Human Globin Gene Research Articles

The Hellenic type of nondeletional hereditary persistence of fetal hemoglobin results from a novel mutation (g.-109G>T) in the HBG2 gene promoter

Nondeletional hereditary persistence of fetal hemoglobin (nd-HPFH), a rare hereditary condition resulting in elevated levels of fetal hemoglobin (Hb F) in adults, is associated with promoter mutations in the human fetal globin (HBG1 and HBG2) genes. In this paper, we report a novel type of nd-HPFH due to a HBG2 gene promoter mutation (HBG2:g.-109G>T). This mutation, located at the 3′ end of the HBG2 distal CCAAT box, was initially identified in an adult female subject of Central Greek origin and results in elevated Hb F levels (4.1%) and significantly increased Gγ-globin chain production (79.2%). Family studies and DNA analysis revealed that the HBG2:g.-109G>T mutation is also found in the family members in compound heterozygosity with the HBG2:g.-158C>T single nucleotide polymorphism or the silent HBB:g.-101C>T β-thalassemia mutation, resulting in the latter case in significantly elevated Hb F levels (14.3%). Electrophoretic mobility shift analysis revealed that the HBG2:g.-109G>T mutation abolishes a transcription factor binding site, consistent with previous observations using DNA footprinting analysis, suggesting that guanine at position HBG2/1:g.-109 is critical for NF-E3 binding. These data suggest that the HBG2:g-109G>T mutation has a functional role in increasing HBG2 transcription and is responsible for the HPFH phenotype observed in our index cases.

Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-Specific Repressor BCL11A

Numerous molecular approaches have been taken to elucidate the regulation of the human β-like globin genes, and particularly the “fetal” (γ- to β-) globin switch, given the role of fetal hemoglobin (HbF) levels on disease severity in the β-hemoglobin disorders. Despite these efforts, no developmental stage-specific nuclear regulators of HbF expression have been identified and validated. Recent genome-wide single nucleotide polymorphism (SNP) association studies by us and others have revealed novel loci that are significantly associated with HbF levels in normal, sickle cell, and thalassemia populations. One variant, lying within intron 2 of the chromosome 2 gene BCL11A, accounts for >10% of the variation in HbF levels. We have now tested the hypothesis that BCL11A, a zinc-finger transcription factor, serves as a stage-specific regulator of HbF expression, rather than merely a genetic marker of HbF status. We found that BCL11A is expressed as two major isoforms (termed XL and L) in human erythroid progenitors. The level of BCL11A expression is inversely correlated with the expression of the HbF gene, γ-globin, in human erythroid cell types representative of different developmental stages. Expression of BCL11A is negligible in embryonic, and high in adult, erythroid cells. Correlation of SNP genotypes with levels of BCL11A RNA in cells derived from individuals of known genotypes indicates that the “high HbF” genotype is associated with reduced BCL11A expression. To better characterize its potential role in erythropoiesis and globin gene regulation, we identified interacting protein partners of BCL11A in erythroid cells through affinity purification and protein microsequencing. We found that the BCL11A protein exists in complexes with the nucleosome remodeling and histone deacetylase (NuRD) corepressor complex, as well as the erythroid transcription factors GATA-1 and FOG-1. Taken together, the genetic, developmental, and biochemical data are most consistent with a model in which BCL11A functions as a repressor of γ-globin gene expression. To directly test this possibility, we modulated expression of BCL11A in primary human erythroid precursors expanded from adult CD34+ progenitors. Transient or persistent knockdown of BCL11A accomplished by siRNA or lentiviral shRNA delivery, respectively, led to robust induction of γ-globin gene expression. Importantly, down-regulation of BCL11A expression did not alter the differentiation state or global transcriptional profile of the cells, suggesting an effect on a limited number of targets, including the γ-globin gene. In summary, these studies establish BCL11A as a potent regulator of human globin switching. As an adult-stage repressor, BCL11A represents a primary target for therapy aimed at reactivating HbF expression in patients with β-hemoglobin disorders. Our studies illustrate the power of an integrative approach to reveal the functional connection between a common genetic variant and a trait that serves as a prominent modifier of disease severity.

Chromatin Structure and Transcription of the Human Alpha Globin Locus during Erythroid Differentation

The human alpha globin genes are controlled by DNase hypersensitive sites (HS) HS-4, HS-8, HS-10, HS-33 and HS-40 upstream of the ζ gene. Among these, HS40 functions as a strong enhancer of the alpha like genes. The alpha globin genes are situated amidst actively transcribing genes, but are transcriptionally silent in non-erythroid cells including hematopoietic progenitor cells We have undertaken an analysis of the chromatin structure of the alpha globin locus, recruitment of transcription factors, and the transcriptional activity of the locus in CD34+ hematopoietic progenitor cells and upon their differentiation into erythroid cells. Chromatin immunoprecipitation (ChIP) followed by PCR analysis of all the regulatory and structural segments of the α-globin locus were performed using antibodies against chemically modified tails of histone H3, the insulator binding factor CTCF, transcription factors such as GATA-1 and NF-E2, and Pol II. Both H3Me2K4 and H3AcK9 modifications were present at HS48 and HS33 in CD34+ cells and substantially increase when these cells are differentiated into erythroid lineage. At the HS40 region, these modifications were present at a low level in CD34+ cells and did not change during erythroid differentiation. Among the α-like gene promoters, we find these modifications at the Mu and theta gene promoters in CD34+ cells and they increase during erythropoiesis. These modifications were absent at the zeta gene promoter consistent with the inactivity of this gene during definitive erythropoiesis. Overall the dominant HS40 enhancer possesses moderate levels of H3Me2K4 and H3AcK9 modifications, and its cognate major a-globin promoter is devoid of these modifications in CD34+ cells even when these cells are differentiated into erythroid lineage. The entire α-globin locus including the HS enhancer regions and a-like gene promoters did not contain the unphosphorylated (initiation) form of Pol II recruitment in CD34+ cells. When these cells differentiated into the erythroid lineage, Pol II was recruited at the HS40 and HS48 regions and at the Mu and theta promoters. Rearrangement of the CTCF binding sites at the α-globin locus occurs during differentiation of CD34+ cells into the erythroid lineage. In CD34+ cells, as in HeLa cells, the α-globin genes are flanked by multiple CTCF binding events at the 5′ and 3′ ends of the locus. At the 5′ end of the locus, the HS40 and HS48 sequences were surrounded by four CTCF binding sites at HS33, HS46, HS55 and HS90. At the 3′ end of the locus CTCF was observed at the theta globin promoter and at the 3′ end of the theta globin gene. Upon differentiation of the CD34+ cells into the erythroid pathway, CTCF recruitment is significantly reduced at HS90 and HS46 sequences, while the sites at HS55 and HS33 show increased CTCF binding. Thus, in contrast to the CD34+ cells, the HS40 and HS48 sequences are y flanked by two CTCF recruitment sites in erythroid cells. Such a differential placement of CTCF binding sites suggests that differential interaction among CTCF sites may regulate the effects of the HS-40 enhancer. In erythroid cells, a strong HS40 enhancer formed by virtue of the recruitment of the enhancer factors can overcome blocking by the downstream flanking CTCF site and this might be mediated by specific interactions between the two flanking insulators. The CTCF binding at the 3′ end of the theta globin gene is abolished during erythropoiesis of CD34+ cells. However, the recruitment of CTCF at the theta globin promoter is unchanged suggesting that the theta globin may be insulated by the influence of the α-globin enhancer sequences. We have detected transcripts from parts of the theta and zeta genes and intergenic regions in HeLa, NB4 and 06990 lymphoblastoid cells and primary erythroid cells in culture. The transcription of the locus was localized to certain regions, suggesting that there may be unappreciated transcriptional regulatory elements within the locus.

Structural Analysis of a Methyl Cytosine-Binding Protein Complex That Regulates Expression of Fetal/Embryonic Globin Genes

DNA methylation is one of the major epigenetic mechanisms for regulating gene expression in eukaryotic organisms. Regions of increased CpG content (CpG islands) are often associated with gene promoters and can recruit regulatory complexes that specifically recognize methyl cytosine and down-regulate expression of the associated gene. One such regulatory complex, MeCPC, has recently been purified from a primary cell source and characterized as binding to the methylated ρ-globin promoter in chickens [Kransdorf et al. Blood 2006; 108:2836–45]. This complex contains the chicken homologues of MBD2, MTA1, HDAC2, p66, and Mi2. Methylation of the ρ-globin promoter and binding of the MeCPC promotes the switch to β-globin during development while siRNA knock-down of cMBD2 leads to a 25-fold increase in ρ-globin expression. Likewise, knockout of MBD2 results in a ~20 fold upregulation of the human gamma globin gene in β.YAC transgenic mice [Rupon et al. PNAS 2006; 103:6617–22]. These observations suggest that disruption of the MeCPC would increase fetal hemoglobin expression; a therapeutically beneficial effect for both sickle cell anemia and β-thalassemia. This possibility is further supported by the observation that DNA methylation inhibitors such as 5-azacitidine can increase the expression of γ-globin in patients. In order to determine the molecular details of the interactions that form the MeCPC, we are pursuing atomic resolution structural analysis of MBD2 and its binding partners. The solution structure of the methyl binding domain from MBD2 bound to a methylated oligonucleotide from the ρ-globin promoter has been determined by NMR. MBD2 recognizes a central methylated CpG in this oligonucleotide through highly conserved arginine and tyrosine amino acids. Interestingly, MBD2 adopts a single orientation on the ρ-globin promoter sequence despite the symmetry of the CpG sequence. This observation indicates that interactions between MBD2 and the nucleotides surrounding the methylated CpG likely orient the protein on this promoter and may help explain why different methyl binding domain proteins are selectively recruited to different promoter regions and CpG islands. This work represents the first atomic resolution structure reported to date of MBD2 bound to a methylated promoter sequence identified as a target in a primary cell source. Additional structural studies reported here build on the observation that MBD2 directly interacts with p66α through two highly conserved regions in p66α and two regions in MBD2 [Brackertz et al. J. Biol. Chem. 2002; 277:40958–66]. The coiled coil domains of each protein have been characterized by gel filtration chromatography and NMR spectroscopy. These studies demonstrate that a specific and stable interaction is formed between the coiled domains from each protein. Intermolecular NOEs have been measured by NMR which highlight the amino residues responsible for direct protein:protein contacts. Ultimately these structural studies will provide a molecular basis for the development of agents that will specifically disrupt the MeCPC complex and increase expression of embryonic and fetal hemoglobin in adult erythrocytes.

Gamma-Globin Gene Expression in Adult Human Erythroblasts Is Associated with Concurrent Changes in the Nuclear Protein Levels of at Least Seven Transcription Factors.

Alterations of globin-gene cluster expression mediated through cytokine signal transduction have been previously established. In adult erythroblasts, ex vivo combinatorial signaling from cytokines such as erythropoietin (EPO), stem cell factor (SCF), and transforming growth factor-beta (TGF-B) causes a robust reactivation of fetal hemoglobin. To determine if cytokine-activated expression of fetal hemoglobin significantly alters the transcript and protein abundance of nuclear transcription factors, profiling studies of transcription factor expression were performed. Primary CD34+ cells were cultured as donor-matched pairs using cytokine combinations that produced low vs. high levels of gamma-globin mRNA and fetal hemoglobin (Bhanu et al. Blood, 2005). To identify candidate genes, total RNA from 15 separate healthy volunteer donors was collected and combined into 5 pools. The pooled RNA was labeled and hybridized to Affymetrix HG-U133 Plus 2.0 GeneChips. Informatics strategies were aimed toward identifying differential expression of those transcription factors with binding motif on any of the 10 human globin gene promoter regions (553 transcription factors; 115 binding motif families), versus all transcription factors demonstrating robust expression with changes of at least 3-fold. Combining both strategies, 11 candidate transcription factors were identified (BCL11A, CBFB, EGR-1, ELK-1, HHEX, ID2, MAFF, MNDA, SOX-6, TCF3, and THRB). RNA-based descriptions of gene activity do not necessarily reflect changes in protein levels; therefore, Western analyses were performed. GATA-1 was utilized as a control, since no significant change in GATA-1 mRNA was detected by expression array profiling. Nuclear protein extracts were obtained from 3 additional donors' CD34+ cells cultured under conditions of low vs. high gamma-globin mRNA synthesis (1.6E+06 ± 3.8E+05 and 1.5E+07 ± 6.3E+06 copies/ng total RNA, respectively). Western blot analysis revealed reproducible and robust differences in protein expression levels for 7 of the 11 candidate transcription factors (EGR-1, ELK-1, HHEX, ID2, MAFF, MNDA, and SOX-6). Two other candidates were expressed below the protein detection limit. GATA-1 and the remaining 2 candidates demonstrated no change in the nuclear protein levels. Among the group with confirmed changes in gene expression, ELK-1 and EGR-1 are downstream targets of the MAP kinase signal transduction cascade. MAFF, ID2, and SOX-6 are known regulators of globin gene expression. The differential expression of HHEX and MNDA warrants further investigation. These data demonstrate that cytokine signal transduction causes changes in the intranuclear levels of at least 7 transcription factors concurrently with activation of gamma-globin gene expression.

Le profil sérologique des molécules d’adhésion chez des patients drépanocytaires

Sickle-cell disease (SCD) is an hemoglobinopathy resulting from a ponctual mutation in the human globin gene. The major related SCD complications are usually unpredictable and reveal the lack of our knowledge of their starting factors. The aim of this study was to evaluate the serologic profile of three adhesion molecules: intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and l-selectin in patients with SCD. Studied adhesion molecules were quantitated by an ELISA sandwich method. In all studied sickle-cell patients, the levels of adhesion molecules were significantly increased. These results outline the important participation of endothelial cells through the overexpression and release of adhesion molecules in case of their activation by the vascular microenvironment. La drépanocytose, qui est une hémoglobinopathie dûe à une mutation ponctuelle sur le gène de la globine humaine, est imprévisible par la survenue des complications majeures, traduisant en fait notre ignorance de leurs facteurs déclenchants. Dans le but de participer à l’ensemble des études réalisées ces dernières années sur la drépanocytose, nous avons étudié le profil sérologique des molécules d’adhésion intercellular adhesion molecule-1, vascular cell adhesion molecule-1 et l-séléctine chez les malades drépanocytaires de différents génotypes. Nous avons réalisé une étude quantitative de ces trois molécules d’adhésion par une technique immunoenzymatique basée sur le principe Elisa de type sandwich. Une augmentation exagérée de ces molécules chez les sujets malades a été observée. Les résultats de notre étude montrent la drépanocytose sous un angle différent avec une participation importante de la cellule endothéliale via ces molécules d’adhésion qui sont exprimées et secrétées en cas d’activation par le microenvironnement vasculaire.

A mechanism for Ikaros regulation of human globin gene switching

The human beta globin locus consists of an upstream LCR and functional genes arranged sequentially in the order of their expression during development: 5'-HBE1, HBG2, HBG1, HBD, HBB-3'. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Here we show that the transcription factor Ikaros plays a major role in the formation of the beta-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation, there is concomitant marked down-regulation of HBB, and up-regulation of HBG expression. We show for the first time Ikaros and its family member Eos, bind to critical cis elements implicated in haemoglobin switching and deletional hereditary persistence of fetal haemoglobin (HPFH). Chromatin conformation capture (3C) data demonstrated that Ikaros facilitates long-distance DNA looping between the LCR and a region upstream of HBD. This study provides new insights into the mechanism of stage-specific assembly of the beta-globin ACH, and HPFH.

Mouse models of sickle cell disease

In the absence of a natural animal model for sickle cell disease, transgenic mouse models have been generated to better understand the complex pathophysiology of the disease and to evaluate potential specific therapies. In the early nineties, the simple addition of human globin genes induced the expression of hemoglobin S (HbS) or HbS-related human hemoglobins in mice still expressing mouse hemoglobin. To increase the proportion of human hemoglobin and the severity of the mouse sickle cell syndrome, the proportion of mouse hemoglobin could be decreased by a combination of mouse α- and β-thalassemic defects, leading to complex genotypes and mild disease. Following the discovery of gene targeting in the mouse embryonic stem cells (ES cells), it was made possible to knock out all mouse adult globin genes ( 2α and 2β) and to add the human homologous genes elsewhere in the mouse genome. In addition, the human γ gene of fetal hemoglobin was protecting the fetus from HbS polymer formation. Accordingly, the resulting adult mouse models obtained in 1997, expressing human HbS-only, had a very severe anemia (Hb = 5–6 g/dL). In order to survive, these “HbS-only mice” had to reduce the HbS concentration within the red blood cells. The phenotype could be less severe by adding modified human γ genes, still expressed in adult mice. In 2006, a last “S-only” model was obtained by homologous knock in, replacing the mouse globin genes by human genes. This array of models contributes to better understand the role of different interacting factors in the complexity of sickle cell events, such as red cell defects, changes in blood flow and vaso-occlusion, hyperhemolysis, vascular tone dysregulation, oxidations, inflammation, activation and adhesion of cells, ischemia, reperfusion… In addition, each model has an appropriate usefulness to evaluate experimental therapies in vivo and to perform preclinical studies.

The 5′ Regulatory Region of the Human Fetal Globin Genes is a Gene Conversion Hotspot

The human fetal globin genes consist of the first mammalian genomic loci for which gene conversion was reported. To date, 14 gene conversions have been described in the human Gγ- and Aγ-globin genes, the vast majority of which are restricted to the coding sequences. Here, we provide evidence for three new gene conversion events in the 5′ regulatory region of the human fetal globin genes, identified during a large genetic screening effort in adult individuals with high fetal hemoglobin (Hb) levels. The sequence variations, resulting from these conversion events, are transcriptionally silent polymorphisms that do not contribute to increased fetal Hb levels. Our results suggest that the 5′ regulatory region of the human fetal globin genes is a gene conversion hotspot that prevent globin gene promoter sequence diversification, further underlining the need for two functional fetal globin genes in fetal erythropoiesis.

Tissue-specific histone modification and transcription factor binding in α globin gene expression

To address the mechanism by which the human globin genes are activated during erythropoiesis, we have used a tiled microarray to analyze the pattern of transcription factor binding and associated histone modifications across the telomeric region of human chromosome 16 in primary erythroid and nonerythroid cells. This 220-kb region includes the alpha globin genes and 9 widely expressed genes flanking the alpha globin locus. This un-biased, comprehensive analysis of transcription factor binding and histone modifications (acetylation and methylation) described here not only identified all known cis-acting regulatory elements in the human alpha globin cluster but also demonstrated that there are no additional erythroid-specific regulatory elements in the 220-kb region tested. In addition, the pattern of histone modification distinguished promoter elements from potential enhancer elements across this region. Finally, comparison of the human and mouse orthologous regions in a unique mouse model, with both regions coexpressed in the same animal, showed significant differences that may explain how these 2 clusters are regulated differently in vivo.

Human Gamma Globin Gene Regulation in Knock-In Mouse Models of Anemia.

The generation of transgenic and gene targeted mouse models of human hemoglobinopathies provides valuable opportunities to test mechanisms of human globin gene regulation and experimental therapies. Yet mice do not naturally have a fetal hemoglobin, challenging our ability to adequately model the developmental onset of disease. Transgenic model systems that contain the entire human β-globin locus present obstacles to the study of human globin gene switching, including a fetal to adult globin gene switch that occurs too early in development. The generation of genetically engineered mice with a delayed human γ to β hemoglobin switch has been a major topic of interest for our laboratory. Delayed γ globin gene expression improves the clinical progression in patients as well as animal models with hemoglobinopathies. However, molecular mechanisms involved in globin gene switching are not well understood. In this study the transcriptional and epigenetic regulation of human γ to β hemoglobin switching are analyzed in novel human knock-in (KI) mouse models that complete the switch from fetal to adult hemoglobin after birth. These KI mice were generated by replacement of the adult mouse β-globin genes by homologous recombination in embryonic stem cells with a delayed switching human γ to β globin gene construct. Quantitative real-time PCR and HPLC were used to measure mouse and human embryonic, fetal, and adult globin genes through development and show that we have given the mouse a true fetal hemoglobin. Heterozygous mice express human β-like globin genes at a high level comparable to the adult mouse β globin genes. Mutations responsible for hereditary persistence of fetal hemoglobin (HPFH) in the γ globin promoter recapitulate the human phenotype in KI mice, with over 50 fold γ globin gene upregulation in adults. These HPFH KI mice also display higher γ globin levels at birth and markedly delayed γ globin gene downregulation in the weeks following birth. These studies in KI mice demonstrate that human β-like globin genes interacting with the mouse LCR are regulated in a manner similar to what is seen in humans and may be used to study the mechanisms of globin gene switching. Greater understanding of γ-globin gene regulation will be required for achieving the therapeutic goal of reactivating silenced γ-globin genes to ameliorate severe human hemoglobinopathies.

Silencing of γ-Globin Gene Expression during Adult Definitive Erythropoiesis Is Mediated by a GATA-1 Repressor Complex.

Autonomous silencing of gene expression is one mechanism operative in the control of human β-like globin gene switching. Experiments using variously truncated Aγ-globin genes linked to LCR sequences suggested that a region of the Aγ-globin gene between -730 to -378 relative to the mRNA CAP site may function as an adult stage-specific silencer element. A marked copy of the Aγ-globin gene (Aγm-globin) was inserted between LCR 5′ HS1 and the ε-globin gene in a human β-globin locus yeast artificial chromosome (Aγm 5′ ε β-YAC). The Aγm-globin gene was autonomously silenced in Aγm 5′ ε β-YAC transgenic mice, even in the absence of an adult β-globin gene. A -730 to -378 deletion of the Aγm-globin gene was introduced into the Aγm 5′ ε β-YAC to produce a Δ1s Aγm 5′ ε β-YAC. Transgenic lines containing intact β-globin loci expressed the Δ1s Aγm-globin gene in embryonic yolk sac, fetal liver, and adult blood. To further delineate the function of the Δ1s fragment, transient transfection assays and protein-DNA interaction assays were performed. The Δ1s fragment was found to act as a repressor of a constitutively active SV40 promoter in K562 cells. DNaseI footprinting analysis and electromobility shift assays demonstrated GATA-1-binding at a site -570 bp upstream of the Aγ-globin CAP site. Recently generated β-YAC transgenic mice containing a T>G point mutation at the -570 GATA site of the normally-located Aγ-globin gene displayed a HPFH phenotype. Together, these data suggested that the -730 to -378 Aγ-globin gene region contains a silencer element at the -570 GATA site that binds a GATA-1 repressor complex during the adult stage of definitive erythropoiesis to silence expression of the Aγ-globin gene. Previous studies suggested that when GATA-1 functions as a repressor, it interacts with components of the MeCp1/NuRD complex. This complex may remodel chromatin into a repressed state, leading to silenced Aγ-globin gene expression during adult definitive erythropoiesis. The presence of components of the MeCP1/NuRD complex was assessed in uninduced (γ-globin repressor present) and induced (γ-globin repressor absent) erythroid cells (K562 and KU812) and non-erythroid cells (HFF) by Western blot analysis using an antibody to Mi2, which is a component of the NuRD complex. Mi2 protein was observed in erythroid cells when the levels of γ-globin were low (uninduced K562 or KU812 cells), whereas only a weak signal was detected when γ-globin expression was induced in these cells. The Mi2 signal in the HFF cells was even weaker. Chromatin immunoprecipitation (ChIP) using fetal liver samples from day E12 and E18 conceptuses of wild-type β-YAC transgenics showed that GATA-1, FOG-1 and Mi2 proteins co-localize to the -570 GATA site of the Aγ-globin gene in samples where γ-globin is silenced (E18 fetal liver), but not in samples where γ-globin is expressed (E12 fetal liver). Our data strongly suggest that the MeCP1/NuRD complex interacts with GATA-1 protein to form a repressor that may be involved in silencing Aγ-globin gene expression. In addition, we show that GATA-1, FOG-1 and Mi2 are recruited to the analogous -567 GATA site of Gγ-globin, in a pattern that parallels that of Aγ-globin. However the binding of these proteins to Gγ-globin is weaker than that observed for Aγ-globin. These data suggest that GATA-1-mediated repression is common to both γ-globin genes, but that other mechanisms function in the differential regulation of the two γ-globin genes.

Efficient Delivery of the A-Gamma Globin Gene in Human Hematopoietic and Non-Hematopoietic Cells by Cationic Phosphonolipid- and Lipophosphoramide-Mediated Transfection.

Liposome-mediated gene transfer is known to exhibit a number of desirable features for gene delivery, such as decreased immunogenicity, lack of mutagenesis, efficient transgene expression, internalization of episomal DNA irrespective of size, reproducibility, compositional variability and ease of use. As we and others recently demonstrated, cationic Phosphonolipids (CPs) have pronounced gene delivery potency into human cells. Based on the well-established dependency of gene transfer efficiency upon lipoplex properties, the superior-performing CP EG.308 and a novel cationic lipophosphoramide vechicle (CL) KLN-5, were evaluated for delivery of the human A-gamma globin gene in K562 human erythroleukemia and 5637 human epithelial carcinoma cells. The plasmid vector used for Aγ-globin gene was pAγLuc (3.3 kb HindIII human Aγ-globin gene). Two other vectors were used as controls:i.pGL3bLuc+, which encodes for luciferase; and,ii.pEGFP-CI, which encodes for the green fluorescent protein.Dynamic Light Scattering experiments underlined the cooperative effect of the liposome vesicle diameter, complex formation medium and charge ratio upon lipoplex size. At optimized lipoplex generation conditions, the initial lipid size was not found to be always predictive of lipofection potency, rather transfection efficiency is a function of the liposome vehicle to generate lipoplexes of enhanced size (300–3500nm). Dot-blot analysis and reporter gene-expression assays revealed that among a number of varying composition solvent-systems, optimal nuclear transgene incorporation and expression was attained in 300mM NaCl. Lipoplexes EG.308- and KLN-5/pAγLuc formed in 300mM NaCl mediated maximal transgene delivery and expression 1 and 3 days-post transfection, respectively (330pg luciferase/ng genomic DNA, 80000 RLU/μg protein, respectively). On day 14, sustained plasmid DNA quantity was still detected in the nucleus. Enhanced plasmid integrity was confirmed by Southern blotting analysis. Both linear and circular pAγLuc DNA delivery resulted in pronounced transgene entry (330pg/ng genomic DNA); however, by introducing the latter, transgene expression was almost 100% higher. As opposed to the effective KLN-5-mediated transfection of K562 cells (330pg luciferase/ng genomic DNA, 80000 RLU/μg protein), transgene targeting and expression in 5637 cells was low (73pg luciferase/ng genomic DNA, 13150 RLU/μg protein). Confocal fluorescence microscopy revealed similar lipoplex-cell membrane association events and overlapping endocytic activity in EG.308- and KLN-5- mediated lipofection of target cells. However, KLN-5/pDNA nuclear co-localization in a greater number of GFP+ transfected cells compared to EG.308/pDNA lipofected ones, support a lipid-specific complex behaviour. These results indicate that effective transgene nuclear targeting is feasible with the tested CP and CL. Human Aγ-globin transgene nuclear incorporation and expression in target cells by CPs lends credence to their use as a vehicle of therapeutic transgene delivery.

Induction of γ-globin mRNA, erythroid differentiation and apoptosis in UVA-irradiated human erythroid cells in the presence of furocumarin derivatives

Psoralens, also known as furocoumarins, are a class of photosensitizers largely used in the therapy of various skin diseases. In this study we have evaluated the combined effects of UVA irradiation and furocoumarins derivatives on (a) erythroid differentiation and apoptosis of human leukemia K562 cells and (b) globin gene expression in cultures of human erythroid progenitors derived from the peripheral blood. To prove the activity of a series of linear and angular furocoumarins derivatives, we employed the human leukemia K562 cell line and the two-phase liquid culture procedure for growing erythroid progenitors. Quantitative real-time reverse transcription polymerase-chain assay (Q-RT-PCR) was employed for quantification of the accumulation of globin mRNAs. The results obtained demonstrate that both linear and angular furocoumarins are strong inducers of erythroid differentiation of K562 cells. From a preliminary screening, we have selected two derivatives, 5-methoxypsoralen (5-MOP) and trimethylangelicin (TMA), for which we have investigated their mechanism of action. The cell cycle analysis showed that these derivatives induce, after irradiation, a cell cycle arrest in the G2/M phase, followed by apoptosis. Mitochondrial depolarisation and caspases activation seem to be involved in the mechanism of cell death. In erythroid precursor cells, psoralens in combination with UVA irradiation, stimulate at very low concentrations a preferential increase of γ-globin mRNA. Altogether, these data suggest that psoralen derivatives warrant further evaluation as potential therapeutic drugs in β-thalassemia and sickle cell anemia.

Point:Counterpoint: Sickle cell trait should/should not be considered asymptomatic and as a benign condition during physical activity

Sickle cell trait (SCT) is found in ∼8% of African Americans and may be found in up to 40% of West Africans. However, most of them are unaware of their status. This observation suggests that SCT may be asymptomatic and remains a benign condition for daily life activities ([5][1]). Indeed, numerous

Maturation and enucleation of primitive erythroblasts during mouse embryogenesis is accompanied by changes in cell-surface antigen expression

AbstractPrimitive erythroblasts (EryPs) are the first hematopoietic cell type to form during mammalian embryogenesis and emerge within the blood islands of the yolk sac. Large, nucleated EryPs begin to circulate around midgestation, when connections between yolk sac and embryonic vasculature mature. Two to 3 days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and rapidly outnumber EryPs in the circulation. The development and maturation of EryPs remain poorly defined. Our analysis of embryonic blood at different stages reveals a stepwise developmental progression within the EryP lineage from E9.5 to E12.5. Thereafter, EryDs are also present in the bloodstream, and the 2 lineages are not easily distinguished. We have generated a transgenic mouse line in which the human ϵ-globin gene promoter drives expression of green fluorescent protein exclusively within the EryP lineage. Here, we have used this line to characterize changes in cell morphology and surface-marker expression as EryPs mature and to track EryP numbers and enucleation throughout gestation. This study identifies previously unrecognized synchronous developmental stages leading to the maturation of EryPs in the mouse embryo. Unexpectedly, we find that EryPs are a stable cell population that persists through the end of gestation.

An Overview of Current Microarray-Based Human Globin Gene Mutation Detection Methods

The panoply of human globin gene mutation detection methods could become significantly enriched with the advent of microarray-based genotyping platforms. The aim of this article is to provide an overview of the current medium and high-throughput microarray-based globin gene mutation detection platforms, namely the microelectronic array, the “thalassochip” arrayed primer extension (APEX) technology and the single base extension methods. This article also outlines an emerging method based on multiple ligation probe amplification (MLPA) and discusses the implications of customized solutions for resequencing of genomic loci in relation to molecular genetic testing of hemoglobinopathies.

Lineage-specific activators affect β-globin locus chromatin in multipotent hematopoietic progenitors

During development, the regulated expression of tissue-specific genes can be preceded by their potentiation, that is, by chromatin activation in progenitor cells. For example, the human β-like globin genes are potentiated in a gene- and developmental-specific manner in hematopoietic progenitors. Developmental regulation of human β-gene expression in erythroid cells is mostly determined by transcriptional activators; however, it is not clear how gene-specific potentiation is set in hematopoietic progenitors. Using human and transgenic multipotent hematopoietic progenitors, we demonstrate that human β-globin locus activation is characterized by TBP, NF-E2, CBP and BRG1 recruitment at both the Locus Control Region and human β-gene promoter. Our results further indicate that in hematopoietic progenitors, EKLF influences chromatin organization at the human β-globin locus and is instrumental for human β-gene potentiation. Thus, we show that lineage-specific transcriptional activators expressed at basal levels in progenitor cells can participate in gene potentiation.

New insights into early human erythropoiesis and globin gene switching from differentiation of human ES cells

New insights into early human erythropoiesis and globin gene switching from differentiation of human ES cells

Novel Insights into the Maturation and Enucleation of Primitive Erythroblasts.

Primitive erythroblasts (EryP), the first blood cell type to form during mammalian embryogenesis, arise in the yolk sac (YS) as a synchronous cohort before the establishment of the embryonic circulation. The large, nucleated EryP begin to circulate around midgestation, when connections between YS and embryo mature. Two to three days later, small cells of the definitive erythroid lineage (EryD) begin to differentiate within the fetal liver and rapidly outnumber EryP within the circulation. Cells of the two erythroid lineages differ not only in size but also in their expression of globin genes. Recently it has been shown that EryP begin to enucleate around E12.5 in the mouse. We have begun to characterize EryP maturation during mouse embryogenesis, with a focus on circulating cells. Analysis of embryonic blood at different stages revealed a stepwise developmental progression within the primitive erythroid lineage: loss of nucleoli (E9.5–10.5); decrease in cell diameter and cross-sectional area (E10.5–11.5); progressive nuclear condensation (E10.5 onwards); and enucleation (E12.5 onwards). Thereafter, EryD are also present in the bloodstream. A major obstacle to the study of primitive erythroid development is the current inability to easily distinguish EryP from EryD and to cleanly separate these populations, particularly given that enucleation can no longer be considered an exclusively definitive erythroid characteristic. No EryP-specific surface antigens have been identified that would permit the detection and isolation of cells of this lineage. We have exploited the restricted expression of the human embryonic beta-like globin gene epsilon to generate EryP lineage-specific transgenic mouse and embryonic stem (ES) cell models in which green fluorescent protein (GFP) reporter expression is driven exclusively in EryP and have used this line to track quantitatively the differentiation and maturation of circulating EryP during embryogenesis. The maturation of primitive erythroblasts was reflected in their dynamic expression of Ter119 and CD71. Up-regulation of a number of nonerythroid-specific cell surface markers, including integrins and other adhesion molecules, was apparent beginning around E12.5, reflecting a previously unrecognized EryP maturation process. Interestingly, enucleation of EryP is accompanied by changes in surface antigen expression. Thus, the phenotype of primitive reticulocytes is distinct from that of their nucleated precursors. In contrast with the prevailing view that EryP are present only transiently in the embryo, we find that they comprise a stable cell population that persists through the end of gestation. The human epsilon-globin::GFP transgenic mouse model provides a novel system for defining, at the cellular and molecular level, the mechanisms involved in development of the primitive erythroid lineage.