Mouse ferritin H subunit gene. Functional analysis of the promoter and identification of an upstream regulatory element active in erythroid cells.

Abstract Ankyrin forms the bridge between the spectrin/actin network of the erythrocyte membrane skeleton and the red cell membrane by binding to both β-spectrin and band 3. The erythrocyte ankyrin promoter (Ank-1E) is active only in erythroid cells, while two other Ank-1 promoters located 20 kb downstream and 40 kb upstream of Ank-1E are active in the cerebellum and muscle cells respectively. We have been studying the mechanism by which the Ank-1E promoter becomes active in erythroid cells by studying the cis acting regulatory elements and the chromatin structure of the Ank-1 promoter region. We have previously shown that the sequences between −296 and −15 of the Ank-1E promoter are fully sufficient for erythroid specific, copy number dependent uniform expression of reporter genes in transgenic mice. We have also mapped a DNase I Hypersensitive site (5′HS) between −300 and −100 of the human and mouse Ank-1E promoters in human K562 and mouse fetal liver cells. Both the mouse and human 5′HS are capable of preventing the silencing of a β-globin/GFP reporter gene in K562 cells, establishing that they function as barrier elements. Consistent with this observation, the human and mouse 5′HS are hyperacetylated in erythroid cells. The chromatin 10 kb 5′ to the 5′HS is DNase I resistant (associated with inactive chromatin) in human and mouse erythroid and non-erythroid cells. Approximately 6 kb 3′ to 5′HS are two adjacent HS (3′HS1, 3′HS2). Beyond 3′HS2 the chromatin is also DNase I resistant in both human and mouse erythroid and non-erythroid cells. Between 5′HS and 3′HS1 the 6kb region is DNase I sensitive (active) in erythroid cells but not in other cell types. We hypothesized that this 6 kb region contains regulatory elements that activate the Ank-1E promoter. To screen for regulatory elements we isolated overlapping segments of a 10 kb region extending from 2 kb upstream of 5′HS to 2 kb downstream of 3′HS2. We inserted these fragments into a plasmid vector containing the Ank-1E promoter linked to a luciferase reporter gene and transfected these constructs into K562 cells. A single region up regulated Ank-1E/luciferase expression. This region mapped to a 211bp segment that included 3′HS1, but did not include 3′HS2. A fragment containing only 3′HS2 did not up regulate an Ank-1E/luciferase reporter gene, but 3′HS2 was capable of preventing the silencing of a β-globin/Green Fluorescent Protein reporter gene in K562 cells, demonstrating barrier activity. The region around 3′HS1 and 2 was also a site of histone hyperacetylation. The sequence of the 211 bp fragment containing 3′HS1 does not contain consensus sequences for any known erythroid-specific transcription factors, but does contain potential binding sites fro Sp1, AP-1 and E-box binding proteins. Using the Chromatin Conformation Capture assay we demonstrated that 5′HS and 3′HS1 and 2 are in close proximity in K562 chromatin, but are not closely associated in chromatin from other cell types. We propose that an erythroid-specific chromatin loop brings 3′HS1 and 2 into proximity with 5′HS, adjacent to the Ank-1E promoter. This interaction translocates the positive regulatory element in 3′HS1 to the Ank-1E promoter allowing the Ank-1E promoter to become active in erythroid cells.

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High-Level Globin Gene Expression Mediated by a Recombinant Adeno-Associated Virus Genome That Contains the 3′ γ Globin Gene Regulatory Element and Integrates as Tandem Copies in Erythroid Cells

Blood ◽

10.1182/blood.v89.6.2167 ◽

1997 ◽

Vol 89 (6) ◽

pp. 2167-2175 ◽

Cited By ~ 47

Author(s):

Phillip W. Hargrove ◽

Elio F. Vanin ◽

Gary J. Kurtzman ◽

Arthur W. Nienhuis

Keyword(s):

Gene Expression ◽

Regulatory Element ◽

Globin Gene ◽

Functional Expression ◽

Single Copy ◽

Erythroid Cells ◽

Erythroid Progenitors ◽

Adeno Associated Virus ◽

Variable Expression ◽

In Erythroid Cells

Abstract Recombinant adeno-associated virus (rAAV) vectors are being evaluated for gene therapy applications. Using purified rAAV containing a mutationally marked globin gene (Aγ*) and sites 2, 3, and 4 from the locus control region (rHS432Aγ*), but lacking a drug-resistance gene, we investigated the relationship between multiplicity of infection (MOI), gene expression, and unselected genome integration in erythroid cells. Most primary erythroid progenitors were transduced as reflected by Aγ* mRNA in mature colonies but only at an MOI of greater than 5 × 107. Using immortalized erythroleukemia cells as a model, we found that fewer than one half of the colonies that contained the Aγ* transcript had an integrated, intact rHS432Aγ* genome. rHS432Aγ* integrated as a single copy with expression at approximately 50% the level of an endogenous γ globin gene. A second vector, rHS32Aγ*3′RE, containing the regulatory element (RE) from 3′ to the chromosomal Aγ globin gene, integrated as an intact, tandem head to tail concatamer with a median copy number of 6 with variable expression per copy ranging from approximately onefold to threefold that of an endogenous γ globin gene. These results establish that purified rAAV can be used to achieve integration and functional expression of a globin gene in erythroid cells, but only when high MOIs are used.

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