Fetal Globin Gene Expression Research Articles

Identification of a Transcription Factor Potentially Involved in γ-Globin Gene Expression.

The switch from fetal to adult globin gene expression occurs around birth when fetal hemoglobin (HbF) production gradually declines within a few months. Much effort is underway to clarify the molecular basis of this mechanism, since impaired hemoglobin switching leading to persistent expression of fetal globin genes in adults (HPFH) offers therapeutic potential for hemoglobinopathies (S.L. Thein. Br. J. Haematol 2004; 124: 264–74).In order to identify and study regulatory factors putatively involved in γ-globin gene expression, we examined the reticulocyte mRNAs differently expressed in three siblings (one brother, 54 years-old and two sisters 35- and 37-years old, respectively). The eldest brother had been referred to Umberto I Hospital for evaluation of a severe condition of β-thalassemia intermedia on chronic transfusion therapy since 1990. Both of his sisters resulted clinically affected by a milder form of thalassemia intermedia, non-transfusion dependent, showing Hb values around 8.4 g/dL, Hb A2 levels from 6.0 to 7.9% and HbF ranging from 14.5 to 27.6%, values higher than those resulted in their brother (5.9% Hb A2 and 7.2% HbF). Molecular analysis was performed on DNA extracted from peripheral leucocytes and revealed the same β-globin gene cluster genotype for all these subjects who resulted homozygous for the β+ IVSI-6 (C→T) mutation associated to haplotype VI chromosomes. Different levels of HbF were thus presumably responsible of different clinical phenotypes. To investigate the possible causes of the variations in γ-globin gene expression, extensive sequence analysis was performed on putative regulatory regions within the β-globin gene cluster (Zhi-Hong Lu et al., Blood 1996; 87: 1604–11). Results showed the same genetic background in all the siblings. It was thus supposed that genetic determinants external to the β-globin gene cluster were responsible of the different γ-globin gene expression. To explore this hypothesis, the reticulocyte transcriptome was analyzed by a differential mRNA display approach. Reticulocytes were isolated from peripheral blood and total RNA extracted for all family members. Our study revealed several bands differentially displayed in the sample from the more severely affected sibling respect to his sisters. Selected bands were cloned in a pGEM T-vector (Promega, WI) and sequenced. Comparative sequence searches were performed using the BLAST algorithm. Preliminary data gave, for two of the clones originated from bands with increased expression in the brother, a complete homology (greater than 95%) with the cDNA sequence of a cold shock domain protein, displaying features of a repressor factor for several hematopoietic genes (Horwitz M. et al. JBC 1994; 269: 14130–39; P. Coles et al. Nucleic Acids Res 1996; 12: 2311–17).

Silencing Reversed: Coordinated Expression of Embryonic and Fetal Globin Genes in Adult Human Erythroblasts.

It was recently demonstrated that erythropoietin combined with stem cell factor and transforming growth factor beta (EST; EPO+SCF+TGF-B) signals a pancellular reversal of gamma-globin gene silencing among ex vivo cultures of CD34+ cells when compared to cultures supplemented with erythropoietin (EPO) alone (Bhanu et al., Blood. 2005; 105(1):387–93). This primary human erythroblast culture model is now being utilized to study the molecular basis of globin gene regulation. In this study, we sought to determine whether EST-signaled effects are restricted to the gamma-globin gene(s). For this purpose, quantitative PCR (QPCR) assays were developed for the alpha-globin (zeta, mu, alpha, and theta) and beta-globin (epsilon, gamma, delta, and beta) gene clusters. EPO versus EST matched cultures from a total of 15 donors (3 single donors and two pools, 6 donors each) were investigated using culture day 7 proerythroblasts and day 14 hemoglobinized precursors. The QPCR products were quantified by comparison with plasmid-generated standard curves (20,000,000 to 200 copies). The patterns of mu-, alpha- and theta-globin gene expression were unchanged under all culture conditions. The delta- and beta-globin transcripts showed a significant reduction in levels in response to EST. In contrast, zeta-, epsilon- and gamma-globins all demonstrated significant increases in response to EST. The increases were consistently detected in the individual and pooled samples. The most dramatic increase in the zeta-, epsilon- and gamma-globins occurred at the proerythroblast stage of differentiation. On day 7, zeta-globin levels showed 190-fold increase (EPO: 6.9E+01±5.5E+01;EST: 1.3E+04±7.7E+03; p=0.01), epsilon-globin levels, a 27-fold increase (EPO: 1.0E+03±5.8E+02; EST: 2.7E+04±1.1E+04; p=0.001), and gamma-globin levels, a 13-fold increase (EPO: 9.1E+05±4.1E+05; EST: 1.2E+07±1.8E+06; p=0.001). Consistent with the 3–4 log difference between the levels of zeta- and epsilon-, versus gamma-globin copy numbers and the low sensitivity of HPLC compared with QPCR, no embryonic proteins were detected by reverse-phase HPLC (day 14 samples studied; 9 donors). However, G-gamma and A-gamma globin peaks confirmed EST-signaled activation of both gamma-globins genes (G-gamma/A-gamma= 1.0 in EPO versus 1.9 in EST; (total gamma)/(total gamma + beta-globin) = 3.7±2.4% in EPO versus 42.4 ±5.8% in EST; p=1.6E-08). These novel data suggest that cytokines can reverse the coordinated developmental silencing of human embryonic and fetal genes in both globin loci.

Enhancement of growth and survival and alterations in Bcl-family proteins in β-thalassemic erythroid progenitors by novel short-chain fatty acid derivatives

Accelerated apoptosis of erythroid progenitors is a characteristic of β-thalassemia which presents a significant barrier to definitive therapeutic approaches utilizing induction of endogenous fetal globin gene expression. γ-globin gene expression may not be inducible in, or may not be able to rescue, erythroid cells in which programmed cell death is initiated early in erythroblast development. In this report, short-chain fatty acid derivatives (SCFADs) which induce fetal globin gene expression were tested for their ability to promote proliferation and survival of erythroid progenitors cultured from β-thalassemic subjects, and of cytokine-dependent erythroid cell lines. Certain SCFADs promoted thalassemic Bfu-e growth and cytokine-independent growth and survival of erythroid cell lines. A 40–80% increase in erythroid Bfu-e colony number was observed in cultures established with any of five mitogenic SCFADs, compared to control or butyrate-treated cultures from the same subjects. Immunoblot analysis demonstrated that these same SCFADs also regulated the expression of specific protein inhibitors of apoptosis. Anti-apoptotic ratios of the proteins Bcl-x L/Bcl-x S in thalassemic Bfu-e were increased by 30–120% with exposure to the SCFDs, compared to the ratios in the same cells cultured under control conditions. Similar anti-apoptotic increases in Mcl-1 L/Mcl-1 S ratios were induced by the SCFADs. These findings suggest that select fetal globin-inducing SCFADs which enhance proliferation of β-thalassemia progenitors may enhance survival of these progenitors by altering levels of Bcl-family protein members. This combination of effects should enhance erythroid cell survival in the β-thalassemia syndromes, allowing fetal globin gene expression to be induced more effectively than currently available, growth-suppressing, fetal globin-inducing agents, such as the butyrates or chemotherapeutic agents.

Spontaneous switch from Aγ- to β-globin promoter activity in a stable transfected dual reporter vector

Here it is analyzed the expression of a mini locus dual reporter construct composed by a micro-LCR and by the promoters for Aγ- and β-globin gene, each one linked to a different Luciferase, in stably transfected GM979 cells for as long as 1–2 years from transfection. The transfected GM979 cells rapidly (within 1 month) evolved into a stable population which expresses constant levels of reporters for more than a year of continuous bulk culture. No silencing of the inserted construct was observed over time. In contrast, after 1 month, the reporter activity (both from Aγ- and β-promoter) expressed per cell increased over time. The analysis of the Luciferase contained in single cell clones indicated that the higher reporter activity was due to increased gene expression per cell rather than to clonal selection of the most expressing clones. Since the activity driven by the β-promoter increased 10-fold more than that driven by the Aγ one, the ratio between Aγ-driven/( Aγ-driven + β-driven) reporter activity in the cells decreased after 1 month and became similar to the γ/(γ + β) globin mRNA ratio expressed by adult erythroid cells. Moreover, although both cells from early and late bulk culture responded to incubation with butyric acid, a known inducer of fetal globin gene expression, by increasing the reporter activity driven by the Aγ-promoter, only cells from late bulk culture decreased, as normal primary erythroblasts do, the activity of the reporter driven by the β-promoter. These results suggest that the rapid changes in activity driven by the Aγ- and β-globin promoters occurring during the first month after transfection may represent a novel in vitro model to study epigenetic regulation of the Aγ- and β-promoter during the fetal to adult hemoglobin switch and confirm GM979 cells stably transfected with the dual reporter construct as a reliable assay for automated screening of chemical inducers of fetal globin gene activation.

Fetal Globin Induction—Can It Cure β Thalassemia?

The beta thalassemias are one of a few medical conditions in which reactivation of a gene product that is expressed during fetal life can functionally replace a deficiency of essential proteins expressed at a later developmental stage. The fetal globin genes are present and normally integrated in hematopoietic stem cells, and at least one fetal gene appears accessible for reactivation, particularly in beta degrees thalassemia. However, rapid cellular apoptosis from alpha globin chain precipitation, and relatively low levels of endogenous erythropoietin (EPO) in some beta(+) thalassemia patients contribute to the anemia in beta thalassemia syndromes. In clinical trials, three classes of therapeutics have demonstrated proof-of-principle of this approach by raising total hemoglobin levels by 1-4 g/dL above baseline in thalassemia patients: EPO preparations, short chain fatty acid derivatives (SCFADs), and chemotherapeutic agents. Although thalassemic erythrocytes survive only for a few days, the magnitude of these responses is similar to those induced by rhu-EPO in anemic conditions of normal erythrocyte survival. New oral therapeutic candidates, which stimulate both fetal globin gene expression and erythropoiesis, and combinations of therapeutics with complementary molecular actions now make this gene-reactivation approach feasible to produce transfusion independence in many patients. Development of the candidate therapeutics is hindered largely by costs of drug development for an orphan patient population.

The Role of p22 NF-E4 in Human Globin Gene Switching

The human stage selector protein, a complex containing the ubiquitous transcription factor CP2 and the erythroid-specific factor p22 NF-E4, facilitates the interaction of the gamma-globin genes with the locus control region in fetal erythroid cells. Enforced expression of p22 NF-E4 in K562 cells and human cord blood progenitors increases fetal globin gene expression, and in progenitors, reduces beta-globin expression. To examine the role of NF-E4 in an in vivo model of hemoglobin switching, we enforced the expression of p22 NF-E4 in transgenic mice carrying the human beta-globin locus yeast artificial chromosome. Although murine erythropoiesis and globin gene expression is unaffected in these mice, the expression profile of the human globin genes is altered. All three transgenic lines displayed an increased gamma:beta-globin ratio in E12.5-14.5 fetal liver, resulting in a delay in the fetal/adult switch. At E12.5, this is primarily due to a reduction of beta-gene expression, whereas at E14.5, the increased gamma:beta ratio is due to enhanced gamma-gene expression. Despite this, the switch in globin subtype is fully completed in the adult bone marrow. These findings indicate that p22 NF-E4 is capable of influencing human globin gene expression in vivo but is incapable of overriding the intrinsic mechanisms governing gamma-gene silencing in this context.

Mutation of a transcriptional motif of a distant regulatory element reduces the expression of embryonic and fetal globin genes.

High-level beta-globin gene expression is dependent on the presence of the locus control region (LCR), a powerful regulatory element physically characterized by five DNase I-hypersensitive sites (HS), designated HS1-HS5. Of these, HS3 contains seven GT motifs that are essential for its activity. One of the motifs, GT6, has been shown by in vivo footprinting to display the largest difference in signal between fetal and adult globin expressing cells. We assessed the contribution of GT6 on the downstream globin gene expression by mutating this motif in a 248 kb beta-globin locus yeast artificial chromosome and measuring the activity of beta-globin genes in GT6m beta-YAC transgenic mice. Seven transgenic lines were established, three of which contained at least one intact copy of the beta-globin locus and were further investigated. The mutation of the GT6 motif reduced the expression of epsilon- and gamma-globin genes during embryonic erythropoiesis. During definitive erythropoiesis, gamma-globin gene expression was significantly reduced while beta-globin gene expression was virtually indistinguishable from wild-type controls. We conclude that the GT6 motif of hypersensitive site 3 of the LCR is required for normal epsilon- and gamma-globin gene expression during embryonic erythropoiesis and for gamma-globin gene expression during definitive erythropoiesis in the fetal liver. Our results provide evidence that mutations of single transcriptional motifs of distant regulatory elements can have profound effects on gene expression.

Hydroxamide derivatives of short-chain fatty acids are potent inducers of human fetal globin gene expression.

To examine whether hydroxamic acids are inducers of fetal hemoglobin expression, we tested the effects on gamma gene expression of butyric and propionic hydroxamic acids and of two other hydroxamic acids (SBHA and SAHA), which are potent inhibitors of histone deacetylase (HDAC). We also investigated whether there is a correlation between HDAC inhibitory activity of the compounds and their ability to induce gamma-globin gene expression. Effects on gamma-globin expression were assessed by two methods: 1) a screening assay in which specific gamma-globin gene inducers are recognized by their ability to increase gamma firefly luciferase activity significantly more than beta-renilla luciferase activity; and 2) measurements of gamma-globin mRNA and the frequency of fetal hemoglobin-positive erythroblasts in cultures of burst-forming unit erythroid (BFU-E) from normal individuals. HDAC in vitro activity was measured with a partially purified rat liver HDAC and a fluorogenic substrate. All compounds tested increased gamma firefly luciferase activity, gamma/gamma+beta mRNA ratios, and percentage of fetal hemoglobin-containing erythroblasts in BFU-E cultures, in a dose-dependent fashion. Butyryl-hydroxamic acid 100 microM increased the gamma/gamma+beta mRNA ratios by 5.8-fold and the frequency of fetal hemoglobin-containing erythroblasts by 4.1-fold. Propionyl-hydroxamic acid 150 microM increased the gamma/gamma+beta ratios by 6.3-fold and the fetal hemoglobin-containing erythroblasts by 3.9-fold. SBHA induced gamma-globin gene expression at very low concentrations, 5 to 20 microM in the luciferase system and 2 to 8 microM in BFU-E cultures; SAHA at 1 to 7.5 microM in the luciferase system and 1 to 2.5 microM in the BFU-E cultures. HDAC in vitro inhibition was observed in the millimolar range for propionate and butyrate. IC(50) determinations led to values of 384 microM for propionyl-hydroxamate, 47 microM for butyryl-hydroxamate, 0.93 microM for SBHA, and 0.26 microM for SAHA. CONCLUCION: Our data indicate that hydroxamic acid-based HDAC inhibitors are potent gamma-globin gene inducers and that the concentration range of their effects on gamma gene expression can be correlated roughly with their HDAC inhibitory potencies.

Mechanism for fetal globin gene expression: role of the soluble guanylate cyclase-cGMP-dependent protein kinase pathway.

Despite considerable concerns with pharmacological stimulation of fetal hemoglobin (Hb F) as a therapeutic option for the beta-globin disorders, the molecular basis of action of Hb F-inducing agents remains unclear. Here we show that an intracellular pathway including soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG) plays a role in induced expression of the gamma-globin gene. sGC, an obligate heterodimer of alpha- and beta-subunits, participates in a variety of physiological processes by converting GTP to cGMP. Northern blot analyses with erythroid cell lines expressing different beta-like globin genes showed that, whereas the beta-subunit is expressed at similar levels, high-level expression of the alpha-subunit is preferentially observed in erythroid cells expressing gamma-globin but not those expressing beta-globin. Also, the levels of expression of the gamma-globin gene correlate to those of the alpha-subunit. sGC activators or cGMP analogs increased expression of the gamma-globin gene in erythroleukemic cells as well as in primary erythroblasts from normal subjects and patients with beta-thalassemia. Nuclear run-off assays showed that the sGC activator protoporphyrin IX stimulates transcription of the gamma-globin gene. Furthermore, increased expression of the gamma-globin gene by well known Hb F-inducers such as hemin and butyrate was abolished by inhibiting sGC or PKG activity. Taken together, these results strongly suggest that the sGC-PKG pathway constitutes a mechanism that regulates expression of the gamma-globin gene. Further characterization of this pathway should permit us to develop new therapeutics for the beta-globin disorders.

Induction of human fetal globin gene expression by a novel erythroid factor, NF-E4.

The stage selector protein (SSP) is a heteromeric complex involved in preferential expression of the human gamma-globin genes in fetal-erythroid cells. We have previously identified the ubiquitous transcription factor CP2 as a component of this complex. Using the protein dimerization domain of CP2 in a yeast two-hybrid screen, we have cloned a novel gene, NF-E4, encoding the tissue-restricted component of the SSP. NF-E4 and CP2 coimmunoprecipitate from extract derived from a fetal-erythroid cell line, and antiserum to NF-E4 ablates binding of the SSP to the gamma promoter. NF-E4 is expressed in fetal liver, cord blood, and bone marrow and in the K562 and HEL cell lines, which constitutively express the fetal globin genes. Enforced expression of NF-E4 in K562 cells and primary erythroid progenitors induces endogenous fetal globin gene expression, suggesting a possible strategy for therapeutic intervention in the hemoglobinopathies.

Hemoglobin S haplotypes among sickle cell patients in the Puerto Rican population

The Hb S mutation has been associated with four major haplotypes of the β-like gene cluster in the African continent, which include the following: haplotype #19 or Benin; #20 or Bantu, or Central African Republic (CAR); #3 or Senegal; and #17 or Cameroon. Neonatal screening in Puerto Rico in recent years has shown an overall incidence of abnormal hemoglobins of 2.24%, including 1.87% Hb S and 0.31% of Hb C. It has been postulated that certain unique mutant sites linked to the β gene cluster haplotypes might be responsible, at least partly, for the differences in the fetal globin gene expression and clinical severity observed among sickle cell patients. The purpose of our study was to identify the haplotypes present in Puerto Rican Hb S patients to better characterize sickle cell disease in Puerto Rico. Blood samples from twenty nine individuals with sickle cell disease and other sickling syndromes were collected at the Neonatal Hemoglobinopathy Screening Program Clinic. All blood samples were analyzed by cellulose acetate, citrate agar and isoelectric focusing electrophoresis. β-globin gene haplotypes were analyzed by allele-specific oligonucleotide (ASO) probe hybridization and/or by PCR RFLP. The predominant haplotypes for the forty eight β5 chromosomes analyzed were the haplotype #19 (33%}, and haplotype #20 (33%). For the haplotype #3,31 mutation, 12.5% of the chromosomes presented the mutation. Mutations characteristic for haplotypes #10 and #11 were 8.34% of β5 chromosomes while 12.5% were mixed or other haplotypes. Comparing Puerto Rico with other countries, we have a prevalence of the Benin and Bantu haplotypes, differing with the United States prevalences in which more than 50% are Benin haplotypes. Our haplotype frequencies are similar to those found in a recent study from Cuba. The predominant haplotype in the SS population we studied (#19/20) is associated with a moderate clinical condition. In our studies a strict correlation with clinical severity and β5 haplotypes does not exist in all of the SS patients analyzed. We acknowledge support from NIH-RCMI G12-RR-03051 and the UPR School of Medicine.

Maturation and developmental stage-related changes in fetal globin gene expression are reproduced in transiently transfected primary adult human erythroblasts

A novel system is described, which uses transfection of primary human erythroblasts for the study of gene regulation in differentiating human red cells. This system includes a protocol for liquid culture of erythroid progenitors, which reproduces developmental differences in globin gene expression found between adult and cord blood as well as the maturation-related changes in fetal globin levels observed in adult cells. Reporter constructs driven by globin gene promoters were electroporated into adult and cord blood-derived erythroblasts at different time points during culture. Both the developmental stage and maturation-related differences in endogenous fetal and adult globin gene expression could be reproduced by the transiently transfected reporter constructs. Transfection of primary human erythroblasts during differentiation provides a previously unavailable opportunity to study dynamic aspects of erythropoiesis.

Understanding fetal globin gene expression: a step towards effective HbF reactivation in haemoglobinopathies.

Understanding fetal globin gene expression: a step towards effective HbF reactivation in haemoglobinopathies.

Abrogation of IL-3 Requirements and Stimulation of Hematopoietic Cell Proliferationin Vitroandin Vivoby Carboxylic Acids

ABSTRACT: Short-chain fatty acids, such as butyrate and propionate, induce fetal globin gene expression and are under clinical investigation in the β-hemoglobinopathies. Limitations of the short-chain fatty acids as therapeutics include their rapid metabolism and a tendency to induce cell growth arrest if administered for prolonged periods. In studies described here, the cellular effects of other inducers of fetal globin, phenoxyacetic acid and derivatives of short-chain fatty acids and cinnamic acids, were investigated in the human erythroid cell line K562, the IL-3 dependent multi-lineage cell line (32D), and in mice and primates. Several test compounds supported 32D cell proliferation despite a 50-fold depletion of IL-3, which resulted in growth arrest and apoptotic death in control cells. The degree of proliferation induced by certain test compounds was similar to the degree of proliferation induced by Erythropoietin and G-CSF in the cells. Eight of ten compounds induced γ globin mRNA in K562 cells. A 2.5 to 6-fold increase in reticulocytosis was observedin vivoin mice treated with two prototype compounds. Pharmacokinetic studies of three prototype compounds demonstrated millimolar plasma concentrations after single oral doses for many hours in primates. These findings identify orally bioavailable compounds which induce γ globin gene expression and hematopoietic cell proliferation through an activity which partially abrogates requirements for IL-3. Such compounds provide potential for oral therapeutics which stimulate proliferation of hematopoietic cells of multiple lineages, as well as inducing fetal globin.

Butyrate in the treatment of sickle cell disease and beta-thalassemia.

The search for, and discovery of, a physiologic model in which the developmentally regulated switch from fetal to adult globin gene expression could be prevented has resulted in the development of a new class of therapeutic agents, consisting of simple fatty acids, such as butyric acid, for the treatment of the beta-hemoglobinopathies. Butyrate and related drugs stimulate fetal (gamma-) globin gene expression in erythroid cells cultured from patients, and in chicken, ovine, and primate animal models. The butyrates are perhaps the first class of drugs designed to transcriptionally activate specific genes--in this particular case, to reactivate the developmentally silenced fetal globin genes. Phase I-II clinical trials resulting from this basic research have been initiated on a small scale during the past 3 years. Analysis of two butyrate-derived therapeutic agents, one delivered intravenously and one orally, has shown initial efficacy in stimulating fetal hemoglobin expression in 50% to 85% of patients. Correction of the anemia from the beta-hemoglobinopathy has followed induction of fetal globin, and has been adequate to eliminate the need for erythrocyte transfusions in some patients with beta-thalassemia. These compounds have been relatively safe and without generalized cytotoxicity in patients, but drug tolerance develops in some patients after prolonged therapy. Third-generation, small two- to five-carbon butyrate derivatives are in development. The molecular basis for butyrate action is being defined. Binding of putative regulatory proteins to a specific region of the gamma-globin promoter is altered in vivo in patients receiving butyrate therapy. Further analysis of the mode of action may contribute to development of other therapeutic agents designed to regulate gene transcription.

Isobutyramide, an orally bioavailable butyrate analogue, stimulates fetal globin gene expression in vitro and in vivo.

Butyrate and other short-chain fatty acids stimulate fetal globin gene expression and have potential for ameliorating the beta globin disorders. Butyrate, however, is rapidly metabolized in vivo and reaches only micromolar concentrations in plasma. We report here that a branched-chain derivative of butyrate, isobutyramide, increases gamma globin gene expression in cultured human erythroid progenitors in vitro and stimulates activity from a minimal gamma globin gene promoter linked to a reporter gene in stable and transient expression assays, with slightly less activity in these in vitro assays than butyrate. In vivo, administration of isobutyramide to anaemic adult baboons rapidly stimulates fetal globin synthesis and F-reticulocyte production. Plasma concentrations at millimolar levels are achieved after a single intravenous or oral dose (500-600 mg/kg), and these concentrations are maintained for 9.5-10.5 h. These results indicate that although isobutyramide has slightly less activity than butyrate in vitro in enhancing fetal globin expression at the cellular and molecular level, its prolonged in vivo half-life may provide superior activity as a therapeutic agent for reactivating fetal globin gene expression in vivo.

Differential induction of adult and fetal globin gene expression in the human CML cell subline KU-812F/33.

Various chemicals which are known to have positive effects on differentiation of some erythroid cell lines were tested on a human chronic myelogenous leukemia cell line, KU-812F. Succinic acid, 5-azacytidine, daunomycin, and hemin showed a positive effect. Among them, hemin and 5-azacytidine were the most effective inducers for erythroid differentiation of KU-812F cells. Dimethylsulfoxide, cytosine arabinofuranoside, and sodium n-butyrate showed no effect. In addition, subclone KU-812F/33 derived from the KU-812F cell line showed differential expression of the beta- and gamma-globin genes in the presence of either 2 microM 5-azacytidine or 40 microM hemin. Hemoglobin synthesis in differentiated KU-812F/33 cells was analyzed by isoelectric focusing gel electrophoresis, and S1 mapping analysis of beta- and gamma-globin mRNA was performed. After treatment with 5-azacytidine, the beta-globin gene expression was predominantly enhanced (18.75-fold higher level of beta-globin mRNA). After treatment with hemin, the most notable increase was in the gamma-globin gene expression (1.83-fold higher level of gamma-globin mRNA), while no increment of beta-globin was observed.

Butyrate-induced reactivation of the fetal globin genes: a molecular treatment for the beta-hemoglobinopathies.

The inherited beta-hemoglobinopathies (sickle cell disease and beta thalassemia) are the result of a mutation in the adult (beta) globin gene. The fetal globin chain, encoded by the gamma globin genes, can substitute for the mutated or defective beta globin chain, but expression of the gamma globin gene is developmentally inactivated prior to birth. Re-inducing expression of the normal fetal globin genes is a preferred method of ameliorating sickle cell disease and the beta thalassemias. Stimulation of as little as 4-8% fetal globin synthesis in the bone marrow can produce > 20% fetal hemoglobin in the peripheral circulation, due to enhanced survival of red blood cells containing both sickle and fetal hemoglobin, compared to those containing sickle hemoglobin alone. Butyric acid and butyrate derivatives are generally safe compounds which induce fetal hemoglobin production by stimulating the promoter of the fetal globin genes. An initial trial with the parent compound, delivered as Arginine Butyrate, has demonstrated rapid stimulation of fetal globin expression to levels that have been shown to ameliorate these conditions. Phase 1 trials of an oral butyrate derivative with a long plasma half-life have just begun. These agents now provide a specific new approach for ameliorating these classic molecular disorders and merit further investigation in larger patient populations.

The 32.6 kb Indian delta beta-thalassaemia deletion ends in a 3.4 kb L1 element downstream of the beta-globin gene.

The Indian delta beta-thalassaemia, with elevated fetal gamma globin gene expression, was previously found to have a large deletion beginning 1 kb 3' of the (A) gamma globin gene at GenBank HUMHBB coordinate 42151, and extending into a new L1 sequence. We have now determined the 3' breakpoint of this deletion, and in doing so we have extended the known beta-globin gene cluster DNA sequence from its end at 73326 to projected GenBank coordinate 79016. These data show that the deletion is 32.6 kb long, terminating 11 kb 3' of the beta-globin gene. This 3' breakpoint is at 74772, within a 3.4 kb partial L1 repeat at 74263-77665; the Black ((A) gamma delta beta)(0)-thalassaemia also terminates in this L1, at 76508. In addition, two Alu sequences were found, at 73692-73816 and 78171-78441. Among the protein-binding DNA sequence motifs 3' to the Indian delta beta-thalassaemia breakpoint, at 76581/76607 there is a TGATAA/ACACCC pair that binds the erythroid-specific GATA-1 and ubiquitous CACCC-box binding proteins. We hypothesize that elevated fetal haemoglobin may be due to an enhancer or enhancers 3' to the deletion breakpoints and may involve the TGATAA/ACACCC pair.

A novel nuclear protein which binds to G gamma and A gamma globin promoters and modulates hemoglobin synthesis in K562 cells.

Nuclear extract of human erythroleukemic cell line K562 contains a 70 kDa protein which is gradually reduced when cells are induced to express globin genes by 25 microM hemin. When globin synthesis was inhibited by cycloheximide (100 micrograms/ml) or Actinomycin D (1 microgram/ml), the disappearance of this protein was prevented. The 70 kDa nuclear protein exhibited strong binding to G gamma and A gamma globin promoters but not to beta-globin promoter. This suggests that this 70 kDa nuclear protein may be involved in the regulation of fetal globin gene expression.