Analysis of gene expression in JAK2 exon 12-mutant erythroblasts
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Anna Godfrey
PROVIDER: E-MTAB-3946 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Early erythroid progenitors were isolated from mouse E14.5 fetal liver. After cell lysing, control IgG or RBP specific antibody were incubated with cell lysis. Immunoprecipitation followed by microarray experiments were carried out to identify transcripts that are immunoprecipitated by either control IgG or RBP specific antibody. Examination of transcripts that are immunoprecipitated by either control IgG or RBP specific antibody in primary mouse early erythroid progenitor
2013-06-14 | E-GEOD-46108 | biostudies-arrayexpress
Project description:Analyses of gene expression by RNA-Seq in mouse E14.5 fetal liver burst-forming unit erythroid (BFU-E) cells untreated or treated by dexamethasone (DEX) with or without PPAR? agonist GW7647. RNA-Seq was performed on enriched populations of mouse BFU-E isolated from E14.5 fetal liver, as well as BFU-E enriched cells treated with Dex ± GW7647.
2015-06-08 | E-GEOD-63836 | biostudies-arrayexpress
Project description:Early erythroid progenitors were isolated from mouse E14.5 fetal liver, infected with viruses encoding either control shRNA or shRNAs targeting the RNA binding protein (RBP), and cultured in a medium containing stem cell factor, erythropoietin, and insulin-like growth factor 1, in the absence or presence of dexamethasone (DEX). After 3 days of culture, microarray experiments were carried out to profile the gene expression pattern of these cells. Examination of mRNA expression profiles in day3 cultured cells
2013-06-14 | E-GEOD-46215 | biostudies-arrayexpress
Project description:Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. We combined mathematical modeling and extensive time-resolved data sets in primary erythroid progenitor cells and dissected the roles of the two transcriptional feedback regulators of the SOCS family, CIS and SOCS3 in JAK2/STAT5 signaling. Our model revealed that both feedbacks are most effective at different ligand concentration ranges. To identify the relevant transcriptional feedback regulators that are involved in attenuation of Epo-induced JAK2-STAT5 signaling in addition to CIS, we performed a time-resolved genome-wide expression profiling of murine erythroid progenitor cells at the colony forming unit erythroid (CFU-E) stage up to 24 and 7 hours after EPO stimulation and in control, repectively. The analysis identified SOCS3 as the only further de novo regulated gene upon Epo-induced JAK2-STAT5 signaling. From subsequent mathematical modeling, we calculated the STAT5 response in previously unobserved Epo concentrations, which provided a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. In conclusion, our combined modeling approach revealed novel insights into the orchestrated action of feedback control to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations. Freshly sorted CFU-E cells were starved for 1 h in Panserin 401 supplemented with BSA and 50 µM β-Mercaptoethanol. Subsequently, cells were stimulated with 0.5 U/ml Epo or left untreated and RNA was extracted at different time points (0,1,2,3,4,5,6,7,8,14,19,24 hrs after Epo stimulation and at 0,1,2,3,4,5,6,7 hrs without Epo treatment) using the RNeasy Mini Plus Kit (Qiagen, Hilden, Germany).
2011-06-30 | E-GEOD-26151 | biostudies-arrayexpress
Project description:Background: Cancers result from accumulation of somatic mutations and their properties are thought to reflect the sum of these mutations. However, little is known about the consequences of altering the order of mutation acquisition. Methods: Mutation order was determined in myeloproliferative neoplasm patients by genotyping hematopoietic colonies or next generation sequencing. Stem and progenitor cells were isolated to study the effect of mutation order on mature and immature hematopoietic cells. Results: Age of presentation, acquisition of JAK2V617F homozygosity and the balance of immature progenitors were all influenced by mutation order. Compared to TET2-first patients, JAK2-first patients had an increased likelihood of presenting with polycythemia vera than essential thrombocythemia, an increased risk of thrombosis and an increased sensitivity of JAK2-mutant progenitors to ruxolitinib in vitro. In studies of single hematopoietic stem and progenitor cells (HSPCs), mutation order influenced the proliferative response to JAK2V617F and the capacity of double-mutant HSPCs to generate colony-forming cells. Moreover the HSPC compartment was dominated by TET2 single-mutant cells in TET2-first patients but by JAK2/TET2 double-mutant cells in JAK2-first patients. Prior mutation of TET2 altered the transcriptional consequences of JAK2V617F in a cell-intrinsic manner, and prevented JAK2V617F from up-regulating genes associated with proliferation. These data demonstrate that mutation order influences progenitor proliferation and terminal cell expansion, thus influencing clinical presentation, thrombosis risk and progenitor response to targeted therapy. Conclusions: The order in which JAK2 and TET2 mutations are acquired influences clinical features, stem/progenitor cell biology and clonal evolution in patients with myeloproliferative neoplasms.
2014-11-17 | E-MTAB-3086 | biostudies-arrayexpress
Project description:This experiment was designed to identify genes differentially expressed in association with the JAK2V617F mutation in polycythemia vera (PV) and essential thrombocythemia (ET). Peripheral blood was obtained from 20 ET and 16 PV patients and erythroid progenitors were grown in semi-solid methylcellulose media supplemented with 0.01 U/ml erythropoietin. Individual clones were plucked and genotyped for the presence of the JAK2V617F mutation, and up to 20 normal and mutant colonies were pooled from each patient, and subjected to expression profiling. In total, 72 expression profiling datasets were generated, representing paired samples of normal and mutant cells from 36 patients.
2011-11-22 | E-MTAB-384 | biostudies-arrayexpress
Project description:Primitive erythropoiesis in the mouse yolk sac is followed by definitive erythropoiesis resulting in adult erythrocytes. In comparison to definitive erythropoiesis little is known about the genes that control the embryonic erythroid program. The purpose of this study was to generate a profile of mouse embryonic yolk sac erythroid cells and identify novel regulatory genes differentially expressed in erythroid compared to non-erythroid (epithelial cells). The identification of these genes will contribute to a greater understanding of how the primitive erythroid program is controlled. This work will have clinical implications for treating sickle cell anemia and β-thalassemia. Activating genes in adult erythroid cells that increase embryonic or fetal globin gene expression may be a therapeutic approach to treat individuals with these disorders. Experiment Overall Design: Embryonic day 9.5 (E9.5) yolk sacs were dissected from the embryos of timed-pregnant FVB/N mice. These tissues were frozen in OCT media and 8-micron frozen sections were obtained. Laser capture microdissection (LCM) was used to isolate primitive erythroid precursors and epithelial cells from these E9.5 yolk sac frozen sections using 2 to 4 yolk sacs from 2 different litters per biological replicate. Paired erythroid and epithelial samples were collected from the same microscope slides. Total RNA was isolated from 4 different pairs of erythroid and epithelial samples and hybridized to Affymetrix 430 A 2.0 microarrays.
2008-06-11 | E-GEOD-10002 | biostudies-arrayexpress
Project description:KLF2 is a Krüppel-like zinc-finger transcription factor required for blood vessel, lung, T-cell, and erythroid development. KLF2-/- mice die by embryonic day 14.5 (E14.5), due to hemorrhaging and heart failure. Embryonic -like globin gene expression is reduced in KLF2-/- embryos compared to wildtype (WT), and E10.5 erythroid cells exhibit abnormal morphology. Other KLF2 target genes were identified by comparing E9.5 KLF2-/- and WT yolk sac erythroid cells, using laser capture microdissection and microarray assays. One hundred and ninety-six genes exhibited significant differences in expression; eighty-nine of these are downregulated in KLF2-/- compared to WT. Genes involved in cell migration, differentiation and development are over-represented in the KLF2-regulated gene list. Previously identified erythroid-enriched regulatory genes such as reelin, adenylate cyclase 7, cytotoxic T lymphocyte-associated protein 2 alpha, and CD24a antigen are downregulated in KLF2-/- compared to WT. SOX2, a pluripotency factor in ES cells, is also a KLF2 target in embryonic erythroid cells. We investigated whether reelin, which has an established role in neuronal migration and proliferation, has a role in embryonic erythropoiesis. Luciferase reporter assays demonstrated that KLF2 directly transactivates the reelin promoter, but reelin mutant mice have no apparent abnormalities in embryonic erythroid morphology or globin gene expression. Timed-pregnant KLF2+/- females were anesthetized and sacrificed. E9.5 yolk sacs were dissected from the embryo, cryoprotected in 20% sucrose in PBS and frozen in OCT media. A small portion of the embryo tail was used for PCR genotyping. Eight micron KLF2-/- frozen yolk sac sections were obtained and laser capture microdissection (LCM) was used to isolate primitive erythroid precursors. For each biological replicate, 2 to 4 yolk sacs from 2 different litters were used. Total RNA was isolated from 4 different KLF2-/- erythroid samples and hybridized to Affymetrix 430 A 2.0 microarrays
2011-03-02 | E-GEOD-27602 | biostudies-arrayexpress
Project description:Erythropoiesis is a tightly regulated process. Development of red blood cells occurs through differentiation of hematopoietic stem cells into more committed progenitors and finally into erythrocytes. Binding of erythropoietin to its receptor (EpoR) is strictly required for erythropoiesis as it promotes survival and late maturation of erythroid progenitors. In vivo and in vitro studies have highlighted the requirement of EpoR signaling through Jak2 tyrosine-kinase and Stat5a/b as a central pathway. Here, we demonstrate that phospholipase C gamma 1 (Plcγ1) is activated downstream of EpoR/Jak2 independently of Stat5. Plcγ1-deficient proerythroblasts and erythroid progenitors exhibited strong impairment in differentiation and colony-forming potential. In vivo, suppression of Plcγ1 in immunophenotypically defined hematopoietic stem cells (Lin-Sca1+KIT+CD48-CD150+) severely reduced erythroid development. To identify Plcγ1 effector molecules involved in regulation of erythroid differentiation we assessed for changes on the level of transcription and DNA methylation after inactivation of Plcγ1. The single common downstream effector was H2AFY2, which encodes for the histone variant macroH2A2 (mH2A2). Suppression of macroH2A2 expression recapitulated the effects of Plcγ1 knockdown on erythroid maturation. Taken together, our findings identify Plcγ1 and its downstream target macroH2A2, as a ânon-canonicalâ signaling pathway essential for erythroid differentiation. MCIP-seq was used to interrogate methylation changes during Epo-induced differentiation of murine I/11 erythroblastic cell line upon knock-down of Plcy1 as compared to a mock control. Two different shRNA constructs that target Plcg1 were investigated at two different time points.
2015-12-31 | E-GEOD-60087 | biostudies-arrayexpress
Project description:Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Enhancers are typically marked by the co-transcriptional activator protein p300 or by groups of cell-expressed transcription factors. We hypothesized that a unique set of enhancers regulates gene expression in human erythroid cells, a highly specialized cell type evolved to provide adequate amounts of oxygen throughout the body. Using chromatin immunoprecipitation followed by massively parallel sequencing, genome-wide maps of candidate enhancers were constructed for p300 and four transcription factors, GATA1, NF-E2, KLF1, and SCL, using primary human erythroid cells. These data were combined with gene expression analyses and candidate enhancers identified. Consistent with their predicted function as candidate enhancers, there was statistically significant enrichment of p300 and combinations of co-localizing erythroid transcription factors within 1-50 kb of the TSS of genes highly expressed in erythroid cells. Candidate enhancers were also enriched near genes with known erythroid cell function or erythroid cell phenotypes. Candidate enhancers exhibited only moderate conservation with mouse and minimal conservation with nonplacental vertebrates. Candidate enhancers were mapped to a data set of erythroid-associated, biologically relevant, SNPs from the GWAS catalog of the NHGRI. Fourteen candidate enhancers, representing 10 genetic loci, mapped to sites associated with biologically relevant erythroid traits. Fragments from these loci directed statistically significant expression in reporter gene assays. Identification of enhancers in human erythroid cells will allow a better understanding of erythroid cell development, differentiation, structure, and function, and provide insights into inherited and acquired hematologic disease. CD34+-selected stem and progenitor cells were expanded for three days in the absence of EPO, and total RNA was isolated from a portion of the cells. The cells were further cultured in the presence of EPO, and RNA was isolated after cells differentiated into R3/R4 nucleated erythroid cells. There were 3 replicates of CD34 cells and 9 replicates of R3/R4 erythroid cells.
2013-01-28 | E-GEOD-43624 | biostudies-arrayexpress