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 7

The mutant ankyrin-1 –108/–153 phenotype in vivo.

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The mutant ankyrin-1 –108/–153 phenotype in vivo. Mice with mutant –108/...
Mice with mutant –108/–153 ankyrin promoter transgenes. (A) Transgene. Transgenic mice were created with a mutant –108/–153 ankyrin erythroid promoter/human γ-globin reporter gene cassette (–296 mutant –108/–153/Aγ) and compared with previously described wild-type ankyrin erythroid promoter/human γ-globin reporter transgenic mice (10). (B) Detection of Aγ-globin mRNA in reticulocytes of transgenic mice. RNA from adult reticulocytes was hybridized to 32P-labeled antisense riboprobes for the human Aγ-globin gene (top band) and the mouse α-globin gene (lower band) and digested with RNase. Protected fragments were separated by PAGE followed by radiography. Transgenic lines are indicated by letters above the lanes corresponding to Table 2. (C) Correlation of transgene copy number with the levels of Aγ-globin mRNA. For comparison, as previously published (10), linear regression analysis of the transgene copy number with the corrected mRNA expression level was performed with wild-type –296 WT/Aγ mice and –296 mutant –108/–153/Aγ mice. There was a linear relationship in –296 WT/ Aγ mice, indicating copy number–dependent expression (10). There was no relationship in –296 mutant –108/–153/Aγ mice, indicating copy number–independent expression.