title: 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.affiliation: Departments of Pediatrics, Yale University School of Medicine, New Haven, Connecticut, USA. patrick.gallagher@yale.edu
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Gallagher
authors.forename: Laurie A
authors.initials: LA
authors.lastname: Steiner
authors.forename: Robert I
authors.initials: RI
authors.lastname: Liem
authors.forename: Ashley N
authors.initials: AN
authors.lastname: Owen
authors.forename: Amanda P
authors.initials: AP
authors.lastname: Cline
authors.forename: Nancy E
authors.initials: NE
authors.lastname: Seidel
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Garrett
authors.forename: David M
authors.initials: DM
authors.lastname: Bodine
mesh_terms: D000818:Animals; D017487:Ankyrins / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D002843:Chromatin / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D003850:Deoxyribonuclease I; D041905:Erythroid Cells / Q000378:metabolism; D015870:Gene Expression; D020868:Gene Silencing; D006801:Humans; D038101:Insulator Elements; D020014:K562 Cells; D051379:Mice; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D050505:Mutant Proteins / Q000235:genetics*; D009154:Mutation; D011401:Promoter Regions, Genetic; D013103:Spherocytosis, Hereditary / Q000097:blood / Q000235:genetics*
chemical_list: C110202:ANK1 protein, human; D017487:Ankyrins; D002843:Chromatin; D017931:DNA Primers; D050505:Mutant Proteins; D003850:Deoxyribonuclease I