Human Globin Research Articles

A Modified ANK-1 Promoter Directs Uniform, Copy Number Dependent Gamma Globin Gene Expression at Therapeutic Levels of a in a Lentivirus Vector.

Abstract 3565Poster Board III-502Successful gene therapy for hemoglobin disorders beta-thalassemia and sickle cell disease (SCD) has been hindered by inefficient expression of beta-like globin genes from viral vectors. It is estimated that transduction of ∼25% of hematopoietic stem cells (HSC) to express a beta-like globin gene at ∼20% the level of endogenous alpha-globin would be therapeutic. The expression of the beta-like globin genes in their native locus is a precisely regulated process that relies on the coordinated activities of several cis-acting enhancer elements that make up the beta-globin locus control region (LCR). Expression of therapeutic levels of beta-like globin in mouse models and patient cells requires inclusion of LCR elements. These LCR elements are susceptible to silencing, reduce virus titer and can potentially activate oncogenes at the site of proviral insertion. To minimize these risks we have developed a novel approach using non-globin erythroid promoters to drive high levels of globin gene expression independent of enhancers. Previously, we showed that a minimal 276-bp promoter of the human ankyrin gene (ANK-1) does not contain an enhancer element, but does contain a barrier element that allows erythroid-specific, uniform, position independent and copy-number dependent expression of a linked gamma-globin gene. However, the level of gamma-globin expression from the ANK-1 promoter (4% of mouse alpha-globin per copy) was sub-therapeutic. TFIID, a component of the transcription initiation complex, binds a 9-bp sequence (TGCGGTGAG) within the wild type ANK-1 promoter. A dinucleotide deletion within this sequence that disrupts the binding of TFIID and results in ankyrin deficient Hereditary Spherocytosis (Gallagher et al. Hum Mol Genet.14: 2501-9. 2005). We hypothesized that sequence modifications of the ANK1 promoter in this region would increase the affinity for TFIID and increase expression. Mutational analysis of the wildtype TFIID site identified a number of sequence variants that increase expression. An ANK-1 promoter sequence variant that differs from the wildtype TFIID sequence in the first three nucleotides (GCGGGTGAG; GC-ANK-1), results in a 7-fold increase in transcriptional activity in K562 cells. We developed five independent transgenic mouse lines that carry this GC-ANK-1 promoter linked to a human gamma globin gene. Expression in all five lines is erythroid-specific, uniform, position independent and copy-number dependent. Moreover, the average level of gamma-globin mRNA is 34 ± 14 % of mouse alpha-globin mRNA per transgene copy, a 8.5-fold increase in gamma-globin expression over mice harboring the wildtype ANK-1 promoter. We developed a simplified lentiviral vector that contains the CG-ANK-1/gamma-globin gene. This vector was produced at high titer and transduced 57% (23/40) of mouse colony forming unit Spleen (CFU-S) cells. In CFU-S containing single copies of the provirus, the level of gamma-globin mRNA was 6.8 ± 2.9 % of mouse alpha-globin per copy. In CFU-S with two and three copies of the GC-ANK-1 gamma-globin provirus, expression of gamma-globin mRNA averaged 20% ± 1.6% and 30% of mouse alpha-globin per copy respectively, demonstrating copy-number dependent of expression. Overall, an analysis of 16 CFU-S foci demonstrated that the CG-ANK-1/gamma-globin vector expressed gamma globin mRNA at levels that average 8.5 ± 2.0% of mouse alpha-globin. Our data demonstrate that just two copies of the simplified CG-ANK-1/gamma-globin lentiviral vector could potentially produce the levels of gamma-globin necessary for effective treatment of the hemoglobin disorders. Disclosures:No relevant conflicts of interest to declare.

Targeting a Methyl Cytosine-Binding Protein Complex to Augment Fetal/Embryonic Globin Expression.

Abstract 974The vertebrate β-type globin genes were among the first genes shown to be regulated, at least in part, by DNA methylation. The mechanism of transcriptional repression by DNA methylation is chiefly through binding of methyl cytosine binding domain (MBD) proteins and their associated co-repressor complexes. The chicken homolog to an MBD2 containing NuRD co-repressor complex (MeCPC) has previously been purified from primary erythroid cells and characterized as binding to the methylated ρ-globin promoter in erythroid cells of adult chickens in which the gene is silent [Kransdorf et al. Blood 2006; 108:2836-45]. Knockdown of MBD2 by siRNA in MEL cells stably transfected with a methylated ρ-globin gene construct leads to a greater than 10-fold increase in ρ-globin gene expression. Likewise, knockout of MBD2 results in a ∼20 fold upregulation of the human gamma globin gene in adult erythroid cells of βYAC transgenic mice [Rupon et al. PNAS 2006; 103:6617-22]. These observations suggest that disruption of the interaction of MBD2 with its co-repressor complex in adult erythropoiesis 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. Based on these studies, we have pursued structural analysis of the interaction between MBD2 and other components from the MeCPC. We have shown that the individual coiled coil regions from MBD2 and a subunit of the NuRD complex, p66α, form a stable heterodimeric complex. Solving the structure of this coiled coil complex by NMR reveals that the interaction involves a combination of hydrophobic and ionic interactions typical of coiled coil complexes as well as a unique charge interaction involving a pair of highly conserved glutamates residues from p66α and arginine residues from MBD2. The key residues involved in binding are conserved across species, between p66α and p66β homologs, as well as between MBD2, MBD3, and the MBD3L1-L5 homologs. We have shown that the p66α coiled coil can stably bind to MBD3 in solution, indicating that similar tertiary interactions are involved in forming both MBD2 and MBD3 containing NuRD complexes. In order to explore this interaction as a potential therapeutic target, we hypothesized that over-expressing the p66α coiled coil region in tissue culture would disrupt the formation of a normal MeCPC and thereby block the function of MBD2. As predicted, expressing this region in both avian (MEL-ρ) and human (CID-βYAC) tissue culture models of globin gene regulation in adult erythroid cells induces embryonic and fetal β-type globin gene expression, respectively. Furthermore, knock-down of p66α induces fetal/embryonic globin gene expression to a similar degree as knock-down of MBD2. These studies suggest a model in which the p66α coiled peptide can bind MBD2 and block recruitment of native p66α to the NuRD complex, thereby acting in a dominant-negative manner to disrupt MBD2 function. We propose that a peptidomimetic of the p66α coiled coil region could be used therapeutically to augment fetal hemoglobin expression. Disclosures:No relevant conflicts of interest to declare.

Compounds of the anthracycline family of antibiotics elevate human γ-globin expression both in erythroid cultures and in a transgenic mouse model

We examined the effect of the anthracyclines aclarubicin, bleomycin, daunorubicin, doxorubicin and idarubicin on human γ- and β-globin promoter activity in an in vitro luciferase assay, ex vivo in erythroid cultures and in vivo in transgenic mice carrying the human γ-globin gene. Effects in erythroid liquid cultures derived from healthy donors were assayed by evaluating HbF production with high performance liquid chromatography and by measuring mRNA levels of the globin genes and the proportion of erythroblasts containing HbF. Compounds testing positive in the in vitro and ex vivo assays were applied to erythroid cultures derived from thalassaemic patients. Doxorubicin, idarubicin and daunorubicin increased HbF production in cultures of both, healthy and thalassaemic donors. Daunorubicin induced HbF in thalassaemic cells ex vivo with the highest statistical significance and, importantly and in contrast to the clinical HbF inducer hydroxyurea, showed specific induction of γ-globin without associated induction of α-globin. Daunorubicin was screened in transgenic mice carrying the human Aγ-globin gene, and it resulted in increased Aγ-globin mRNA levels. Our results indicate that anthracyclines are a promising group of compounds with the potential to provide lead substances for the synthesis of new agents with clinical applications as γ-globin gene inducers. In parallel, future studies of the epigenetic effects of the five anthracyclines on the β-globin locus will generate possible mechanistic leads on the regulation of the globin genes.

Persistent Fetal γ-Globin Expression in Adult Transgenic Mice following Deletion of Two Silencer Elements Located 3′ to the Human Aγ-Globin Gene

Natural deletions of the human gamma-globin gene cluster lead to specific syndromes characterized by increased production of fetal hemoglobin in adult life and provide a useful model to delineate novel cis-acting elements involved in the developmental control of hemoglobin switching. A hypothesis accounting for these phenotypic features assumes that silencers located within the Agamma-to delta-gene region are deleted in hereditary persistence of fetal hemoglobin (HPFH) and deltabeta-thalassemias, leading to failure of switching. In the present study, we sought to clarify the in vivo role of two elements, termed Enh and F, located 3' to the Agamma-globin, in silencing the fetal genes. To this end, we generated three transgenic lines using cosmid constructs containing the full length of the globin locus control region (LCR) linked to the 3.3-kb Agamma-gene lacking both the Enh and F elements. The Enh/F deletion resulted in high levels of Agamma-globin gene expression in adult mice in all single copy lines, whereas, the LCR-Agamma single copy lines which retain the Enh and F elements exhibited complete normal switching of the fetal Agamma-gene. Our study documents directly for the first time the in vivo role of these two gene-proximal negative regulatory elements in silencing the fetal globin gene in the perinatal period, and thus these data may permit their eventual exploitation in therapeutic approaches for thalassemias.

Influence of the polymorphisms of the α-major regulatory element HS-40 on in vitro gene expression

The alpha-MRE is the major regulatory element responsible for the expression of human alpha-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the alpha-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the alpha-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different alpha-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type alpha-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G-->A polymorphism at positions +130, +199, and +209, separately, were also tested. The different alpha-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C-->A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of alpha-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

Differential requirement of a distal regulatory region for pre-initiation complex formation at globin gene promoters

Although distal regulatory regions are frequent throughout the genome, the molecular mechanisms by which they act in a promoter-specific manner remain to be elucidated. The human β-globin locus constitutes an extremely well-established multigenic model to investigate this issue. In erythroid cells, the β-globin locus control region (LCR) exerts distal regulatory function by influencing local chromatin organization and inducing high-level expression of individual β-like globin genes. Moreover, in transgenic mice expressing the entire human β-globin locus, deletion of LCR-hypersensitive site 2 (HS2) can alter β-like globin gene expression. Here, we show that abnormal expression of human β-like globin genes in the absence of HS2 is associated with decreased efficacy of pre-initiation complex formation at the human ɛ- and γ-promoters, but not at the β-promoter. This promoter-specific phenomenon is associated with reduced long-range interactions between the HS2-deleted LCR and human γ-promoters. We also find that HS2 is dispensable for high-level human β-gene transcription, whereas deletion of this hypersensitive site can alter locus chromatin organization; therefore the functions exerted by HS2 in transcriptional enhancement and locus chromatin organization are distinct. Overall, our data delineate one mechanism whereby a distal regulatory region provides promoter-specific transcriptional enhancement.

Developmentally regulated extended domains of DNA hypomethylation encompass highly transcribed genes of the human β-globin locus

DNA methylation has long been implicated in developmental beta-globin gene regulation. However, the mechanism underlying this regulation is unclear, especially because these genes do not contain CpG islands. This has led us to propose and test the hypothesis that, just as for histone modifications, developmentally specific changes in human beta-like globin gene expression are associated with long-range changes in DNA methylation. Bisulfite sequencing was used to determine the methylation state of individual CpG dinucleotides across the beta-globin locus in uncultured primary human erythroblasts from fetal liver and bone marrow, and in primitive-like erythroid cells derived from human embryonic stem cells. beta-globin locus CpGs are generally highly methylated, but domains of DNA hypomethylation spanning thousands of base pairs are established around the most highly expressed genes during each developmental stage. These large domains of DNA hypomethylation are found within domains of histone modifications associated with gene expression. We also find hypomethylation of a small proportion of gamma-globin promoters in adult erythroid cells, suggesting a mechanism by which adult erythroid cells produce fetal hemoglobin. This is one of the first reports to show that changes in DNA methylation patterns across large domains around non-CpG island genes correspond with changes in developmentally regulated histone modifications and gene expression. These data support a new model in which extended domains of DNA hypomethylation and active histone marks are coordinately established to achieve developmentally specific gene expression of non-CpG island genes.

Erythroid Kruppel‐like factor (EKLF) is recruited to the γ‐globin gene promoter as a co‐activator and is required for γ‐globin gene induction by short‐chain fatty acid derivatives

The erythroid Kruppel-like factor (EKLF) is an essential transcription factor for beta-type globin gene switching, and specifically activates transcription of the adult beta-globin gene promoter. We sought to determine if EKLF is also required for activation of the gamma-globin gene by short-chain fatty acid (SCFA) derivatives, which are now entering clinical trials. The functional and physical interaction of EKLF and co-regulatory molecules with the endogenous human globin gene promoters was studied in primary human erythroid progenitors and cell lines, using chromatin immunoprecipitation (ChIP) assays and genetic manipulation of the levels of EKLF and co-regulators. Knockdown of EKLF prevents SCFA-induced expression of the gamma-globin promoter in a stably expressed microLCRbeta(pr)R(luc) (A)gamma(pr)F(luc) cassette, and prevents induction of the endogenous gamma-globin gene in primary human erythroid progenitors. EKLF is actively recruited to endogenous gamma-globin gene promoters after exposure of primary human erythroid progenitors, and murine hematopoietic cell lines, to SCFA derivatives. The core ATPase BRG1 subunit of the human SWI/WNF complex, a ubiquitous multimeric complex that regulates gene expression by remodeling nucleosomal structure, is also required for gamma-globin gene induction by SCFA derivatives. BRG1 is actively recruited to the endogenous gamma-globin promoter of primary human erythroid progenitors by exposure to SCFA derivatives, and this recruitment is dependent upon the presence of EKLF. These findings demonstrate that EKLF, and the co-activator BRG1, previously demonstrated to be required for definitive or adult erythropoietic patterns of globin gene expression, are co-opted by SCFA derivatives to activate the fetal globin genes.

Evidence for a Bigenic Chromatin Subdomain in Regulation of the Fetal-to-Adult Hemoglobin Switch

During development, human beta-globin locus regulation undergoes two critical switches, the embryonic-to-fetal and fetal-to-adult hemoglobin switches. To define the role of the fetal (A)gamma-globin promoter in switching, human beta-globin-YAC transgenic mice were produced with the (A)gamma-globin promoter replaced by the erythroid porphobilinogen deaminase (PBGD) promoter (PBGD(A)gamma-YAC). Activation of the stage-independent PBGD(A)gamma-globin strikingly stimulated native (G)gamma-globin expression at the fetal and adult stages, identifying a fetal gene pair or bigenic cooperative mechanism. This impaired fetal silencing severely suppressed both delta- and beta-globin expression in PBGD(A)gamma-YAC mice from fetal to neonatal stages and altered kinetics and delayed switching of adult beta-globin. This regulation evokes the two human globin switching patterns in the mouse. Both patterns of DNA demethylation and chromatin immunoprecipitation analysis correlated with gene activation and open chromatin. Locus control region (LCR) interactions detected by chromosome conformation capture revealed distinct spatial fetal and adult LCR bigenic subdomains. Since both intact fetal promoters are critical regulators of fetal silencing at the adult stage, we concluded that fetal genes are controlled as a bigenic subdomain rather than a gene-autonomous mechanism. Our study also provides evidence for LCR complex interaction with spatial fetal or adult bigenic functional subdomains as a niche for transcriptional activation and hemoglobin switching.

Humanized Mouse Model of Cooley's Anemia

A novel humanized mouse model of Cooley's Anemia (CA) was generated by targeted gene replacement in embryonic stem (ES) cells. Because the mouse does not have a true fetal hemoglobin, a delayed switching human gamma to beta(0) globin gene cassette (gammabeta(0)) was inserted directly into the murine beta globin locus replacing both adult mouse beta globin genes. The inserted human beta(0) globin allele has a mutation in the splice donor site that produces the same aberrant transcripts in mice as described in human cells. No functional human beta globin polypeptide chains are produced. Heterozygous gammabeta(0) mice suffer from microcytic anemia. Unlike previously described animal models of beta thalassemia major, homozygous gammabeta(0) mice switch from mouse embryonic globin chains to human fetal gamma globin during fetal life. When bred with human alpha globin knockin mice, homozygous CA mice survive solely upon human fetal hemoglobin at birth. This preclinical animal model of CA can be utilized to study the regulation of globin gene expression, synthesis, and switching; the reactivation of human fetal globin gene expression; and the testing of genetic and cell-based therapies for the correction of thalassemia.

HBA Mutations Detected By MLPA In Beta-Thalassemia Patients

Thalassemia syndromes are the most common monogenic disorders in humans and b-thalassemia major is one of the most important public health problems in Turkey. Many genetic factors play roles in modifying the disease severity such as mutations in the alpha globin gene (HBA), UGT1 gene (bilirubin metabolism) or HFE gene (iron metabolism). Deletions or duplications in HBA gene determine the accumulation of a chain in b-thalassemia major patients. The human alpha globin gene cluster is located on chromosome 16 and deletions or duplications are the most important mutations. Multiple ligation dependent probe amplification (MLPA) technique is a simple and rapid technique and used in many genetic disorders in which deletions and duplications are common. In this study we used a commercial MLPA kit for detecting alpha globin gene mutations. The kit contains 24 different probes in the HBA region. In this study we aimed to investigate the incidence of alpha globin gene mutations using MLPA technique in 19 b-thalassemia major patients which were diagnosed in our department.Alpha globin gene deletions were detected in 2 out of 19 (10.5%) b-thalassemia major patients. In one of the patients the deletion was located between the HBA2 gene and HBA1P gene. The deletion detected in the other patient was located between the HBA1 gene and HBA2 gene. This result suggests that alpha globin gene mutations in b-thalassemia major patients are not rare. MLPA technique is suitable to investigate the mutations in this complex gene cluster.

Numerical Characterization of Protein Sequences and Application to Voltage-Gated Sodium Channel α Subunit Phylogeny

We propose a new method to compare sequences of protein families by generating numerical characterizations through a 20D representation. Using a walk along the axes representing the amino acids we generate a vector for each sequence whose components can be used to derive distance matrices between sequences and whose magnitudes can be used to compare the similarities/dissimilarities between the different sequences. The distance matrices enable creation of phylogenetic trees without need for multiple alignments or any other model dependencies. In this paper we test this technique with human globin gene sequences and then apply the method to a contemporary issue of evolutionary relationships of rat and human voltage-gated sodium channel alpha subunits and compare with published literature. The close match of the results demonstrates the reliability and ease of use of this method.

Synthesis and Secretion of Hemoglobin by Retinal Pigment Epithelium

To demonstrate the production of hemoglobin by human retinal pigment epithelium (RPE). Proteomic analysis using 10 donor eyes identified hemoglobin as a major constituent of soluble human RPE proteome. Western blot analysis, RT-PCR, and immunocytochemistry were used to confirm the The presence of erythrocyte-specific proteins within primary human RPE cytosol was investigated to rule out phagocytosis as the source of hemoglobin. ELISA was used to determine the rate of hemoglobin secretion from human RPE cells. Globin mRNA expression of human RPE was studied in comparison with a human erythroblast cell line and a spontaneously transformed human RPE cell line (ARPE-19). results. Hemoglobin is a regular constituent of soluble human RPE proteome. RT-PCR and Western blot analysis confirmed the presence of hemoglobin in human RPE. No other erythrocyte-specific proteins were detected within human RPE cytosol. Hemoglobin expression persisted up to seven passages in vitro. Human RPE globin expression exceeded that in human erythroblast and ARPE-19 cells. Immunohistochemistry revealed the presence of hemoglobin within RPE and Bruch's membrane. The hemoglobin release rate was calculated to be 1.9+/-1.2 attomoles per cell per hour. Hemoglobin expression by human RPE brings a new perspective to the understanding of oxygen transport to the outer retina. Malfunction of RPE-hemoglobin production may underlie the pathophysiology of ocular diseases characterized by subfoveal hypoxia and VEGF upregulation, such as age-related macular degeneration and diabetic retinopathy. Pharmacologic modulations of local hemoglobin production in RPE cells will create new opportunities to interfere with the course of these diseases.

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.

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.

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.

Analysis of DNA Methylation at the Human Alpha Globin Cluster during Hematopoiesis.

The analysis of globin gene regulation has elucidated many of the principles underlying mammalian gene expression. To understand how the epigenetic program that unfolds during hematopoiesis impacts on alpha (α)-globin gene expression, we analyzed DNA methylation at over 200 CpG dinucleotides in 130 kb around the human α-globin gene cluster focussing particularly on a region (12 kb) covering the α2 and α1-globin genes (HBA2 and HBA1), and the upstream regulatory sites (DNAse I hypersensitive sites (HS) -48, HS-40, HS-33, and HS-10); so-called multi-species conserved sequencesregulatory element1-4 (MCS-R1-4). Cultures of primary adult erythroid cells (at three stages: early, intermediate, and late) were analyzed together with non-erythroid human cells (neutrophils, peripheral blood mononuclear cells, and a human embryonic stem (ES) cell line). Using a recently published method involving bisulphite modification and MALDI-TOF Mass Spectrometry we quantified the level of DNA methylation across the alpha globin cluster. Most sequences outside of the CpG islands were methylated. However, the DNA methylation levels between erythroid and non-erythroid cells differed at regulatory elements. MCS-R1 contained only 1 CpG site and the average percentage of DNA methylation for erythroid and non-erythroid was 8% and 40%, respectively. While they were 8% vs. 42% (p=0.428), 11% vs. 99% (p=0.054), and 14% vs. 97% (p=0.001) in MCS-R2 to 4, respectively. To study changes in DNA methylation in a comprehensive, developmental and tissue-specific manner, we used a humanized-mouse model, in which the conserved α-globin syntenic region of mouse genomic sequence is replaced by that of human. This contains all cis-acting sequences required for fully regulated expression of the α-globin genes. Although the alpha globin cluster is expressed efficiently in this model, DNA methylation patterns differed in the 20 kb region encompassing the α-globin genes when comparing primary human cells and primary humanized cells. DNA in humanized cells were relatively hypomethylated both in erythroid and non-erythroid tissues. The methylation levels were higher in testis of the humanized mouse than in other tissues but still not as high as those in normal human non-erythroid cells. The cause of these differences is being investigated. Despite these differences, the humanized-mouse mimicked the patterns of methylation found in regulatory elements in erythroid and nonerythroid cells. This study shows that despite appropriate regulation of gene expression, epigenetic templating may differ between species (human and mouse). DNA methylation at the upstream regulatory elements might be involved in the regulation of α-globin gene expression during erythropoiesis, although it is equally possible that these changes in methylation are generated by passive demethylation secondary to transcription factor binding in erythroid cells. The mechanism(s) underlying demethylation of regulatory elements during differentiation remains to be clarified.

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.