Mouse Α-globin Research Articles

On-microscope staging of live cells reveals changes in the dynamics of transcriptional bursting during differentiation

Determining the mechanisms by which genes are switched on and off during development is a key aim of current biomedical research. Gene transcription has been widely observed to occur in a discontinuous fashion, with short bursts of activity interspersed with periods of inactivity. It is currently not known if or how this dynamic behaviour changes as mammalian cells differentiate. To investigate this, using an on-microscope analysis, we monitored mouse α-globin transcription in live cells throughout erythropoiesis. We find that changes in the overall levels of α-globin transcription are most closely associated with changes in the fraction of time a gene spends in the active transcriptional state. We identify differences in the patterns of transcriptional bursting throughout differentiation, with maximal transcriptional activity occurring in the mid-phase of differentiation. Early in differentiation, we observe increased fluctuation in transcriptional activity whereas at the peak of gene expression, in early erythroblasts, transcription is relatively stable. Later during differentiation as α-globin expression declines, we again observe more variability in transcription within individual cells. We propose that the observed changes in transcriptional behaviour may reflect changes in the stability of active transcriptional compartments as gene expression is regulated during differentiation.

Thalassemia Gene Therapy By In Vivo Transduction of Mobilized Hematopoietic Stem Cells (HSCs) with an Integrating Hybrid Adenovirus Vector System

To overcome the cost and complexity of current thalassemia ex vivogene therapy protocols, we developed a minimally invasive and readily translatable approach for in vivo HSC gene delivery which abrogates the need for HSC leukapheresis, CD34+ cell selection, ex vivo HSC culture, myeloablation and ultimately, transplantation. Our approach involves HSC mobilization with G-CSF/AMD3100 andintravenous injections of a hybrid vector system consisting ofa CD46-targeting, helper-dependent adenoviral vector and the hyperactive Sleeping Beauty transposase (SB100x) that mediates integration of thevector-encoded γ-globin and mgmtP140K genes. Pretreatment with glucocorticoids before virus injectionsis used to blockthe release of pro-inflammatory cytokines andimmunosuppression is applied in order to avoid responses against human g-globin- and MGMT protein-expressing cells. We tested our approach in a mouse model recapitulating the phenotypeof human β-thalassemia intermedia (Hbbth-3/hCD46++ mice). At week 8 post transduction, hCD46+/+/Hbbth-3 mice expressed HbF in 31.2±2.7% of circulating erythrocytes. Due to a significant drop in HbF expression by week 16 (11.9±3.0%), a 4-dose O6BG/BCNU treatment was administeredin order to in vivo select forgene corrected hematopoietic progenitors, thus recovering the HbF expression in76.0±5.7% of the circulating erythrocytes, by week 29 post in vivo transduction. With an average vector copy number of 1.4/cell, the human γ-globin to mouse α-globin expression was ~10% by HPLC and the human γ-globin to mouse β-globin mRNA ratio ~10%, by qRT-PCR. Hematological parameters (RBCs, Ht, Hb, MCV, RDW, Reticulocytes) at week 29 post in vivo transduction, were significantly improved over baseline or were indistinguishable from normal values, suggesting near to complete phenotypic correction. Treated mice showed significant reduction of spleen size, extramedullary erythropoiesis and parenchymal hemosiderosis. After secondary transplantation and without in vivo selection, more than 90% of donor-derived erythrocytes (CD46+) were g-globin-positive, up to 20 weeks post-transplant. Safety was demonstrated by the good tolerability of treatment, the absence of alterations in hematopoiesis, the normal colony-forming potential of bone marrow cells and the random integration pattern of our vector system. Overall, we present a simplified platform for gene therapy of thalassemia, which can serve as a cost-efficient and “portable” approach to make gene therapy accessible even to resource-poor regions where thalassemia major is endemic but only minimally complex strategies could be adopted. DisclosuresNo relevant conflicts of interest to declare.

Tissue-specific CTCF-cohesin-mediated chromatin architecture delimits enhancer interactions and function in vivo.

The genome is organised via CTCF/Cohesin binding sites, which partition chromosomes into 1-5Mb topologically associated domains (TADs), and further into smaller sub-domains (sub-TADs). Here we examined in vivo an ~80kb sub-TAD, containing the mouse α-globin gene cluster, lying within a ~1Mb TAD. We find that the sub-TAD is flanked by predominantly convergent CTCF/cohesin sites which are ubiquitously bound by CTCF but only interact during erythropoiesis, defining a self-interacting erythroid compartment. Whereas the α-globin regulatory elements normally act solely on promoters downstream of the enhancers, removal of a conserved upstream CTCF/cohesin boundary extends the sub-TAD to adjacent upstream CTCF/cohesin binding sites. The α-globin enhancers now interact with the flanking chromatin, upregulating expression of genes within this extended sub-TAD. Rather than acting solely as a barrier to chromatin modification, CTCF/cohesin boundaries in this sub-TAD delimit the region of chromatin to which enhancers have access and within which they interact with receptive promoters.

Decrease of α-Chains in β-Thalassemia

In the pathophysiology of β-thalassemia, globin chain imbalance plays a central role in predicting red blood cell (RBC) life span and disease severity. Strategies to improve globin chain imbalance are therefore a legitimate target in the management of this incurable genetic disorder. Classical gene addition with the available retroviral vectors can alter one of the two variables while combined reduction of achains could provide a more potent therapeutic effect. We developed foamy virus (FV) vectors for the production of β-globin and vectors targeting the a-globin transcript using the shRNA technology. Using FVderived vectors, we expressed human anti-a-globin short hairpin RNAs, off a potent PolIII promoter (H1); of the 4 different shRNAs tested, α-globin mRNA reduction varied from 6.3 to 54% of the control CD34+ cells. Similarly, vectors developed for the mouse α-globin, resulted in a significant reduction (range 15-28% of the control) of aglobin in erythroid colonies of Lin- cells. To assay vector performance in vivo, we investigated the hematological parameters in thal3+/- mice transpalnted with FV-transduced Lin- cells, transduced with anti-alpha-globin shRNA. Despite low chimerism and low vector copy numbers (<0.5 per cell), we observed a 10% reduction in red cell distribution width, a marker for distorted erythropoiesis. We finally developed a combination FV vector expressing β-globin off a HS40 enhancer and anti-α-globin shRNA and tested its performance in human CD34+ cells from a thalassemic patient. Globin chain imbalance was ameliorated from a β/α ratio of 0.12 to the level of 0.5, clearly indicating a therapeutic benefit. Overall, shRNA control of α-globin excess is a feasible target but requires improvements since the RNAi effect is tough to predict and should ideally be combined with controllable elements.

The Role of DNA Damage/Stress Response Pathways in the Mechanism of Action of Decitabine

The DNA methyltransferase (DNMT) inhibitor decitabine increases fetal hemoglobin (HbF) in experimental primates and to therapeutic levels in patients with sickle cell disease. Decitabine-DNMT covalent adducts formed following incorporation of decitabine into newly synthesized DNA can activate DNA damage/stress response signal transduction pathways. To test the hypothesis that DNA damage/stress response pathways are involved in the ability of decitabine to increase γ-globin expression, experiments were performed in a chemical inducer of dimerization (CID)-dependent mouse fetal bone marrow (BM)-derived cell line containing the human β-globin gene locus in the context of a yeast artificial chromosome and in primary baboon erythroid progenitors derived from CD34+ BM cells grown in Iscove's media containing 30% fetal bovine serum, 2U/ml epo, 200ng/ml SCF, and 1uM dexamethasone. Addition of decitabine (1μM) to both the CID-dependent mouse BM cell line and erythroid progenitors on day 7 of culture increased phospho-H2A.X and phospho-p38 in Western blot assays demonstrating activation of the ATM/ATR and p38 MAP kinase signal transduction pathways. Decitabine increased γ-globin 280 fold and ε-globin 38 fold in the mouse BM cell line as determined by real time PCR using the ΔΔCT method with mouse α-globin as the standard. Co-addition of the p38 MAP kinase inhibitor SB203580 reduced γ-globin induction 93.4% and ε-globin induction 88.4% in the mouse BM cell line. In primary erythroid progenitors, decitabine increased γ-globin 2.8 fold and ε-globin 44 fold. SB203580 reduced decitabine-mediated ε-globin induction 46% while no significant effect on the level of γ-globin induction was observed. Decitabine also increased phospho-CREB in the mouse BM cell line, a target of the p38 MAP kinase pathway required for γ-globin induction by histone deacetylase inhibitors. These results suggest that activation of the p38 MAP kinase pathway is required for induction of ε- and γ-globin expression by decitabine in the mouse fetal BM cell line and may contribute to increased ε-globin expression in primary erythroid progenitors. Additional Western blot analysis showed that decitabine treatment of erythroid progenitors increased p21WAF1 and p27KIP1 48–72 hours following addition of drug. Examination of Wright's stained cytospin preparations from decitabine-treated cultured erythroid progenitors showed that orthochromatic erythroblasts were increased >2 fold and basophilic erythroblasts were decreased 5 fold compared to untreated controls 96–120 hours post-drug addition at doses ranging from 0.125 to 1.0mM. These results demonstrate that decitabine increases terminal erythroid differentiation within this cell population. As RB protein is required for cell cycle exit and terminal erythroid differentiation, we suggest that induction of terminal differentiation by decitabine is mediated by the effect of increased p21WAF1 and p27KIP1 on RB phosphorylation. Because of the known asynchrony of γ- and β-globin expression during erythroid differentiation, the ability of decitabine to induce terminal erythroid differentiation could play a role in its ability to increase γ-globin expression. We conclude that activation of DNA damage/stress response pathways by decitabine may contribute to its ability to increase γ-globin expression and induce terminal erythroid differentiation.

Rules of gene attraction

Active genes can be sociable, snuggling up to one another. Brown et al. offer a new explanation for this clustering, suggesting that genes gather for the services of RNA splicing enzymes Figure 1 Genes that are activated during red blood cell differentiation (green and red) cozy up in a human erythroblast. A gene's location in the nucleus often reflects its activity. Hard-working genes tend to congregate in the interior of the nucleus, whereas their lazier counterparts hang out at the edge. Moreover, active genes on different chromosomes sometimes bunch up. How often active genes come together is uncertain. Whether the associations serve a purpose is also unclear, although some researchers propose that genes converge at so-called transcription factories that contain RNA polymerase. To address these issues, Brown et al. pinpointed five genes that crank up during the differentiation of human red blood cells. Some of the genes were friendlier than others. For example, two α-globin genes were about five times more likely to be near each other than were two β-globin genes. Next, the researchers tested whether a gene's chromatin environment affects its tendency to cluster. In human cells, β-globin sits in a tightly packaged chromatin region, whereas α-globin's neighborhood is looser. But in mouse cells, α-globin resides in a condensed region. The team replaced the mouse α-globin gene with the human version, so that the ordinarily loose human gene was now in a condensed chromatin environment. Like the β-globin gene in human cells, the inserted α-globin gene in mouse cells was aloof, suggesting that a gene's surroundings do influence its position relative to other genes. However, the team found that the inserted gene worked normally, showing that associations aren't essential for normal transcription. The results also indicate that genes aren't sharing transcription factories. The average distance between associating active genes, the researchers determined, was about 10 times the diameter of a factory. Instead, the genes were congregating at nuclear speckles, much larger structures than factories that harbor enzymes for splicing RNA after transcription. The team concludes that genes associate because they sometimes happen to be drawn to the same speckle. Brown, J.M., et al. 2008. J. Cell Biol. doi:.10.1083/jcb.200803174 [PubMed] [Cross Ref]

Association between active genes occurs at nuclear speckles and is modulated by chromatin environment

Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin. By replacing the mouse α-globin gene cluster in situ with its human counterpart, we demonstrate a direct effect of the regional chromatin environment on the frequency of association, whereas nascent transcription from the human α-globin gene appears unaffected. We see no evidence that cotranscribed erythroid genes associate at shared transcription foci, but we do see stochastic clustering of active genes around common nuclear SC35-enriched speckles (hence the apparent nonrandom association between genes). Thus, association between active genes may result from their location on decondensed chromatin that enables clustering around common nuclear speckles.

A Humanized Cooley's Anemia Mouse Model.

Cooley's Anemia (CA) is a heterogeneous group of inherited disorders all marked by the complete absence of adult β-globin chains in red blood cells (RBCs). Newborns with CA are healthy because of the high level of fetal hemoglobin (HbF) present at birth. As HbF levels decline during the first year of life, the absence of functional adult β-globin genes results in a severe anemia that necessitates the initiation of regular blood transfusions for the remainder of life. CA has been difficult to study in murine models due to the lack of a human fetal hemoglobin equivalent in the mouse. This study reports a novel preclinical animal model of CA that survives solely on human fetal hemoglobin at birth and is blood transfusion dependent for life upon completion of the hemoglobin switch after birth. These humanized CA mice were generated by targeted gene replacement in embryonic stem cells of the adult mouse α-globin genes with human α-globin and the adult mouse β-globin genes with a delayed switching γδβ0-globin gene cassette. The nonfunctional human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1. Both wild-type and hereditary persistence of fetal hemoglobin (HPFH) promoter mutations were tested in the human γ-globin knock-in allele. Heterozygous knock-in mice exhibit β thalassemia intermedia. Newborn homozygous knock-in mice express 100% human hemoglobin in their RBCs, suffer from ineffective erythropoiesis, survive from one to ten days after birth, and are blood transfusion dependent for adult life. This is the first CA mouse model that recapitulates the temporal onset of the disease in human patients. This CA disease model is useful for the study of the regulation of globin gene expression, synthesis, and switching; development of transfusion and iron chelation therapies; induction of fetal hemoglobin synthesis; and the testing of novel genetic and cell-based therapies for the correction of thalassemia.

Lentiviral Insertion Site Analysis of Corrected Mouse Model of β Thalassemia Following Lentiviral Transduction of Embryonic Stem Cells.

The delivery of therapeutic transgenes via lentiviral transduction of stem cells holds great promise for future cell based therapies for inherited genetic disorders. The regulation of any transgene integrated into patient derived embryonic stem (ES) cells (generated via nuclear transfer or reprogramming) needs to be studied in great detail prior to their differentiation and transplantation back into the patient. Because genetically manipulated ES cells can be clonally isolated, expanded to great numbers in an undifferentiated state, and differentiated to specific cell types, they can be carefully tested in vitro prior to their reintroduction back into patients. ES cells derived from a mouse model of β thalassemia were transduced with a recombinant, self inactivating (SIN) lentiviral vector containing a 2.3 kb human β-globin gene and a 3.2 kb Locus Control Region composed of regulatory elements; HS2, HS3, and HS4. Clonal populations of transfected β thalassemic ES cells were isolated, archived, and utilized to produce genetically identical offspring by injection into tetraploid blastocysts or eight-cell embryos. Real-Time QPCR expression analyses demonstrated high levels of human β-globin gene expression in the peripheral blood of five of the six lines produced. The average expression level per transgene copy number ranged from 13% to 62% of endogenous mouse α-globin levels. Peripheral blood hemolysates analyzed by HPLC confirmed the high level production of human β-globin chains. Measurement of the red blood indices in these five lines showed that the anemia was corrected. In order to examine the influence of chromosomal position effects on individual transgene expression, the lentiviral insertion sites were mapped by linear amplification (LAM) PCR. Cloned mice from the six lines were bred in order to segregate each of their individual lentiviral transgenes. Offspring containing single insertion sites were analyzed for human β-globin transgene expression and synthesis by Real-Time QPCR of blood RNA and by HPLC of hemolysates, respectively. These analyses demonstrate that there is a preference for provirus integration in ES cells; all transgene insertions are not expressed equally; lentiviral transduction of a human β-globin gene into ES cells can cure a severe hemoglobinopathy; and that the delivery of therapeutic transgenes via lentiviral transduction of ES cells did not harm the developmental potential of the cells to produce an entire mouse.

Associated chromatin assay of mouse α-globin gene cluster and identification of an a-globin gene-specific silencer

Associated chromatin assay of mouse α-globin gene cluster and identification of an a-globin gene-specific silencer

Novel γ-Globin Gene Lentiviral Insulated Vectors Containing the HPFH-2 Enhancer and the Activating -117 HPFH Aγ-Promoter Mutation.

Lentiviral vectors for γ-globin genes are being developed as an efficient tool for the gene therapy of β-chain hemoglobinopathies. The γ-globin gene has been chosen as a therapeutic gene based on the potent anti-sickling properties of γ-globins and on their ability to bind free α-chains. However, their development has been hampered by low titers, variable expression and gene silencing. To address these problems, we have initiated a strategy to exploit novel regulatory elements of the β-cluster conferring high level and sustained globin gene expression. To this end, we have successfully used the HPFH-2 enhancer combined with a 210 bp Aγ-globin gene promoter harboring the Greek HPFH -117 mutation and the HS-40 enhancer from the α-globin locus, in a series of oncoretrovirus vectors (Fragkos et al. Gene Ther 12:1591–1600, 2005). Based on the high level of expression of the Aγ-gene (248 ± 99 % per copy of mouse α-globin) and the absence of vector silencing of these vectors and to further exploit the superior transducing efficiency of hematopoietic stem cells by lentiviral vectors, in the present study we have generated two novel self-inactivating lentiviral vectors containing the above regulatory elements. Specifically, vector GGHI contains an expression cassette for Aγ-globin gene linked to the 210 bp Aγ-gene promoter with the Greek HPFH -117 point mutation, the HS-40 enhancer at its 5′ end and the HPFH-2 enhancer at its 3′ end, as well as the cHS4 insulator in the 3′ LTR. The second vector, designated GGHI/PM is essentially similar to GGHI but carries also the MGMT-140K cDNA selectable marker under the control of PGK promoter, to enrich for genetically modified cells. Both vectors exhibited high titers of 108 TU/ml, for GGHI and 107 TU/ml, for GGH/PM. Their efficiency was tested in MEL-585 cells transduced at an MOI of 1–100 and a series of independent clones were generated. The clones were further induced to differentiate using hemin and HMBA and the level of expression of the Aγ-globin transgene was determined by Real Time PCR and by flow cytometry. Vector GGHI was expressed at 237 ± 369 % per copy of mouse α-globin with a mean copy number of 19.3 in 8 individual clones, while GGHI/PM was expressed only at 10 ± 16 % per copy of mouse α-globin, with a mean copy number of 60 in 10 individual clones of unselected cells. FACS analysis using an anti-γ-globin antibody, revealed a pancellular expression of γ-globin (mean MFI 69.7 for GGHI and mean MFI 40.15 for GGHI/PM), while there was no expression of the transgene in undifferentiated MEL-585 cells, suggesting that both vectors are erythroid-specific. Moreover, there was no sign of transgene silencing in any of the above clones. The results for the novel GGHI vector, are consistent with our previous studies and reflect a) the robust synergistic capacity of the HS-40 and HPFH-2 elements to enhance transcription, b) the ability of HPFH-2 to reduce the rate of gene silencing and c) the ability of the -117 point mutation to support the Aγ-globin gene expression in the adult erythroid environment, for the first time, in the context of lentiviral vectors. This extremely high level of expression if achieved in vivo, would clearly exceed the proposed therapeutic threshold for the β-chain hemoglobinopathies. Current studies combine their assessment on CD34+ cells from patients with β-thalassemia as well as their evaluation in vivo using the Hbthal3+/− thalassemic mouse model.

Active Chromatin Hub of the Mouse α-Globin Locus Forms in a Transcription Factory of Clustered Housekeeping Genes

RNA polymerases can be shared by a particular group of genes in a transcription "factory" in nuclei, where transcription may be coordinated in concert with the distribution of coexpressed genes in higher-eukaryote genomes. Moreover, gene expression can be modulated by regulatory elements working over a long distance. Here, we compared the conformation of a 130-kb chromatin region containing the mouse alpha-globin cluster and their flanking housekeeping genes in 14.5-day-postcoitum fetal liver and brain cells. The analysis of chromatin conformation showed that the active alpha1 and alpha2 globin genes and upstream regulatory elements are in close spatial proximity, indicating that looping may function in the transcriptional regulation of the mouse alpha-globin cluster. In fetal liver cells, the active alpha1 and alpha2 genes, but not the inactive zeta gene, colocalize with neighboring housekeeping genes C16orf33, C16orf8, MPG, and C16orf35. This is in sharp contrast with the mouse alpha-globin genes in nonexpressing cells, which are separated from the congregated housekeeping genes. A comparison of RNA polymerase II (Pol II) occupancies showed that active alpha1 and alpha2 gene promoters have a much higher RNA Pol II enrichment in liver than in brain. The RNA Pol II occupancy at the zeta gene promoter, which is specifically repressed during development, is much lower than that at the alpha1 and alpha2 promoters. Thus, the mouse alpha-globin gene cluster may be regulated through moving in or out active globin gene promoters and regulatory elements of a preexisting transcription factory in the nucleus, which is maintained by the flanking clustered housekeeping genes, to activate or inactivate alpha-globin gene expression.

Generation of a Novel Mouse Model of Cooley's Anemia.

A novel mouse model of Cooley's Anemia (CA) has been generated by targeted gene replacement of the adult murine α-globin genes with human α-globin and the adult mouse β-globin genes with a human γ- to β-globin gene switching cassette containing a β0 thalassemic allele. A positive-negative gene replacement construct was designed to simultaneously delete both of the adult mouse α-globin genes by inserting a 3.8kb human α1-globin gene and a hygromycin marker gene flanked by loxP sites in murine embryonic stem (ES) cells. Both adult murine β-globin genes were deleted by insertion of an Hprt marker gene that was later replaced by a 5.6kb human Aγ-globin gene, 4.1kb human β0-globin gene, and a loxP flanked hygromycin marker gene by a “tag and exchange” strategy. The human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1 [β0-IVS1(GtoA)-globin]. This single base change destroys the splice donor site of IVS-1 resulting in the recruitment of several cryptic splice sites. Use of these cryptic splice sites produces a frameshift in the mRNA that results in no functional β-globin polypeptide synthesis from this allele. This β0-IVS1(GtoA)-globin gene mutation is a naturally occurring β0 thalassemia allele found in Mediterranean populations. Chimeric mice were generated from both the α- and β-globin targeted cells lines. After germline transmission the α- and β-globin targeted mice were bred to cre recombinase transgenic mice to remove the marker genes. Heterozygous CA mice exhibit β thalassemia intermedia. The α- and β-globin targeted mice were interbred to produce animals homozygous for the human α1- and γβ0-globin knock-in alleles. Instead of dying early in fetal life as all current homozygous β0 thalassemia mouse models, these novel homozygous CA mice survive solely on high levels of human fetal hemoglobin (α2γ2) throughout fetal development. Newborn homozygous CA mice are blood transfusion dependent similar to β thalassemia major infants. This novel model of CA has multiple improvements over existing models of β thalassemia. Namely, CA mice express 100% human hemoglobin in their RBCs, mimic the human γ- to β-globin gene switch, synthesize no functional β-globin chains after birth, have a single mutant human β0-globin knock-in allele at each β-globin locus, and are blood transfusion dependent for life after birth.

Isolation and characterization of the bovine corticotropin/beta-lipotropin precursor gene.

The entire bovine corticotropin/beta-lipotropin precursor gene has been isolated as a set of overlapping genomic DNA fragments which extend over a length of approximately 17000 base pairs. Restriction mapping of the cloned DNA fragments and nucleotide sequence analysis of the whole mRNA-coding segments and their surrounding regions have established that the corticotropin/beta-lipotropin precursor gene is approximately 7300-base-pairs long and contains two intervening sequences; one with an approximate length of 4000 base pairs is located within the segment encoding the 5'-untranslated region of the mRNA, and the other with an approximate length of 220 base pairs interrupts the protein-coding sequence near the signal peptide region. Sequence analysis of more than 200 base pairs preceding the proximal end of the corticotropin/beta-lipotropin precursor gene has revealed a 'Hogness box' and a variant of the model sequence d(G-G-TC-C-A-A-T-C-T) as well as palindrome structures as observed in other eukaryotic genes. Furthermore, some sequence similarities in the 5'-flanking region are found between the corticotropin/beta-lipotropin precursor gene and the mouse alpha-globin and beta-globin genes, all of which are negatively regulated by glucocorticoids. At least four homologous repetitive sequences are distributed at 3000-5000-base-pair distances in the corticotropin/beta-lipotropin precursor gene region; two such sequences are located in the 5'-flanking region, and one within each intervening sequence. Blot hybridization analysis of bovine pituitary nuclear RNA has indicated that the entire corticotropin/beta-lipotropin precursor gene is transcribed into a primary hnRNA product, which is then spliced to form the mature mRNA.

Chromatin Remodeling of the Mouse AHSP Gene Requires EKLF.

Alpha-Hemoglobin Stabilizing Protein (AHSP) is an erythroid-specific protein that binds to α-globin, preventing precipitation of α-hemoglobin tetramers. Our interest has focused on how the AHSP gene is specifically expressed in erythroid cells, and we have investigated the roles of cis acting DNA sequences, the transcription factor EKLF and chromatin structure on AHSP gene expression. We have previously shown that the AHSP gene has a single mRNA initiation site followed by a non-coding exon. Putative promoter sequences from −904, −479 or −170 were active in luciferase reporter assays only when the constructs contained the downstream 269 bp containing exon 1 and intron 1. The −904/+269, −479/+269 and −170/+269 constructs gave 53.3+2.0 to 122.1+8.8 -fold increased levels of luciferase expression in K562 cells compared to plasmids without exon 1 and intron 1 (p<0.001; Gallagher et al., BLOOD 102, 267a, 2003). In vitro DNase I footprinting and EMSA assays revealed two regions (−75 to −67 and +153 to +164) that bound GATA-1. Analysis of 5 lines of transgenic mice with between 1 and 11 copies of the −170/+269 promoter fused to the human γ-globin gene demonstrated position independent expression (5/5 lines express) of γ-globin mRNA, at levels that were 4.6% the level of mouse α-globin mRNA per transgene copy. There was no correlation between transgene copy number and the level of γ-globin mRNA and 3/5 lines exhibited variegated expression. We concluded that sequences upstream of −170 or downstream of +269 are required for authentic expression from the AHSP promoter. To examine the role of EKLF in AHSP expression, we used subtractive hybridization, microarray and RNase protection analysis to compare AHSP mRNA levels in fetal liver cells from wild type and EKLF−/− mice. EKLF −/− fetal liver cells had 9 -fold less AHSP mRNA than wild type fetal liver cells, which we propose would exacerbate the moderate β- thalassemia described in EKLF −/− mice. Based on the observation that EKLF associates with an erythroid chromatin remodeling complex (Armstrong et al. Cell 95, 93–104, 1998), we hypothesized that EKLF was involved in chromatin remodeling at the AHSP locus. We assayed for DNase I Hypersensitive sites (HS) in chromatin from 13.5 day wild type and EKLF−/− mouse fetal liver nuclei. We demonstrated a strong DNase I HS between −200 and −400 of the AHSP gene, just upstream of the minimal −170 promoter, that was not present in chromatin from EKLF−/− fetal liver cells. To examine histone acetylation across the 3.5 kb AHSP locus we performed Chromatin Immune Precipitation analysis on wild type and EKLF −/− fetal liver chromatin using 13 different primer pairs (~300 bp intervals). In wild type chromatin there are two regions where histones H3 and H4 were hyperacetylated relative to a control region from the mouse α-globin gene promoter. The 5′ region corresponded to the DNase I HS at −400 to −200, while the second region maps 3′ to the AATAAA signal in the AHSP gene. Histones H3 and H4 were also acetylated in the interval between the hyperacetylated regions, while the chromatin upstream and downstream (~1 kb in each direction) of these regions was hypoacetylated. In contrast, all sites were hypoacetylated in chromatin from EKLF−/− fetal liver cells, correlating with the severe reduction in AHSP gene expression. We conclude that EKLF is required for remodeling the chromatin of the AHSP locus and that EKLF could be a modifier gene for the thalassemia syndromes.

Sequences Downstream of the Erythroid Promoter Promote High-Level Expression of the Human α-Spectrin Gene by Providing Boundary Activity and Positive Regulatory Elements.

Characterization of the regulatory elements that control α-spectrin (ASp) gene expression is important for understanding the pathogenesis of ASp-linked hemolytic anemia. Our previous studies demonstrated that the ASp promoter directs low levels of expression, and, addition of a downstream region of noncoding exon 1 and intron 1 containing 2 GATA-1 sites conferred a 10-fold increase in activity in transient transfection assays. Transgenic (TG) mouse lines with the ASp promoter, the Asp promoter-exon 1-intron 1, or ASp promoter-exon 1-intron 1 with mutations of both splice sites linked to the human Aγ-globin gene as reporter were created. In reticultocytes, no expression was detected in any of the 8 lines transmitting the ASp promoter-Aγ-globin transgene. TG mice with the ASp promoter-exon1-intron 1 demonstrated significant levels of Aγ-globin gene expression in reticulocytes, with levels of Aγ-globin mRNA of ~0.4% of mouse α-globin mRNA/transgene copy #. This expression was nearly position independent, as 22/24 lines expressed the transgene. Using a FACS-based assay, γ-globin protein was present in 100% of erythrocytes. Expression levels comparable to the Asp promoter-exon 1-intron 1 TG were detected in 9/9 lines with the mutated splice sites, indicating splicing did not contribute to changes in expression. DNaseI hypersensitive site (HS) mapping identified a broad, erythroid-specific HS across exon 1 and intron 1. The presence of a DNaseI HS site suggested the presence of a positive regulatory element or a chromatin modification such as a boundary element. Analysis of a positive regulatory element in vivo was sought by stably transfecting the following luciferase (luc) plasmids into K562 cells: ASp promoter, ASp promoter-exon 1-intron 1, ASp promoter-exon 1, ASp promoter-intron 1, and ASp promoter-exon 1-intron 1 with both GATA-1 sites mutated. Clones with copy # ≤5 were analyzed; ≥9 independent clones/line were analyzed. Normalized luc activity of the ASp promoter-exon 1-intron 1 was significantly higher than the ASp promoter in stably transfected cells, 86±15 v 28±3 (p<0.001). Mutation of both GATA-1 sites in the exon 1-intron 1 plasmid reduced activity to background. Normalized luc activity from the promoter-exon was 46±6; from the promoter-intron 101±31, suggesting the intron functions as a positive regulatory element. A barrier assay was performed by flanking a β-globin promoter-EGFP gene using wild type (WT) exon 1, exon 1 with the GATA site abolished, or WT intron 1, and stably transfecting the plasmids into K562 cells. The WT exon 1 and mutant exon 1 expressed GFP in 10/12 and 7/8 lines, respectively, indicating a barrier function for exon 1 independent of GATA-1 activity. Only 1/9 lines expressed EGFP when the cassette was flanked by the ASp intron and 0/8 expressed EGFP when there were no sequences flanking the β-globin promoter. TG mouse lines with the Asp promoter-exon 1 or the Asp promoter-intron 1 linked to the Aγ-globin gene were created. 1/5 TG lines with ASp promoter-exon 1 expressed at low levels and 3/7 TG lines with ASp promoter-intron 1 expressed at levels comparable to the ASp promoter-exon 1-intron 1. These results demonstrate that 2 elements downstream of the ASp promoter are required for high-level, erythroid-specific expression. Exon 1 has barrier activity and intron 1 functions as a positive regulatory element. This is an excellent candidate region for mutations associated with ASp-linked inherited hemolytic anemia.

Disruption of the Mechanism of Long Range Activation within the Human α-Globin Complex

The human alpha-globin complex lies at the tip of the short arm of chromosome 16. It comprises three functional globin genes (5'-zeta2-alpha2-alpha1-3'), the expression of which is strictly dependent on a positive regulatory element located 40-kb upstream, HS-40. This DNase I-hypersensitive site is the only known regulatory element displaying strong erythroid-specific enhancer activity within the human alpha-globin complex. How this enhancer activity is shared among different erythroid genes present in the same cluster without affecting the ubiquitous genes present within and around the complex is poorly understood. To address this issue, we used hybrid murine erythroleukemia cells containing a single copy of human chromosome 16 and targeted the insertion of different sequences downstream of HS-40 by recombinase-mediated cassette exchange. We thus demonstrate that (i). HS-40-mediated erythroid-specific activation of the alpha-globin genes is impaired solely by the insertion of a promoter sequence and not a coding sequence, unless it is methylated, and that (ii). the degree of transcriptional repression observed seems to be related directly to the transcriptional rate of the inserted promoter. Taken together, these results emphasize the importance of promoter sequences as the main targets for the activation mechanism of the human alpha-globin genes by HS-40.

Development of virus vectors for gene therapy of β chain hemoglobinopathies: flanking with a chromatin insulator reduces γ-globin gene silencing in vivo

AbstractWe have previously described the development of oncoretrovirus vectors for human γ-globin using a truncated β-globin promoter, modified γ-globin cassette, and α-globin enhancer. However, one of these vectors is genetically unstable, and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. To address these problems, we identified and removed the vector sequences responsible for genetic instability and flanked the resultant vector with the chicken β-globin HS4 chromatin insulator to protect expression from chromosomal position effects. After determining that flanking with the cHS4 element allowed higher, more uniform levels of γ-globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the γ-globin cassettes from the uninsulated vectors were expressed in only 2% to 5% of red blood cells (RBCs) long term, indicating they are highly sensitive to epigenetic silencing. In contrast, when present the γ-globin cassette from the insulated vector was expressed in 49% ± 20% of RBCs long term. RNase protection analysis indicated that the insulated γ-globin cassette was expressed at 23% ± 16% per copy of mouse α-globin in transduced RBCs. These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which in turn allows for γ-globin expression approaching the therapeutic range for sickle cell anemia and β thalassemia.

The expression of human alpha -like globin genes in transgenic mice mediated by bacterial artificial chromosome.

After screening a bacterial artificial chromosome of human genomic DNA library with human HS-40, zeta-, alpha-, and theta-globin probes, a 110-kb clone bearing the whole human alpha-globin gene cluster was obtained and rare restriction endonuclease mapping was performed. The bacterial artificial chromosome DNA was isolated, and transgenic mice were generated. Three founders were detected from 35 newborn mice. The copy numbers were 1, 2, and 2, and the expression of human alpha-globin genes in various tissues at different developmental stages in the transgenic mice was assayed. The human alpha-globin mRNA can be detected in bone marrow, kidney, liver, brain, but not in muscle, testis, or thymus. The human zeta-globin genes were switched off, and the alpha-globin genes were switched at day 11.5 in mouse embryo, indicating that developmental stage-specific expression of the alpha-like globin genes was properly regulated. The human alpha-globin mRNA ranged between 17-68% of the endogenous mouse alpha-globin, suggesting that the expression of human alpha-globin genes is integration site-dependent in transgenic mice. The ratio of human alpha(2)- and alpha(1)-globin gene expression in adult transgenic mouse is about 2.5:1 similar to the expression in human.

High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral vectors

AbstractUse of oncoretroviral vectors in gene therapy for hemoglobinopathies has been impeded by low titer vectors, genetic instability, and poor expression. Fifteen self- inactivating (SIN) lentiviral vectors using 4 erythroid promoters in combination with 4 erythroid enhancers with or without the woodchuck hepatitis virus postregulatory element (WPRE) were generated using the enhanced green fluorescent protein as a reporter gene. Vectors with high erythroid-specific expression in cell lines were tested in primary human CD34+ cells and in vivo in the murine bone marrow (BM) transplantation model. Vectors containing the ankyrin-1 promoter showed high-level expression and stable proviral transmission. Two vectors containing the ankyrin-1 promoter and 2 erythroid enhancers (HS-40 plus GATA-1 or HS-40 plus 5-aminolevulinate synthase intron 8 [I8] enhancers) and WPRE expressed at levels higher than the HS2/β-promoter vector in bulk unilineage erythroid cultures and individual erythroid blast-forming units derived from human BM CD34+ cells. Sca1+/lineage− Ly5.1 mouse hematopoietic cells, transduced with these 2 ankyrin-1 promoter vectors, were injected into lethally irradiated Ly5.2 recipients. Eleven weeks after transplantation, high-level expression was seen from both vectors in blood (63%-89% of red blood cells) and erythroid cells in BM (70%-86% engraftment), compared with negligible expression in myeloid and lymphoid lineages in blood, BM, spleen, and thymus (0%-4%). The I8/HS-40–containing vector encoding a hybrid human β/γ-globin gene led to 43% to 113% human γ-globin expression/copy of the mouse α-globin gene. Thus, modular use of erythroid-specific enhancers/promoters and WPRE in SIN-lentiviral vectors led to identification of high-titer, stably transmitted vectors with high-level erythroid-specific expression for gene therapy of red cell diseases.