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PubMed1999

Correlation between IL-3 receptor expression and growth potential of human CD34+ hematopoietic cells from different tissues.

CD123 (alpha-subunit of IL-3 receptor) expression on primitive and committed human hematopoietic cells was studied by multicolor sorting and single-cell culture. The sources of cells included fetal liver (FLV), fetal bone marrow, umbilical cord blood, adult bone marrow and mobilized peripheral blood. Three subsets of CD34+ cells were defined by the levels of surface CD123: CD123negative, CD123low, and CD123bright. Coexpression of lineage markers showed that a majority of CD34+CD123bright cells were myeloid and B-lymphoid progenitors, while erythroid progenitors were mainly in the CD34+CD123negative subset. The CD34+CD123low subset contained a heterogeneous distribution of early and committed progenitor cells. Single CD34+ cells from the CD123 subsets were cultured in a cytokine cocktail of stem cell factor, interleukin 3 (IL-3), IL-6, GM-CSF, erythropoietin, insulin-like growth factor-1, and basic fibroblast growth factor. After 14 days of incubation, a higher cloning efficiency (CE) was observed in the CD34+CD123negative and CD34+CD123low fractions (37+/-23% and 44+/-23%, respectively) than in the CD34+CD123bright fraction (15+/-21%). Using previously published criteria that colonies containing dispersed, translucent cells (dispersed growth pattern, DGP) were derived from primitive cells and that colonies composed solely of clusters were from committed cells, early precursors were distributed evenly in the CD34+CD123negative and CD34+CD123low subsets. When CD38 and CD90 (Thy-1) were used for further characterization of CD34+ cells from FLV, CE increased from 37+/-23% in CD123negative to 70+/-19% in CD123negativeCD38- and from 44+/-23% in CD123low to 66+/-19% in CD123lowCD38-. No significant increase in CE or DGP progenitors was observed when CD34+ cells were sorted by CD90 and CD123. We concluded that: A) high levels of CD123 were expressed on B-lymphoid and myeloid progenitors; B) early erythroid progenitors had little or no surface CD123, and C) primitive hematopoietic cells are characterized by CD123negative/low expression.

ADP-ribosyl Cyclase↗
PubMed1996

Overexpression of the ferritin H subunit in cultured erythroid cells changes the intracellular iron distribution.

To test the hypothesis that variations in H- and L-subunit composition in the ferritin shell affect intracellular iron metabolism, we established stable transfectants of mouse erythroleukemia cells overexpressing the H-ferritin subunit. Analyses were performed on individual clones of transfected cells induced to differentiate with hexamethylenbisacetamide (HMBA). The results showed that there was a reduction in the amount of hemoglobin produced, in inverse relationship with the level of H-subunit overexpression. Incorporation of [2-14C]glycine into heme was reduced by 20% t0 30% in the clones overexpressing H-ferritin subunit compared with control clone. However, the reduction in hemoglobin production was not reversed by addition of heme precursors (delta-aminolevulinic acid or iron) or by hemin itself. A reduced accumulation of beta-globin mRNA was also observed, which could account for the impaired hemoglobin synthesis. Furthermore, synthesis of the endogenous L-ferritin subunit was greatly repressed. Gel retardation assays performed on cytoplasmic extracts of transfected cells using an iron-responsive element (IRE) as a probe revealed that in overexpressing cells, the iron-regulatory protein (IRP) had a conformation with a high RNA-binding affinity, thus leading to translational repression of the endogenous L-ferritin synthesis. These data suggest that an increased formation of H-rich isoferritins leads to a rapid chelation of the regulatory iron pool. While the mechanism underlying the reduction in beta-globin mRNA remains to be elucidated, this study provides direct evidence for the role of IRP-mediated regulation of ferritin expression in erythroid cell metabolism.

Acetamides↗
PubMed1995

Identification of a major positive regulatory element located 5' to the human zeta-globin gene.

The function of the zeta-globin promoter was studied using a series of zeta-globin promoter deletion constructs to drive luciferase expression in transiently transfected human erythroleukemia cells. The promoters were used without enhancers, or with enhancers derived from the beta-globin locus control region and the alpha-globin HS-40 enhancer. When transfected into K562 cells, which express zeta-globin, comparable amounts of activity were obtained from the -557 and -417 zeta-luciferase constructs and the alpha-luciferase constructs when no enhancers or the alpha-globin locus enhancers were used. When the constructs were transfected into OCIM1 cells, which do not express zeta-globin, the zeta-globin promoters were at best 20% as active as the alpha-globin promoters. When sequences from -417 to -207 5' to the zeta-globin mRNA cap site were deleted, up to 95% of the zeta-globin promoter activity was lost in K562 cells. Reinsertion of these sequences into zeta-luciferase constructs missing the -417 to -207 region showed that the sequences lack classical enhancer activity. Point mutation of a GATA-1 site at -230 reduced promoter activity by 37%. Point mutation of a CCACC site at -240 had no effect. Electrophoretic mobility shift assays indicated that the -230 GATA-1 site has a relatively low affinity for GATA-1. These experiments show the presence of a strong positive-acting element, located between -417 and -207 bp 5' to the zeta-globin mRNA cap site, is necessary for high-level promoter activity in K562 cells. This element requires GATA-1 and additional unknown factors for maximal activity.

Base Sequence↗
PubMed1995

Expression of erythroid-specific genes in acute megakaryoblastic leukaemia and transient myeloproliferative disorder in Down's syndrome.

Acute megakaryoblastic leukaemia (M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. To clarify properties of the blast cells in M7 and TMD cases, we examined erythroid markers expression in blasts from six cases with M7 and seven cases with TMD in this study. Erythroid-specific mRNAs encoding gamma-globin and erythroid delta-aminolevulinate synthase were found to be expressed in blasts from most of these cases, indicating that majorities of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. We also found that mRNAs encoding GATA-1 and GATA-2 are expressed in all these cases. These results suggest that M7 blasts and TMD blasts correspond to the erythroid/megakaryocytic bipotential progenitor cells.

5-Aminolevulinate Synthetase↗
PubMed2005

Hemopoietic precursors: mechanisms of regulation under conditions of chronic stress.

We studied the content of hemopoietic precursor cells in the bone marrow, production of IL-1, IL-3, and colony-stimulating and erythropoietic activities by bone marrow cells under conditions of chronic stress. It was shown that in mice subjected to 15-h immobilization no hyperplasia of the bone marrow developed against the background of increased content of committed erythro- and granulomonocytopoietic precursors and enhanced production of short-distant humoral regulators of hemopoiesis, which reflects dysregulation of hemopoietic cells proliferation and differentiation processes in stressed animals.

Animals↗
PubMed2002

Erythroid expression of the human alpha-spectrin gene promoter is mediated by GATA-1- and NF-E2-binding proteins.

alpha-Spectrin is a highly expressed membrane protein critical for the flexibility and stability of the erythrocyte. Qualitative and quantitative defects of alpha-spectrin are present in the erythrocytes of many patients with abnormalities of red blood cell shape including hereditary spherocytosis and elliptocytosis. We wished to determine the regulatory elements that determine the erythroid-specific expression of the alpha-spectrin gene. We mapped the 5' end of the alpha-spectrin erythroid cDNA and cloned the 5' flanking genomic DNA containing the putative alpha-spectrin gene promoter. Using transfection of promoter/reporter plasmids in human tissue culture cell lines, in vitro DNase I footprinting analyses, and gel mobility shift assays, an alpha-spectrin gene erythroid promoter with binding sites for GATA-1- and NF-E2-related proteins was identified. Both binding sites were required for full promoter activity. In transgenic mice, a reporter gene directed by the alpha-spectrin promoter was expressed in yolk sac, fetal liver, and erythroid cells of bone marrow but not adult reticulocytes. No expression of the reporter gene was detected in nonerythroid tissues. We conclude that this alpha-spectrin gene promoter contains the sequences necessary for low level expression in erythroid progenitor cells.

5' Flanking Region↗
PubMed1991

Inhibition by 1,25 dihydroxyvitamin D3 of chemically induced erythroid differentiation of K562 leukemia cells.

The physiologically active form of vitamin D, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], was found to inhibit erythroid differentiation of human leukemic K562 cells. Differentiation was induced by 1 mumol/L arabinocytosine (Ara-C), 40 mumol/L tiazofurin, 1 mumol/L aphidicolin, or 1 mumol/L hydroxyurea, and was monitored daily by the appearance of hemoglobin in an increasing proportion of cells. Pretreatment for 48 hours with 2.4 x 10(-8) mol/L 1,25(OH)2D3, a concentration that is also optimal for induction of monocytic differentiation of HL-60 cells, reproducibly inhibited subsequent induction of erythroid differentiation by all of the above inducers, and modified the morphologic changes that Ara-C produced in these cells. The inhibition of hemoglobinization was approximately 50% irrespective of the degree of differentiation produced by the various inducers, but growth inhibition associated with exposure to the inducers was not affected by 1,25(OH)2D3. Similar inhibition of differentiation by 1,25(OH)2D3 was observed in mouse erythroleukemia cells MEL-D1B treated with 5 mmol/L hexamethylenebisacetamide. The inhibitory effect of 1,25(OH)2D3 on erythroid differentiation of K562 cells was abrogated by cyclohexamide (20 micrograms/mL), an inhibitor of protein synthesis. The mRNA for 1,25(OH)2D3 receptor (VDR) was detected in K562 cells, and was downregulated by a 96-hour exposure to 1,25(OH)2D3 or a 48-hour exposure to Ara-C. The presence of VDR mRNA suggests a physiologic role for 1,25(OH)2D3 in K562 cells that are precursors of erythroid cells. This role is perhaps to shift the pathways of differentiation from the erythroid to the monocytic lineage.

Animals↗
PubMed1985

Carbonic anhydrase I is an early specific marker of normal human erythroid differentiation.

The expression of carbonic anhydrase (CA) as a marker of erythroid differentiation was investigated by immunologic and enzymatic procedures. A polyclonal anti-CA antibody was obtained by immunizing rabbits with purified CA I isozyme. This antibody is reactive with CA I but not with CA II. Within blood cells, CA I was only present in erythrocytes, whereas CA II was also detected in platelet lysates by enzymatic assay. Concerning marrow cells, identifiable erythroblasts and some blast cells expressed CA I. Most of the glycophorin A-positive marrow cells were clearly labeled by the anti-CA I antibody. However, rare CA I-positive cells were not reactive with anti-glycophorin A antibodies. We therefore investigated whether these cells were erythroid precursors or progenitors. In cell sorting experiments of marrow cells with the FA6 152 monoclonal antibody, which among hematopoietic progenitors is reactive only with CFU-E and a part of BFU-E, was performed, CA I+ cells were found mainly in the positive fraction. The percentage of CA I+ cells nonreactive with anti-glycophorin A antibodies contained in the two fractions was in the same range as the percentage of erythroid progenitors identified by their capacity to form colonies. In addition, the anti-CA I antibody labeled blood BFU-E-derived colonies as early as day 6 of culture, whereas in similar experiments with the anti-glycophorin A antibodies, they were stained three or four days later. No labeling was observed in CFU-GM- or CFU-MK-derived colonies. The phenotype of the day 6 cells expressing CA I was similar to that of erythroid progenitors (CFU-E or BFU-E): negative for glycophorin A and hemoglobin, and positive for HLA-DR antigen, the antigen identified by FA6 152, and blood group A antigen. Among the cell lines tested, only HEL cells expressed CA I, while K562 was unlabeled by the anti-CA I antibody. In contrast, HEL and K562 cells expressed CA II as detected by a biochemical technique. Synthesis of CA I, as with other erythroid markers such as glycophorin A and hemoglobin, was almost abolished after 12-O-tetradecanoyl-phorbol-13 acetate treatment of HEL cells. In conclusion, CA I appears to be an early specific marker of the erythroid differentiation, expressed by a cell with a similar phenotype as an erythroid progenitor.

Antibody Specificity↗
PubMed2000

An intrinsic but cell-nonautonomous defect in GATA-1-overexpressing mouse erythroid cells.

GATA-1 is a tissue-specific transcription factor that is essential for the production of red blood cells. Here we show that overexpression of GATA-1 in erythroid cells inhibits their differentiation, leading to a lethal anaemia. Using chromosome-X-inactivation of a GATA-1 transgene and chimaeric animals, we show that this defect is intrinsic to erythroid cells, but nevertheless cell nonautonomous. Usually, cell nonautonomy is thought to reflect aberrant gene function in cells other than those that exhibit the phenotype. On the basis of our data, we propose an alternative mechanism in which a signal originating from wild-type erythroid cells restores normal differentiation to cells overexpressing GATA-1 in vivo. The existence of such a signalling mechanism indicates that previous interpretations of cell-nonautonomous defects may be erroneous in some cases and may in fact assign gene function to incorrect cell types.

Anemia↗
PubMed1993

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.

Base Sequence↗
PubMed2002

A transgenic mouse model for non-immune hydrops fetalis induced by the NS1 gene of human parvovirus B19.

Human parvovirus B19 (B19) infection during pregnancy is associated with the adverse foetal outcome known as non-immune hydrops fetalis (NIHF). Although B19 is known to infect erythroid-lineage cells in vivo as well as in vitro, the mechanism leading to the occurrence of NIHF is not clear. To investigate the possible involvement of the B19 non-structural protein NS1 in NIHF, three independent lines of transgenic mice were generated that expressed NS1 under the control of the Cre-loxP system and the GATA1 promoter. Two of the three lines expressed NS1 in erythroid-lineage cells. Most of the transgenic mice died at the embryonic stage, some of which developed hydropic changes caused by severe anaemia at embryonic day 15.5 (E15.5). Histological examination of embryos at E15.5 showed significantly fewer erythropoietic islands in the liver parenchyma, whereas their hearts showed no abnormal signs, such as cardiomegaly and apoptotic cells. The NS1-transgenic mouse lines established here provide an animal model for human NIHF and suggest that NS1 plays a crucial role in the adverse outcome associated with intrauterine B19 infection in humans.

Animals↗
PubMed1995

Lineage-restricted regulation of the murine SCL/TAL-1 promoter.

The SCL/TAL-1 gene encodes a basic helix-loop-helix transcription factor that is expressed in multipotent hematopoietic progenitors before lineage commitment. Its expression is maintained during differentiation along erythroid, mast, and megakaryocytic lineages, but is repressed after commitment to nonexpressing lineages. To begin to address the molecular mechanisms underlying this complex pattern of expression, we have studied the regulation of the murine SCL promoter in erythroid and T-cell lines. Analysis of the methylation and chromatin structure of the SCL promoter region showed that SCL mRNA expression correlated with DNase hypersensitive sites and methylation status of the promoter. Transient reporter assays showed that promoter 1a was active in erythroid cells but not in T cells. Sequences between -187 and +26 were sufficient for lineage-restricted activity of promoter 1a. A joint promoter construct containing both promoter 1a and promoter 1b also exhibited lineage-restricted activity. Conserved GATA (-37), MAZ (+242), and ETS (+264) motifs were all shown to contribute to SCL promoter activity in erythroid cells, but several other motifs were not required for full promoter activity. The pattern of complexes binding to the +242 MAZ and +264 ETS sites were the same in erythroid and T cells. However, GATA-1 bound the -37 GATA site in erythroid cells, whereas in T cells GATA-3 was only able to bind weakly, if at all. Moreover, GATA-1 but not GATA-2 or GATA-3 was able to transactivate SCL promoter 1a in a T-cell environment. These results suggest that inactivity of SCL promoter 1a in T cells reflected the absence of GATA-1 rather than the presence of trans-dominant negative regulators.

Amino Acid Sequence↗
PubMed1997

In vitro toxicity of trichothecenes on human erythroblastic progenitors.

Trichothecenes are mycotoxins produced by various species of fungi which can occur on various agricultural products. Among these compounds, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS) and deoxynivalenol (DON) are the most naturally encountered and potent trichothecenes. Consumption of trichothecene contaminated foods by farm animals and humans leads to mycotoxicoses. Trichothecenes are known to induce haematologic disorders such as neutropenia, thrombopenia, and aplastic anemia in human and animals. The aim of our investigations is to explore the effects of trichothecenes on the haematopoietic progenitors. The four trichothecenes previously demonstrated to be strongly cytotoxic for human CFU-GM have been tested on human BFU-E. For this purpose, a culture model of human erythroblastic progenitors (BFU-E) optimized for toxicological studies was used to determine the effects of T-2, HT-2, diacetoxyscirpenol (DAS) and deoxynivalenol (DON) on red blood cell precursor proliferation and differentiation. Results showed that human BFU-E are as sensitive to trichothecenes as human CFU-GM, except for DON, in the range of concentrations tested. Differentiation of erythroblastic progenitors could be perturbed by these mycotoxins. Human erythroblastic progenitors are also a target of trichothecenes.

Cell Differentiation↗
PubMed1997

Epo-induced hemoglobinization of SKT6 cells is mediated by minimal cytoplasmic domains of the Epo or prolactin receptors without modulation of GATA-1 or EKLF.

Interaction of erythropoietin with its type 1 receptor is essential to the development of late erythroid progenitor cells. Through the ectopic expression of receptor mutants in lymphoid and myeloid cell lines, insight has been gained regarding effectors that regulate Epo-induced proliferation. In contrast, effectors that regulate Epo-induced differentiation events (e.g. globin gene expression) are largely undefined. For in vitro studies of this pathway, erythroleukemic SKT6 cell sublines have been isolated which stably and efficiently hemoglobinize in response to Epo. Epo rapidly activated Jak2, STAT5 and detectably STATs 1 and 3, while no effects on GATA-1, EKLF or STAT5 expression were observed. Finally, efficient hemoglobinization of SKT6 cells was shown to be mediated by chimeric receptors comprised of the EGF receptor extracellular domain and truncated cytoplasmic subdomains of either the Epo receptor or the prolactin Nb2 receptor. This work further establishes SKT6 cells as an important model for studies of Epo-stimulated differentiation, and shows that this signaling pathway is promoted by a limited set of membrane-proximal receptor domains and effectors.

Amino Acid Sequence↗
PubMed1996

Expression of erythroid-specific genes in megakaryoblastic disorders.

Currently available data indicate that erythroid and megakaryocytic differentiation pathways are closely related to each other, and there may exist progenitor cells common to those two lineages may exist. Acute megakaryoblastic leukemia (AML-M7) and transient myeloproliferative disorder in Down's syndrome (TMD) are characterized by rapid growth of abnormal blast cells which express megakaryocytic markers. These blast cells express lineage-specific transcription factors such as GATA-1 common to these lineages and frequently express erythroid-specific mRNAs such as gamma-globin and erythroid delta-aminolevulinate synthase (ALAS-E), indicating that most of the blasts in M7 and TMD cases have erythroid and megakaryocytic phenotypes. These results suggest that blasts in M7 and TMD may correspond to progenitors of both erythroid and megakaryocytic lineages.

DNA-Binding Proteins↗
PubMed1995

The role of the erythroid-specific delta-aminolevulinate synthase gene expression in erythroid heme synthesis.

Using antisense technology, the effects of suppressed gene expression of the erythroid-specific delta-aminolevulinate (ALA) synthase (ALAS-E) on heme synthesis, expression of mRNAs encoding an erythroid-specific transcription factor NF-E2, other heme pathway enzymes, and beta-globin were examined in murine erythroleukemia (MEL) cells. In MEL cells in which an antisense ALAS-E RNA was expressed (AS clone), sense ALAS-E mRNA levels in both untreated and dimethylsulfoxide (DMSO)-treated cells were decreased compared with their respective controls. Heme synthesis in AS clones was decreased in proportion to the suppressed levels of ALAS-E mRNA. In addition, mRNAs for ALA dehydratase, porphobilinogen deaminase, ferrochelatase (FeC), and beta-globin were also decreased in AS clones. There was a strong correlation between the level of ALAS-E mRNA and most of the mRNAs of the heme pathway enzymes and beta-globin. There was a decrease in the mRNA level of p45, but not of mafK, which are the large and the small subunits of NF-E2, respectively, in AS clones. Treatment of AS cells with hemin and ALA in the presence of DMSO partially restored the suppressed mRNA levels for beta-globin and FeC and heme content, respectively. These findings thus indicate that heme formation, which is determined by the level of ALAS-E, plays an essential role on gene expression of many proteins necessary for erythroid development.

5-Aminolevulinate Synthetase↗
PubMed1993

Increased colony growth in peripheral blood cultures from patients with ALL depends on immunological subtype.

The proliferative capacity of precursor cells in bone marrow and peripheral blood of 19 patients with acute lymphoblastic leukaemia (ALL) at diagnosis was studied and results were compared with the immunophenotype of the leukaemic population. Bone marrow proliferative capacity in these patients was strongly diminished, low or absent, independent of the immunophenotype, compared with control values (p < 0.0002). In contrast, the growth pattern in peripheral blood cultures from the same patients varied widely according to the subtype of ALL: whereas in patients with undifferentiated ALL [TdT+, HLA-DR+, CD19+ or-, CD10-, (n = 4) or CD7+, CD5+, CD1-, CD4- and CD8- (n = 1)] PB had strongly reduced proliferative capacity compared with control (p < 0.05), there was excess growth of normal neutrophil and erythroid colonies in BP cultures from patients with a more mature immunophenotype of either B-[CD10+, (n = 11)] or T [CD1+, CD4+ and/or CD8+, (n = 3)] phenotype. This phenomenon was only seen in patients who had circulating lymphoblasts: If their number was low, growth was so prolific that single colonies could not be identified. In the presence of a high blast count, colony growth was less prolific--probably due to a "dilution" effect--but still higher than normal (p < 0.05). We conclude that, in relatively mature ALL of the B- and the T-cell line, the presence of circulating lymphoblasts is associated with increased PB proliferative capacity.

Adolescent↗
PubMed1992

The developmental switch in embryonic rho-globin expression is correlated with erythroid lineage-specific differences in transcription factor levels.

During chicken embryogenesis, the rho-globin gene is expressed only in the early developmental stages. We have examined the mechanisms that are responsible for this behavior. The transcription of the rho-globin gene is strongly correlated with the presence during development of primitive erythroid lineage cells, consistent with the idea that the expression of the rho-globin gene is restricted to that lineage. The "switching off" of rho-globin during development thus reflects the change from primitive to definitive cell lineages which occurs during erythropoiesis in chicken. We use transient expression assays in primary erythroid and other cells to show that the information for lineage- and tissue-specific expression of the rho-globin gene is contained in a 456 bp region upstream of the gene's translational start site. DNA-binding studies, coupled with analysis of the effect on expression of deletions and binding site mutations, were used to identify important control elements within this 456 bp region. We find that binding sites for the ubiquitous transcription factor Sp1, and the specific hematopoietic factor GATA-1, are crucial for expression of the gene in primitive erythroid cells. Quantitative analysis shows that nuclei of the primitive erythroid lineage contain 10-fold more of these factors than do the nuclei of definitive cells. We show that in principle these differences in factor concentration are sufficient to explain the lineage-specific behavior that we observe in our assays. We suggest that this may be an important part of the mechanism for lineage-restricted rho-globin expression during chicken erythroid development. Similar mechanisms may be involved in regulation of other (but not all) members of the globin family.

Animals↗