Adult Erythroblasts Research Articles

Methyl Binding Domain Protein 2 Mediates gamma-Globin Silencing in Adult beta-YAC Transgenic Mice.

The genes of the vertebrate beta-globin locus undergo a switch in expression during development whereby embryonic/fetal genes of the cluster are sequentially silenced and the adult genes are activated during erythroid development. DNA methylation has been shown to be associated with developmentally silenced globin genes, and compounds that inhibit cytosine methylation have been shown to activate transcription from developmentally silenced globin genes in several species, including humans. Previously, we have shown that the methyl domain binding protein 2 (MBD2) is involved in maintaining embryonic rho-globin gene silencing in adult avian erythroid cells. We describe here a role for MBD2 in the DNA methylation mediated silencing and maintenance of silencing of the human fetal gamma-globin gene in a transgenic mouse model. We confirmed the previously published report by Pace et al that the gamma-globin gene is reactivated, upon treatment with the DNA methyltransferase inhibitor, 5-azacytidine, of mice containing the entire beta-globin locus as a yeast artificial chromosome (BetaYAC) transgene. In order to elucidate the mechanism through which DNA methylation represses the gamma-globin gene in adult erythroid cells, betaYAC/MBD2−/− mice were generated by breeding BetaYAC mice with MBD2−/− mice. Anemic adult betaYAC/MBD2−/− mice continue to express the gamma-globin gene at a level commensurate with animals treated with 5-azacytidine, which is10–20 fold over those treated with 1-acetyl-2-phenylhydrazine alone, as measured by both quantitative PCR and by RNase protection assays. In addition, the level of gamma-globin gene expression is consistently several fold higher in MBD2−/− compared to wild type BetaYAC mice in 14.5 and 16.5 dpc fetal liver erythroblasts. Furthermore, transcriptional activation of the gamma-globin gene in adult erythroblasts is associated with a modest decrease in DNA methylation around the gamma-globin promoters and a ~4-fold enrichment of histone H3 trimethylated at lysine 4 (TriK4), as measured by chromatin immunoprecipitation assay using quantitative PCR. Finally, treatment of MBD2 null mice with 5-azacytidine induces only a small, non-additive induction of gamma-globin expression indicating that DNA methylation acts primarily through MBD2 to maintain gamma-globin suppression in adult erythroid cells. These results suggest that MBD2 is a potential target for therapeutic induction of gamma-globin gene expression in the settings of sickle cell anemia and beta-thalassemia.

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.

Synergistic and Additive Properties of the Beta-Globin Locus Control Region (LCR) Revealed by 5′HS3 Deletion Mutations: Implication for LCR Chromatin Architecture

Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5'HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (beta-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5'HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5'HS3 deletion abolished histone acetylation throughout the beta-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5' DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5'HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5' DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5'HS3 and HS3 core deletions.

A sustained and pancellular reversal of gamma-globin gene silencing in adult human erythroid precursor cells

AbstractWe systematically compared cytokine-mediated increases or decreases in proliferation with globin gene and protein expression in adult human erythroblasts. Despite their opposite effects on growth, stem cell factor (SCF) and transforming growth factorbeta (TGF-B) had synergistic effects with respect to fetal hemoglobin (HbF): average HbF/HbF + adult hemoglobin (HbA) ratio in erythropoietin (EPO) = 1.4 ± 1.0%; EPO + TGF-B = 10.8 ± 1.9%; EPO + SCF = 19.1 ± 6.2%; and EPO + SCF + TGF-B (EST) = 39.3 ± 6.3%. Polymerase chain reaction (PCR) revealed significant increases in gamma-globin transcripts that were balanced by reduced beta-globin transcripts. Single-cell quantitative PCR demonstrated a complete reversal of gamma-globin gene silencing with detectable gamma-globin mRNA in more than 95% of the cells. Immunostaining with HbF antibodies also showed a pancellular distribution in EST (96.2 ± 0.01% HbF positive) compared with a heterocellular distribution in EPO (42.9 ± 0.01% HbF positive). As shown here for the first time, a robust and pancellular reversal of gamma-globin gene silencing among hemoglobinized erythroblasts from adult humans may be achieved in the absence of hereditary mutation or direct genomic manipulation. (Blood. 2005;105:387-393)

Inhibition of Fetal Hemoglobin Reactivation in Adult Human Erythroblasts.

The highly variable levels of fetal hemoglobin (HbF) reactivation among patients with sickle cell disease is not well understood. We previously determined that pancellular reversal of gamma-globin gene silencing is achievable through the activation of specific signal transduction pathways using stem cell factor (SCF) and transforming growth factor beta (TGF-B). Based upon the reliability of this culture assay for HbF reactivation, the culture system is additionally being utilized to screen for inhibitors of HbF reactivation. As an initial screening strategy, published reports, high-throughput sequencing efforts, and array-based transcription profiles were examined to identify erythroblast-expressed receptors with known ligands that may serve a signaling role in the regulation of globin expression. A dose-escalation strategy was used to screen seventeen ligands that bind to a subset of G-protein coupled receptors (GPCR). Among those ligands, oleoyl-lysophosphatidic acid (OLPA) and neurokinin A (NKA) were identified as potential inhibitors of HbF reactivation. Lysophosphatidic acid is a lipid metabolite that is released after tissue injury and may play a role in vascular remodeling. Neurokinin A is a small neuropeptide mainly involved in pain signaling. OLPA inhibited HbF reactivation in a dose-dependent manner (HbF/HbF+HbA ratios: EPO: 0.7±0.01%; EPO+SCF+TGF-B (EST): 36.3±0.1%; EST+10nM OLPA: 36.5±0.1%; EST+100nM OLPA: 31.6±0.1%; EST+1uM OLPA: 12.7±0.1%, p=1.9E-03). NKA also inhibited HbF reactivation, but the magnitude of the NKA effect was donor-specific. Cells from one donor demonstrated a robust response (HbF/HbF+HbA ratios: EPO: 1.7%; EST: 26.7%; EST+10nM NKA: 24.5%; EST+100nM NKA: 8.8%; EST+1uM NKA: 7.8%). Since OLPA and NKA may both act through adenylate cyclase, the role of this enzyme was examined further using the activator, forskolin (0.8 uM-20uM) and inhibitor, SQ22536 (40uM – 600uM). While we were unable to demonstrate a significant increase in HbF among cultures grown in forskolin, the addition of SQ22536 resulted in a profound, dose-dependent inhibitory effect on the expression of HbF (EPO: 1.3±1.2%; EST: 36.7±11.3%; EST+40uM SQ: 30.3±10.2%; EST+200 uM SQ : 14.5±4.5%, p=1.9E-02; EST+600uM SQ: 2.3±2.7%, p=4.1E-03). Flow cytometry revealed that the inhibitory effects of SQ22536 on HbF were pancellular. Importantly, inhibition of the main downstream target of adenylate cyclase (protein kinase A) with H89 (10nM-1.0 uM) did not result in a significant reduction in HbF. However, phosphorylation studies demonstrated that SQ22536 altered the kinetics of MEK activation. MEK is a kinase that helps mediate the HbF-activating effect of SCF [Blood 2004, 1;103(5):1929–33]. These data suggest that inhibitory effects upon HbF reactivation in adult erythroblasts can be mediated by endogenous signaling molecules. Based upon these results, we propose that patient-specific responses to tissue injury and pain may play a negative role in HbF reactivation in sickle-cell disease.

Histone Acetylation at the β-Globin Locus Changes with Developmental Age.

The β-globin locus contains a number of developmentally regulated genes that undergo switching of expression. In the embryo the ε-globin gene is expressed with a subsequent switch to γ-globin in the fetal liver, followed by a final switch to β-globin in definitive cells. Recently, the importance of chromatin structure in regulating gene expression has been recognized, and it has been proposed that modifications of histones (i.e. acetylation, methylation) have a direct effect on gene regulation. This hypothesis would suggest that changes in the histone code underlie the differences in globin gene expression during development. To address this we studied the pattern of histone acetylation across the β-globin locus at different stages of human development. Two different time points of human fetal erythropoiesis (approximately 55dpc and 98dpc) were studied. At these stages the level of γ mRNA is approximately 98%, there is no detectable ε mRNA, while β mRNA is about 2% of total mRNA. The adult stage of erythropoiesis was represented by erythroblasts derived from cultured adult CD34+ cells where γ mRNA was approx. 25% of total mRNA. Nuclei from the fetal and adult erythroblasts were analyzed by Chromatin Immunoprecipitation (ChiP) using antibodies to histone H3 acetylation and H4 acetylation. Quantification of enrichment was carried out by SYBR green real-time PCR, with PCR primers spanning the globin locus. The LCR region showed high levels of enrichment for acetylation in all stages of development. The ε-globin gene showed minimal levels of enrichment in all stages of development. In the 55dpc human erythroblasts γ-globin showed the highest level of enrichment of all globin genes, with the acetylation spreading across the promoter as well as the whole γ-coding region. In the 98dpc fetal erythroblasts, the acetylation at γ-globin was also enriched.In the cultured adult erythroblasts a reduced level of γ-globin acetylation was present, which was expected since there was less γ-globin synthesis in these samples. There was a higher level of acetylation at the δ- and β-globin genes in the adult cells, as would be expected for the adult stage of development. These results demonstrate that dynamic changes in histone acetylation take place as the human β-globin gene region undergoes its developmental switches. Whether these changes represent primary events in the course of globin gene switching remains to be determined.

Aroyl-Pyrrolyl-Hydroxy-Amides (APHAs), a Novel Family of Synthetic Histone Deacetylases Inhibitors, Are Potent Inducers of Human g-Globin Gene Expression.

Post-natal pharmacological reactivation of HbF, by restoring the unbalanced α/non-α globin chain production in red cells of patients affected by β-thalassemia or sickle cell anemia, represents a potential cure for these diseases. Many classes of compounds have been identified capable to induce Hb F synthesis in vitro by acting at different levels of the globin gene expression regulatory machinery. One of these classes is represented by inhibitors of a family of enzymes, the histone deacetylases (HDACs), involved in chromatin remodelling and gene transcription regulation. HDACs act in multi-protein complexes that remove acetyl groups from lysine residues on several proteins, including histones and are divided into three distinct structural classes, depending on whether their catalytic activity is zinc (class I/II)- or NAD+ (class III)-dependent. The effects of the HDACs inhibitors identified so far on HbF synthesis is, however, modest and often associated with high toxicity. Therefore, the potential of their clinical use is unclear. We have recently described a new family of synthetic HDACs inhibitors, the Aroyl-pyrrolyl-hydroxy-amides (APHAs), that induce differentiation, growth arrest and/or apoptosis of transformed cell in culture [Mai A et al, J Med Chem 2004; 47:1098]. In this study, we investigate the capability of 10 different APHA compounds to induce Hb F in two in vitro assays. One assay is based on the ability of APHA compounds to activate either the human Aγ-driven Firefly (Aγ-F) or the β-promoter drives Renilla Luciferase (β-R) reporter in GM979 cells stably transfected with a Dual Luciferase Reporter construct. The second assay is represented by the induction of γ-globin expression (by quantitative RT-PCR) in primary adult erythroblasts obtained in HEMA cultures of mononuclear cells from normal donors. The majority of the compounds tested did not significantly increased the Aγ−F (Aγ−F+β−R) reporter ratio in GM979 cells. However, the compound MC1575 increased by 3-fold (from 0.09 to 0.30) the reporter ratio in GM979 cells at a concentration of 20 μM, with modest effects of the proliferation activity of GM979 cells over the three days of the assay. When MC1575 was added at a concentration of 2–10 μM in cultures of primary adult erythroblasts induced to differentiate in serum-free media for 4 days, it induced a three fold increase of the γ/(γ+β) globin ratio (from 0.04 to 0.12), with no apparent cellular toxicity. Among the HDAC inhibitors tested in this study, MC1575 was not the most potent inhibitor of total enzyme activity. However, it was the compound that most selectively inhibited the activity of the maize homologue of mammalian class IIa HDAC enzymes [Mai et al, J Med Chem 2003; 46:4826]. These results are consistent with the hypothesis that each class of histone deacetylases might have a specific biological function and indicate that those of class IIa might represent the enzymes most specifically involved in globin gene regulation. We suggest that, by targeting the chemical inhibitors toward the catalytic domain of this class of enzymes, it should be possible to identify more specific, more potent and less toxic compounds for pharmacological treatment of β-thalassemia or sickle cell anemia.

Developmentally Specific Role of the CCAAT Box in Regulation of Human γ-Globin Gene Expression

The CCAAT box is a widespread motif in eukaryotic promoters. In this study we demonstrate that the effects of the CCAAT box on gamma-globin gene activation are developmentally distinct. Although this promoter element is essential for high level gamma gene expression in adult erythropoiesis, it plays little role in embryonic erythroid cells. The CCAAT mutation in the human gamma-globin gene promoter impairs recruitment of TATA-binding protein (TBP), TFIIB, and RNA polymerase II in adult splenic erythroblasts but not in embryonic erythroid cells. We also show that the efficiency of gamma gene transcription is correlated with recruitment of TBP on the TATA box but that the level of TBP recruitment is not nuclear factor Y (NF-Y)-dependent. Our data also suggest that it is unlikely that transcriptional stimulation by the CCAAT box is exerted through direct protein-protein interaction between NF-Y and TBP.

A new anti-hemoglobin F antibody against synthetic peptides for the detection of F-cell precursors (F-blasts) in bone marrow.

To date there are few antibodies available for the detection of fetal hemoglobin (HbF)-containing erythroblasts (F-blasts) in paraffin-embedded hematopoietic tissues. Recently, we developed a new polyclonal antibody specific to F-blasts by immunization of a rabbit with synthetic peptides of human HbF. Specificity and reactivity of the antibody were confirmed by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry. ELISA confirmed that the antibody showed strong immunoreactivity to fetal hemoglobin but no reaction to adult hemoglobin. The antibody could detect the presence of fetal blood hemolysates (10 microg/mL) at a dilution of 10(-5). According to immunohistochemical analysis, there were strong positive reactions to fetal erythroblasts in the liver and the spleen at 29 weeks of gestation and to erythroblasts from patients with myelodysplastic syndromes and erythroleukemia but no reactions to normal adult erythroblasts in bone marrow. Fetal erythrocytes in fetal blood vessels of placental tissues were strictly distinguished from maternal erythrocytes in the same sections by their positive reactions, indicating the presence of HbF. This new antibody will be a useful tool for studying fetal hemoglobin synthesis and for detecting F-blasts in archival specimens of various hematological diseases.

Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization

AbstractIsolating fetal erythroblasts from maternal blood offers a promising noninvasive alternative for prenatal diagnosis. The current immunoenzymatic methods of identifying fetal cells from background maternal cells postenrichment by labeling γ-globin are problematic. They are nonspecific because maternal cells may produce γ-globin, give poor hybridization efficiencies with chromosomal fluorescence in situ hybridization (FISH), and do not permit simultaneous visualization of the fetal cell identifier and the FISH signal. We describe a novel technique that allows simultaneous visualization of fetal erythroblast morphology, chromosomal FISH, and ε-globin labeled with AMCA (7-amino-4-methylcoumarin-3-acetic acid). AMCA was chosen as the fluorescent label to circumvent the problem of heme autofluorescence because the mean difference in relative fluorescence intensity between fetal erythroblasts stained positive for antiglobin antibody and autofluorescence of unstained cells was greater with AMCA (mean 43.2; 95% confidence interval [CI], 34.6-51.9; SD = 14.0) as the reporting label compared with fluorescein isothiocyanate (mean 24.2; 95% CI, 16.4-31.9; SD = 12.4) or phycoerythrin (mean 9.8; 95% CI, 4.8-14.8; SD = 8.0). Median FISH hybridization efficiency was 97%, comparable to the 98% (n = 5 paired samples) using Carnoy fixative. One ε-positive fetal erythroblast was identified among 105 maternal nucleated cells in 6 paired mixture experiments of fetal erythroblasts in maternal blood (P < .001). Male ε-positive fetal erythroblasts were clearly distinguishable from adult female ε-negative erythroblasts, with no false positives (n = 1000). The frequency of fetal erythroblasts expressing ε-globin declines linearly from 7 to 14 weeks' gestation (y = −15.8 × + 230.8;R2 = 0.8; P < .001). We describe a rapid and accurate method to detect simultaneously fetal erythroblast morphology, intracytoplasmic ε-globin, and nuclear FISH.

Cloning and Characterization of a Potential Transcriptional Activator of Human γ-Globin Genes

ABSTRACTHybrids produced by fusing human fetal erythroblasts (HFE) with mouse erythroleukemia (MEL) cells initially produce predominantly or exclusively human γ-globin and switch to human β globin expression as time in culture advances. One explanation for the initially predominant expression of γ-globin gene in these hybrids is the presence of trans-acting factors that activate γ-globin gene transcription. We used differential display of hybrids before and after the γ to β switch as well as fetal liver and adult erythroblasts to identify cDNAs that could be candidates for potential γ gene activators. Identically sized amplicons which were present in fetal liver erythroblasts and in the hybrids expressing only γ-globin but were absent in the adult erythroblasts and in the same hybrids after they had switched to β globin expression were cloned and sequenced. Fifty pairs of cDNAs fitting these criteria were chosen for further analysis. The sequences of the two members of 48 pairs differed from each other, revealing the low efficiency of this experimental approach. One clone pair coded for human proteosome subunit X. The second pair coded for a protein containing an acidic domain in the N-terminus and three consecutive CDC10/SW16/ankyrin repeats in the C-terminus. Transactivation assays in the yeast hybrid system and transient transfection assays in COS cells showed that a potent trans-activating domain resides in the N-terminus of this protein. Northern blot and RT-PCR assays showed that this gene is expressed in several fetal tissues but not in adult tissues. Stable transfection assays provided evidence that the product of this gene may increase the level of γ mRNA in HFE × MEL cell hybrids that undergo the γ to β switch, suggesting that this new gene encodes a protein that may function as γ gene activator.

T3-hydrocortisone synergism on adult-type erythroblast proliferation and T3-mediated apoptosis of larval-type erythroblasts during erythropoietic conversion in Xenopus laevis.

The conversion of an erythropoietic system from larval to adult type in anuran amphibia may possibly come about through cell replacement. The hormonal regulation of apoptosis of larval-type precursor cells and adult-type cell proliferation has yet to be examined in detail. In amphibians, corticoids synergize T3 action during metamorphosis. In the present study, examination was made of the process of larval-to-adult conversion in the liver erythropoietic site of Xenopus laevis, with special attention to how these metamorphic hormones, T3 and corticoid, regulate programmed cell death specific for larval erythroblasts and the proliferation of adult cells. Immunohistochemical analysis of liver sections indicates that the number of larval erythroblasts decreased to less than 50% at the early climax stage (stages 59-60) of metamorphosis. Overall liver morphology greatly changed subsequent to the climax stage from the three-lobe to the two-lobe shape. The addition of T3 (10(-8) M) to premetamorphic tadpoles induced considerable liver morphological change and a 50% decrease in larval-type erythroblasts. These erythroblast decreases seem to take place through the apoptotic process, since double-staining experiments with in situ DNA nick-end labeling (TUNEL) and hemoglobin immunostaining revealed that DNA breakage of nuclei, a well-known feature of apoptosis, occurred specifically in larval erythroblasts during prometamorphosis. Hydrocortisone (HC), which modulates T3 action during metamorphosis, was found not to be a factor in larval cell decrease. But adult erythroblasts increased by 8 times as much through the action of T3 and 32 times as much by the action of T3 plus HC, indicating the important action of T3-HC synergism. It thus follows that the erythropoietic system is converted during metamorphosis effectively by two distinct hormonal mechanisms, T3-HC synergism on adult erythroblast proliferation and T3-mediated programmed death of larval precursor cells.

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.

High expression of the tec gene product in murine testicular germ cells and erythroblasts.

Tec is a novel non-receptor-type protein tyrosine kinase that was originally identified from a murine liver cDNA library. While the function of Tec remains unknown, it was shown recently that two Tec-related kinases are involved directly in the growth and differentiation of bone marrow stem cells. As the localization of Tec protein has not been reported yet, immunohistochemical and immunochemical studies of various murine organs were conducted in the present study to clarify which cells express this kinase protein. An intense immunohistologic reaction was observed in neonatal and adult testicular germ cells, and neonatal and fetal hepatic erythroblasts. In addition, a clear immunostaining was noted in neonatal and adult tubal epithelial cells, hepatocytes, basal cells of the non-glandular stomach, foveolar epithelium of the glandular stomach, sebaceous cells of the skin and fetal cartilage. The immune reaction of germ cells and erythroblasts was observed in the cell membrane, although this protein does not have a transmembrane domain. Supportive western blotting of testis, adult liver, spleen and heart of adult C.B-17 mice with the use of anti-Tec antibody demonstrated a heavy 70 kDa band in the liver and testis, and a much weaker, small band in the heart and spleen. These results suggest that Tec protein has a specific role in testicular germ cells and erythroblasts.

Genomic footprinting and sequencing of human beta-globin locus. Tissue specificity and cell line artifact.

In order to gain further insights of the regulatory mechanisms of human beta-like globin gene switch during erythroid development, we have studied protein-DNA interaction in vivo at the human adult beta and fetal gamma globin promoters and their upstream enhancer, 5'HS-2, in purified human adult erythroblasts, in which the beta, but not gamma or epsilon, globin gene is actively transcribing. This genomic footprinting analysis of adult erythroblasts was carried out in conjunction with those of different non-erythroid human tissues, an embryonic/fetal erythroid cell line K562, and several non-erythroid human cell lines. Protein-DNA binding in the beta globin promoter, in particular at the two CACC promoter boxes and the CCAAT box, is detectable only in the adult erythroblasts. As expected, the gamma globin promoters were bound with specific nuclear factors in the expressing K562 cells, but not in non-erythroid tissues or cell lines. Relatively weak protein binding could also be detected in the vicinities of the two CCAAT boxes of the inactive gamma globin promoters in the adult erythroblasts. Although the patterns of nuclear factor-DNA interaction in vivo at the NF-E2/AP1, GATA-1, and GT-I motifs of 5'HS-2 enhancer in adult erythroblasts are similar to those in K562 cells, we have identified a previously undetected factor-binding motif of 5'HS-2 that is protected only in the adult erythroblasts. This motif is identical in sequence to the 3'-CACC box of the human beta globin promoter, and it is well conserved at the same location among all mammalian 5'HS-2 enhancers, suggesting an important regulatory role of this element in human beta globin gene transcription in adult erythroblasts. All of the above four motifs of 5'HS-2 are free of nuclear factor binding in non-erythroid tissues, but two of them, NF-E2/AP1 and GT-I, are bound with factors in some non-erythroid cell lines but not in others. The functional implications of these genomic footprinting data and the tissue-specific CpG methylation patterns of the beta-like globin promoters we obtained by genomic sequencing are discussed in terms of positive and negative regulation of the human beta-like globin switch during erythroid development.

Transcriptional activation of human zeta 2 globin promoter by the alpha globin regulatory element (HS-40): functional role of specific nuclear factor-DNA complexes.

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

Transcriptional Activation of Human ζ2 Globin Promoter by the α Globin Regulatory Element (HS-40): Functional Role of Specific Nuclear Factor-DNA Complexes

We studied the functional interaction between human embryonic zeta 2 globin promoter and the alpha globin regulatory element (HS-40) located 40 kb upstream of the zeta 2 globin gene. It was shown by transient expression assay that HS-40 behaved as an authentic enhancer for high-level zeta 2 globin promoter activity in K562 cells, an erythroid cell line of embryonic and/or fetal origin. Although sequences located between -559 and -88 of the zeta 2 globin gene were dispensable for its expression on enhancerless plasmids, they were required for the HS-40 enhancer-mediated activity of the zeta 2 globin promoter. Site-directed mutagenesis demonstrated that this HS-40 enhancer-zeta 2 globin promoter interaction is mediated by the two GATA-1 factor binding motifs located at -230 and -104, respectively. The functional domains of HS-40 were also mapped. Bal 31 deletion mapping data suggested that one GATA-1 motif, one GT motif, and two NF-E2/AP1 motifs together formed the functional core of HS-40 in the erythroid-specific activation of the zeta 2 globin promoter. Site-directed mutagenesis further demonstrated that the enhancer function of one of the two NF-E2/AP1 motifs of HS-40 is mediated through its binding to NF-E2 but not AP1 transcription factor. Finally, we did genomic footprinting of the HS-40 enhancer region in K562 cells, adult nucleated erythroblasts, and different nonerythroid cells. All sequence motifs within the functional core of HS-40, as mapped by transient expression analysis, appeared to bind a nuclear factor(s) in living K562 cells but not in nonerythroid cells. On the other hand, only one of the apparently nonfunctional sequence motifs was bound with factors in vivo. In comparison to K562, nucleated erythroblasts from adult human bone marrow exhibited a similar but nonidentical pattern of nuclear factor binding in vivo at the HS-40 region. These data suggest that transcriptional activation of human embryonic zeta 2 globin gene and the fetal/adult alpha globin genes is mediated by erythroid cell-specific and developmental stage-specific nuclear factor-DNA complexes which form at the enhancer (HS-40) and the globin promoters.

Clastogenic activity of urethane in mice

Single intraperitoneal (i.p.) treatment of male and female BDF1 (C57B1 × DBA2) mice with urethane (0.5 or 1.0 g/kg) caused a significant increase in micronucleated polychromatic erythrocytes (MNPCE) in bone marrow after 24 h. The clastogenic effect observed was dose-, sex- and age-dependent, the male and younger (6–8 weeks old) animals being more susceptible than the female and older (6 months of age) mice. 3-week oral treatment of female Balb/c mice with urethane (3 g/l added to the drinking water) caused an up to 4-fold increase in the number of micronucleated normochromatic erythrocytes (MNNCE) in mouse peripheral blood. In a month after the carcinogen treatment was stopped, the number of MNNCE dropped to the control values. In addition, a single i.p. treatment of pregnant BDF1 mice on day 17 of gestation with urethane (1.0 g/kg) caused a 514.3% ( p < 0.001) elevation of MNPCE in mouse fetal liver after 24 h as well as a 154.4% ( p < 0.05) increase in MNPCE frequency in the fetal peripheral blood. At this time point, the clastogenic response in mouse fetal liver erythroblasts was less pronounced than that detected in the maternal bone marrow cells. Urethane is a strong clastogen in mice when administered either intraperitoneally or orally and the micronucleus test applied to adult and fetal erythroblasts is a convenient method of choice for studying the acute and subchronic clastogenicity of this carcinogen, its transplacental effects as well as the influence of modifying factors on these processes.

Molecular cloning and protein structure of a human blood group Rh polypeptide.

cDNA clones encoding a human blood group Rh polypeptide were isolated from a human bone marrow cDNA library by using a polymerase chain reaction-amplified DNA fragment encoding the known common N-terminal region of the Rh proteins. The entire primary structure of the Rh polypeptide has been deduced from the nucleotide sequence of a 1384-base-pair-long cDNA clone. Translation of the open reading frame indicates that the Rh protein is composed of 417 amino acids, including the initiator methionine, which is removed in the mature protein, lacks a cleavable N-terminal sequence, and has no consensus site for potential N-glycosylation. The predicted molecular mass of the protein is 45,500, while that estimated for the Rh protein analyzed in NaDodSO4/polyacrylamide gels is in the range of 30,000-32,000. These findings suggest either that the hydrophobic Rh protein behaves abnormally on NaDodSO4 gels or that the Rh mRNA may encode a precursor protein, which is further matured by a proteolytic cleavage of the C-terminal region of the polypeptide. Hydropathy analysis and secondary structure predictions suggest the presence of 13 membrane-spanning domains, indicating that the Rh polypeptide is highly hydrophobic and deeply buried within the phospholipid bilayer. In RNA blot-hybridization (Northern) analysis, the Rh cDNA probe detects a major 1.7-kilobase and a minor 3.5-kilobase mRNA species in adult erythroblasts, fetal liver, and erythroid (K562, HEL) and megakaryocytic (MEG01) leukemic cell lines, but not in adult liver and kidney tissues or lymphoid (Jurkat) and promyelocytic (HL60) cell lines. These results suggest that the expression of the Rh gene(s) might be restricted to tissues or cell lines expressing erythroid characters.

Interferon-γ Modulates Fetal Hemoglobin Synthesis in Sickle Cell Anemia and Thalassemia

Interferon-gamma (IFN-gamma) has been shown to influence globin gene expression in cord blood and normal adult progenitor-derived erythroblasts. To explore the influence of IFN-gamma on fetal hemoglobin (HbF) synthesis in the hemoglobinopathies, erythroid progenitors (BFU-E, burst forming unit-erythroid) from patients with sickle cell anemia (SCA) and thalassemia were co-cultured with or without IFN-gamma. Hemoglobin content in progenitor-derived erythroblasts was assessed by radioligand assay (RIA). Co-culture of erythroid progenitors from 12 SCA patients with 200-400 U/ml of IFN-gamma resulted in a significant decrease in picograms of HbF and percent HbF per BFU-E-derived erythroblast. The mean decrease (+/- SEM) of picograms of HbF per cell and percent of HbF was by 42 +/- 9% and 35 +/- 8% of control cultures, respectively. Co-culture of erythroid progenitors from 10 patients with thalassemia major or thalassemia variant (HPFH/thalassemia, sickle/beta 0-thalassemia) with 200 U/ml IFN-gamma also resulted in a significant decrease in picograms and percent of HbF per BFU-E-derived erythroblast. IFN-gamma treatment also inhibited the enhancement in gamma-globin synthesis induced in culture by butyric acid. Erythroid progenitors from 2 patients with SCA, 1 patient with sickle/beta 0-thalassemia, and 1 patient with HbE/beta 0-thalassemia were co-cultured with IFN-gamma, L-alpha-amino-n-butyric acid, or both. HbF content (expressed as picograms HbF/cell) was decreased in samples co-cultured with IFN, increased in cultures with L-alpha-amino-n-butyric acid, but remained at control values in cultures treated with IFN plus L-alpha-amino-n-butyric acid. These data demonstrate that IFN-gamma is an environmental factor that influences gamma-globin gene expression in the beta hemoglobinopathies in vitro.