Erythroid Ankyrin Research Articles

Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

Analysis of the Mobilities of Band 3 Populations Associated with Ankyrin Protein and Junctional Complexes in Intact Murine Erythrocytes

Current models of the erythrocyte membrane depict three populations of band 3: (i) a population tethered to spectrin via ankyrin, (ii) a fraction attached to the spectrin-actin junctional complex via adducin, and (iii) a freely diffusing population. Because many studies of band 3 diffusion also distinguish three populations of the polypeptide, it has been speculated that the three populations envisioned in membrane models correspond to the three fractions observed in diffusion analyses. To test this hypothesis, we characterized band 3 diffusion by single-particle tracking in wild-type and ankyrin- and adducin-deficient erythrocytes. We report that ∼40% of total band 3 in wild-type murine erythrocytes is attached to ankyrin, whereas ∼33% is immobilized by adducin, and ∼27% is not attached to any cytoskeletal anchor. More detailed analyses reveal that mobilities of individual ankyrin- and adducin-tethered band 3 molecules are heterogeneous, varying by nearly 2 orders of magnitude and that there is considerable overlap in diffusion coefficients for adducin and ankyrin-tethered populations. Taken together, the data suggest that although the ankyrin- and adducin-immobilized band 3 can be monitored separately, significant heterogeneity still exists within each population, suggesting that structural and compositional properties likely vary considerably within each band 3 complex.

Ankyrin and band 3 differentially affect expression of membrane glycoproteins but are not required for erythroblast enucleation

During late stages of mammalian erythropoiesis the nucleus undergoes chromatin condensation, migration to the plasma membrane, and extrusion from the cytoplasm surrounded by a segment of plasma membrane. Since nuclear condensation occurs in all vertebrates, mammalian erythroid membrane and cytoskeleton proteins were implicated as playing important roles in mediating the movement and extrusion of the nucleus. Here we use erythroid ankyrin deficient and band 3 knockout mouse models to show that band 3, but not ankyrin, plays an important role in regulating the level of erythroid cell membrane proteins, as evidenced by decreased cell surface expression of glycophorin A in band 3 knockout mice. However, neither band 3 nor ankyrin are required for enucleation. These results demonstrate that mammalian erythroblast enucleation does not depend on the membrane integrity generated by the ankyrin-band 3 complex.

Insulator Elements in Erythrocyte Membrane Genes

Insulators are DNA sequences and associated binding proteins that establish and/or maintain the boundaries between euchromatin and heterochromatin. One type of insulator establishes chromatin domains to separate enhancers and promoters and prevent their interaction (enhancer-blocking insulators) whereas another type creates a barrier to protect against heterochromatin-mediated gene silencing (barrier insulators). In the well-characterized chicken beta-globin LCR 5′HS4 insulator, binding of CTCF mediates enhancer-blocking functions and USF proteins mediate barrier activity. Because varying transcripts of erythrocyte membrane protein genes are expressed in both erythroid and nonerythroid cells, we hypothesized that that insulator elements that bind USF and CTCF participate in their regulation. Advances in technology have permitted rapid identification of DNA sequences bound by transcription factors and other DNA-associated proteins on a genome-wide scale. Coupling chromatin immunoprecipitation to microarrays that contain genomic regions (ChIP-chip) is a high resolution technique available for mapping protein-DNA interactions in vivo. We used ChIP-chip to identify CTCF and USF factor binding sites with potential insulator function that regulate membrane protein gene expression in erythroid cells. ChIP was performed with K562 and HeLa cells using antibodies against CTCF, USF1, and USF2. DNA obtained from these IPs was hybridized to a custom NimbleGen high-density human genomic DNA microarray. Chip probes were ~50bp in length, Tm ≥76°C, tiled ~65bp apart. Regions of repetitive DNA excluded. The chip included 15 erythrocyte membrane protein genes, most encoding complex loci with multiple tissue-, cell-, and developmental stage-specific transcripts, including alpha spectrin, beta spectrin, ankyrin, spectrin, band 3, ƒnalpha adducin, beta adducin, gamma adducin, ICAM4, erythroid associated membrane protein, protein 4.1R, protein 4.2, dematin, beta actin, tropomodulin, and tropomyosin. Each gene plus 50 to 100kb of flanking DNA were included on the chip. Binding sites on the custom DNA array were identified using the Tamalpais peak calling algorithm using L1–L3 level of stringency (Genom Res 16:595, 2006) or Tilescope using a signal cutoff of 0.9 (Genome Biol 8:R81, 2007). In K562 chromatin, 117 sites of CTCF occupancy were identified. There were 49 sites in 5′ flanking DNA, 3 in promoters, 9 in coding sequence, 20 in introns, 1 in 3′ untranslated region, and 35 in 3′ flanking DNA. Sites of USF1 and USF2 occupancy were identified in 7/15 and 9/15 genes, respectively. 15 USF1 sites were identified; 10 in 5′ flanking DNA, 1 in a promoter (<500bp from transcription start site), and 4 in 3′ flanking DNA. 22 sites of USF2 occupancy were identified; 9 in 5′ flanking DNA, 1 in a promoter, 4 in introns, and 8 in 3′ flanking DNA. USF1 and USF2 frequently heterodimerize and 10 sites bound both USF1 and USF2. To validate ChIP-chip results, a site binding USF2 near the ankyrin gene erythroid promoter was further analyzed. This site was chosen because the USF2-associated sequence directs uniform, copy-number dependent expression of a linked reporter gene in transgenic mice, the erythroid ankyrin promoter is positioned between 2 alternate, nonerythroid ankyrin promoters, and DNaseI mapping revealed that this region is contained within an erythroid-specific chromatin domain. Quantitative ChIP confirmed USF2 occupancy, as well as USF1 occupancy, in this region. For functional analysis, we used a gene silencing/position-effect variegation (PEV) assay to determine whether this region possess barrier element function. Using a HS2-beta globin promoter-GFP cassette as negative control and a cHS4-HS2-beta globin promoter-GFP-cHS4 cassette as positive control, the ankyrin promoter region fragment (p<0.001) functioned as barrier elements in this PEV assay. These data demonstrate that there are numerous binding sites for insulator-associated DNA binding proteins in erythrocyte membrane protein genes and indicate that these elements likely play a significant role in the regulation of tissue-specific expression of many genes expressed in erythroid cells.

Molecular epitopes of the ankyrin-spectrin interaction.

Isoforms of ankyrin and its binding partner spectrin are responsible for a number of interactions in a variety of human cells. Conflicting evidence, however, had identified two different, non-overlapping human erythroid ankyrin subdomains, Zu5 and 272, as the minimum binding region for beta-spectrin. Complementary studies on the ankyrin-binding domain of spectrin have been somewhat more conclusive yet have not presented binding in terms of well-phased, integral numbers of spectrin repeats. Thus, the objective of this study was to clearly define and characterize the minimal ankyrin-spectrin binding epitopes. Circular dichroism (CD) wavelength spectra of the aforementioned ankyrin subdomains show that these fragments are 30-60% unstructured. In contrast, human erythroid beta-spectrin repeats 13, 14, 15, and 16 (prepared in all combinations of two adjacent repeats) demonstrated proper folding and stability as determined by CD and tryptophan wavelength and heat denaturation scans. Native polyacrylamide gel electrophoresis (PAGE) gel shifts as well as affinity pull-down assays implicated Zu5 and beta-spectrin repeats 14-15 as the minimum binding epitopes. These results were confirmed by analytical ultracentrifugation to sedimentation equilibrium by which a 1:1 complex was obtained if and only if Zu5 was mixed with beta-spectrin constructs containing repeats 14 and 15 in tandem. Surface plasmon resonance yielded a K D of 15.2 nM for binding of beta-spectrin fragments to the ankyrin subdomain Zu5, accounting for all of the binding observed between the intact molecules. Collectively, these results show the 14th and 15th beta-spectrin repeats comprise the minimal, phased region of beta-spectrin, which binds ankyrin at the Zu5 subdomain with high affinity.

Identification of a TFIID Binding Sequence That Increases Expression from the Ankyrin-1 Promoter: Application to Globin Gene Therapy

Effective Gene Therapy of the hemoglobin β-chain disorders β-thalassemia or Sickle Cell Disease (SCD), requires that viral vectors deliver β-like globin gene to hematopoietic stem cells (HSC) and express β-globin at levels >20% of that of endogenous α-globin. However, the β-globin gene is poorly expressed without sequences from the Locus Control Region (LCR). The LCR contains sequences that contribute to inefficient production and low titers of the recombinant virus, and pose a significant risk of insertional activation of leukemia genes. We have taken an alternative approach using enhancer independent promoters from genes expressed in erythrocytes to express sufficient β-like globin. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the four different tissue specific promoters used to express the ANK-1 gene. We have shown that a double copy of Moloney Leukemia Virus (MLV) vector in which the ANK-1E promoter linked to a γ-globin gene replaced the promoter and enhancer sequences in the MLV Long Terminal Repeat was produced at high titer. We have also demonstrated that in mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein were expressed at a uniform level of 7.5% of α-globin per vector copy. To obtain the 3–4 fold increase in γ-globin expression needed to reach therapeutic levels we have taken advantage of our recent demonstration that patients with a deletion of a TG dinucleotide in the transcribed region (+4 or +32 relative to the proximal or distal mRNA start sites) of the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID. We hypothesized that introducing sequences with higher TFIID binding affinity into the region of the ANK-1E promoter would result in increased level of ANK-1E/γ-globin transcription needed for an effective therapy for β-thalassemia and SCD. An ANK-1E promoter library was generated using degenerate sequence in the TFIID binding region while preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5′RACE and analyzed by sequencing. Four different sequences were obtained: wild type (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones ((G/T)(G/C)(G/C)GGTGAG) was found at two other locations in the ANK-1E promoter (−5 and −54 relative to the distal mRNA start site) as well as in 25% of 5′UTR across the human genome (p=2.2e−16). 64% of GC rich promoters contain the consensus sequence, which is enriched at +50 and −70 relative to the mRNA start site. ANK-1E promoters containing the novel sequences were linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCCGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels of the luciferase than the wild type promoter (p=0.001; 0.005 respectively). Electrophoretic mobility shift assays demonstrated that the two more active promoters bind more TFIID than the wild type promoter. We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.

Multiple Erythroid Ankyrin Isoforms in the Mouse Cerebellum: Differential Localization in Purkinje Cells.

The erythroid ankyrin gene (Ank1) produces a large and varied number of isoforms due to alternative splicing of the mRNA. In addition to expression in erythroid tissues, some of these Ank1 proteins are highly expressed in the Purkinje cells (PKC) of the mouse cerebellum. Mice deficient in Ank1 as a result of a mutation in the Ank1 gene (normoblastosis, nb) show a progressive loss of PKCs with an attendant ataxia. We have generated a panel of Ank1 antibodies to aid in sorting out the expression pattern and function of Ank1 proteins in the cerebellum. Two of these antibodies are specific to the alternatively spliced A and B COOH-terminal segments of Ank1. Immunohistochemical (IHC) experiments using these antibodies show strikingly different patterns of localization. Anti-C-termA (α-A) stains the PKC cell body and dendrites while anti-C-termB (α-B) is restricted to the PKC membrane. Both antibodies stain structures in the granule cell layer (GCL) including the granule cell membrane (α-B) and structures known as glomeruli where granule cell dendrites synapse with mossy fiber axons (α-A and α-B). Mossy fibers are a major afferent system that inputs to the cerebellum. α-A, α-B, antibodies to the α-1 subunit of Na+/K+ATPase (NaK-α1) and anti-Synapsin 1, a specific marker for synaptic vesicles, all co-localize in the glomeruli, suggesting a possible functional link. PKC membrane staining with α-B is absent in nb/nb cerebellum whereas PKC staining with α-A is unaffected. GCL staining with both antibodies is reduced in the mutant and this deficit may be important to PKC survival since granule cell axons are a major input system to PKC dendrites. Immunoblots stained with α-A and α-B are consistent with the IHC findings. In addition to the typical large isoforms (∼210kD) that are deficient in the nb mutant, immunoblots of cerebellar lysates reveal a number of small Ank1 related proteins ranging in size from 17 to 50 kD. The α-A and α-B banding patterns are unaffected by the nb mutation suggesting that they may be produced by splicing out the exon containing the nb mutation (E36) or by using an alternative promoter in the 3′ end of the gene as was found for the small Ank1 isoforms in skeletal muscle. Additional IHC findings using GFP-tagged PKC show a PKC axonopathy in nb/nb cerebellum. PKC axons exhibit multiple swellings that accumulate with age raising the possibility that axonal transport is abnormal in the nb PKCs. In summary 1) immunoblots reveal multiple previously undescribed small Ank1 isoforms in cerebellum, 2) two of the alternate Ank1 COOH-termini show very different localization in PKC suggesting distinct functions for the Ank1 proteins carrying them, 3) in the GCL, antibodies to the two COOH-termini co-localize with antibodies to the Na+/K+ATPase α-1 subunit in synaptic densities, 4) deficiencies of Ank1 in the GCL of nb/nb mice may influence PKC survival and 5) axonal transport may be affected in nb/nb PKC. These findings indicate that Ank1 proteins play a more varied role in the cerebellum than previously suspected and suggest new directions for the study of Ank1 function.

Identification of a Novel Core Promoter Element That Enhances Transcription: Application to Ankyrin Promoters for Globin Gene Therapy.

Gene Therapy for the hemoglobin β-chain disorders, Sickle Cell Disease (SCD) or beta thalassemia (β-thal), requires both efficient globin gene transfer into hematopoietic stem cells (HSC) as well as expression of globin mRNA and protein at levels that are >20% of the level of α-globin. Expression of the β-like globin genes in either transgenic mice or from RNA virus vectors integrated into the genome of erythroid cells requires sequences from the Locus Control Region (LCR). The LCR sequences contain cryptic splicing and polyadenylation sequences that contribute to inefficient production and low titers of recombinant virus particles. In addition, the powerful LCR enhancer elements pose a significant risk of insertional activation of leukemia genes. We hypothesized that enhancer-independent promoters from other genes expressed in erythrocytes could be used to express sufficient amounts of β-like globin to treat SCD or β-thal. We have focused on the erythroid ankyrin (ANK-1E) promoter, a compact GC-rich promoter with no conserved sequences that is one of the 4 different tissue-specific promoters used to express the ANK-1 gene. We have previously demonstrated that a “double copy” Moloney Leukemia Virus (MLV) vector in which the ANK-1E promoter linked to a γ-globin gene replaced the promoter and enhancer sequences in the MLV Long Terminal Repeat was produced at high titer. In mice repopulated with HSC transduced with the ANK-1E/γ-globin double copy vector, γ-globin mRNA and protein was expressed at uniform level of 7.5 % of α-globin per vector copy (Sabatino et al. PNAS 97:13294–9, 2000). To obtain the 3–4 fold increase in γ-globin expression needed to reach therapeutic levels we have taken advantage of our recent demonstration that patients with a deletion of a TG (−73/−72) dinucleotide in the ANK-1E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID (Gallagher et al. Hum Mol Gen 14:2501–9, 2005). We hypothesized that altering the sequence in the TFIID binding region of the ANK-1E promoter would increase TFIID binding, resulting in the increased level of ANK-1E/γ-globin transcription needed for an effective therapy for SCD and β-thal. We generated a library of ANK-1E promoters with degenerate sequence in the TFIID binding region, preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5′RACE and analyzed by sequencing. Four different sequences were obtained from the sequencing results: the wild type sequence (TGCGGTGAG), GGCGGTGAG, GCCGGTGAG and GGGGGTGAG. The consensus sequence derived from these clones: (T/G)(G/C)(G/C)GG(T/A)GAG was found in similar locations relative to the transcriptional start site in the three other ANK-1 promoter regions, as well as in 22% of >4000 human loci with GC-rich promoters that lack TATA boxes and other consensus promoter sequences. ANK-1E promoters containing the novel sequences were linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCCGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels of the luciferase mRNA and protein than the wild type promoter (p=0.001; 0.005 respectively). We are evaluating the ability of these promoters to direct higher levels of γ-globin expression in primary mouse erythroid cells.

Association of small ankyrin 1 with the sarcoplasmic reticulum

Small ankyrin 1, or sAnk1, is a small, alternatively spliced product of the erythroid ankyrin gene, ANK1, that is expressed in striated muscle and concentrated in the network sarcoplasmic reticulum (SR) surrounding the Z disks and M lines. We have characterized sAnk1 in muscle homogenates and SR vesicles, and have identified the region that targets it to the network SR. Selective extractions and partitioning into Triton X-114 show that sAnk1 behaves like the SR Ca-ATPase and so is an integral protein of the SR membrane. Mild proteolytic treatment of isolated SR vesicles indicates that sAnk1 is oriented with its hydrophilic, C-terminal sequence exposed to the solution, which is equivalent to the cytoplasmic face of the SR membrane in situ. SDS-PAGE in non-reducing gels suggests that sAnk1 is present as dimers and larger oligomers in the native SR. These results suggest that sAnk1 is oligomeric and oriented with its C-terminus exposed to the cytoplasm, where it may interact with proteins of the contractile apparatus. The N-terminal 29 amino acid hydrophobic sequence of sAnk1, which is predicted to span the SR membrane, is sufficient to target proteins to and anchor them in internal membranes of HEK 293 cells. It also targets reporter proteins to the network SR of skeletal myofibers and is thus the first example of a sequence that targets proteins to a particular compartment of the SR.

Defining the Active Chromatin Domain of the Erythroid Ankyrin Promoter.

Erythrocyte ankyrin forms the bridge between the spectrin/actin cytoskeleton and the red cell membrane protein band 3. Mutations resulting in defective or deficient ankyrin are the most common cause of Hereditary Spherocytosis. The erythroid ankyrin promoter (ankR) lies between two other ankyrin promoters, one that is active in neuronal cells and the other in many cell types. Active genes are generally found in DNase I sensitive regions of chromatin while regulatory sequences (promoters, enhancers, boundary elements) are associated with DNase I Hypersensitive Sites (HS). We hypothesized that the chromatin surrounding ankR determines its activity. In human K562 cells and mouse fetal liver cells a DNase I HS (5′HS) maps between −300 and −100 of ankR. 5′HS marks the end of a DNase I resistant chromatin extends at least 10 kb upstream, indicating that this chromatin is inactive in erythroid cells. We have previously shown that a 300 bp fragment containing ankR was sufficient to direct erythroid specific, copy number dependent, position independent and uniform expression of a linked γ-globin gene in transgenic mice. Identical results were obtained with mice containing −2.7 kb and −656 bp ankyrin promoters, as expected, and 5′ HS was present in fetal liver chromatin of all 8 lines examined. Downstream of 5′HS is a 5.7 kb DNase I sensitive region that includes a 500 bp enhancer-like element near the 3′ end. At the 3′ end of the DNase I sensitive domain are 2 HS (3′HS) that mark the beginning of a DNase I resistant region that extends at least 3 kb downstream. AnkR is inactive in non-erythroid HeLa, Jurkat and Sh-SY5Y (neuronal) cells. Although both 5′HS and 3′HS are present in these cells, the intervening 5.7 kb region is DNase I resistant indicating that erythroid specific factors are required for the activation of ankR. Boundary elements (insulators) are found between DNase I resistant and DNase I sensitive chromatin and have the ability to prevent the transgene silencing at ectopic sites in the genome. To test whether the 5′HS and 3′HS function as boundary elements, a β-globin promoter/EGFP transgene was flanked by 200 bp fragments containing either 5′HS or 3′HS. While 0/8 K562 cell lines with the transgene alone expressed GFP, GFP expression was observed in 12/12 5′HS and 11/12 3′HS K562 cell lines (χ2=12.0, p<0.001; χ2=9.0, p<0.01 respectively), indicating boundary function. We have previously shown that mutations at positions −153 and −108 in 5′HS associated with Hereditary Spherocytosis cause position dependent and variegated expression of the γ-globin transgene in mice. When the −153/−108 sequence was used to flank the β-globin/EGFP transgene gene silencing was observed in 5/10 K562 cell lines (χ2=5.7,p<0.02). Because boundary elements are sites of high levels of histone acetylase (HA) activity we hypothesized that the −153/−108 mutations disrupted histone acetylation in ankR. EMSA analysis showed that the −153/−108 mutations disrupted the binding of a complex that includes both CTCF (a zinc finger protein associated with boundary elements in the β-globin cluster) and Brg-1 (the ATP dependent subunit of the HA complex). Chomatin Immune Precipitation analysis of transfected K562 cells showed that histone acetylation in sequences containing the −153/−108 mutations was 4-fold less than in wild type sequences (p<0.02). These studies have defined the active chromatin domain of the ankR and have demonstrated that the −153/−108 mutations cause Hereditary Spherocytosis by inhibiting histone acetylation in ankR.

701. A SIN Lentiviral Vector That Allows In Vivo Drug-Selection of Hematopoietic Stem Cells yet Retains Erythroid-Specific Expression of the Ferrochelatase Gene and Results in Cure of Murine Protoporphyria

Erythropoietic Protoporphyria (EPP) is an inherited defect of the ferrochelatase (FC) gene characterized by accumulation of toxic protoporphyrin mainly in the bone marrow (BM) erythroid lineage. We previously described successful gene therapy of EPP mice with erythroid-specific (ES) SIN lentiviral vectors without pre-selection of corrected cells (Richard E et al Mol Ther 2001). However, high-level gene transfer in mice is not translatable to large animal models and humans if there is no selective advantage to genetically modified hematopoietic stem cells (HCSs) in vivo. We first developed a bipromoter vector, with erythroid specific human ankyrin promoter/HS-40 enhancer driving EGFP expression and the MND promoter driving the mutant methylguanine-methyltransferase (MGMT) expression (HAGMM vector). Although we achieved in vivo selection of GFP+ RBC in mice transplanted with HAGMM transduced cells, there was loss of ES. We next developed bipromoter-SIN-lentiviral vectors encoding EGFP/FC based on human PGK promoter driving MGMT expression (HAGPM/HAFPM). We hypothesized that these vectors would allow ubiquitous expression of MGMT and yet retain ES expression of EGFP/FC genes. We also developed bicistronic vectors with the MND promoter driving expression of both genes (EGFP/FC and MGMT linked with an IRES) as control. Normal or EPP HSCs were transduced with EGFP or FC-expressing bicistronic vectors, respectively, and transplanted into non-myeloablated (4 Gy irradiation) EPP mice. Following a low-level of engraftment into EPP mice, we were able to expand the transduced cell population in vivo by 3 cycles O6-benzylguanine (BG)/ BCNU administration, resulting in a 10-fold increase of normal or corrected EPP cells in the bone marrow (BM) and peripheral blood (PB) (8 ± 5 vs 87 ± 21% EGFP+ normal RBCs without or with BG/BCNU, respectively). Selection of EPP HSC transduced with the FC expressing vector led to 4 fold increase of BM FC activity as compared with normal mice [22 ± 14 vs. 5 ± 2 FC specific activity (SA)] and complete correction of the disease phenotype. Southern blot analysis of secondary transplant and tertiary CFU-S confirmed that HSCs were selectively expanded. However, expression was not lineage-specific. EPP HSCs were then transduced with the HAFPM vector and BM transplants were performed. After BG/BCNU injection, we observed a 25-fold increase of PB corrected RBCs associated with phenotypic correction of the disease. A 4-fold increase of FC activity was seen but limited to BM Ter119+ lineage (2.65 ± 0.5 vs. 0.7 ± 0.3 SA in Ter119+ cells for transduced EPP/BG/BCNU vs. untransduced EPP respectively). Experiments with HAGPM vector demonstrated also ES expression. In conclusion, although MGMT-based in vivo selection can efficiently expand lentiviral vector transduced-HSCs transplanted without myeloablation in a murine model of EPP, we can demonstrate MGMT-based HSC selection associated with ES expression of a therapeutic gene. These results can form the basis of new gene therapy strategies for other RBC disorders such as hemoglobinopathies.

Normoblastosis, a murine model for ankyrin-deficient hemolytic anemia, is caused by a hypomorphic mutation in the erythroid ankyrin gene Ank1.

Ankyrin deficiency is one of the most common causes of hereditary spherocytosis in humans. A spontaneous mutation, normoblastosis (Ank1nb), discovered in 1969 in a mouse stock maintained at the Jackson Laboratory, provides an important animal model for these human ankyrin-deficient anemias. Study of this model has led to the finding of multiple isoforms of Ank1 as well as Ank1nb-related pathology in nonerythroid tissues. To enhance the usefulness of this model, we have identified the Ank1nb mutation as the deletion of a guanosine residue in exon 36 of the erythroid ankyrin gene (Ank1). This results in a frame shift that introduces a stop 13 codons downstream and predicts a 157 kDa nb-ankyrin lacking the regulatory domain but including intact membrane- and spectrin-binding domains. By epitope scanning on immunoblots, we show that a previously reported protein (p150) found in nb reticulocytes is the predicted nb-ankyrin. Existing evidence indicates that this protein is functional, making the normoblastosis mutation a hypomorph rather than a null as originally thought. The nb-ankyrin provides an explanation for the milder phenotype displayed by nb/nb animals relative to the murine spectrin-deficient anemias, spherocytosis (Spna1(sph), Spna1(sph-1J), Spna1(sph-2BC), Spna1(sph-DEM)) and jaundiced (Spnb1(ja)), and suggests that truncated ankyrins could be useful in gene replacement therapy.

CK2 constitutively associates with and phosphorylates chicken erythroid ankyrin and regulates its ability to bind to spectrin.

Previous analyses have shown that the phosphorylation state of chicken erythroid ankyrin regulates its association with the spectrin cytoskeleton in vivo. Treatment of erythroid cells with serine and threonine phosphatase inhibitors stimulates the hyperphosphorylation of ankyrin and its dissociation from spectrin. In this study, we demonstrate that a kinase that directs the phosphorylation of ankyrin in vivo coprecipitates with ankyrin-containing complexes and has properties identical to CK2. Studies using CK2-specific inhibitors have indicated that all of the phosphorylation events associated with erythroid ankyrin in vivo are CK2 dependent. Furthermore, inhibitor studies combined with in vitro binding analyses have indicated that the phosphorylation of erythroid ankyrin by CK2 regulates its ability to associate with spectrin. Additional analyses revealed that CK2 coprecipitates with ankyrin-3-containing complexes isolated from Madin Darby canine kidney epithelial cells and phosphorylates this epithelial ankyrin isoform in vivo. These results are the first demonstration of a kinase constitutively associating with the ankyrin-spectrin cytoskeleton in erythroid and kidney epithelial cells. This association provides a mechanism for rapidly reorganizing the membrane cytoskeleton in these cell types through the phosphorylation of ankyrin.

Dynamics of ankyrin-containing complexes in chicken embryonic erythroid cells: role of phosphorylation.

Chicken erythroid ankyrin undergoes a fairly rapid cycle of cytoskeletal association, dissociation, and turnover. In addition, the cytoskeletal association of ankyrin is regulated by phosphorylation. Treatment of erythroid cells with serine and threonine phosphatase inhibitors stimulated the hyperphosphorylation of the 225- and 205-kDa ankyrin isoforms, and dissociated the bulk of these isoforms from cytoskeletal spectrin. In vitro binding studies have shown that this dissociation of ankyrin from spectrin in vivo can be attributed to a reduced ability of hyperphosphorylated ankyrin to bind spectrin. Interestingly, a significant fraction of detergent insoluble ankyrin accumulates in a spectrin-independent pool. At least some of this spectrin-independent pool of ankyrin is complexed with the AE1 anion exchanger, and the solubility properties of this pool are also regulated by phosphorylation. Treatment of cells with serine and threonine phosphatase inhibitors had no effect on ankyrin/AE1 complex formation. However, these inhibitors were sufficient to shift ankyrin/AE1 complexes from the detergent insoluble to the soluble pool. These analyses, which are the first to document the in vivo consequences of ankyrin phosphorylation, indicate that erythroid ankyrin-containing complexes can undergo dynamic rearrangements in response to changes in phosphorylation.

An Alternative First Exon in the Distal End of the Erythroid Ankyrin Gene Leads to Production of a Small Isoform Containing an NH2-Terminal Membrane Anchor

Mouse erythroid ankyrin is encoded by theAnk1gene on Chromosome 8. The best studied isoform is 210 kDa and contains three large functional domains. We have recently reported a small Ank1 isoform (relative mobility 25 kDa) that localizes to the M and Z lines in skeletal muscle. Analyses of cDNA and genomic clones show that three transcripts of 3.5, 2.0, and 1.6 kb code for this protein. The different transcript sizes are due to their 3′-untranslated regions. They are encoded by a new first exon located in intron 39 of theAnk1gene and three previously describedAnk1exons (40, 41, and 42). The 5′-flanking region contains a putative muscle-specific promoter. The sequence of the first 72 amino acids is novel and is predicted to form a transmembrane helix at the NH2-terminus. Functional testing of the putative transmembrane segment indicates that it acts as a membrane anchor, suggesting that the new Ank1 isoform may play an important role in organizing the contractile apparatus within the cell.

Ankyrin Bugey: a de novo deletional frameshift variant in exon 6 of the ankyrin gene associated with spherocytosis.

We describe a case of spherocytosis in a French child splenectomized at age 10 years. The parents were devoid of any clinical, hematological, or biochemical abnormalities. Following splenectomy, the proposita exhibited a reduction of red cell membrane ankyrin. The variable number of dinucleotide repeats associated with the erythroid ankyrin gene (ANK1) were studied at the genomic level. The father, the mother, and the proposita had the AC14/AC11, AC14/AC14, and AC14/AC11 genotypes, respectively, although the proposita exhibited a pattern consistent with an AC14,-combination at the cDNA level. We thought there could be a de novo mutation in the ANK1 allele of paternal origin (AC11). A false paternity seemed most unlikely. Based on PCR-amplification of exons, SSCP analysis, and, when appropriate, nucleotide sequencing, we found a one-nucleotide deletion in codon 146 (exon 6): 521delC, ACG-->AG. This placed in phase a TAG triplet normally overlapping codons 150 and 151. Early interruption of translation presumably accounted for the premature degradation of mutant mRNA. Restriction analysis confirmed the presence of the mutation in the proposita and its absence in the parents. The variant was designated ankyrin Bugey.

Small, membrane-bound, alternatively spliced forms of ankyrin 1 associated with the sarcoplasmic reticulum of mammalian skeletal muscle.

We have recently found that the erythroid ankyrin gene, Ank1, expresses isoforms in mouse skeletal muscle, several of which share COOH-terminal sequence with previously known Ank1 isoforms but have a novel, highly hydrophobic 72-amino acid segment at their NH2 termini. Here, through the use of domain-specific peptide antibodies, we report the presence of the small ankyrins in rat and rabbit skeletal muscle and demonstrate their selective association with the sarcoplasmic reticulum. In frozen sections of rat skeletal muscle, antibodies to the spectrin-binding domain (anti-p65) react only with a 210-kD Ank1 and label the sarcolemma and nuclei, while antibodies to the COOH terminus of the small ankyrin (anti-p6) react with peptides of 20 to 26 kD on immunoblots and decorate the myoplasm in a reticular pattern. Mice homozygous for the normoblastosis mutation (gene symbol nb) are deficient in the 210-kD ankyrin but contain normal levels of the small ankyrins in the myoplasm. In nb/nb skeletal muscle, anti-p65 label is absent from the sarcolemma, whereas anti-p6 label shows the same distribution as in control skeletal muscle. In normal skeletal muscle of the rat, anti-p6 decorates Z lines, as defined by antidesmin distribution, and is also present at M lines where it surrounds the thick myosin filaments. Immunoblots of the proteins isolated with rabbit sarcoplasmic reticulum indicate that the small ankyrins are highly enriched in this fraction. When expressed in transfected HEK 293 cells, the small ankyrins are distributed in a reticular pattern resembling the ER if the NH2-terminal hydrophobic domain is present, but they are uniformly distributed in the cytosol if this domain is absent. These results suggest that the small ankyrins are integral membrane proteins of the sarcoplasmic reticulum. We propose that, unlike the 210-kD form of Ank1, previously localized to the sarcolemma and believed to be a part of the supporting cytoskeleton, the small Ank1 isoforms may stabilize the sarcoplasmic reticulum by linking it to the contractile apparatus.

Isoform Cloning, Actin Binding, and Chromosomal Localization of Human Erythroid Dematin, a Member of the Villin Superfamily

Dematin is an actin-bundling protein of the erythroid membrane skeleton and is abundantly expressed in human brain, heart, skeletal, muscle, kidney, and lung. The 48-kDa subunit of dematin contains a headpiece domain which was originally identified in villin, and actin-binding protein of the brush-border cytoskeleton. The head-piece domain of villin is essential for its morphogenic function in vivo. Here we report the primary structure of 52-kDa subunit of dematin which differs from the 48-kDa subunit by a 22-amino-acid insertion within its headpiece domain. A unique feature of the insertion sequence of the 52-kDa subunit is its homology to erythrocyte protein 4.2. The insertion sequence also includes a cysteine residue which may explain the formation of sulfhydryl-linked trimers of dematin. Actin binding measurements using recombinant fusion proteins revealed that each monomer of dematin contains two F-actin binding sites: one in the headpiece domain and the other in the undefined N-terminal domain. Although the actin bundling activity of intact dematin was abolished by phosphorylation, no effect of phosphorylation was observed on the actin binding activity of fusion proteins. Using somatic cell hybrid panels and fluorescence in situ hybridization, the dematin gene was localized on the short arm of chromosome 8. The dematin locus, 8p21.1, is distal to the known locus of human erythroid ankyrin (8p11.2) and may contribute to the etiology of hemolytic anemia in a subset of patients with severe hereditary spherocytosis.

A nonsense mutation 1669Glu--&gt;Ter within the regulatory domain of human erythroid ankyrin leads to a selective deficiency of the major ankyrin isoform (band 2.1) and a phenotype of autosomal dominant hereditary spherocytosis.

We describe a nonsense mutation in the regulatory domain of erythroid ankyrin associated with autosomal dominant hereditary spherocytosis with a selective deficiency of the ankyrin isoform 2.1 (55% of normal), a deficiency of spectrin (58% of normal) proportional to the decrease in ankyrin 2.1, and a normal content of the other main ankyrin isoform, protein 2.2. PCR amplification of cDNA encoding the regulatory domain of ankyrin revealed a marked decreased in the ratio of ankyrin 2.1 mRNA to the ankyrin 2.2 mRNA. Sequencing of ankyrin gene in the region where the 2.1 and 2.2 mRNA differ detected a nonsense mutation 1669Glu-->Ter (GAA-->TAA) in one ankyrin allele. Only normal ankyrin 2.1 mRNA was detected in the reticulocyte RNA. Since the alternative splicing within the regulatory domain of ankyrin retains codon 1669 in ankyrin 2.1 mRNA and removes it from ankyrin 2.2 mRNA, we propose that the 1669Glu-->Ter mutation decreases the stability of the abnormal ankyrin 2.1 mRNA allele leading to a decreased synthesis of ankyrin 2.1 and a secondary deficiency of spectrin.

Combined Spectrin and Ankyrin Deficiency Is Common in Autosomal Dominant Hereditary Spherocytosis

The common autosomal dominant form of hereditary spherocytosis (HS) has been genetically linked to defects of the erythroid ankyrin gene in a few families; however, the frequency of ankyrin deficiency and its relationship to red blood cell (RBC) spectrin content are unknown. To test these questions, we measured RBC spectrin and ankyrin by radioimmunoassay in 39 patients from 20 families with dominant HS. Normal RBCs contained 242,000 +/- 20,500 spectrin heterodimers and 124,500 +/- 11,000 ankyrins per cell. In dominant HS, RBC spectrin and ankyrin ranged from about 40% to 100% of normal and were continuously distributed. Measurements in the same patient on different occasions were reproducible (+/- 5% to 10%) and RBCs from affected members of a kindred contained similar amounts of spectrin and ankyrin (+/- 3% to 4%). Spectrin and ankyrin levels were almost always less than the assay controls, but were less than the normal range in only 75% and 80% of kindreds, respectively. Remarkably, the degree of RBC spectrin and ankyrin deficiency was very similar in 19 of 20 HS kindreds. One otherwise typical family differed, with marked ankyrin deficiency (45% of control) and a relatively mild spectrin deficit (81%). We conclude that most patients with dominant HS have combined ankyrin and spectrin deficiency and that the two proteins are usually about equally deficient, suggesting that defects in ankyrin expression, ankyrin stability, or ankyrin band 3 (AE1) interactions may be common in dominant HS.