Mechanisms of terminal erythroid differentiation defect in EKLF-deficient mice
ABSTRACT: EKLF is a Krüppel-like transcription factor identified as a transcriptional activator and chromatin modifier in erythroid cells. EKLF-deficient (Eklf -/-) mice die at day 14.5 of gestation from severe anemia. In this study, we demonstrate that early progenitor cells fail to undergo terminal erythroid differention in Eklf -/- embryos. To discover potential EKLF target genes responsible for the failure of erythropoiesis, transcriptional profiling was performed with RNA from wild type and Eklf -/- early erythroid progenitor cells. These analyses identified significant perturbation of a network of genes involved in cell cycle regulation, with the critical regulator of the cell cycle, E2f2, at a hub. E2f2 mRNA and protein levels were markedly decreased in Eklf -/- early erythroid progenitor cells, which showed a delay in the G1-to-S-phase transition. Chromatin immunoprecipitation analysis demonstrated EKLF occupancy at the proximal E2f2 promoter in vivo. Consistent with the role of EKLF as a chromatin modifier, EKLF binding-sites in the E2f2 promoter were located in a region of EKLF-dependent DNase I sensitivity in early erythroid progenitor cells. We propose a model in which EKLF-dependent activation and modification of the E2f2 locus is required for cell cycle progression preceding terminal erythroid differentiation. RNA was isolated from flow-sorted early erythroid progenitors in 13.5 day old fetal livers from EKLF knock out mice (n=3 fetal livers) and wild-type control mice (n=3 fetal livers) for gene expression analysis
Project description:Erythropoiesis is dependent on the activity of transcription factors, including the erythroid-specific erythroid Kruppel-like factor (EKLF). ChIP followed by massively parallel sequencing (ChIP-Seq) is a powerful, unbiased method to map transfactor occupancy. We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts. We correlated these results with the nuclear distribution of EKLF, RNA-Seq analysis of the transcriptome, and the occupancy of other erythroid transcription factors. In progenitor cells, EKLF is found predominantly at the periphery of the nucleus, where EKLF primarily occupies the promoter regions of genes and acts as a transcriptional activator. In erythroblasts, EKLF is distributed throughout the nucleus, and erythroblast-specific EKLF occupancy is predominantly in intragenic regions. In progenitor cells, EKLF modulates general cell growth and cell cycle regulatory pathways, whereas in erythroblasts EKLF is associated with repression of these pathways. The EKLF interactome shows very little overlap with the interactomes of GATA1, GATA2, or TAL1, leading to a model in which EKLF directs programs that are independent of those regulated by the GATA factors or TAL1. (Blood.2011;118(17):e139-e148) We used ChIP-Seq to study the interactome of EKLF in mouse erythroid progenitor cells and more differentiated erythroblasts and RNA-Seq analysis of the transcriptome.
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
Project description:This SuperSeries is composed of the following subset Series: GSE30380: Expression profile of hemin induced erythroid differentiation, over-expressing and knocking-down GATA-1, EKLF and NF-E2 in K562s GSE30808: ChIP-chip from 48h hemin induced K562 cells with GATA-1, EKLF, and NF-E2 antibodies Refer to individual Series
Project description:Exosc8 and Exosc9 are components of the exosome that establish a barricade to erythroid maturation. Here, we knocked down Exosc8 in fetal liver-derived erythroid progenitor cells to determine the cohort of Exosc8-regulated genes in erythroid cells. Freshly isolated fetal liver progenitor cells were infected with retrovirus expressing shRNA targeting either luciferase or Exosc8. Total RNA was isolated from these cells after 3 days ex-vivo culture, during which the cells underwent erythroid maturation.
Project description:The transcription factor GATA-1, EKLF and NF-E2 promotes erythroid differentition by regulating their target genes, however, the intricate interplays between these key TFs and microRNA genes are largely unknown. Chromatin immunoprecipitation (ChIP) of GATA-1, EKLF and NF-E2 together with microRNA genomic promoter profiling by ChIP-on-chip analysis demonstrated that GATA-1, EKLF and NF-E2 collaborately regulate a series of microRNA genes. Comparison of microRNA promoter arrays of GATA-1 VS EKLF VS NF-E2 in K562 cells suffering with hemin induced erythroid differentiation
Project description:In order to elucidate the molecular mechanism giving rise to the rare In(Lu) type of Lu(a-b-) blood group phenotype we compared the transcriptome of normal and In(Lu) erythroblasts at different stages of maturation. Many erythroid-specific genes had reduced transcript levels suggesting the phenotype resulted from a transcription factor abnormality. A search for mutations in erythroid transcription factors revealed mutations in the promoter or coding sequence of EKLF in 21 of 24 individuals with the In(Lu) phenotype. In all cases the mutant EKLF allele occurred in the presence of a normal EKLF allele. Individuals with the In(Lu) phenotype have no reported pathology indicating that one functional EKLF allele is sufficient to sustain human erythropoiesis. These data provide the first description of inactivating mutations in human EKLF and the first demonstration of a blood group phenotype resulting from mutations in a transcription factor. Experiment Overall Design: Time course study looking at changes in gene expression during erythropoiesis in two cell types: cultured erythroblasts (days 4, 6 and 11) from normal blood donors and from those with the rare blood group In(Lu) phenotype. There are 20 samples in total (10 for each cell type). For each cell type there are 2 samples at day 4 (1 biological replicate, 2 technical replicates, and 4 samples at each of days 6 and 11 (2 biological replicates, 2 technical replicates).
Project description:We over-expressed ESlncRNA (AK148461) in fetal liver erythroid progenitor cells (Lin-cells), followed by microarray analysis to examine the global changes of gene expression level. We showed that ESlncRNA has an anti-apoptotic activity during mouse erythropoiesis. Compare the gene expression level in vector transduced fetal liver erythroid progenitor cells (Lin-cells) with that in ESlncRNA transduced fetal liver erythroid progenitor cells (Lin-cells).
Project description:Klf1 (formerly known as Eklf) regulates the development of erythroid cells from bi-potent progenitor cells via the transcriptional activation of a diverse set of genes. Mice lacking Klf1 die in utero prior to E15 from severe anemia due to the inadequate expression of genes controlling hemoglobin production, cell membrane and cytoskeletal integrity, and the cell cycle and proliferation. We have recently described the full repertoire of Klf1 binding sites in vivo by performing Klf1 ChIP-seq in primary erythroid tissue (E14.5 fetal liver). Here we describe the Klf1-dependent erythroid transcriptome by comparing mRNA-seq from Klf1+/+ and Klf1-/- erythroid tissue. This has revealed novel target genes not previously obtainable by traditional microarray technology and provided novel insights into the function of Klf1 as a transcriptional activator such as interactions with Gata1, Scl/Tal1 and p300. We also describe a set of erythroid specific promoters not previously identified that drive high level expression of otherwise ubiquitously expressed genes in erythroid cells. Additionally, our study has identified for the first time two novel lnc-RNAs that are dynamically expressed during erythroid differentiation as well as a role for Klf1 in directing apoptotic gene expression to drive the terminal stages of erythroid maturation. Examination of mRNA expression in 3 Klf1-/- and 3 Klf1+/+ fetal livers This submission represents mRNA-Seq component of study.
Project description:Background: It has been reported that the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway regulates erythropoietin (EPO)-induced survival, proliferation, and maturation of early erythroid progenitors. Erythroid cell proliferation and survival has also been related to activation of the JAK-STAT pathway. The goal of this study was to observe the function of EPO activation of JAK-STAT and PI3K/AKT pathways in the development of erythroid progenitors from hematopoietic CD34+ progenitor cells, as well as to distinguish early EPO target genes in human erythroid progenitors during ontogeny. Methods: Hematopoietic CD34+ progenitor cells, isolated from fetal and adult hematopoietic tissues, were differentiated into erythroid progenitor cells. We have used microarray analysis to examine JAK-STAT and PI3K/AKT related genes, as well as broad gene expression modulation in these human erythroid progenitor cells. Results: In microarray studies, a total of 1755 genes were expressed in fetal liver, 3844 in cord blood, 1770 in adult bone marrow, and 1325 genes in peripheral blood-derived erythroid progenitor cells. The erythroid progenitor cells shared 1011 common genes. Using the Ingenuity Pathways Analysis software, we evaluated the network pathways of genes linked to hematological system development, cellular growth and proliferation. The KITLG, EPO, GATA1, PIM1 and STAT3 genes represent the major connection points in the hematological system development linked genes. Some JAK-STAT signaling pathway-linked genes were steadily upregulated throughout ontogeny (PIM1, SOCS, MYC, PTPN11), while others were downregulated (PTPN6, PIAS, SPRED2). In addition, some JAK-STAT pathway related genes have specific expression just at certain stages of ontogeny (STATs, GRB2, CREBB). Beside continuously upregulated (AKT1, PPP2CA, CHUK, NFKB1) and downregulated (FOXO1, PDPK1, PIK3CG) genes in the PI3K-AKT signaling pathway, we also observed intermittently regulated gene expression (NFKBIA, YWHAH). Conclusions: This broad overview of gene expression in erythropoiesis revealed transcription factors with a prevalence at certain stages of ontogenesis. Finally, our results show that EPO-mediated proliferation and survival of erythroid progenitors occurs mainly through modulation of JAK-STAT pathway associated STATs, GRB2 and PIK3 genes, as well as AKT pathway- coupled NFKBIA and YWHAH genes. Adult peripheral blood mononuclear cells were isolated from buffy coats of 3 healthy donors using Lymphocyte Separation Medium (BioWhittaker, Walkersville, MD). We washed mononuclear cells twice with Dulbecco's phosphate-buffered saline (PBS, Invitrogen Corporation, Carlsbad, CA), and CD34+ cells were purified by positive immunomagnetic selection using the MACS cell isolation system (Miltenyi Biotec, Auburn, CA). Fresh bone marrow CD34+ cells were collected (AllCells LLC, Berkeley, CA). Cord blood CD34+ cells (AllCells LLC) and fetal liver CD34+ cells (Cambrex Bio Science, Inc., Walkersville, MD) were collected and frozen. For analysis, CD34+ cells were resuspended in medium, which contained 30% FBS, 2 mmol/L glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% deionized BSA, 10 mmol/L beta-mercaptoethanol, 1 mmol/L dexamethasone, 33 µg/ml holo-transferrin, 10 ng/ml SCF, 1 ng/ml IL-3 and 1 ng/ml GM-CSF (Sigma, St. Louis, MO), and 1 U/ml human recombinant EPO (Amgen Inc, Thousand Oaks, CA) Erythroid progenitor cells differentiated from hematopoietic CD34+ progenitor cells of fetal liver, cord blood, bone marrow and peripheral blood origin Biological replicates: 2 fetal liver, 3 cord blood, 3 bone marrow, 3 peripheral blood origin
Project description:Hematopoietic progenitor cells were isolated from 13.5 day mouse fetal livers by lineage depletion and expanded for three days. Fetal livers were isolated from both wild type and Gata-1 knock embryos. Gata-1 knock embryos contain a deletion of the Gata-1 promoter sequence that results in undetectable levels of Gata-1 protein specifically in the megakaryocyte lineage. Following progenitor outgrowth megakaryocytes were enriched in a differentiation media for three days and isolated on a discontinuous BSA gradient. The resulting megakaryocytes were >90% pure as determined by acetylcholinesterase staining. These cells were lysed in Trizol and the resulting RNA was used for hybridization.