not subject to position effect

“…One problem of the study with transgenic mice is that the expression pattern of a transgene could be different between transgenic lines (Boyer et al, 1997;Grosveld et al, 1987;Jones et al, 1995;Frazar et al, 2003). This is usually because of position effect, which makes it difficult to interpret the results and to understand the genuine promoter activity.…”