Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E)

Terszowski, Grzegorz; Waskow, Claudia; Conradt, Peter; Lenze, Dido; Koenigsmann, Jessica; Carstanjen, Dirk; Horak, Ivan D.; Rodewald, Hans Reimer

doi:10.1182/blood-2004-09-3459

Cited by 68 publications

(68 citation statements)

References 37 publications

(42 reference statements)

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“…The first specific progenitors to be formed are referred to as erythroid burst-forming units (BFU-Es) (17,18). More committed erythroid precursor cells give rise to erythroid colony-forming units (CFU-Es) and can be prospectively isolated (19). Later on in erythroid differentiation, erythroblasts extrude their nuclei and become reticulocytes, which further loose specific organelles to generate the fully mature circulating erythrocytes.…”

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confidence: 99%

The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220

Stumpf

Waskow

Krötschel

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The Mediator complex forms the bridge between transcriptional activators and RNA polymerase II. Mediator subunit Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors. Med1 deficiency causes lethality at embryonic day 11.5 because of defects in heart and placenta development. Here we show that Med1-deficient 10.5 days postcoitum embryos are anemic but have normal numbers of hematopoietic progenitor cells. Med1-deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E), but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. In transcription assays, Med1 deficiency leads to a defect in GATA-1-mediated transactivation. In chromatin immunoprecipitation experiments, we find Mediator components at GATA-1-occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development.

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mentioning

confidence: 99%

The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220

Stumpf

Waskow

Krötschel

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…No other types of colonies were detected. Considering that the cloning (or plating) efficiency of previously isolated mouse CFU-Es using different strategies ranges from 40 to 70% (18,26,27), these results suggest that the c-Kit 1 CD71 high Ter-119 2 cell population obtained is highly enriched in CFU-Es.…”

Section: Cultures Supplemented With Scf or Epo Alone Support A Similamentioning

confidence: 98%

Measurement of generation‐dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation

Akbarian¹,

Wang²,

Audet³

2012

Cytometry Pt A

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Herein, we describe an experimental and computational approach to perform quantitative carboxyfluorescein diacetate succinimidyl ester (CFSE) cell-division tracking in cultures of primary colony-forming unit-erythroid (CFU-E) cells, a hematopoietic progenitor cell type, which is an important target for the treatment of blood disorders and for the manufacture of red blood cells. CFSE labeling of CFU-Es isolated from mouse fetal livers was performed to examine the effects of stem cell factor (SCF) and erythropoietin (EPO) in culture. We used a dynamic model of proliferation based on the Smith-Martin representation of the cell cycle to extract proliferation rates and death rates from CFSE time-series. However, we found that to accurately represent the cell population dynamics in differentiation cultures of CFU-Es, it was necessary to develop a model with generation-specific rate parameters. The generation-specific rates of proliferation and death were extracted for six generations (G 0 -G 5 ) and they revealed that, although SCF alone or EPO alone supported similar total cell outputs in culture, stimulation with EPO resulted in significantly higher proliferation rates from G 2 to G 5 and higher death rates in G 2 , G 3 , and G 5 compared with SCF. In addition, proliferation rates tended to increase from G 1 to G 5 in cultures supplemented with EPO and EPO 1 SCF, while they remained lower and more constant across generations with SCF. The results are consistent with the notion that SCF promotes CFU-E self-renewal while EPO promotes CFU-E differentiation in culture. ' 2012 International Society for Advancement of Cytometry Key terms red blood cells; carboxyfluorescein diacetate succinimidyl ester; cell differentiation; cell division; erythropoiesis; cell division; CFSE CARBOXYFLUORESCEIN diacetate succinimidyl ester (CFSE) cell-division tracking is widely used to determine the number of divisions cells have undergone in cultures or in vivo. This flow cytometric assay is based on the redistribution and dilution of the CFSE fluorescent dye between mother and daughter cells during cell division, resulting in distinct fluorescence intensity for each cell generation (1-3). Originally developed for homogenous lymphocyte populations (1), Nordon et al. (4) extended this approach to track the divisions of primary hematopoietic progenitor cell populations that are composed of multiple cell subsets (5-7). Qualitative examination of flow cytometric histograms of CFSE fluorescence is useful but does not allow a complete interpretation of time-series experiments. However, quantitative CFSE celldivision tracking consists of using computational tools and a working model of the dynamics of cell division to extract parameters that characterize the rates of cell activation, proliferation, and death from CFSE labeling experiments (8-13).The ''transition probability'' model of the cell cycle developed by Smith and Martin (14) has been useful to develop mathematical models to extract proliferation rates from CFSE data (8-10,13,15,...

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“…Specifically, the earliest erythropoietin (EPO)-dependent progenitors (the colonyforming unit-erythroid, CFU-E) are CD71 high Ter119 À IL3Ra À , proerythroblasts are CD71 high ter119 med and terminally differentiated erythroblasts are Ter119 high . [19][20][21] Accordingly, CD71 high Ter119 À IL3Ra À cells purified from the bone marrow of wild-type (wt) mice and grown in methylcellulose supplemented with EPO gave rise to CFU-E, as indicated by their clonogenicity, morphology, EPO dependence and haemoglobinization (blue staining with benzidine) (Figure 4a). Compared with wt mice, the bone marrow ( Figure 4a) and spleen ( Figure 4b) of Spi-1 transgenic leukaemic mice contained a higher fraction of CD71 high Ter119 À cells (around 30 and 55 versus 10% and 1%, respectively) that were mainly IL3Ra À (around 80%).…”

Section: Spi-1 Decreases Bim Expression In Preleukaemic Cellsmentioning

confidence: 99%

Epigenetic silencing of Bim transcription by Spi-1/PU.1 promotes apoptosis resistance in leukaemia

et al. 2013

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Deregulation of transcriptional networks contributes to haematopoietic malignancies. The transcription factor Spi-1/PU.1 is a master regulator of haematopoiesis and its alteration leads to leukaemia. Spi-1 overexpression inhibits differentiation and promotes resistance to apoptosis in erythroleukaemia. Here, we show that Spi-1 inhibits mitochondrial apoptosis in vitro and in vivo through the transcriptional repression of Bim, a proapoptotic factor. BIM interacts with MCL-1 that behaves as a major player in the survival of the preleukaemic cells. The repression of BIM expression reduces the amount of BIM-MCL-1 complexes, thus increasing the fraction of potentially active antiapoptotic MCL-1. We then demonstrate that Spi-1 represses Bim transcription by binding to the Bim promoter and by promoting the trimethylation of histone 3 on lysine 27 (H3K27me3, a repressive histone mark) on the Bim promoter. The PRC2 repressive complex of Polycomb is directly responsible for the deposit of H3K27me3 mark at the Bim promoter. SUZ12 and the histone methyltransferase EZH2, two PRC2 subunits bind to the Bim promoter at the same location than H3K27me3, distinct of the Spi-1 DNA binding site. As Spi-1 interacts with SUZ12 and EZH2, these results indicate that Spi-1 modulates the activity of PRC2 without directly recruiting the complex to the site of its activity on the chromatin. Our results identify a new mechanism whereby Spi-1 represses transcription and provide mechanistic insights on the antiapoptotic function of a transcription factor mediated by the epigenetic control of gene expression. Cell Death and Differentiation (2013) 20, 1268-1278; doi:10.1038/cdd.2013.88; published online 12 July 2013Acute myeloid leukaemia (AML) develops through a multistep process driven by the progressive accumulation of mutations, leading to deregulation of cell proliferation and differentiation. Most frequently, abnormalities in cell differentiation are the result of loss-of-function mutations in lineage-specific transcription factors, whereas alterations in cell proliferation are associated with gain-of-function mutations in signalling pathways. 1 Mutations that target components of the spliceosomal machinery 2 and epigenetic regulators 3 have been identified as well, although their functional consequences in leukaemogenesis are not clear. To date, AML treatments are largely determined by the nature of the identified mutated proteins and a major challenge is to design molecules that can target each molecular alteration contributing to transformation. For that, understanding the molecular mechanisms controlled by an oncoprotein is one essential step. We have previously described a mouse model of erythroleukaemia (Spi-1 transgenic mice) that recapitulates the multistep development of human AML. 4 Spi-1 is a master transcription factor of haematopoiesis that is also involved in pre-mRNA splicing regulation. 5 During the preleukaemic stage of the disease, the differentiation blockage of the erythroid progenitors is the first oncogenic mark assoc...

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Prospective isolation and global gene expression analysis of the erythrocyte colony-forming unit (CFU-E)

Cited by 68 publications

References 37 publications

The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220

The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220

Measurement of generation‐dependent proliferation rates and death rates during mouse erythroid progenitor cell differentiation

Epigenetic silencing of Bim transcription by Spi-1/PU.1 promotes apoptosis resistance in leukaemia

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