Globin Gene Regulation Research Articles

Does Transcription of Remote α-Globin Regulatory Elements Affect Their Function?.

Abstract 4060Poster Board III-995Expression of many tissue and developmental stage specific genes is controlled by remote regulatory elements. The mammalian a-globin gene cluster includes four previously characterised remote regulatory elements (MCS-R1 to R4) upstream of the a-globin genes. Naturally occurring deletions and experiments in transgenic mice, have shown that one or more of these elements are necessary for fully regulated expression of the a-like globin genes. At present it is unclear exactly how these elements interact with the a-globin genes to enhance their expression, although ultimately we know that they physically interact with the promoters forming a chromatin loop. Of interest, three of the MCS-R sequences are located within introns of a widely expressed gene C16orf35, which lies adjacent to the a-globin locus and we now know that such complex, intermingled arrangements are common in the mammalian genome. We have asked two questions: can the MCS-R elements be transcribed while activating the globin genes, and does transcription of the MCS-R elements play any role in their activation?We have analysed nascent transcription of C16orf35 and a-globin, and shown that both genes are simultaneously transcribed in a high proportion of erythroid cells. Thus it appears that the MCS-R elements can interact with the globin genes while being transcribed. We next asked whether transcription of the C16orf35 gene (through the upstream regulatory elements) is necessary for their activation. The promoter region and a putative erythroid-specific promoter of the C16orf35 gene were deleted (singly and in combination) by homologous recombination in mice. Provisional analysis has shown no evidence of a-thalassaemia in these mice, suggesting that activation of the MCS-R elements occurs independently of their transcription. In addition to their importance in globin gene regulation, these findings have general implications for the relationship between structure and function in the mammalian genome and the mechanisms by which long-range elements interact with their cognate promoters. Disclosures:No relevant conflicts of interest to declare.

A Novel EKLF Mutation in a Patient with Dyserythropoietic Anemia: The First Association of EKLF with Disease in Man.

Abstract 162We describe a single-point mutation in the transcription factor EKLF associated with dyserythropoietic anemia. The female Danish patient was extensively studied in the early 1990's (Wickramasinghe et al. Br J Haem 1991,79:322; Tang et al. Blood 1993,81:1636; Parsons et al. Blood 1994,83:860; Agre et al. J Clin Invest 1994,94:1050). The patient was severely anemic at birth and required repeated transfusions during childhood. Notable features included persistent expression of epsilon and zeta embryonic globins, an HbF level of 40%, novel intra-erythroblastic and intra-erythrocytic inclusions and deficiency of erythroid proteins CD44 and Aquaporin 1. Evidence that EKLF plays a major role in globin gene regulation in particular and erythroid gene expression in general led us to examine EKLF in this patient, her unaffected sister, and her parents. The coding sequence of EKLF was normal wild-type in both healthy parents and sister, but the patient had a mutation on one allele of EKLF in the second zinc finger domain (Glu325Lys). This mutation is at a site that is central to the expected site of interaction of EKLF with DNA. However, modelling revealed that reversal of the charge at this position in a critical DNA-binding domain was likely to create a novel direct contact between Lys325 and a phosphate on the DNA backbone, hence maintaining rather than disrupting binding of EKLF to promoter regions. To test this, we created recombinant forms of the EKLF zinc finger domain, corresponding to the wild-type, Glu325Lys and 3 presumed loss-of-function mutations previously associated with the rare blood group In(Lu) phenotype (Singleton et al. Blood 2008,112:2081). Using fluorescence-based binding assays, mutant EKLF proteins Arg328Leu, Arg328His, and Arg331Gly showed virtually no binding to the beta globin (HBB) promoter sequence, as expected. In contrast, there was no significant difference in the binding of EKLF Glu325Lys and wild-type EKLF to the promoter sequence. We then transfected full-length wild-type and mutated EKLF into K562 cells and measured the effect on expression of several genes by quantitative real-time PCR. Transfectants with wild-type EKLF showed an average 13.7 fold increase in EKLF mRNA expression (SD 4.8, n=5) compared with a clone transfected with the empty vector. This was associated with an elevation in HBB and CD44H mRNA expression (average 12.8 (SD 10.9, n=5) and 27.0 (SD 21.7, n=5) fold respectively, compared with the empty vector clone). In contrast, transfectants with EKLF Glu325Lys, although expressing slightly lower levels of EKLF mRNA than the wild-type clones (average 8.0 fold compared with the empty vector clone, SD 5.2, n=11), showed much reduced HBB and CD44H expression (average 1.9 (SD 1.6, n=11) and 1.4 (SD 1.4, n=11) fold respectively, compared with the empty vector clone). Our findings indicate that EKLF Glu325Lys has a reduced ability to activate HBB and CD44H expression, thus establishing a link between the mutation and the patient's phenotype. This reduction, however, does not appear to be explained by differences in the ability of the mutant EKLF to bind to the HBB promoter, implying that other mechanisms of gene regulation must be affected in the patient. 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.

Cytokine-mediated increases in fetal hemoglobin are associated with globin gene histone modification and transcription factor reprogramming

AbstractTherapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34+ cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

Organization of a β and α Globin Gene Set in the Teleost Atlantic Cod, Gadus morhua

Developmental globin gene expression and gene switching in vertebrates have been extensively studied. Globin gene regions have been characterized in some fish species and show linked α and β loci. Understanding coordinated expression between α and β globin genes in fish is of importance for further insights into globin gene regulation in teleosts and higher vertebrates. We characterize linked β and α globin genes in Atlantic cod, pulled from the Atlantic cod genome with a PCR research strategy, by screening a genomic λ library and primer walking. The genes are oriented tail-to-head (5'-3'), differing from the head-to-head orientation in transcriptional polarity characteristic of teleostean globin genes. Four tandem repeats are found in an intergenic region of 1500 base pairs. One microsatellite, which consists primarily of atg tandem repeats, has an open reading frame. The globin genes and open reading frame have a CCAAT promoter element and TATA boxes. The promoters of the open reading frame and the β gene share an 89-bp block (with 100% identity) that probably regulates transcription.

Ikaros and GATA-1 Combinatorial Effect Is Required for Silencing of Human γ-Globin Genes

During development and erythropoiesis, globin gene expression is finely modulated through an important network of transcription factors and chromatin modifying activities. In this report we provide in vivo evidence that endogenous Ikaros is recruited to the human beta-globin locus and targets the histone deacetylase HDAC1 and the chromatin remodeling protein Mi-2 to the human gamma-gene promoters, thereby contributing to gamma-globin gene silencing at the time of the gamma- to beta-globin gene transcriptional switch. We show for the first time that Ikaros interacts with GATA-1 and enhances the binding of the latter to different regulatory regions across the locus. Consistent with these results, we show that the combinatorial effect of Ikaros and GATA-1 impairs close proximity between the locus control region and the human gamma-globin genes. Since the absence of Ikaros also affects GATA-1 recruitment to GATA-2 promoter, we propose that the combinatorial effect of Ikaros and GATA-1 is not restricted to globin gene regulation.

Mir-144 selectively regulates embryonic α-hemoglobin synthesis during primitive erythropoiesis

AbstractPrecise transcriptional control of developmental stage-specific expression and switching of α- and β-globin genes is significantly important to understand the general principles controlling gene expression and the pathogenesis of thalassemia. Although transcription factors regulating β-globin genes have been identified, little is known about the microRNAs and trans-acting mechanism controlling α-globin genes transcription. Here, we show that an erythroid lineage-specific microRNA gene, miR-144, expressed at specific developmental stages during zebrafish embryogenesis, negatively regulates the embryonic α-globin, but not embryonic β-globin, gene expression, through physiologically targeting klfd, an erythroid-specific Krüppel-like transcription factor. Klfd selectively binds to the CACCC boxes in the promoters of both α-globin and miR-144 genes to activate their transcriptions, thus forming a negative feedback circuitry to fine-tune the expression of embryonic α-globin gene. The selective effect of the miR-144-Klfd pathway on globin gene regulation may thereby constitute a novel therapeutic target for improving the clinical outcome of patients with thalassemia.

A novel β-thalassemic allele due to a thirteen nucleotide deletion: codons 54–58 (-T ATG GGC AAC CCT)

Dear Editor, The β-thalassemia is one of the most common monogenetic diseases in the world. It is characterized by reduced or absent synthesis of β-globin chains of hemoglobin. Severe cases of β-thalassemia result in pronounced anemia which needs lifelong blood transfusion. Worldwide control of thalassemia is achieved through population screening, counseling of carriers and prenatal diagnosis of high-risk couples. Over 200 mutations causing β-thalassemia have been described from different parts of the world though relatively small numbers of these mutations can be found for each population. For example, in the Chinese population, five mutations of the 30 known, account for more than 90% of all cases [1]. Delineation of rare mutations is important for prenatal diagnosis in affected families. They may also provide insight into the underlying processes of globin gene regulation and in the studies of genotype– phenotype relationship. Here, we report a novel βthalassemic mutation due to a thirteen nucleotide deletion in the β-globin gene at codons 54–58. The family was from Maoming city of Guangdong province in southern China. They were referred to our center for prenatal diagnosis at the mother’s first trimester of gestation. Their first daughter had been diagnosed with β-thalassemia major at 4 months of age and had been regularly transfused. The child died at the age of 3 years. Blood samples were obtained from the parents, and blood count and morphology showed red blood cell microcytosis, with the following hematological findings: RBC 5.40× 10/L, Hb 11.8 g/L, MCV 72.1 fL, MCH 21.6 pg, Hb A 92.9%, and Hb A2 7.1% in the father, and RBC 4.57×10 / L, Hb 10.8 g/L, MCV 76.1 fL, MCH 22.4 pg, Hb A 94.5%, and Hb A2 5.5% in the mother. No abnormal hemoglobin was present in routine electrophoresis or in high-performance liquid chromatography analyses. β-Globin gene mutation analysis for the parents was performed by using polymerase chain reaction (PCR)-based reverse dot blots (RDB) hybridization technique [2]. After a screening of 17 known mutations in Chinese population, the father was revealed to be a heterozygote for the mutation of codon 17 (A→T), and the mother failed to identify any of the known mutations. The mother was then assumed to have an uncommon β-thalassemic allele. The β-globin gene was amplified from DNA of the mother by PCR and the PCR product was analyzed by direct nucleotide sequencing using the BigDyeTM terminator Cycle Sequencing Kit and the ABI PRISMTM 310 genetic analyzer. Sequence analysis of the entire β-globin gene revealed a novel β-thalassemia mutation in the mother. The β-thalassemic mutation was found to be a deletion of 13 nucleotides (-T ATG GGC AAC CCT) from codon 54 through codon 58 (Fig. 1). Gap-PCR was also undertaken in the couple to exclude the coinheritance of αthalassemia [3], and this did not reveal any of the three common Chinese α-globin gene deletion mutations (−, −α, and −α). The clarification of the mutations in the parents made the prenatal diagnosis easy. At 12 weeks of Ann Hematol (2009) 88:799–801 DOI 10.1007/s00277-008-0658-6

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

Differences in the amount of fetal hemoglobin (HbF) that persists into adulthood affect the severity of sickle cell disease and the beta-thalassemia syndromes. Genetic association studies have identified sequence variants in the gene BCL11A that influence HbF levels. Here, we examine BCL11A as a potential regulator of HbF expression. The high-HbF BCL11A genotype is associated with reduced BCL11A expression. Moreover, abundant expression of full-length forms of BCL11A is developmentally restricted to adult erythroid cells. Down-regulation of BCL11A expression in primary adult erythroid cells leads to robust HbF expression. Consistent with a direct role of BCL11A in globin gene regulation, we find that BCL11A occupies several discrete sites in the beta-globin gene cluster. BCL11A emerges as a therapeutic target for reactivation of HbF in beta-hemoglobin disorders.

The alpha thalassaemias.

Recent work in the alpha thalassaemia field has started to provide some indication of the mechanisms involved in the very high frequency of the different forms of alpha thalassaemia among the populations of tropical countries, and, at the same time, is starting to define at least some of the mechanisms for its remarkable phenotypic heterogeneity. These diseases continue to provide extremely valuable models for the better understanding of the regulation of the alpha globin genes, and for human molecular pathology in general. The much less common disorders, ATR-16 and ATR-X are also providing valuable information about the spectrum of molecular lesions associated with different forms of mental retardation and about the molecular mechanisms involved in their varying phenotypes.

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.

GATA Factor Mechanisms and Globin Gene Regulation.

Abstract Key steps in hematopoiesis and the expression of genes encoding hemoglobin subunits are critically dependent upon specific members of the GATA factor family of transcription factors. Our recent efforts have focused on elucidating how GATA factors select functional sites in chromatin and how they function combinatorially with additional regulatory factors. GATA motifs are often arranged in close proximity to E-boxes, and such composite elements commonly mediate GATA factor- and Scl/TAL1-dependent transcriptional responses. Only a small fraction of these composite elements in chromatin are occupied by GATA factors and Scl/TAL1, and a specific epigenetic signature distinguishes occupied versus unoccupied elements genome-wide. In the context of hemoglobin synthesis, we are using genetic and molecular approaches to dissect the multistep mechanism underlying the control of β-globin transcription. GATA-1-containing complexes assemble at the β-globin Locus Control Region (LCR) prior to the murine adult βmajor promoter. Though the LCR physically interacts with the βmajor promoter, this interaction is not required for the binding of several trans-acting factors to the LCR or the promoter. A hypomorphic mutation of the chromatin remodeler BRG1 limits the extent to which RNA Polymerase II (Pol II) is recruited to the promoter and also abrogates the LCR-promoter interaction. Whereas looping is not required for assembly of the full complement of promoter complex components, looping is linked to the establishment of maximal levels of Pol II at the promoter. Collectively, these results provide insights into the relationship between, and importance of, individual steps in the multi-step activation mechanism. I will discuss progress on unraveling mechanisms underlying GATA-1-mediated activation of the adult β-like globin genes as well as fundamental aspects of GATA factor function, which have broad relevance in diverse systems. promoter. Though the LCR physically interacts with the β promoter, this interaction is not required for the binding of several -acting factors to the LCR or the promoter. A hypomorphic mutation of the chromatin remodeler BRG1 limits the extent to which RNA Polymerase II (Pol II) is recruited to the promoter and also abrogates the LCR-promoter interaction. Whereas looping is not required for assembly of the full complement of promoter complex components, looping is linked to the establishment of maximal levels of Pol II at the promoter. Collectively, these results provide insights into the relationship between, and importance of, individual steps in the multi-step activation mechanism. I will discuss progress on unraveling mechanisms underlying GATA-1-mediated activation of the adult β-like globin genes as well as fundamental aspects of GATA factor function, which have broad relevance in diverse systems.

The G9A Histone Methyltransferase BIX-01294 Reactivates ε-Globin Expression and Blocks Erythroid Differentiation in Primary Baboon Erythroid Progenitor Cell Cultures

The development of new therapies to increase fetal hemoglobin (HbF) levels in patients with sickle cell disease and β-thalassemia depends on an increased understanding of the mechanism responsible for the developmental regulation of globin gene expression. A role for epigenetic modifications in the mechanism of of globin gene regulation is suggested by the presence of high levels of DNA methylation near the 5' regions of developmentally silenced ε- and γ-globin genes and the ability of pharmacological inhibitors of DNA methyltransferase (DNMTase) to reactivate ε- and γ-globin expression in adults. Whether additional epigenetic modifications associated with gene silencing and DNA methylation, such as histone H3 (lys9) dimethylation, are also involved is unknown. To investigate the hypothesis that histone H3 (lys9) dimethylation may function in the mechanism of developmental globin gene silencing, chromatin immunopreciptation assays were performed to determine the distribution of histone H3 (lys9) dimethyl and histone H3 (lys9) acetyl throughout the β-globin gene complex in purified primary baboon bone marrow (BM) erythroid cells from phlebotomized baboons expressing low levels (5–10%) of HbF and purified erythroid cells from erythroid progenitor cell cultures expressing high levels of HbF (30–50%). In BM erythroid cells, the level of histone H3 (lys9) acetyl associated with the β-globin gene was 10–20 fold higher than with the ε- and γ-globin genes, while the level of histone H3 (lys9) dimethyl associated with the ε- and γ-globin genes was 2–4 fold higher than with the β-globin gene. In erythroid cells from day 12 erythroid progenitor cell cultures, the level of histone H3 (lys9) acetyl associated with the highly expressed γ- and β-globin genes was 10–20 fold higher than with the silent ε-globin gene, while the level of histone H3 (lys9) dimethyl associated with the ε-globin gene was 2–4 fold higher than with the γ- and β-globin genes. Therefore a reciprocal relationship was observed between levels of histone H3 (lys9) acetylation and dimethylation associated with active and inactive globin genes. Experiments were performed to further investigate the role of histone H3 (lys9) dimethyl in ε-globin gene silencing by determining the effect of the G9A histone methyltransferase inhibitor BIX-01294 on ε-globin expression. Erythroid progenitor cell cultures derived from CD34+ BM cells of three individual baboons were treated with the varying doses of the DNMTase inhibitor decitabine (0.125–1.0μM), and BIX-01294 (1.25–5μM), alone and in combination. Changes in ε- globin were assessed by real time PCR using the ΔΔCT method with α-globin as the standard. Decitabine (0.5μM) increased ε-globin 25.8±7.7 fold while BIX-01294 (2.5μM) increased ε-globin 3.09±1.16 fold. Decitabine (1μM) and BIX-01294 (2.5μM) in combination increased ε-globin 55.7±24.9 fold. BIX-01294 enhanced ε-globin expression approximately twofold at all decitabine doses ranging from 0.125–1.0μM (mean increase=103± 44.7%). BIX-01294 also blocked terminal erythroid differentiation and allowed expansion of more primitive cells as evidenced by the presence of a large population of basophilic erythroblasts at late stages of culture (day 14). These results demonstrate that BIX-01294 reactivates expression of the silenced ε-globin gene and that synergistic reactivation can be achieved using combinations of BIX-01294 and decitabine. While these results are consistent with the hypothesis that epigenetic modifications are important in the mechanism of developmental globin gene silencing, the observation that BIX-01294 blocks erythroid differentiation suggests the possible involvement of a reprogramming mechanism.

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.

First report of a nonsense mutation at codon 15(TGG→TAG) in exon 1 of the β globin gene in a β thalassemia trait in State of Orissa, India

Prevention of β thalassemia requires knowledge of the molecular spectrum occurring in the population at risk. This knowledge is particularly necessary when prevention control is applied to a multiethnic population. For this purpose, we are analyzing different populations of Orissa (India). During the study we encountered a β thalassemia major patient (a child) who was doubly heterozygous for IVS I-5(G→C) and codon 15(G→A) alleles where codon 15(G→A) was for the first time found in the father of the patient. Also, the patient showed severe clinical abnormalities because of the severe nature of these β thalassemia alleles. This will provide further insights into β globin gene regulation and the genotype–phenotype relationship.

Ikaros Drives Human Haemoglobin Switching by Facilitating Active Chromatin Hub Formation.

The human β globin locus consists of an upstream locus control region (LCR) and five functional genes arranged sequentially in the order of their expression during development: 5′-ε-Gγ-Aγ- δ- β-3′. Haemoglobin switching entails the successive recruitment of these genes into an active chromatin hub (ACH). Although much is known about the cis elements and transcription factors involved in globin gene regulation, less is known about ACH formation. Here we show that the transcription factor Ikaros plays an essential role in both the formation of the β-globin ACH, and in haemoglobin switching. In Plastic mice, where the DNA-binding region of Ikaros is disrupted by a point mutation (H191R), there is concomitant marked (10 fold) down-regulation of human β-globin, and up-regulation of γ-globin gene expression. We show Ikaros binds to a critical cis elements in the LCR near the HS3 core and upstream of the δ-globin gene in the β-globin locus by electormobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) and that this DNA binding activity is lost in Plast mice. This latter site is implicated in deletional hereditary persistence of fetal haemoglobin (HPFH). Furthermore, chromatin conformation capture (3C) data suggest Ikaros facilitates long range looping between the LCR and a region upstream of the δ-globin gene. This study provides new insights into the mechanism of adult stage-specific assembly of the β-globin ACH. In addition the findings could lead to the development of novel drugs to reactivate HbF in adults with β-thalassemia and sickle cell disease.

Mitosis Is Not Required for Coordinated Changes in Iron Metabolism and High-Level Globin Gene Transcription in Erythropoietin-Responsive CD34+ Cells.

Within the erythroid lineage, erythropoietin (EPO) responsiveness is manifested by cell division and growth as well as specific changes in heme and globin production that ultimately result in the production of erythrocytes. However, the nascent relationship between EPO-associated mitosis and globin gene regulation has not been fully defined. In this study, cultured adult human CD34+ cells from peripheral blood were used to investigate early cellular responses to erythropoietin in the context of mitosis. Matched cultures were performed in replicate using human cells from at least two healthy adults. To detect mitosis, one million cells were labeled with 2uM carboxyfluorescein diacetate succinamidyl ester (CFSE). The CFSE-labeled cells were then cultured in the presence [EPO(+)] or absence [EPO(−)] of 4U/mL EPO, and analyzed using flow cytometry. No cell divisions were detected in either condition during the first 24 hours in culture, and multiple cell divisions were noted on subsequent culture days only in the EPO(+) cultures. Remarkably, dual-staining with CFSE and CD71 revealed a small (<1%) population of the undivided cells at 24 hours with very high surface levels of transferrin receptor [CD71(++++)] exclusively in the EPO(+) cultures. Further analysis of those rare EPO-responsive, pre-mitotic cells revealed DNA synthesis and entry into the cell cycle in 62.5±4.7% compared with 1.5±1.8% among the cells with lower CD71 expression. None expressed glycophorin A. Based upon their distinct phenotype, single cells were sorted into 96-well plates, with sorting confirmation by quantitative RT-PCR of GAPDH mRNA (20 copies/cell detection limit). Next, gamma- and beta-globin transcripts were amplified for comparison (4 separate experiments). Among the pre-mitotic, CD71(++++) population, 20 of 182 total sorted cells (11%) lacked detectable levels of gamma- and beta-globin mRNA. Only 1 of 182 cells (0.5%) expressed gamma-globin mRNA and no detectable beta globin mRNA. 138 of 182 cells (76%) expressed only beta-globin mRNA, and 23 of 182 cells (13%) expressed both globin mRNAs. The median and mean levels of gamma-globin mRNA among the 24 gamma(+) cells were 92 and 245±1015 copies/cell respectively. In contrast, the median and mean levels of beta-globin mRNA among the 161 beta(+) cells were 1624 and 3999±5892 copies/cell respectively. By comparison, the CD34+ cells with low levels of surface CD71 in either EPO(+) or EPO(−) cultures demonstrated detectable levels of gamma globin mRNA in only 7 of 145 sorted cells (5%), and beta-globin mRNA in 85 of 145 cells (59%), with total (gamma+beta) globin mRNA under 200 copies/cell in >90% of those cells. These novel results suggest that the frequency and levels of gamma-globin transcripts are quite low at the earliest stages of an EPO response among adult human CD34+ cells. However, it is clearly demonstrated that the cells can increase their capacity to import iron and transcribe beta-globin mRNA at very high levels prior to their first EPO-dependent cell division.

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.

Role of epigenetic modifications in normal globin gene regulation and butyrate-mediated induction of fetal hemoglobin

AbstractButyrate is a prototype of histone deacetylase inhibitors that is believed to reactivate silent genes by inducing epigenetic modifications. Although butyrate was shown to induce fetal hemoglobin (HbF) production in patients with hemoglobin disorders, the mechanism of this induction has not been fully elucidated. Our studies of the epigenetic configuration of the β-globin cluster suggest that DNA methylation and histone H3 acetylation are important for the regulation of developmental stage-specific expression of the β-like globin genes, whereas acetylation of both histones H3 and H4 seem to be important for the regulation of tissue-specific expression. These studies suggest that DNA methylation may be important for the silencing of the β-like globin genes in nonerythroid hematopoietic cells but may not be necessary for their silencing in nonhematopoietic cells. Furthermore, our studies demonstrate that butyrate exposure results in a true reversal of the normal developmental switch from γ- to β-globin expression. This is associated with increased histone acetylation and decreased DNA methylation of the γ-globin genes, with opposite changes in the β-globin gene. These studies provide strong support for the role of epigenetic modifications in the normal developmental and tissue-specific regulation of globin gene expression and in the butyrate-mediated pharmacologic induction of HbF production.

Epigenetic analysis of the human α- and β-globin gene clusters

K562 erythroleukemia cells have been widely used as a model for the study of globin gene regulation. A number of agents have been shown to activate or suppress globin gene expression in these cells. However, the molecular effects of these agents on the epigenetic configuration of the α- and γ-globin genes that encode HbF are not known. In this report, we investigated the relationship between globin expression and histone acetylation of the human α- and β-globin clusters in the fetal erythroid environment of K562 cells. Our studies suggest that acetylation of histone H3 may be important in regulating developmental stage-specific expression of the different β-like globin genes while acetylation of both histones H3 and H4 may be important for the regulation of tissue-specific expression of these genes. In contrast, acetylation of both histones H3 and H4 at the α-like globin promoters appears to be important for both developmental stage- and tissue-specific expression. Interestingly, butyrate-induced activation of α-globin gene expression in K562 cells is associated with significant increase in histone acetylation levels while TPA-induced inhibition is associated with decreased histone acetylation at its promoters. In contrast, changes in histone acetylation and DNA methylation do not appear to be important in the regulation of γ-globin gene expression by the same agents. These data suggest that the butyrate-mediated induction of the fetal γ-globin genes in K562 cells is not a direct result of its histone deacetylase inhibitor activity of butyrate on the chromatin of the γ-globin promoters, while the induction of the α-globin genes could be a result of a direct effect of butyrate on chromatin at its promoters. This is another example of the important differences in the molecular mechanisms of regulation of the genes of the α- and β-like globin clusters.