scholarly journals OncogenicGata1causes stage-specific megakaryocyte differentiation delay

2019 ◽  
Author(s):  
Gaëtan Juban ◽  
Nathalie Sakakini ◽  
Hedia Chagraoui ◽  
Qian Cheng ◽  
Kelly Soady ◽  
...  
Keyword(s):  
Gene Expression ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Detailed Examination ◽  
S Phase ◽  
Myeloproliferative Disorder ◽  
Specific Gene ◽  
Specific Stage

AbstractThe megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively in hemopoiesis. GATA1s causes an arrest late in erythroid differentiationin vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41himegakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.Scientific CategoryHaematopoiesis and Stem CellsKey PointsGATA1s-induced stage-specific differentiation delay increases immature megakaryocytesin vivoandin vitro, during development.Differentiation delay is associated with increased numbers of cells in S-phase and reduced apoptosis.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

scholarly journals A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

2008 ◽  
Vol 22 (12) ◽  
pp. 2677-2688 ◽  
Author(s):  
Paul G. Tiffen ◽  
Nader Omidvar ◽  
Nuria Marquez-Almuina ◽  
Dawn Croston ◽  
Christine J. Watson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Oncostatin M ◽  
Specific Gene ◽  
Mammary Involution

Abstract Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes β-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed β-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

scholarly journals Epigenetic regulation of neural cell differentiation plasticity in the adult mammalian brain

2008 ◽  
Vol 105 (46) ◽  
pp. 18012-18017 ◽  
Author(s):  
Jun Kohyama ◽  
Takuro Kojima ◽  
Eriko Takatsuka ◽  
Toru Yamashita ◽  
Jun Namiki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Epigenetic Modification ◽  
Mammalian Brain ◽  
Cytokine Signaling ◽  
Specific Gene ◽  
Neural Cells ◽  
Specific Gene Expression

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in fate specification of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which are expressed predominantly in neurons in the central nervous system, inhibit astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vivo (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

Analyses of Heme-Regulated Genes during Erythoid Differentiation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2660-2660
Author(s):  
Tohru Fujiwara ◽  
Yoko Okitsu ◽  
Tsuyoshi Ikura ◽  
Shinichiro Takahashi ◽  
Kazumichi Furuyama ◽  
...  
Keyword(s):  
Es Cells ◽  
Erythroid Differentiation ◽  
Surface Expression ◽  
Nuclear Extract ◽  
Luciferase Assay ◽  
Wild Type ◽  
Acetyl Coa ◽  
Cis Element

Abstract (Introduction) During erythroid differentiation, the level of erythroid-specific genes increases synchronizing with the intracellular heme content. In addition, heme has been shown to play a role in transcription and protein synthesis. Based on these evidences, it is possible that heme widely regulates the expression of erythroid specific genes. With this hypothesis, we compared the gene expression profile between wild-type and heme-deficient erythroblasts generated from wild-type and ALAS2 (−) ES cells in in vitro, and identified 4 heme-regulated erythroid-specific genes (UCP2, CNBP, NuSAP and unknown EST1), (BBRC2006;340:105–110). Among them, unknown EST1 is consisted of 110 a.a. with a conserved acetyl-CoA binding domain, which was characteristic of GNAT (GCN5-related N-acetyltransferase) superfamily. Thus, it is likely that EST1 is a novel acetyltransferase. In the present study, we focused two genes, EST1 and NuSAP, and investigated their function during erythoid differentiation. (Methods) First, the expression and regulation of NuSAP gene during erythroid differentiation was examined. For the expression analysis, in vivo erythroblasts were fractionated according to the surface expression of TER119/CD71, and the level of expression of NuSAP mRNA was examined by quantitative RT-PCR. For the promoter analysis, the promoter region of mouse NuSAP gene was cloned, and the regulatory cis-element was determined by luciferase assay and EMSA. Next, for defining the properties of EST1, EST1 was constitutively expressed using Flag/HA tagged retroviral vector into mouse erythroleukemia (MEL) cell line, and nuclear extract of EST1-expessed MEL cells was purified by affinity chromatography, which was loaded on an SDS/PAGE gel and subjected to electrophoresis. In addition, for in vitro histone acetyltransferase (HAT) assay, free histone and purified EST1 protein were incubated with [3H]acetyl-CoA, and acetyltransferase activity was measured by scintigraphy. (Results) (1) NuSAP mRNA was more significantly abundant in the subset corresponding to immature erythroblasts (TER119+CD71high) than mature erythroblasts (TER119+CD71low), and it was significantly increased in TER119+ cells from in vivo phlebotomized mice compared with control mice. Furthermore, during erythroid maturation of MEL cells by dimethylsulfoxide, NuSAP mRNA was increased at 24–72 hrs. Promoter analyses of NuSAP gene demonstrated that duplicated CCAAT boxes located at −81/−85 and −30/−34 were essential for promoter activity, which was trans-activated by NF-YA. These results suggested that NuSAP might contribute to the expansion of immature erythroblast pool under the control of NF-Y. (2) By the gel electrophoresis, it was revealed that EST1 protein forms a multimeric protein complex. Furthermore, whereas recombinant EST1 protein did not show HAT activity, EST1 complex could acetylate free histones in vitro, suggesting that EST1 might be a component of HAT complex. (Conclusion) The novel functions of EST1 and NuSAP suggest that heme regulates erythroid differentiation by controlling the expression of variety of genes.

Murine gastrulation requires HNF-4 regulated gene expression in the visceral endoderm: tetraploid rescue of Hnf-4(−/−) embryos

Development ◽  
1997 ◽  
Vol 124 (2) ◽  
pp. 279-287 ◽  
Author(s):  
S.A. Duncan ◽  
A. Nagy ◽  
W. Chan
Keyword(s):  
Gene Expression ◽  
Genetic Manipulation ◽  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Retinol Binding Protein ◽  
Specific Gene ◽  
Visceral Endoderm ◽  
Regulated Gene Expression

Immediately prior to gastrulation the murine embryo consists of an outer layer of visceral endoderm (VE) and an inner layer of ectoderm. Differentiation and migration of the ectoderm then occurs to produce the three germ layers (ectoderm, embryonic endoderm and mesoderm) from which the fetus is derived. An indication that the VE might have a critical role in this process emerged from studies of Hnf-4(−/−) mouse embryos which fail to undergo normal gastrulation. Since expression of the transcription factor HNF-4 is restricted to the VE during this phase of development, we proposed that HNF-4-regulated gene expression in the VE creates an environment capable of supporting gastrulation. To address this directly we have exploited the versatility of embryonic stem (ES) cells which are amenable to genetic manipulation and can be induced to form VE in vitro. Moreover, embryos derived solely from ES cells can be generated by aggregation with tetraploid morulae. Using Hnf-4(−/−) ES cells we demonstrate that HNF-4 is a key regulator of tissue-specific gene expression in the VE, required for normal expression of secreted factors including alphafetoprotein, apolipoproteins, transthyretin, retinol binding protein, and transferrin. Furthermore, specific complementation of Hnf-4(−/−) embryos with tetraploid-derived Hnf-4(+/+) VE rescues their early developmental arrest, showing conclusively that a functional VE is mandatory for gastrulation.

GATA-1 Forms Distinct Activating and Repressive Complexes in Erythroid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 356-356
Author(s):  
John Strouboulis ◽  
Patrick Rodriguez ◽  
Edgar Bonte ◽  
Jeroen Krijgsveld ◽  
Katarzyna Kolodziej ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Chromatin Remodeling ◽  
Gene Activation ◽  
Erythroid Differentiation ◽  
Specific Gene ◽  
Erythroid Cells ◽  
In Erythroid Cells

Abstract GATA-1 is a key transcription factor essential for the differentiation of the erythroid, megakaryocytic and eosinophilic lineages. GATA-1 functions in erythropoiesis involve lineage-specific gene activation and repression of early hematopoietic transcription programs. GATA-1 is known to interact with other transcription factors, such as FOG-1, TAL-1 and Sp1 and also with CBP/p300 and the SWI/SNF chromatin remodeling complex in vitro. Despite this information the molecular basis of its essential functions in erythropoiesis remains unclear. We show here that GATA-1 is mostly present in a high (> 670kDa) molecular weight complex that appears to be dynamic during erythroid differentiation. In order to characterize the GATA-1 complex(es) from erythroid cells, we employed an in vivo biotinylation tagging approach in mouse erythroleukemic (MEL) cells1. Briefly, this involved the fusion of a small (23aa) peptide tag to GATA-1 and its specific, efficient biotinylation by the bacterial BirA biotin ligase which is co-expressed with tagged GATA-1 in MEL cells. Nuclear extracts expressing biotinylated tagged GATA-1 were bound directly to streptavidin beads and co-purifying proteins were identified by mass spectrometry. In addition to the known GATA-1-interacting transcription factors FOG-1, TAL-1 and Ldb-1, we describe novel interactions with the essential hematopoietic transcription factor Gfi-1b and the chromatin remodeling complexes MeCP1 and ACF/WCRF. Significantly, GATA-1 interaction with the repressive MeCP1 complex requires FOG-1. We also show in erythroid cells that GATA-1, FOG-1 and MeCP1 are stably bound to repressed genes representing early hematopoietic (e.g. GATA-2) or alternative lineage-specific (e.g. eosinophilic) transcription programs, whereas the GATA-1/Gfi1b complex is bound to repressed genes involved in cell proliferation. In contrast, GATA-1 and TAL-1 are bound to the active erythroid-specific EKLF gene. Our findings on GATA-1 complexes provide novel insight as to the critical roles that GATA-1 plays in many aspects of erythropoiesis by revealing the GATA-1 partners in the execution of specific functions.

scholarly journals Regulation of Id3 cell cycle function by Cdk-2-dependent phosphorylation.

1997 ◽  
Vol 17 (12) ◽  
pp. 6815-6821 ◽  
Author(s):  
R W Deed ◽  
E Hara ◽  
G T Atherton ◽  
G Peters ◽  
J D Norton
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Complex Formation ◽  
S Phase ◽  
Regulatory Function ◽  
Specific Gene ◽  
Wild Type ◽  
E Box ◽  
Phase Entry

The functions of basic helix-loop-helix (bHLH) transcription factors in activating differentiation-linked gene expression and in inducing G1 cell cycle arrest are negatively regulated by members of the Id family of HLH proteins. These bHLH antagonists are induced during a mitogenic signalling response, and they function by sequestering their bHLH targets in inactive heterodimers that are unable to bind to specific gene regulatory (E box) sequences. Recently, cyclin E-Cdk2- and cyclin A-Cdk2-dependent phosphorylation of a single conserved serine residue (Ser5) in Id2 has been shown to occur during late G1-to-S phase transition of the cell cycle, and this neutralizes the function of Id2 in abrogating E-box-dependent bHLH homo- or heterodimer complex formation in vitro (E. Hara, M. Hall, and G. Peters, EMBO J. 16:332-342, 1997). We now show that an analogous cell-cycle-regulated phosphorylation of Id3 alters the specificity of Id3 for abrogating both E-box-dependent bHLH homo- or heterodimer complex formation in vitro and E-box-dependent reporter gene function in vivo. Furthermore, compared with wild-type Id3, an Id3 Asp5 mutant (mimicking phosphorylation) is unable to promote cell cycle S phase entry in transfected fibroblasts, whereas an Id3 Ala5 mutant (ablating phosphorylation) displays an activity significantly greater than that of wild-type Id3 protein. Cdk2-dependent phosphorylation therefore provides a switch during late G1-to-S phase that both nullifies an early G1 cell cycle regulatory function of Id3 and modulates its target bHLH specificity. These data also demonstrate that the ability of Id3 to promote cell cycle S phase entry is not simply a function of its ability to modulate bHLH heterodimer-dependent gene expression and establish a biologically important mechanism through which Cdk2 and Id-bHLH functions are integrated in the coordination of cell proliferation and differentiation.

scholarly journals Vimentin downregulation is an inherent feature of murine erythropoiesis and occurs independently of lineage

Development ◽  
1990 ◽  
Vol 110 (1) ◽  
pp. 85-96
Author(s):  
F. Sangiorgi ◽  
C.M. Woods ◽  
E. Lazarides
Keyword(s):  
Gene Expression ◽  
Intermediate Filament ◽  
Structural Changes ◽  
Erythroid Differentiation ◽  
Inherent Feature ◽  
Vimentin Gene ◽  
Cells Cultured ◽  
Vimentin Filaments

In mammalian erythropoiesis, the mature cells of the primitive lineage remain nucleated while those of the definitive lineage are anuclear. One of the molecular and structural changes that precedes enucleation in cells of the definitive lineage is the cessation in the expression of the gene for the intermediate filament (IF) protein vimentin and the removal of all vimentin filaments from the cytoplasm. We show here that in immature primitive cells vimentin is synthesized and forms a cytoplasmic network of IFs. As differentiation proceeds in vivo, vimentin gene expression is downregulated in these cells; this is accompanied by the loss of vimentin filaments from the cytoplasm. This loss temporally coincides with the nucleus becoming freely mobile within the cytoplasm, suggesting that, while IF removal is not directly linked to the physical process of enucleation, it may be a prerequisite for the initiation of nuclear mobility in both lineages. These changes are also observed in early primitive cells cultured in vitro, suggesting that they constitute an intrinsic part of the murine erythroid differentiation program independent of lineage and hematopoietic microenvironment.

Hemogenic and nonhemogenic endothelium can be distinguished by the activity of fetal liver kinase (Flk)–1promoter/enhancer during mouse embryogenesis

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 886-893 ◽  
Author(s):  
Hideyo Hirai ◽  
Minetaro Ogawa ◽  
Norio Suzuki ◽  
Masayuki Yamamoto ◽  
Georg Breier ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Specific Gene ◽  
Vascular Endothelial ◽  
Myb Genes ◽  
Definitive Hematopoiesis

Abstract Accumulating evidence in various species has suggested that the origin of definitive hematopoiesis is associated with a special subset of endothelial cells (ECs) that maintain the potential to give rise to hematopoietic cells (HPCs). In this study, we demonstrated that a combination of 5′-flanking region and 3′ portion of the first intron of the Flk-1 gene (Flk-1 p/e) that has been implicated in endothelium-specific gene expression distinguishes prospectively the EC that has lost hemogenic activity. We assessed the activity of this Flk-1 p/e by embryonic stem (ES) cell differentiation culture and transgenic mice by using theGFP gene conjugated to this unit. The expression ofGFP differed from that of the endogenous Flk-1gene in that it is active in undifferentiated ES cells and inactive in Flk-1+ lateral mesoderm. Flk-1 p/e becomes active after generation of vascular endothelial (VE)–cadherin+ ECs. Emergence of GFP− ECs preceded that of GFP+ ECs, and, finally, most ECs expressed GFP both in vitro and in vivo. Cell sorting experiments demonstrated that only GFP− ECs could give rise to HPCs and preferentially expressed Runx1 and c-Myb genes that are required for the definitive hematopoiesis. Integration of both GFP+ and GFP− ECs was observed in the dorsal aorta, but cell clusters appeared associated only to GFP−ECs. These results indicate that activation of Flk-1 p/e is associated with a process that excludes HPC potential from the EC differentiation pathway and will be useful for investigating molecular mechanisms underlying the divergence of endothelial and hematopoietic lineages.

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