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 1

Mapping DNase I HSs in the erythroid promoter region of the ankyrin-1 locus in erythroid cells.

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Mapping DNase I HSs in the erythroid promoter region of the ankyrin-1 lo...
(A) The map indicates the position of ankyrin exon 1E, flanking restriction enzyme sites, and probes used in DNase I mapping. A close-up view of the BamHI fragment with appropriate restriction enzyme sites, the –108 and –153 mutations, and exon 1E is also shown. Corresponding coordinates are (GRCh37/hg19) BamHI 41657070; StuI 41656031; SacI 41655737; EagI 41655310; 153 mutation 41655209; –108 mutation 41655164; BmgBI 41655152; ATG. (B) Mapping 5′ of exon 1E. Nuclei from K562 cells were treated with increasing amounts of DNase I, digested with BamHI, and subjected to Southern blot analysis using the 5′ probe in A. This yielded the expected 3.5-kb BamHI fragment and a smaller, 2.0-kb fragment corresponding to a DNase I HS. (C) Fine mapping the 5′ HS. Human genomic DNA digested with BamHI (B) or with BamHI and the indicated second enzyme. DNA from DNase I–treated K562 nuclei was also digested with BamHI (B+DNase). These digests were subjected to Southern blot analysis using the 5′ probe in A. Dashed lines indicate the estimated upper and lower limits of 5′HS. The undigested parent band is indicated (PB). (D) Mapping 3′ of exon 1E. Nuclei from K562 cells were treated with increasing amounts of DNase I, digested with NsiI, and subjected to Southern blot analysis using the 3′ probe in A. This yielded the expected 5.3-kb NsiI fragment and two smaller, 1.8-kb and 1.6-kb fragments, corresponding to DNase I HSs.