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.

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.

scholarly journals Quantitative Transcription Factor Analysis of Undifferentiated Single Human Embryonic Stem Cells

2009 ◽  
Vol 55 (12) ◽  
pp. 2162-2170 ◽  
Author(s):  
Anders Ståhlberg ◽  
Martin Bengtsson ◽  
Martin Hemberg ◽  
Henrik Semb
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Cell Line ◽  
Human Embryonic Stem Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Measurement Noise ◽  
Sox2 Expression

Abstract Background: Human embryonic stem cells (hESCs) require expression of transcription factor genes POU5F1 (POU class 5 homeobox 1), NANOG (Nanog homeobox), and SOX2 [SRY (sex determining region Y)-box 2] to maintain their capacity for self-renewal and pluripotency. Because of the heterogeneous nature of cell populations, it is desirable to study the gene regulation in single cells. Large and potentially important fluctuations in a few cells cannot be detected at the population scale with microarrays or sequencing technologies. We used single-cell gene expression profiling to study cell heterogeneity in hESCs. Methods: We collected 47 single hESCs from cell line SA121 manually by glass capillaries and 57 single hESCs from cell line HUES3 by flow cytometry. Single hESCs were lysed and reverse-transcribed. Reverse-transcription quantitative real-time PCR was then used to measure the expression POU5F1, NANOG, SOX2, and the inhibitor of DNA binding genes ID1, ID2, and ID3. A quantitative noise model was used to remove measurement noise when pairwise correlations were estimated. Results: The numbers of transcripts per cell varied >100-fold between cells and showed lognormal features. POU5F1 expression positively correlated with ID1 and ID3 expression (P < 0.05) but not with NANOG or SOX2 expression. When we accounted for measurement noise, SOX2 expression was also correlated with ID1, ID2, and NANOG expression (P < 0.05). Conclusions: We demonstrate an accurate method for transcription profiling of individual hESCs. Cell-to-cell variability is large and is at least partly nonrandom because we observed correlations between core transcription factors. High fluctuations in gene expression may explain why individual cells in a seemingly undifferentiated cell population have different susceptibilities for inductive cues.

Large-Scale Liquid Culture Production of Erythroid Cells from Human Embryonic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3631-3631
Author(s):  
Emmanuel N. Olivier ◽  
Caihong Qiu ◽  
Eric E. Bouhassira
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Red Blood Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Liquid Culture ◽  
Embryonic Stem ◽  
Erythroid Cells ◽  
Early Human

Abstract Early human erythropoiesis is difficult to study because the material is hard to access experimentally. Hence, relatively little is known about the gene expression profiles or the mechanism of globin gene expression in these early cells. We report here a system to produce large quantities in liquid culture of virtually pure erythroid cells starting from H1 human embryonic stem cells (hESCs). The system is adapted from methods to produce enucleated red blood cells from cord blood and consist of five steps. During the first step, hESCs are differentiated by co-culture on immortalized human fetal hepatocytes (FH-B-hTERT) for two weeks to produce hematopoietic cells. CD34 positive cells are then magnetically sorted and placed in step 2 for seven days in serum free medium in the presence of SCF, Epo, hydro-cortisone, flt-3 ligand, BMP-4 and IL3. In step 3, the cells are incubated for seven days in the same medium and cytokine cocktail but with IGF-1 and without flt-3-ligand. In step 4, the cells are incubated with Epo for 3 days, and in step 5 the cells are incubated without cytokine on a feeder layer of MS-5 cells. In a typical experiment, 2 millions hESCs (two 10cm2 wells) yield 50,000 sorted CD34 positive cells. Culture of these cells for about three weeks yields about 5 millions erythroid cells. This corresponds to a 5 to 10,000-fold amplification of the sorted hematopoietic cells since we estimate that only a few percent of the cells recovered with the CD34 magnetic beads are hematopoietic. Flow cytometry analysis revealed that at the beginning of the second step the CD34+ cells are CD45−, CD71low and CD235a−. After 7 days in liquid culture CD34 expression is less than 10%, CD45 and CD71 expressions are more than 95% and CD235a is less than 20%. Eight days later the cells are 95% CD34− CD45− CD71high and CD235a+. Finally at the end of the culture the cells become CD34−, CD45−, CD71− and CD235a+. Morphological analysis by Wright-Giemsa staining revealed that the differentiation process in the liquid culture is relatively synchronous and that at the end of the culture the majority of the cells are orthochromatic erythroblasts. In contrast to cord blood derived cells placed in similar differentiation conditions, very few enucleated red blood cells could be obtained from hESCs. Hemoglobin can first be detected spectrophotometrically after day 10 of liquid culture and reach a concentration of 20 pmol/106 cells at the end of the culture. Globin chain analysis by PCR and HPLC reveals that ξ, α, ε, and γ globin chains are synthesized by these cells but not β-globin could be detected. A detailed analysis of globin expression in early human erythroid cells will be presented in an accompanying abstract. This experimental system will be useful to study early erythropoiesis, to test gene therapy vectors, and to create genetically modified red blood cells.

scholarly journals Sequential Analysis of α- and β-Globin Gene Expression During Erythropoietic Differentiation from Primate Embryonic Stem Cells

Stem Cells ◽  
2006 ◽  
Vol 24 (12) ◽  
pp. 2627-2636 ◽  
Author(s):  
Katsutsugu Umeda ◽  
Toshio Heike ◽  
Mami Nakata-Hizume ◽  
Akira Niwa ◽  
Masato Arai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Sequential Analysis ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Globin Gene Expression

scholarly journals The pluripotency transcription factor Nanog represses glutathione reductase gene expression in mouse embryonic stem cells

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Claudia Solari ◽  
María Victoria Petrone ◽  
Ayelén Toro ◽  
Camila Vazquez Echegaray ◽  
María Soledad Cosentino ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Glutathione Reductase ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Reductase Gene

Genomic profiling of developing cardiomyocytes from recombinant murine embryonic stem cells reveals regulation of transcription factor clusters

2009 ◽  
Vol 38 (1) ◽  
pp. 7-15 ◽  
Author(s):  
Michael J. Seewald ◽  
Peter Ellinghaus ◽  
Astrid Kassner ◽  
Ines Stork ◽  
Martina Barg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Expression Pattern ◽  
Embryonic Stem ◽  
Gene Expression Pattern ◽  
Left Ventricular ◽  
Special Focus ◽  
Regulation Of Transcription

Cardiomyocytes derived from pluripotent embryonic stem cells (ESC) have the advantage of providing a source for standardized cell cultures. However, little is known on the regulation of the genome during differentiation of ESC to cardiomyocytes. Here, we characterize the transcriptome of the mouse ESC line CM7/1 during differentiation into beating cardiomyocytes and compare the gene expression profiles with those from primary adult murine cardiomyocytes and left ventricular myocardium. We observe that the cardiac gene expression pattern of fully differentiated CM7/1-ESC is highly similar to adult primary cardiomyocytes and murine myocardium, respectively. This finding is underlined by demonstrating pharmacological effects of catecholamines and endothelin-1 on ESC-derived cardiomyocytes. Furthermore, we monitor the temporal changes in gene expression pattern during ESC differentiation with a special focus on transcription factors involved in cardiomyocyte differentiation. Thus, CM7/1-ESC-derived cardiomyocytes are a promising new tool for functional studies of cardiomyocytes in vitro and for the analysis of the transcription factor network regulating pluripotency and differentiation to cardiomyocytes.

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.

scholarly journals Regulatory networks specifying cortical interneurons from human embryonic stem cells reveal roles for CHD2 in interneuron development

2017 ◽  
Vol 114 (52) ◽  
pp. E11180-E11189 ◽  
Author(s):  
Kesavan Meganathan ◽  
Emily M. A. Lewis ◽  
Paul Gontarz ◽  
Shaopeng Liu ◽  
Edouard G. Stanley ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Chromatin Remodeling ◽  
Human Embryonic Stem Cells ◽  
Regulatory Networks ◽  
Embryonic Stem ◽  
Molecular Networks ◽  
Cortical Interneurons

Cortical interneurons (cINs) modulate excitatory neuronal activity by providing local inhibition. During fetal development, several cIN subtypes derive from the medial ganglionic eminence (MGE), a transient ventral telencephalic structure. While altered cIN development contributes to neurodevelopmental disorders, the inaccessibility of human fetal brain tissue during development has hampered efforts to define molecular networks controlling this process. Here, we modified protocols for directed differentiation of human embryonic stem cells, obtaining efficient, accelerated production of MGE-like progenitors and MGE-derived cIN subtypes with the expected electrophysiological properties. We defined transcriptome changes accompanying this process and integrated these data with direct transcriptional targets of NKX2-1, a transcription factor controlling MGE specification. This analysis defined NKX2-1–associated genes with enriched expression during MGE specification and cIN differentiation, including known and previously unreported transcription factor targets with likely roles in MGE specification, and other target classes regulating cIN migration and function. NKX2-1–associated peaks were enriched for consensus binding motifs for NKX2-1, LHX, and SOX transcription factors, suggesting roles in coregulating MGE gene expression. Among the NKX2-1 direct target genes with cIN-enriched expression was CHD2, which encodes a chromatin remodeling protein mutated to cause human epilepsies. Accordingly, CHD2 deficiency impaired cIN specification and altered later electrophysiological function, while CHD2 coassociated with NKX2-1 at cis-regulatory elements and was required for their transactivation by NKX2-1 in MGE-like progenitors. This analysis identified several aspects of gene-regulatory networks underlying human MGE specification and suggested mechanisms by which NKX2-1 acts with chromatin remodeling activities to regulate gene expression programs underlying cIN development.

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