A Minimal Ankyrin Promoter Linked to a Human γ-Globin Gene Demonstrates Erythroid Specific Copy Number Dependent Expression with Minimal Position or Enhancer Dependence in Transgenic Mice

Sabatino, Denise E.; Wong, Clara; Cline, Amanda P.; Pyle, Louise C.; Garrett, Lisa; Gallagher, Patrick G.; Bodine, David M.

doi:10.1074/jbc.m004043200

Cited by 43 publications

(58 citation statements)

References 35 publications

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“…This may result from certain transcription factors such as GATA-1 and BKLF, which activate minimal ankyrin-1 promoter (14) and are expressed in hepatoma cells (40). Moreover, in transgenic mice, the minimal ankyrin-1 promoter produced low levels of transcripts in both liver and lung (26). However, despite the robust mRNA levels, there was no detectable -globin protein expression in the non-erythroid cells, consistent with previous reports that IHK is erythroid-specific in both cell culture and in vivo (5).…”

Section: Discussionmentioning

confidence: 99%

Erythroid-Specific Expression of β-Globin by the Sleeping Beauty Transposon for Sickle Cell Disease

et al. 2007

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Sickle cell disease (SCD) results predominately from a single monogenic mutation that affects thousands of individuals worldwide. Gene therapy approaches have focused on using viral vectors to transfer wild-type -or γ-globin transgenes into hematopoietic stem cells for long-term expression of the recombinant globins. In this study, we investigated the use of a novel nonviral vector system, the Sleeping Beauty (SB) transposon (Tn) to insert a wild-type -globin expression cassette into the human genome for sustained expression of -globin. We initially constructed a -globin expression vector composed of the hybrid cytomegalovirus (CMV) enhancer chicken -actin promoter (CAGGS) and full-length -globin cDNA, as well as truncated forms lacking either the 3′ or 3′ and 5′ untranslated regions (UTRs), to optimize expression of -globin. -Globin with its 5′ UTR was efficiently expressed from its cDNA in K-562 cells induced with hemin. However, expression was constitutive and not erythroid-specific. We then constructed cis SB-Tn--globin plasmids using a minimal -globin gene driven by hybrid promoter IHK (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human RLCR, ankyrin-1 promoter), IH p (human ALAS2 intron 8 erythroid-specific enhancer, HS40 core element from human RLCR, -globin promoter), or HS3 p (HS3 core element from human LCR, -globin promoter) to establish erythroidspecific expression of -globin. Stable genomic insertion of the minimal gene and expression of the -globin transgene for >5 months at a level comparable to that of the endogenous γ-globin gene were achieved using a SB-Tn -globin cis construct. Interestingly, erythroid-specific expression of -globin driven by IHK was regulated primarily at the translational level, in contrast to post-transcriptional regulation in non-erythroid cells. The SB-Tn system is a promising nonviral vector for efficient genomic insertion conferring stable, persistent erythroid-specific expression of -globin.

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Section: Discussionmentioning

confidence: 99%

Erythroid-Specific Expression of β-Globin by the Sleeping Beauty Transposon for Sickle Cell Disease

et al. 2007

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“…This is in contrast to the erythroid promoters of the ankyrin, band 3, and ␤-spectrin genes, which direct significantly higher levels of expression at all stages of erythroid development (41)(42)(43)(44)(45). This observation was surprising as previous studies had demonstrated that the ␣-spectrin gene is expressed at high levels in erythroid cells and that the expression was controlled at the transcriptional level (22)(23)(24)(25).…”

Section: Discussionmentioning

confidence: 96%

Sequences Downstream of the Erythroid Promoter Are Required for High Level Expression of the Human α-Spectrin Gene

Wong

Lin

Forget

et al. 2004

Journal of Biological Chemistry

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␣-Spectrin is a membrane protein critical for the flexibility and stability of the erythrocyte. We are attempting to identify and characterize the molecular mechanisms controlling the erythroid-specific expression of the ␣-spectrin gene. Previously, we demonstrated that the core promoter of the human ␣-spectrin gene directed low levels of erythroid-specific expression only in the early stages of erythroid differentiation. We have now identified a region 3 of the core promoter that contains a DNase I hypersensitive site and directs high level, erythroid-specific expression in reporter gene/ transfection assays. In vitro DNase I footprinting and electrophoretic mobility shift assays identified two functional GATA-1 sites in this region. Both GATA-1 sites were required for full activity, suggesting that elements binding to each site interact in a combinatorial manner. This region did not demonstrate enhancer activity in any orientation or position relative to either the ␣-spectrin core promoter or the thymidine kinase promoter in reporter gene assays. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of this region and occupancy by GATA-1 and CBP (cAMP-response element-binding protein (CREB)-binding protein). These results demonstrate that a region 3 of the ␣-spectrin core promoter contains a GATA-1-dependent positive regulatory element that is required in its proper genomic orientation. This is an excellent candidate region for mutations associated with decreased ␣-spectrin gene expression in patients with hereditary spherocytosis and hereditary pyropoikilocytosis.Spectrin, the most abundant protein of the erythrocyte membrane skeleton, exists in the erythrocyte as a heterodimer of two homologous proteins, ␣-spectrin and ␤-spectrin (1, 2). ␣-and ␤-spectrin are composed primarily of 106-amino acid repeats that fold into three antiparallel ␣-helices connected by short non-helical segments (3-7). ␣␤-Spectrin heterodimers self-associate to form tetramers and higher order oligomers, forming a lattice-like structure that provides stability and deformability to the erythrocyte membrane (8 -12). In the erythrocyte, spectrin functions include maintenance of cellular shape, regulation of the lateral mobility of integral membrane proteins, and provision of structural support for the lipid bilayer (2, 13).Quantitative and qualitative disorders of spectrin have been associated with abnormalities of erythrocyte shape including hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis (12, 14 -19). Structural abnormalities of ␣ spectrin in the region of the ␣␤ self-association site are the most common defects associated with hereditary elliptocytosis and hereditary pyropoikilocytosis. However, in many patients with ␣-spectrin-linked hereditary spherocytosis and hereditary pyropoikilocytosis, the precise genetic defect(s) is unknown. Studies suggest that these patients have a defect in ␣-spectrin mRNA accumulation, which has been termed a "thalassemialike" defect...

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“…This promoter was chosen for its ability to provide erythroid specific expression with copy number dependence and minimal position dependence [41]. The transgenic mice are viable, fertile and appear phenotypically normal.…”

Section: Fetal Liver Erythroid Cells From Eng/znf Transgenic Mice Demmentioning

confidence: 99%

“…ENG/ZnF was cloned downstream of the minimal ankyrin promoter (−291 to −20) [41] to generate 573Eng transgenic mice as described [42]. Founder animals were identified by PCR analysis of genomic DNA and were crossed for developmental studies.…”

Section: Studies In Transgenic Micementioning

confidence: 99%

Altered regulation of β-like globin genes by a redesigned erythroid transcription factor

Manwani¹,

Galdass²,

Bieker³

2007

Experimental Hematology

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Objective-Targeted regulation of β-like globin genes was studied using designer zinc finger transcription factors containing the DNA binding domain of the red cell specific transcription factor EKLF fused to repression domains.Methods-Globin gene expression was analyzed after introduction of the modified transcription factors into cell lines, embryonic stem cells and transgenic mice.Results-As would be predicted, when introduced transiently into cells these transcription factors were effective in repressing the adult β-globin promoter CACCC element which is the natural target for EKLF. In murine erythroleukemia cells repression of the adult β-globin gene was accompanied by a reactivation of the endogenous embryonic βH1-globin gene. Studies in differentiated embryonic stem cells and transgenic mice confirmed the reactivation of embryonic gene expression during development.Conclusion-Our studies support a competition model for β-globin gene expression and underscore the importance of EKLF in the embryonic/fetal to adult globin switch. They also demonstrate the feasibility of designer zinc finger transcription factors in the study of transcriptional control mechanisms at the β-globin locus and as potential gene therapy agents for sickle cell disease and related hemoglobinopathies.

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A Minimal Ankyrin Promoter Linked to a Human γ-Globin Gene Demonstrates Erythroid Specific Copy Number Dependent Expression with Minimal Position or Enhancer Dependence in Transgenic Mice

Cited by 43 publications

References 35 publications

Erythroid-Specific Expression of β-Globin by the Sleeping Beauty Transposon for Sickle Cell Disease

Erythroid-Specific Expression of β-Globin by the Sleeping Beauty Transposon for Sickle Cell Disease

Sequences Downstream of the Erythroid Promoter Are Required for High Level Expression of the Human α-Spectrin Gene

Altered regulation of β-like globin genes by a redesigned erythroid transcription factor

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