GATA-1 and Erythropoietin Cooperate to Promote Erythroid Cell Survival by Regulating bcl-xL Expression

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 87-96 ◽  
Author(s):  
Todd Gregory ◽  
Channing Yu ◽  
Averil Ma ◽  
Stuart H. Orkin ◽  
Gerd A. Blobel ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Survival ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Downstream Effector ◽  
Apoptotic Protein ◽  
Related Proteins

The transcription factor GATA-1 is essential for normal erythropoiesis. By examining in vitro–differentiated embryonic stem cells, we showed previously that in the absence of GATA-1, committed erythroid precursors fail to complete maturation and instead undergo apoptosis. The mechanisms by which GATA-1 controls cell survival are unknown. Here we report that in erythroid cells, GATA-1 strongly induces the expression of the anti-apoptotic protein bcl-xL, but not the related proteins bcl-2 and mcl-1. Consistent with a role for bcl-xL in mediating GATA-1–induced erythroid cell survival, in vitro–differentiated bcl-xL−/− embryonic stem cells fail to generate viable mature definitive erythroid cells, a phenotype resembling that of GATA-1 gene disruption. In addition, we show that erythropoietin, which is also required for erythroid cell survival, cooperates with GATA-1 to stimulate bcl-xL gene expression and to maintain erythroid cell viability during terminal maturation. Together, our data show that bcl-xL is essential for normal erythroid development and suggest a regulatory hierarchy in which bcl-xL is a critical downstream effector of GATA-1 and erythropoietin-mediated signals.

Positive regulators of the lineage-specific transcription factor GATA-1 in differentiating erythroid cells

1994 ◽  
Vol 14 (5) ◽  
pp. 3108-3114
Author(s):  
M H Baron ◽  
S M Farrington
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Cultured Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Targeted Mutagenesis ◽  
Erythroid Cell ◽  
Erythroid Cells

The zinc finger transcription factor GATA-1 is a major regulator of gene expression in erythroid, megakaryocyte, and mast cell lineages. GATA-1 binds to WGATAR consensus motifs in the regulatory regions of virtually all erythroid cell-specific genes. Analyses with cultured cells and cell-free systems have provided strong evidence that GATA-1 is involved in control of globin gene expression during erythroid differentiation. Targeted mutagenesis of the GATA-1 gene in embryonic stem cells has demonstrated its requirement in normal erythroid development. Efficient rescue of the defect requires an intact GATA element in the distal promoter, suggesting autoregulatory control of GATA-1 transcription. To examine whether GATA-1 expression involves additional regulatory factors or is maintained entirely by an autoregulatory loop, we have used a transient heterokaryon system to test the ability of erythroid factors to activate the GATA-1 gene in nonerythroid nuclei. We show here that proerythroblasts and mature erythroid cells contain a diffusible activity (TAG) capable of transcriptional activation of GATA-1 and that this activity decreases during the terminal differentiation of erythroid cells. Nuclei from GATA-1- mutant embryonic stem cells can still be reprogrammed to express their globin genes in erythroid heterokaryons, indicating that de novo induction of GATA-1 is not required for globin gene activation following cell fusion.

Primitive and Definitive Erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-37-SCI-37
Author(s):  
James Palis
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Erythroid Cells ◽  
Transient Wave ◽  
Self Renewal ◽  
Primitive Erythropoiesis ◽  
Definitive Erythropoiesis

Abstract Abstract SCI-37 Studies in mammalian and nonmammalian vertebrate embryos indicate that erythropoiesis comes in two flavors: primitive and definitive. The primitive erythroid lineage in mammalian embryos is characterized by a transient wave of lineage-committed progenitors that emerge from the yolk sac and generate a wave of precursors that synchronously mature in the bloodstream. Primitive erythroid precursors dynamically regulate embryonic globin gene expression and ultimately enucleate to form erythrocytes. Primitive erythropoiesis is superseded by definitive erythroid cells that mature extravascularly in association with macrophage cells. Studies in the mouse embryo indicate that definitive erythropoiesis has two distinct developmental origins. The first is a transient wave of erythro-myeloid progenitors (EMP) that emerge from the yolk sac and seed the early fetal liver. The second is a long-term program of erythropoiesis derived from hematopoietic stem cells. Erythropoietin is the central regulator of definitive erythropoiesis, in part by regulating the survival of committed progenitors. In contrast, the role of erythropoietin in primitive erythropoiesis remains poorly understood. Recent studies indicate that erythropoietin does not regulate the primitive erythroid progenitor compartment, but rather plays a critical role in establishing an antiapoptotic state during the terminal maturation of primitive erythroblasts. EMP-derived proerythroblasts are capable of extensive self-renewal in vitro, while primitive erythroid progenitors are incapable of self-renewal under the same conditions. These studies, taken together, indicate that the primitive and definitive forms of erythropoiesis have fundamental differences in the regulation of red cell output. The overlapping emergence of primitive and definitive erythroid lineages in differentiating embryonic stem cells suggests that the transient yolk-sac-derived primitive and EMP-derived definitive erythroid programs are recapitulated in vitro. These studies offer the hope that human embryonic stem cells can serve as a source of functional definitive erythroid cells for transfusion therapy. Disclosures: No relevant conflicts of interest to declare.

Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3568-3577
Author(s):  
Scott T. Magness ◽  
Antonio Tugores ◽  
David A. Brenner
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Translational Control ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Egfp Expression ◽  
Cell Stage ◽  
Cis Element

Ferrochelatase, the last enzyme in the heme pathway, chelates protoporphyrin IX and iron to form heme and is mutated in protoporphyria. The ferrochelatase gene is expressed in all tissues at low levels to provide heme for essential heme-containing proteins and is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF–E2 sites, all of which bind their cognatetrans-acting factors in vitro. To investigate the role of these elements during erythropoiesis, we introduced expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells. EGFP expression was monitored during hematopoietic differentiation in vitro using flow cytometry. We show that a promoter fragment containing the Sp1 sites, the NF–E2 and GATA elements, was sufficient to confer developmental-specific expression of the EGFP transgene, with an expression profile identical to that of the endogenous gene. In this system the −0.275 kb NF–E2 cis-element is required for erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between −0.375kb and −1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria. There was no effect of the G haplotype on transcriptional activity of the −1.1 kb transgene.

scholarly journals Positive regulators of the lineage-specific transcription factor GATA-1 in differentiating erythroid cells.

1994 ◽  
Vol 14 (5) ◽  
pp. 3108-3114 ◽  
Author(s):  
M H Baron ◽  
S M Farrington
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Cultured Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Targeted Mutagenesis ◽  
Erythroid Cell ◽  
Erythroid Cells

The zinc finger transcription factor GATA-1 is a major regulator of gene expression in erythroid, megakaryocyte, and mast cell lineages. GATA-1 binds to WGATAR consensus motifs in the regulatory regions of virtually all erythroid cell-specific genes. Analyses with cultured cells and cell-free systems have provided strong evidence that GATA-1 is involved in control of globin gene expression during erythroid differentiation. Targeted mutagenesis of the GATA-1 gene in embryonic stem cells has demonstrated its requirement in normal erythroid development. Efficient rescue of the defect requires an intact GATA element in the distal promoter, suggesting autoregulatory control of GATA-1 transcription. To examine whether GATA-1 expression involves additional regulatory factors or is maintained entirely by an autoregulatory loop, we have used a transient heterokaryon system to test the ability of erythroid factors to activate the GATA-1 gene in nonerythroid nuclei. We show here that proerythroblasts and mature erythroid cells contain a diffusible activity (TAG) capable of transcriptional activation of GATA-1 and that this activity decreases during the terminal differentiation of erythroid cells. Nuclei from GATA-1- mutant embryonic stem cells can still be reprogrammed to express their globin genes in erythroid heterokaryons, indicating that de novo induction of GATA-1 is not required for globin gene activation following cell fusion.

Analysis of ferrochelatase expression during hematopoietic development of embryonic stem cells

Blood ◽  
2000 ◽  
Vol 95 (11) ◽  
pp. 3568-3577 ◽  
Author(s):  
Scott T. Magness ◽  
Antonio Tugores ◽  
David A. Brenner
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Translational Control ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Egfp Expression ◽  
Cell Stage ◽  
Cis Element

Abstract Ferrochelatase, the last enzyme in the heme pathway, chelates protoporphyrin IX and iron to form heme and is mutated in protoporphyria. The ferrochelatase gene is expressed in all tissues at low levels to provide heme for essential heme-containing proteins and is up-regulated during erythropoiesis for the synthesis of hemoglobin. The human ferrochelatase promoter contains 2 Sp1 cis-elements and GATA and NF–E2 sites, all of which bind their cognatetrans-acting factors in vitro. To investigate the role of these elements during erythropoiesis, we introduced expression of the green fluorescent protein (EGFP) transgenes driven by various ferrochelatase promoter fragments into a single locus in mouse embryonic stem cells. EGFP expression was monitored during hematopoietic differentiation in vitro using flow cytometry. We show that a promoter fragment containing the Sp1 sites, the NF–E2 and GATA elements, was sufficient to confer developmental-specific expression of the EGFP transgene, with an expression profile identical to that of the endogenous gene. In this system the −0.275 kb NF–E2 cis-element is required for erythroid-enhanced expression, the GATA cis-element functions as a stage-specific repressor and enhancer, and elements located between −0.375kb and −1.1kb are necessary for optimal levels of expression. Ferrochelatase mRNA increased before the primitive erythroid-cell stage without a concomitant increase in ferrochelatase protein, suggesting the presence of a translational control mechanism. Because of the sensitivity of this system, we were able to assess the effect of an A-to-G polymorphism identified in the promoters of patients with protoporphyria. There was no effect of the G haplotype on transcriptional activity of the −1.1 kb transgene.

High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules

2010 ◽  
Vol 432 (1) ◽  
pp. 21-35 ◽  
Author(s):  
Paul D. Andrews ◽  
Melissa Becroft ◽  
Anders Aspegren ◽  
Jane Gilmour ◽  
Martyn J. James ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Survival ◽  
Small Molecules ◽  
Protein Kinases ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Basic Research ◽  
Rock Inhibitor

The propensity of human embryonic stem cells to die upon enzymatic disaggregation or low-density plating is an obstacle to their isolation and routine use in drug discovery and basic research. Equally, the very low rate of establishment of implanted cells hinders cell therapy. In the present study we have developed a high-content assay for human embryonic stem cell survival and used this to screen a range of libraries of ‘lead-like’ small molecules and known bioactives. From this we identified 18 confirmed hits with four structural classes being represented by multiple compounds: a series of 5-(acyl/alkyl-amino)indazoles, compounds with a 4-(acylamino)pyridine core, simple N6,N6-dialkyladenines and compounds with a 5-(acylamino)indolinone core. In vitro kinase profiling indicated that the ROCK (Rho-associated kinase)/PRK2 (protein kinase C-related kinase 2) protein kinases are of pivotal importance for cell survival and identified previously unreported compound classes that inhibited this important biological activity. An evaluation using an extensive panel of protein kinases showed that six of our hit compounds exhibited better selectivity for ROCK inhibition than the routinely used commercially available ROCK inhibitor Y-27632. In this screen we also identified the K+-ATP channel opener pinacidil and show that it probably promotes cell survival, by ‘off-target’ inhibition of ROCK/PRK2. We have therefore identified novel pro-survival compounds of greater specificity, equivalent potency and reduced toxicity relative to the routinely employed ROCK inhibitor Y-27632.

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