scholarly journals The SCL +40 Enhancer Targets the Midbrain Together with Primitive and Definitive Hematopoiesis and Is Regulated by SCL and GATA Proteins

2007 ◽  
Vol 27 (20) ◽  
pp. 7206-7219 ◽  
Author(s):  
S. Ogilvy ◽  
R. Ferreira ◽  
S. G. Piltz ◽  
J. M. Bowen ◽  
B. Göttgens ◽  
...  
Keyword(s):  
Neural Development ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Enhancer Activity ◽  
Helix Loop Helix ◽  
E Box ◽  
Subsequent Activation ◽  
Definitive Erythropoiesis ◽  
Definitive Hematopoiesis

ABSTRACT The SCL/Tal-1 gene encodes a basic helix-loop-helix transcription factor with key roles in hematopoietic and neural development. SCL is expressed in, and required for, both primitive and definitive erythropoiesis. Thus far, we have identified only one erythroid SCL enhancer. Located 40 kb downstream of exon 1a, the +40 enhancer displays activity in primitive erythroblasts. We demonstrate here that a 3.7-kb fragment containing this element also targets expression to the midbrain, a known site of endogenous SCL expression. Although the 3.7-kb construct was active in primitive, but not definitive, erythroblasts, a larger 5.0-kb fragment, encompassing the 3.7-kb region, was active in both fetal and adult definitive hematopoietic cells. This included Ter119+ erythroid cells along with fetal liver erythroid and myeloid progenitors. Unlike two other SCL hematopoietic enhancers (+18/19 and −4), +40 enhancer transgenes were inactive in the endothelium. A conserved 400-bp core region, essential for both hematopoietic and midbrain +40 enhancer activity in embryos, relied on two GATA/E-box motifs and was bound in vivo by GATA-1 and SCL in erythroid cells. These results suggest a model in which the SCL +18/19 and/or −4 enhancers initiate SCL expression in early mesodermal derivatives capable of generating blood and endothelium, with subsequent activation of the +40 enhancer via an autoregulatory loop.

Emp Null Mice Are Non-Viable and Exhibit Erythroid Differentiation Defect.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 806-806 ◽  
Author(s):  
Shivani Soni ◽  
Shashi Bala ◽  
Babette Gwynn ◽  
Kenneth E. Sahr ◽  
Luanne L. Peters ◽  
...  
Keyword(s):  
Fetal Liver ◽  
Direct Sequencing ◽  
Embryonic Stem ◽  
Gene Trap ◽  
Physical Contact ◽  
Erythroid Cells ◽  
Wild Type ◽  
Definitive Erythropoiesis

Abstract Emp, erythroblast macrophage protein, was originally detected in erythroblasts and macrophages, which form erythroblastic islands during erythropoiesis in the human bone marrow. The physical contact between erythroblasts and macrophages was suggested to promote the terminal maturation of erythroblasts, leading to their enucleation in vitro. To evaluate the function of Emp in vivo, we employed gene targeting studies to develop an Emp(−/−) mouse model. Mouse embryonic stem cells containing a gene-trap insertion in Emp were obtained from BayGenomics. Insertion of the gene-trap vector into Emp was verified by direct sequencing of cDNA obtained by 5′RACE. Chimeric mice generated by blastocyst microinjection were intercrossed, and the offspring were genotyped by PCR and Southern hybridization. The Emp (+/−) mice were healthy and fertile. However, no live Emp (−/−) mice were found among the progeny of the Emp (+/−) intercrosses. Analysis of timed pregnancies revealed that Emp (−/−) embryos were present at a frequency roughly consistent with Mendelian inheritance throughout the embryonal stages. Homozygous Emp (−/−) embryos were small and pale compared to their littermates, and they survived embryonic development but died at birth. To determine the effect, if any, of Emp gene deletion on definitive hematopoiesis, livers of +/+, +/−, and −/− embryos at E15.5 were examined after H&E and Giemsa staining of paraffin-embedded serial sections, and cytospins. We found few mature erythroid cells in the sinusoids of homozygotes, in contrast to those of either wild-type or heterozygotes, where abundant enucleated red blood cells were observed. Although nucleated erythrocytes were found in both wild-type and mutant embryos, their relative proportions were very different: the less mature forms (proerythroblasts) predominated in the −/− embryos whereas the more mature forms (polychromatophilic/orthochromatic and enucleated erythrocytes) were most common in +/+ and +/− embryos. Furthermore, erythroblastic islands consisting of a central macrophage surrounded by developing erythroblasts were seen in the cytospin preparations of wild-type and heterozygote livers but not in those of homozygous null livers. Since fetal liver macrophages (FLMs) are indispensable for definitive erythropoiesis, we investigated the effect that Emp’s absence might have on development of FLMs. The E15.5 fetal liver sections were stained with the macrophage-specific F4/80 antigen. Numerous F4/80-positive macrophages were present throughout the liver of normal embryos whereas, the number was substantially reduced in Emp (−/−) liver. In summary, in the absence of Emp, FLMs are significantly reduced and terminal maturation of erythroid cells is negatively affected. Thus, the availability of Emp(−/−) embryos will provide a unique experimental model to study the function of macrophages in definitive erythropoiesis.

scholarly journals Hypoxic stress underlies defects in erythroblast islands in the Rb-null mouse

Blood ◽  
2007 ◽  
Vol 110 (6) ◽  
pp. 2173-2181 ◽  
Author(s):  
Benjamin T. Spike ◽  
Benjamin C. Dibling ◽  
Kay F. Macleod
Keyword(s):  
Fetal Liver ◽  
Cell Maturation ◽  
Null Mouse ◽  
Hypoxic Stress ◽  
Red Cell ◽  
Exogenous Stress ◽  
Erythroid Maturation ◽  
Definitive Erythropoiesis

Abstract Definitive erythropoiesis occurs in islands composed of a central macrophage in contact with differentiating erythroblasts. Erythroid maturation including enucleation can also occur in the absence of macrophages both in vivo and in vitro. We reported previously that loss of Rb induces cell-autonomous defects in red cell maturation under stress conditions, while other reports have suggested that the failure of Rb-null erythroblasts to enucleate is due to defects in associated macrophages. Here we show that erythropoietic islands are disrupted by hypoxic stress, such as occurs in the Rb-null fetal liver, that Rb−/− macrophages are competent for erythropoietic island formation in the absence of exogenous stress and that enucleation defects persist in Rb-null erythroblasts irrespective of macrophage function.

scholarly journals Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 930-936 ◽  
Author(s):  
I Max-Audit ◽  
U Testa ◽  
D Kechemir ◽  
M Titeux ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Cell Lines ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Low Concentrations ◽  
Erythroleukemia Cell ◽  
Erythroleukemic Cell ◽  
High Level

To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

scholarly journals The SCL/TAL-1 gene is expressed in progenitors of both the hematopoietic and vascular systems during embryogenesis

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1200-1208 ◽  
Author(s):  
AR Kallianpur ◽  
JE Jordan ◽  
SJ Brandt
Keyword(s):  
T Cell ◽  
Neural Development ◽  
Fetal Liver ◽  
Chromosomal Rearrangements ◽  
Chromosomal Translocation ◽  
Cell Types ◽  
Hematopoietic Differentiation ◽  
Helix Loop Helix ◽  
Adult Mice ◽  
Rare Cells

Activation of the SCL (or TAL-1) gene as a result of chromosomal translocation or deletion is a frequent molecular lesion in acute T- cell leukemia. By virtue of its membership in the basic helix-loop- helix family of transcription factors, the SCL gene is a candidate to regulate events in hematopoietic differentiation. We have used polyclonal antibody raised against a bacterial expressed malE-SCL fusion protein to characterize SCL protein expression in postimplantation embryos and in neonatal and adult mice. SCL protein was detected at day 7.5 post coitum at both embryonic and extraembryonic sites, approximately 24 hours before the formation of recognizable hematopoietic elements. Expression then localized to blood islands of the yolk sac followed by localization to fetal liver and spleen, paralleling the hematopoietic activity of these tissues during development. SCL protein was detected in erythroblasts in fetal and adult spleen, myeloid cells and megakaryocytes in spleen and bone marrow, mast cells in skin, and in rare cells in fetal and adult thymus. In addition, SCL protein was noted in endothelial progenitors in blood islands and in endothelial cells and angioblasts in a number of organs at times coincident with their vascularization. SCL expression was also observed in other nonhematopoietic cell types in the developing skeletal and nervous systems. These results show that SCL expression is one of the earliest markers of mammalian hematopoietic development and are compatible with a role for this transcription factor in terminal differentiation of the erythroid and megakaryocytic lineages. SCL expression by cells in the thymus suggests that the gene may be active at some stage of T-cell differentiation and may be relevant to its involvement by chromosomal rearrangements in T- lymphoid leukemias. Finally, expression of the gene in developing brain, cartilage, and vascular endothelium indicates SCL may have actions in neural development, osteogenesis, and vasculogenesis, as well as in hematopoietic differentiation.

NeuroM, a neural helix-loop-helix transcription factor, defines a new transition stage in neurogenesis

Development ◽  
1997 ◽  
Vol 124 (17) ◽  
pp. 3263-3272 ◽  
Author(s):  
T. Roztocil ◽  
L. Matter-Sadzinski ◽  
C. Alliod ◽  
M. Ballivet ◽  
J.M. Matter
Keyword(s):  
Nervous System ◽  
Transcription Factor ◽  
Mitotic Cycle ◽  
Ventricular Zone ◽  
Helix Loop Helix ◽  
E Box ◽  
Genes Encoding ◽  
Developing Nervous System

Genes encoding transcription factors of the helix-loop-helix family are essential for the development of the nervous system in Drosophila and vertebrates. Screens of an embryonic chick neural cDNA library have yielded NeuroM, a novel neural-specific helix-loop-helix transcription factor related to the Drosophila proneural gene atonal. The NeuroM protein most closely resembles the vertebrate NeuroD and Nex1/MATH2 factors, and is capable of transactivating an E-box promoter in vivo. In situ hybridization studies have been conducted, in conjunction with pulse-labeling of S-phase nuclei, to compare NeuroM to NeuroD expression in the developing nervous system. In spinal cord and optic tectum, NeuroM expression precedes that of NeuroD. It is transient and restricted to cells lining the ventricular zone that have ceased proliferating but have not yet begun to migrate into the outer layers. In retina, NeuroM is also transiently expressed in cells as they withdraw from the mitotic cycle, but persists in horizontal and bipolar neurons until full differentiation, assuming an expression pattern exactly complementary to NeuroD. In the peripheral nervous system, NeuroM expression closely follows cell proliferation, suggesting that it intervenes at a similar developmental juncture in all parts of the nervous system. We propose that availability of the NeuroM helix-loop-helix factor defines a new stage in neurogenesis, at the transition between undifferentiated, premigratory and differentiating, migratory neural precursors.

scholarly journals Pattern of pyruvate kinase isozymes in erythroleukemia cell lines and in normal human erythroblasts

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 930-936 ◽  
Author(s):  
I Max-Audit ◽  
U Testa ◽  
D Kechemir ◽  
M Titeux ◽  
W Vainchenker ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Cell Lines ◽  
Fetal Liver ◽  
Erythroid Cells ◽  
Low Concentrations ◽  
Erythroleukemia Cell ◽  
Erythroleukemic Cell ◽  
High Level

Abstract To further investigate the erythroid nature of the two human erythroleukemia cell lines, K562 and HEL-60, and to define the ontogeny of pyruvate kinase (PK) isozymes (R, M2) in developing human erythroid cells, we have studied the isozymic alterations, if any, during differentiation of these cell lines in vitro and normoblasts isolated from fetal liver in vivo. PK activity of erythroleukemic cell lines was intermediate between that observed in leukocytes and in fetal liver erythroblasts. These cell lines contained a high level of M2-PK, but R- PK was always present, albeit at low concentrations, in all the clones or subclones we studied. Erythroblasts from fetal liver were separated according to density on a Stractan gradient. R-PK levels were nearly constant in the different fractions, whereas M2-PK levels markedly decreased as the erythroblasts became mature and almost completely disappeared in late erythroid cells. Thus, these results clearly demonstrate the erythroid origin of these cell lines.

Migratory path of definitive hematopoietic stem/progenitor cells during zebrafish development

Blood ◽  
2007 ◽  
Vol 109 (12) ◽  
pp. 5208-5214 ◽  
Author(s):  
Hao Jin ◽  
Jin Xu ◽  
Zilong Wen
Keyword(s):  
Progenitor Cells ◽  
Fetal Liver ◽  
Embryo Cell ◽  
Migration Route ◽  
Hematopoietic Stem ◽  
Hematopoietic Organ ◽  
Blood Island ◽  
Adult Kidney ◽  
Definitive Hematopoiesis

Abstract The development of vertebrate definitive hematopoiesis is featured by temporally and spatially dynamic distribution of hematopoietic stem/progenitor cells (HSPCs). It is proposed that the migration of definitive HSPCs, at least in part, accounts for this unique characteristic; however, compelling in vivo lineage evidence is still lacking. Here we present an in vivo analysis to delineate the migration route of definitive HSPCs in the early zebrafish embryo. Cell-marking analysis was able to first map definitive HSPCs to the ventral wall of dorsal aorta (DA). These cells were subsequently found to migrate to a previously unappreciated organ, posterior blood island (PBI), located between the caudal artery and caudal vein, and finally populate the kidney, the adult hematopoietic organ. These findings demonstrate that the PBI acts as an intermediate hematopoietic organ in a manner analogous to the mammalian fetal liver to sustain definitive hematopoiesis before adult kidney hematopoiesis occurs. Thus our study unambiguously documents the in vivo trafficking of definitive HSPCs among developmentally successive hematopoietic compartments and underscores the ontogenic conservation of definitive hematopoiesis between zebrafish and mammals.

GATA-1 Regulates Growth and Differentiation of Definitive Erythroid Lineage Cells During In Vitro ES Cell Differentiation

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4108-4118 ◽  
Author(s):  
Naruyoshi Suwabe ◽  
Satoru Takahashi ◽  
Toru Nakano ◽  
Masayuki Yamamoto
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Es Cell ◽  
Globin Mrna ◽  
Differentiated Cells ◽  
Vitro Analysis ◽  
Definitive Hematopoiesis

Abstract Although the importance of GATA-1 in both primitive and definitive hematopoietic lineages has been shown in vivo, the precise roles played by GATA-1 during definitive hematopoiesis have not yet been clarified. In vitro differentiation of embryonic stem (ES) cells using OP9 stroma cells can generate primitive and definitive hematopoietic cells separately, and we have introduced a method that separates hematopoietic progenitors and differentiated cells produced in this system. Closer examination showed that the expression of erythroid transcription factors in this system is regulated in a differentiation stage-specific manner. Therefore, we examined differentiation of GATA-1 promoter-disrupted (GATA-1.05) ES cells using this system. Because the GATA-1.05 mice die by 12.5 embryonic days due to the lack of primitive hematopoiesis, the in vitro analysis is an important approach to elucidate the roles of GATA-1 in definitive hematopoiesis. Consistent with the in vivo observation, differentiation of GATA-1.05 mutant ES cells along both primitive and definitive lineages was arrested in this ES cell culture system. Although the maturation-arrested primitive lineage cells did not express detectable amounts of ɛy-globin mRNA, the blastlike cells accumulated in the definitive stage showed β-globin mRNA expression at approximately 70% of the wild type. Importantly, the TER119 antigen was expressed and porphyrin was accumulated in the definitive cells, although the levels of both were reduced to approximately 10%, indicating that maturation of definitive erythroid cells is arrested by the lack of GATA-1 with different timing from that of the primitive erythroid cells. We also found that the hematopoietic progenitor fraction of GATA-1.05 cells contains more colony-forming activity, termed CFU-OP9. These results suggest that theGATA-1.05 mutation resulted in proliferation of proerythroblasts in the definitive lineage.

Control of Hemoglobin Switching by BCL11A.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 5-5
Author(s):  
Jian Xu ◽  
Vijay G. Sankaran ◽  
Yuko Fujiwara ◽  
Stuart H. Orkin
Keyword(s):  
Gene Silencing ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Association Studies ◽  
Globin Gene ◽  
Clinical Severity ◽  
Genome Wide Association Studies ◽  
Erythroid Cells ◽  
B Locus

Abstract Abstract 5 All vertebrates switch expression of globin chains during development. In humans b-like globins switch from embryonic to fetal to adult, whereas in the mouse a single switch from embryonic to adult occurs. The switch from human fetal (g) to adult (b) expression is especially critical in the b-hemoglobin disorders, such as sickle cell anemia and the b-thalassemias. Delay of the switch or reactivation of the fetal gene in the adult stage greatly ameliorates clinical severity. Despite intensive molecular studies of the human b-globin cluster over more than two decades, the proteins regulating the switch, and the mechanisms controlling the process, have been largely elusive. Recently, genome-wide association studies identified genetic variation at a chromosome 2 locus that correlates with the level of HbF in different populations. The most highly associated single nucleotide polymorphisms (SNPs) reside in an intron of the BCL11A gene, which encodes a zinc-finger repressor protein. Previously we showed that shRNA-mediated ex vivo knockdown of BCL11A in cultured human CD34-derived erythroid precursors leads to robust HbF expression, consistent with a role for BCL11A in maintaining g-genes in a silenced state in adult cells. To address in vivo roles of BCL11A either in development or in globin gene silencing in an intact individual, we have employed stringent genetic tests of function in mice that carry a complete human b-globin gene cluster as a yeast artificial chromosome transgene (b-locus mice). Knockout of BCL11A in mice leads to failure to silence the endogenous b-like embryonic genes in adult erythroid cells of the fetal liver (>2500-fold derepression). The ratio of human g to b globin RNA in the fetal liver of BCL11A knockout mice is inverted compared to controls, such that g constitutes >90% of the b-like human expression at embryonic day (E)14.5 and >75% at E18.5. These quantitatively striking findings indicate that BCL11A controls developmental silencing of g-globin gene expression. To address by formal genetics the contribution of BCL11A to g silencing in adult animals we have employed conditional inactivation of BCL11A through hematopoietic- and erythroid-specific Cre-alleles. These experiments reveal that BCL11A is also required in vivo for g-gene silencing in adults. We observed that human g-globin expression is persistently derepressed >2000-fold (as compared to littermate controls) in bone marrow erythroblasts of 15-20 week old b-locus mice upon erythroid-specific deletion of BCL11A. Taken together, these findings establish BCL11A as the first genetically validated transcriptional regulator of both developmental control of globin switching and silencing of g-globin expression in adults. The recognition of these roles for BCL11A now permits focused mechanistic studies of the switch. In human erythroid cells, BCL11A physically interacts with at least two corepressor complexes, Mi-2/NuRD and LSD1/CoREST, as well as the erythroid transcription factor GATA-1 and the HMG-box protein SOX6. Rather than binding to the promoters of the g- or b-globin genes as do these latter factors, BCL11A protein occupies the upstream locus control and g-d-intergenic regions of the b-globin cluster (as determined by high resolution ChIP-Chip analysis), suggesting that BCL11A mediates long-range interactions and/or reconfigures the locus during different stages. An in-depth mechanistic understanding of globin switching offers the prospect for design of target-based activation of HbF in adult erythroid cells of patients with hemoglobin disorders. Disclosures: No relevant conflicts of interest to declare.

Genetic Determinants of the Definitive Hematopoietic Stem/Progenitor Cell Compartment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1226-1226
Author(s):  
Kirby D Johnson ◽  
Xin Gao ◽  
Rajendran Sanalkumar ◽  
Amy P Hsu ◽  
Myung-Jeom Ryu ◽  
...  
Keyword(s):  
Progenitor Cell ◽  
Fetal Liver ◽  
Chromatin Accessibility ◽  
Complete Loss ◽  
Cell Compartment ◽  
Hematopoietic Stem ◽  
Composite Element ◽  
Fold Reduction ◽  
E Box ◽  
Definitive Hematopoiesis

Abstract Abstract 1226 How transcriptional and post-transcriptional mechanisms control the levels/activities of master developmental regulators has fundamental importance for understanding complex developmental processes such as hematopoiesis and associated pathological disorders. GATA-2 is an essential regulator of hematopoiesis, and GATA-2 mutations characterize heritable disease associated with myelodysplastic syndrome and acute myeloid leukemia, including MonoMAC (syndrome of monocytopendia, B and NK cell lymphopenia, and mycobacterial, fungal and viral infection). However, many questions remain unanswered regarding mechanisms underlying GATA-2 regulation and function. We demonstrated that a MonoMAC patient harbors a 28 bp deletion within GATA2 intron 5 that eliminates a conserved E-box and 5 base pairs of an 8 base pair spacer between the E-box and a conserved GATA motif, which constitutes an E-box-GATA composite element. This composite element resides within the +9.5 kb “GATA switch site” that binds GATA-2 and GATA-1 in the transcriptionally active and repressed states, respectively, and confers hematopoietic and vascular endothelial enhancer activities in transgenic mouse embryos. Importantly, this patient lacked mutations in the GATA2 coding sequence characteristic of other MonoMAC patients, but exhibited prototypical MonoMAC. To elucidate the mechanism underlying the function of the +9.5 composite element, we generated a targeted deletion of the murine element, which yielded embryonic lethality at E13 to E14. Prior to death, +9.5−/− mice exhibit reduced liver size, hemorrhaging, and edema. Nucleated primitive red cells are abundant in the +9.5−/− embryos, in contrast to Gata2 knockout mice, which die at approximately E10.5 from anemia due to failure of primitive and definitive hematopoiesis. Furthermore, primitive erythroid (EryP) colony assays conducted with yolk sacs revealed that the mutation does not affect primitive erythroid precursor functionality. However, the +9.5 deletion strongly reduced Gata2 expression at sites of definitive hematopoiesis, including the fetal liver (8.1 fold, P < 0.004) and cultured explants of the hematopoietic stem cell-generating Aortic Gonadal Mesonephric (AGM) region (4.0 fold, P < 0.001). The homozygous mutant animals exhibited a nearly complete loss of hematopoietic stem cells as determined by flow cytometry (20-fold reduction of Lin-Mac1+CD41-CD48-CD150+Sca+Kit+ cells, P < 0.005) and competitive repopulation (complete loss, P < 0.02) assays, as well as progenitors as determined by colony assays (BFU-E, 60-fold reduction, P < 0.002; CFU-GM, 8.8-fold reduction, P < 0.0001; CFU-GEMM, 19-fold reduction, P < 0.001). To investigate the underlying mechanisms, we developed an allele-specific Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay with heterozygous fetal liver cells to test whether the deletion influences Gata2 chromatin accessibility at the +9.5 region. The deletion significantly reduced (8.4 fold reduction, P < 0.001) chromatin accessibility at this region within the mutant allele, while the wild type allele was unaffected. Thus, any potential remaining cis-elements are insufficient to confer chromatin accessibility, supporting a model in which the transcription factors that normally occupy this GATA switch site lose the capacity to access their respective cis-elements in the context of the mutant allele. Our human and murine studies have therefore revealed a cis-element indispensable for the regulation of Gata2 expression in multiple developmental contexts and necessary for the generation of the definitive hematopoietic stem/progenitor cell compartment. As additional elements are likely to confer Gata2 expression in distinct contexts, including primitive erythropoiesis, we have implemented a multi-faceted effort to identify such elements and to compare their mechanisms with that of the +9.5 site, which will provide fundamental insights into genetic mechanisms controlling normal and malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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