Amanda P. Cline

Substitution of the Human β-Spectrin Promoter for the Human Aγ-Globin Promoter Prevents Silencing of a Linked Human β-Globin Gene in Transgenic Mice

ABSTRACT During development, changes occur in both the sites of erythropoiesis and the globin genes expressed at each developmental stage. Previous work has shown that high-level expression of human β-like globin genes in transgenic mice requires the presence of the locus control region (LCR). Models of hemoglobin switching propose that the LCR and/or stage-specific elements interact with globin gene sequences to activate specific genes in erythroid cells. To test these models, we generated transgenic mice which contain the human Aγ-globin gene linked to a 576-bp fragment containing the human β-spectrin promoter. In these mice, the β-spectrin Aγ-globin (βsp/Aγ) transgene was expressed at high levels in erythroid cells throughout development. Transgenic mice containing a 40-kb cosmid construct with the micro-LCR, βsp/Aγ-, ψβ-, δ-, and β-globin genes showed no developmental switching and expressed both human γ- and β-globin mRNAs in erythroid cells throughout development. Mice containing control cosmids with the Aγ-globin gene promoter showed developmental switching and expressed Aγ-globin mRNA in yolk sac and fetal liver erythroid cells and β-globin mRNA in fetal liver and adult erythroid cells. Our results suggest that replacement of the γ-globin promoter with the β-spectrin promoter allows the expression of the β-globin gene. We conclude that the γ-globin promoter is necessary and sufficient to suppress the expression of the β-globin gene in yolk sac erythroid cells.

A human beta-spectrin gene promoter directs high level expression in erythroid but not muscle or neural cells.

β-Spectrin is an erythrocyte membrane protein that is defective in many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. It is expressed not only in erythroid tissues but also in muscle and brain. We wished to determine the regulatory elements that determine the tissue-specific expression of the β-spectrin gene. We mapped the 5′-end of the β-spectrin erythroid cDNA and cloned the 5′-flanking genomic DNA containing the putative β-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, a β-spectrin gene erythroid promoter with two binding sites for GATA-1 and one site for CACCC-related proteins was identified. All three binding sites were required for full promoter activity; one of the GATA-1 motifs and the CACCC-binding motif were essential for activity. The β-spectrin gene promoter was able to be transactivated in heterologous cells by forced expression of GATA-1. In transgenic mice, a reporter gene directed by the β-spectrin promoter was expressed in erythroid tissues at all stages of development. Only weak expression of the reporter gene was detected in muscle and brain tissue, suggesting that additional regulatory elements are required for high level expression of the β-spectrin gene in these tissues.

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

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.

Variegated Expression from the Murine Band 3 (AE1) Promoter in Transgenic Mice Is Associated with mRNA Transcript Initiation at Upstream Start Sites and Can Be Suppressed by the Addition of the Chicken β-Globin 5′ HS4 Insulator Element

ABSTRACT The anion exchanger protein 1 (AE1; band 3) is an abundant erythrocyte transmembrane protein that regulates chloride-bicarbonate exchange and provides an attachment site for the erythrocyte membrane skeleton on the cytoplasmic domain. We analyzed the function of the erythroid AE1 gene promoter by using run-on transcription, RNase protection, transient transfection, and transgenic mouse assays. AE1 mRNA was transcribed at a higher level and maintained at a higher steady-state level than either ankyrin or β-spectrin in mouse fetal liver cells. When linked to a human γ-globin gene, two different AE1 promoters directed erythroid-specific expression of γ-globin mRNA in 18 of 18 lines of transgenic mice. However, variegated expression of γ-globin was observed in 14 of 18 lines. While there was a significant correlation between transgene copy number and the amount of γ-globin mRNA in all 18 lines, the transgene mRNAs initiated upstream of the start site of the endogenous AE1 mRNA. Addition of the insulator element from 5′HS4 of the chicken β-globin cluster to the AE1/γ-globin transgene allowed position-independent, copy-number-dependent expression at levels similar to the AE1 transcription rate in six of six lines of transgenic mice. The mRNA from the insulated AE1/γ-globin transgene mapped to the start site of the endogenous AE1 mRNA, and γ-globin protein was expressed in 100% of erythrocytes in all lines. We conclude that the chicken β-globin 5′HS4 element is necessary for full function of the AE1 promoter and that position effect variegation is associated with RNA transcription from the upstream start sites.

Improved Amphotropic Retrovirus‐Mediated Gene Transfer into Hematopoietic Stem Cells

Abstract: The efficiency of amphotropic retrovirus‐mediated gene transfer into human Hematopoietic Stem Cells (HSC) is less than 1%. This has impeded gene therapy for hematopoietic diseases. 1–3 In this study we demonstrate that populations of mouse and human HSC contain low to undetectable levels of the amphotropic virus receptor mRNA (ampho R mRNA), and are resistant to transduction with amphotropic retroviral vectors. In a subpopulation of mouse HSC expressing 7‐fold higher levels of ampho R mRNA, transduction with amphotropic retrovirus vectors was 30‐fold higher. We conclude that retrovirus transduction of HSC correlates with ampho R mRNA levels. Our results predict that alternative sources of HSC or retroviruses will be required for human gene therapy of hematopoietic diseases. One alternative source of stem cells is from individuals treated with cytokines. We have previously shown that mice treated with G‐CSF and SCF have an immediate increase in peripheral blood HSC immediately after treatment, followed by a 10‐fold increase in bone marrow HSC 14 days after treatment. 4 In this report we show that when rhesus monkey bone marrow cells collected 14 days after G‐CSF and SCF treatment were transduced with amphotropic retroviruses, gene transfer levels were approximately 10%, which was easily detected by Southern blot analysis. We conclude that the increased gene transfer may be the result of increased expression of the amphotropic retrovirus receptor, increased numbers of cycling HSC or both.

An 11-amino acid β-hairpin loop in the cytoplasmic domain of band 3 is responsible for ankyrin binding in mouse erythrocytes

The best-studied cytoskeletal system is the inner surface of the erythrocyte membrane, which provides an erythrocyte with the structural support needed to be stable yet flexible as it passes through the circulation. Current structural models predict that the spectrin–actin-based cytoskeletal network is attached to the plasma membrane through interactions of the protein ankyrin, which binds to both spectrin and the cytoplasmic domain of the transmembrane protein band 3. The crystal structure of the cytoplasmic domain of band 3 predicted that the ankyrin binding site was located on a β-hairpin loop in the cytoplasmic domain. In vitro, deletion of this loop eliminated ankyrin affinity for band 3 without affecting any other protein–band 3 interaction. To evaluate the importance of the ankyrin–band 3 linkage to membrane properties in vivo, we generated mice with the nucleotides encoding the 11-aa β-hairpin loop in the mouse Slc4a1 gene replaced with sequence encoding a diglycine bridge. Mice homozygous for the loop deletion were viable with mildly spherocytic and osmotically fragile erythrocytes. In vitro, homozygous ld/ld erythrocytes were incapable of binding ankyrin, but contrary to all previous predictions, abolishing the ankyrin–band 3 linkage destabilized the erythrocyte membrane to a lesser degree than complete deficiencies of either band 3 or ankyrin. Our data indicate that as yet uncharacterized interactions between other membrane proteins must significantly contribute to linkage of the spectrin–actin-based membrane cytoskeleton to the plasma membrane.

Development of a Stable Retrovirus Vector Capable of Long‐Term Expression of γ‐Globin mRNA in Mouse Erythrocytes

Abstract: Gene therapy for patients with hemoglobin disorders such has been hampered by the inability of retrovirus vectors to transfer globin genes and the locus control region (LCR) into hematopoietic stem cells without rearrangement. In addition, the expression from intact globin gene vectors has been variable in red blood cells as a result of position effects and retrovirus silencing. We hypothesized that by substituting the globin gene promoter for the promoter of another gene expressed in red blood cells, we could generate stable retrovirus vectors that would express globin at sufficient levels to treat hemoglobinopathies. Transgenic mice containing the human ankyrin (Ank) gene promoter fused to the human γ‐globin gene showed position‐independent, copy number‐dependent expression of a linked γ‐globin mRNA. We generated a “double‐copy” Ank/Aγ‐globin retrovirus vector that transferred two copies of the Ank/Aγ‐globin gene into target cells. Stable gene transfer was observed in primary primary mouse progenitor cells and long‐term repopulating hematopoietic stem cells. Expression of Ank/Aγ‐globin mRNA in mature red blood cells was approximately 8% of the level of mouse α‐globin mRNA. We conclude that this novel retrovirus vector may be valuable for treating a variety of hemoglobinopathies by gene therapy if the level of expression can be further increased.

Isolation of Stem Cell-Specific cDNAs from Hematopoietic Stem Cell Populations

Abstract: We have begun to isolate gene sequences that are specifically expressed in hematopoietic stem cells (HSCs). There are at least three fundamental requirements for the isolation of HSC‐specific transcripts. First, highly enriched populations of HSCs, and an HSC‐depleted cell population for comparison must be isolated. Secondly, the gene isolation procedures must be adapted to accommodate the small amounts of RNA obtained from purified HSCs. Finally, a defined screening strategy must be developed to focus on sequences to be examined in more detail. In this report, we describe the characterization of populations of HSCs that are highly enriched (Lin− c‐kitHI) or depleted (Lin− c‐kitNEG) of HSCs. We compared two methods for gene isolation, differential display polymerase chain reaction (DD‐PCR) and subtractive hybridization (SH), and found that the latter was more powerful and efficient in our hands. Lastly we describe the strategy that we have developed to screen clones for further study.