Expansion and Differentiation of Immature Mouse and Human Hematopoietic Progenitors

Dolznig, Helmut; Kolbus, Andrea; Leberbauer, Cornelia; Schmidt, Uwe; Deiner, Eva-Maria; Müllner, Ernst W.; Beug, Hartmut

doi:10.1385/1-59259-826-9:323

Cited by 31 publications

(63 citation statements)

References 22 publications

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“…ES-EP were cultured as previously described (21). Briefly, cells were grown in StemPro34 medium (Invitrogen) supplemented with 2 units/mL Epogen (Amgen), 40 ng/mL hIGF-1 (Sigma), 1 μM dexamethasone (Sigma), 100 ng/mL murine SCF (R&D Systems/Invitrogen), and 0.1% β-mercaptoethanol (Gibco) at 37°C in a humidified 10% CO 2 atmosphere.…”

Section: Methodsmentioning

confidence: 99%

“…To begin to investigate the possible existence of a "core erythroid network," we carried out ChIP-Seq experiments on endogenous GATA-1 in normal, murine ES cell-derived erythroid progenitors (ES-EP) (21) in both proliferating and differentiating conditions. The following results indicate that our ChIP-Seq data are of high quality.…”

Section: Gata-1 Preferentially Bindsmentioning

confidence: 99%

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A core erythroid transcriptional network is repressed by a master regulator of myelo-lymphoid differentiation

Wontakal¹,

Smith²,

MacCarthy

et al. 2012

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

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Two mechanisms that play important roles in cell fate decisions are control of a “core transcriptional network” and repression of alternative transcriptional programs by antagonizing transcription factors. Whether these two mechanisms operate together is not known. Here we report that GATA-1, SCL, and Klf1 form an erythroid core transcriptional network by co-occupying >300 genes. Importantly, we find that PU.1, a negative regulator of terminal erythroid differentiation, is a highly integrated component of this network. GATA-1, SCL, and Klf1 act to promote, whereas PU.1 represses expression of many of the core network genes. PU.1 also represses the genes encoding GATA-1, SCL, Klf1, and important GATA-1 cofactors. Conversely, in addition to repressing PU.1 expression, GATA-1 also binds to and represses >100 PU.1 myelo-lymphoid gene targets in erythroid progenitors. Mathematical modeling further supports that this dual mechanism of repressing both the opposing upstream activator and its downstream targets provides a synergistic, robust mechanism for lineage specification. Taken together, these results amalgamate two key developmental principles, namely, regulation of a core transcriptional network and repression of an alternative transcriptional program, thereby enhancing our understanding of the mechanisms that establish cellular identity.

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Section: Methodsmentioning

confidence: 99%

Section: Gata-1 Preferentially Bindsmentioning

confidence: 99%

A core erythroid transcriptional network is repressed by a master regulator of myelo-lymphoid differentiation

Wontakal¹,

Smith²,

MacCarthy

et al. 2012

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

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“…Hemoglobin assay was performed as previously. 15,16 The quantification results were normalized by cell number and cell volume. Both protocols are described in detail in supplemental Methods.…”

Section: Heme and Hemoglobin Assaymentioning

confidence: 99%

Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme

Garcia-Santos

Schranzhofer

Horváthová

et al. 2014

Blood

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Key Points• Heme oxygenase-1 levels increase during erythroid differentiation.• Heme oxygenase-1 actively participates in maintaining appropriate hemoglobinization rates.Heme is essential for the function of all aerobic cells. However, it can be toxic when it occurs in a non-protein-bound form; cells maintain a fine balance between heme synthesis and catabolism. The only physiological mechanism of heme degradation is by heme oxygenases (HOs). The heme-inducible isoform, HO-1, has been extensively studied in numerous nonerythroid cells, but virtually nothing is known about the expression and potential significance of HO-1 in developing red blood cells. We have demonstrated that HO-1 is present in erythroid cells and that its expression is upregulated during erythroid differentiation. Overexpression of HO-1 in erythroid cells impairs hemoglobin synthesis, whereas HO-1 absence enhances hemoglobinization in cultured erythroid cells. Based on these results, we conclude that HO-1 controls the regulatory heme pool at appropriate levels for any given stage of erythroid differentiation. In summary, our study brings to light the importance of HO-1 expression for erythroid development and expands our knowledge about the fine regulation of hemoglobin synthesis in erythroid cells. Our results indicate that HO-1 plays an important role as a coregulator of the erythroid differentiation process. Moreover, HO-1 expression must be tightly regulated during red blood cell development. (Blood.

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“…Semiconfluent C2C12 myoblasts were differentiated by incubation in medium with 1% FCS for 10 days. Erythroid progenitors from fetal livers of embryonic day (E)14.5 mouse embryos were cultivated and differentiated as described previously (Dolznig et al, 2005). Transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.…”

Section: Subcellular Fractionation Gel Electrophoresis and Immunoblomentioning

confidence: 99%

“…Mouse fetal-liver-derived primary erythroid progenitors (EPs) have been shown to undergo highly synchronous in vivo-like erythroid differentiation upon replacement of self renewal factors with physiological differentiation factors, resulting in enucleated erythrocytes (Dolznig et al, 2005). As proliferating EPs expressed both LINT-25 and LAP2␣ proteins at high levels, we used these cells as an in vitro differentiation model.…”

mentioning

confidence: 99%

LAP2α-binding protein LINT-25 is a novel chromatin-associated protein involved in cell cycle exit

Naetar

Hutter

Dorner

et al. 2007

Journal of Cell Science

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Lamina-associated polypeptide 2α (LAP2α) is a nuclear protein dynamically associating with chromatin during the cell cycle. In addition, LAP2α interacts with A-type lamins and retinoblastoma protein and regulates cell cycle progression via the E2F-Rb pathway. Using yeast two-hybrid analysis and three independent in vitro binding assays we identified a new LAP2α interaction partner of hitherto unknown functions, which we termed LINT-25. LINT-25 protein levels were upregulated during G1 phase in proliferating cells and upon cell cycle exit in quiescence, senescence and differentiation. Upon cell cycle exit LINT-25 accumulated in heterochromatin foci, and LAP2α protein levels were downregulated by proteasomal degradation. Although LAP2α was not required for the upregulation and reorganization of LINT-25 during cell cycle exit, transient expression of LINT-25 in proliferating cells caused loss of LAP2α and subsequent cell death. Our data show a role of LINT-25 and LAP2α during cell cycle exit, in which LINT-25 acts upstream of LAP2α.

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Expansion and Differentiation of Immature Mouse and Human Hematopoietic Progenitors

Cited by 31 publications

References 22 publications

A core erythroid transcriptional network is repressed by a master regulator of myelo-lymphoid differentiation

A core erythroid transcriptional network is repressed by a master regulator of myelo-lymphoid differentiation

Heme oxygenase 1 is expressed in murine erythroid cells where it controls the level of regulatory heme

LAP2α-binding protein LINT-25 is a novel chromatin-associated protein involved in cell cycle exit

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