Mutation of a barrier insulator in the human ankyrin-1 gene is associated with hereditary spherocytosis
Patrick G. Gallagher, … , Lisa J. Garrett, David M. Bodine
Patrick G. Gallagher, … , Lisa J. Garrett, David M. Bodine
Published November 22, 2010
Citation Information: J Clin Invest. 2010;120(12):4453-4465. https://doi.org/10.1172/JCI42240.
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Research Article

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

Abstract

Defects of the ankyrin-1 gene are the most common cause in humans of hereditary spherocytosis, an inherited anemia that affects patients of all ethnic groups. In some kindreds, linked –108/–153 nucleotide substitutions have been found in the upstream region of the ankyrin gene promoter that is active in erythroid cells. In vivo, the ankyrin erythroid promoter and its upstream region direct position-independent, uniform expression, a property of barrier insulators. Using human erythroid cell lines and primary cells and transgenic mice, here we have demonstrated that a region upstream of the erythroid promoter is a barrier insulator in vivo in erythroid cells. The region exhibited both functional and structural characteristics of a barrier, including prevention of gene silencing in an in vivo functional assay, appropriate chromatin configuration, and occupancy by barrier-associated proteins. Fragments with the –108/–153 spherocytosis-associated mutations failed to function as barrier insulators in vivo and demonstrated perturbations in barrier-associated chromatin configuration. In transgenic mice, flanking a mutant –108/–153 ankyrin gene promoter with the well-characterized chicken HS4 barrier insulator restored position-independent, uniform expression at levels comparable to wild-type. These data indicate that an upstream region of the ankyrin-1 erythroid promoter acts as a barrier insulator and identify disruption of the barrier element as a potential pathogenetic mechanism of human disease.

Authors

Patrick G. Gallagher, Laurie A. Steiner, Robert I. Liem, Ashley N. Owen, Amanda P. Cline, Nancy E. Seidel, Lisa J. Garrett, David M. Bodine

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Figure 8

Rescue of the mutant ankyrin-1 –108/–153 phenotype in vivo.

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Rescue of the mutant ankyrin-1 –108/–153 phenotype in vivo. The cHS4 bar...
The cHS4 barrier insulator rescues the mutant –108/–153 phenotype, restoring position-independent, copy number–dependent, uniform expression to the mutant –108/–153 ankyrin erythroid promoter transgene. (A) Transgenic mice with a mutant –108/–153 ankyrin erythroid promoter/human γ-globin reporter gene cassette flanked by the cHS4 insulator (cHS4-296 mutant –108/–153/Aγ cHS4). (B) Detection of Aγ-globin mRNA in reticulocytes of transgenic mice using 32P-labeled riboprobes for the Aγ-globin gene (top band) and the mouse α-globin gene (lower band). Transgenic lines are indicated with letters above the lanes corresponding to Table 3. (C) Correlation of transgene copy number with the levels of Aγ-globin mRNA. Linear regression analysis of transgene copy number with corrected mRNA expression level was performed with –296 WT/Aγ and cHS4-296 –108/–153/Aγ cHS4 mice. (D) Expression of γ-globin protein in erythrocytes of cHS4-296 mutant –108/–153/Aγ cHS4 transgenic mice using an FITC-conjugated antibody correlated with number of erythrocytes counted. Thick lines represent transgenic mice, and thin lines represent nontransgenic littermate controls. Single peaks represent uniform expression of γ-globin. Bimodal peaks represent nonuniform/variegated expression of γ-globin. Top panels show results from a control mouse with uniform (i.e., 100% of erythrocytes) γ-globin expression and a control with nonuniform/variegated expression, similar to that seen in –296 mutant –108/–153/Aγ transgenic mice (10). The bottom panels represent uniform expression in cHS4-296 mutant –108/–153/Aγ cHS4 transgenic mice. Representative transgenic lines are shown, with the letters corresponding to Table 3.