Differentiation status dependent function of FOG‐1

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GATA-2 is a zinc finger transcription factor essential for differentiation of immature hematopoietic cells. We analyzed the function of GATA-2 by a combined method of tetracycline-dependent conditional gene expression and in vitro hematopoietic differentiation from mouse embryonic stem (ES) cells using OP9 stroma cells (OP9 system). In the presence of macrophage colony-stimulating factor (M-CSF), the OP9 system induced macrophage differentiation. GATA-2 expression in this system inhibited macrophage differentiation and redirected the fate of hematopoietic differentiation to other hematopoietic lineages. GATA-2 expression commencing at day 5 or day 6 induced megakaryocytic or erythroid differentiation, respectively. Expression levels of PU.1, a hematopoietic transcription factor that interferes with GATA-2, appeared to play a critical role in differentiation to megakaryocytic or erythroid lineages. Transcription of PU.1 was affected by histone acetylation induced by binding of GATA-2 to the PU.1 promoter region. This study demonstrates that the function of GATA-2 is modified in a context-dependent manner by expression of PU.1, which in turn is regulated by GATA-2.

Section: Introductionmentioning

confidence: 82%

Redirecting differentiation of hematopoietic progenitors by a transcription factor, GATA-2

Kitajima

Tanaka

Zheng

et al. 2006

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“…DNA content analysis was performed using propidium iodide (PI) as described previously. 41 Cell-cycle distribution was analyzed using ModFit LT software (Becton Dickinson).…”

Section: Flow Cytometry Analysis and Cell Sortingmentioning

confidence: 99%

“…The functions of transcription factors are presumably diversified based on the cell differentiation status. 41 An experimental system combining a null mutation with the conditional expression of the gene is the most suitable way to analyze these functions. In vivo rescue experiments have been reported for only a few genes, presumably because the experiments are difficult to perform.…”

Section: Merits and Limitations Of The Experimental System Using Es Cmentioning

confidence: 99%

Differential effects of GATA-1 on proliferation and differentiation of erythroid lineage cells

Zheng

Kitajima

Sakai

et al. 2006

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The zinc finger transcription factor GATA-1 is essential for both primitive (embryonic) and definitive (adult) erythropoiesis. To define the roles of GATA-1 in the production and differentiation of primitive and definitive erythrocytes, we established GATA-1-null embryonic stem cell lines in which GATA-1 was able to be conditionally expressed by using the tetracycline conditional gene expression system. The cells were subjected to hematopoietic differentiation by coculturing on OP9 stroma cells. We expressed GATA-1 in the course of primitive and definitive erythropoiesis and analyzed the ability of GATA-1 to rescue the defective erythropoiesis caused by the GATA-1 null mutation. Our results show that GATA-1 functions in the proliferation and maturation of erythrocytes in a distinctive manner. The early-stage expression of GATA-1 during both primitive and definitive erythropoiesis was sufficient to promote the proliferation of red blood cells. In contrast, the late-stage expression of GATA-1 was indispensable to the terminal differentiation of primitive and definitive erythrocytes. Thus, GATA-1 affects the proliferation and differentiation of erythrocytes by different mechanisms. IntroductionHematopoiesis is the sequential proliferation and differentiation process that produces more than 8 distinct, mature blood cells. The process is tightly controlled by various lineage-specific transcription factors. 1 Among them, GATA transcription factors are one of the most well-studied transcription factors. The GATA protein is in the zinc finger transcription factor family and is categorized by recognizing the consensus motif WGATAR in a conserved multifunctional domain consisting of 2 C4-type zinc fingers. [2][3][4][5][6] The GATA family includes GATA-1, GATA-2, 5,7-9 and GATA-3, 10 which are essential hematopoietic factors, and GATA-4, GATA-5, and GATA-6, which regulate heart, lung, and gut cell development. 11,12 GATA-1, the founding member of the GATA family, has been mapped to the X chromosome, and the GATA-1 protein has been detected in erythroid, megakaryocytic, eosinophilic, and mast cell lines within the hematopoietic system. [13][14][15][16][17][18][19] From a developmental point of view, erythropoiesis consists of 2 waves of red blood cell production, primitive and definitive erythropoiesis. 20 The differences between primitive and definitive erythropoiesis are attributable to not only the emerging time but also to the origin, morphology, and globin gene expression. Primitive erythropoiesis, the first wave of erythroid production, generates nucleated, primitive erythrocytes (EryPs) that express embryonic hemoglobin at the blood island in the embryonic yolk sac. A recent work reported that murine primitive erythroblasts also enucleated and continued to circulate through late gestation and even into the postnatal period, indicating that the primitive erythropoiesis in mammals shared many processes with its definitive counterpart. 21 Definitive erythropoiesis, the second wave of erythropoiesis, commences in the fe...

“…Conditional manipulation of gene expression enables us to analyze gene function at distinctive stages of hematopoietic differentiation from hESCs, which has not been shown previously, although it was reported by several groups in mouse embryonic stem cells. 32,33 By sequentially infecting H1 cells with rtTA and inducible miRs-126/126* expressing lentiviral vectors ( Figure 3A), we established several hESC lines conditionally expressing miRs-126/ 126* (I-miRs-126/126*). All experiments were performed on 2 independently derived lines, and they produced comparable results.…”

Section: Generation Of Inducible Mirs-126/126* Overexpressing Hescsmentioning

confidence: 99%

Regulated expression of microRNAs-126/126* inhibits erythropoiesis from human embryonic stem cells

Huang

Gschweng

Handel

et al. 2011

IntroductionThe hematopoietic system is hierarchically organized, with a rare population of hematopoietic stem cells giving rise to specific progenitor cells and ultimately to diverse mature blood cell types. Lineage-specific fate decisions are intricately controlled by sets of master transcription factors. 1 For example, erythroid versus myeloid lineage differentiation is based on transcriptional crossantagonism between GATA-1 and PU.1. 2 PU.1 and C/EBP␣ are required for the generation of both macrophages and neutrophils. 3 In addition to transcription factors that are pivotal for hematopoietic cell lineage specification, several developmentally conserved signaling pathways, including Notch, Wnt, Sonic hedgehog, and TGF-␤, have also been implicated in regulating lineage determination. 4 MicroRNAs (miRs) are a recently discovered class of small (20-22 nt) noncoding RNAs that control gene expression posttranscriptionally by regulating mRNA translation or stability. 5 The roles of miRs in regulating the hematopoietic system are still poorly understood, although a series of recent studies have provided some perspective on this important issue. 6 There are several miRs, including miR-142, miR-181a, and miR-223, that are prominently expressed in hematopoietic cells. 7,8 Ectopic expression of miR-181a in hematopoietic stem/progenitor cells promotes B-cell differentiation, whereas overexpression of miR-142 and miR-223 leads to an increase in the proportion of T-lineage cells. 7 Some studies have not only delineated a role for miRs in hematopoiesis but also uncovered their molecular mechanisms of action involving regulation of lineage determining transcription factors. For example, the combination of gain-of-function and loss-of-function strategies has defined the requirement of miR-150 in B-cell differentiation by targeting the transcription factor c-Myb. 9 During monocytopoiesis, down-regulation of miRs 17-5p-20a-106a relieves the suppression of AML1, thus facilitating monocytic differentiation and maturation. 10 In addition to the role of specific miRs, the general requirement of miRs in hematopoiesis has been demonstrated, as deletion of Dicer in the thymus results in aberrant T-cell development in the murine system. 11,12 There is expanding literature suggesting that miRs also play an important role in the pathology of hematologic malignancies as tumor suppressor genes or oncogenes. For example, both miR-15a and miR-16 are deleted or down-regulated in the majority of chronic lymphocytic leukemia cases, 13 whereas C57BL6 mice transplanted with murine hematopoietic progenitor cells overexpressing miR-155 developed a myeloproliferative disorder. 14 Given compelling evidence that miRs are essential in the hematopoietic system, we sought to elucidate the potential roles of miRs in early human hematopoietic development. Insights into hematopoietic development have largely relied on findings from gene knockout and transgenic mouse models as well as in other model organisms. Because of the inaccessibility of human embryos,...