Exosc8-Regulated Genes in Fetal Liver-Derived Erythroid Progenitor Cells
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ABSTRACT: 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 ensemble of Foxo3-regulated genes in the erythroid G1E-ER-GATA-1 cell line was determined by knocking down Foxo3 using siRNA, and measuring genome wide transcription by microarray analysis G1E-ER-GATA-1 cells were treated with control or Foxo3-specific siRNA by nucleofection at t = 0 h and t = 24 h. At t = 24 h, cells were treated with M-CM-^_-estradiol to activate ER-GATA-1. RNA was harvested at t = 48 h and processed for microarray analysis.
Project description:Knockdown of HSPA9 causes a dose-dependent decrease in erythroid maturation of CD34+ cells differentiated in culture. Due to differences in the degree of differentiation, a more homogeneous population was selected for using FACS and the gene expression profile of these cells was compared. We used a lentiviral vector (pLKO.1) expressing short hairpin RNAs targeting either luciferase (control shLUC) or HSPA9 (shHSPA9-433) to knock down expression of HSPA9. We isolated CD34+ cells from human cord blood (Day 0), transduced cells with a lentiviral vector (Day 1), selected for transduced cells with puromycin and differentiated them in erythroid culture media before FACS isolation of the CD34+/CD71- population (Day 5). Four independent CD34+ populations were isolated, differentiated and sorted for biologic replicates.
Project description:The establishment and maintenance of cell type-specific transcriptional programs require an ensemble of broadly expressed chromatin remodeling and modifying enzymes. Many questions remain unanswered regarding the contributions of these enzymes to specialized genetic networks that control critical processes such as lineage commitment and cellular differentiation. We have been addressing this problem in the context of erythrocyte development driven by the transcription factor GATA-1 and its coregulator Friend of GATA-1 (FOG-1). As certain GATA-1 target genes have little to no FOG-1 requirement for expression, presumably additional coregulators can mediate GATA-1 function. Using a genetic complementation assay and RNA interference in GATA-1-null cells, we demonstrate a vital link between GATA-1 and the histone H4 lysine 20 methyltransferase PR-Set7/SetD8 (SetD8). GATA-1 selectively induced H4 monomethylated lysine 20 at repressed, but not activated, loci, and endogenous SetD8 mediated GATA-1-dependent repression of a cohort of its target genes. GATA-1 utilized different combinations of SetD8, FOG-1, and the FOG-1-interacting Nucleosome Remodeling and Deacetylase (NuRD) complex component Mi2b to repress distinct target genes. Implicating SetD8 as a context-dependent GATA-1 corepressor expands the repertoire of coregulators mediating establishment/maintenance of the erythroid cell genetic network and provides a biological framework for dissecting the cell type-specific functions of this important coregulator. We propose a coregulator matrix model in which distinct combinations of chromatin regulators are required at different GATA-1 target genes, and the unique attributes of the target loci mandate these combinations. 3 SetD8 knockdown samples were compared to 3 control samples
Project description:Terminal differentiation of mammalian erythroid progenitors involves 4-5 cell divisions and induction of many erythroid important genes, followed by chromatin and nuclear condensation and enucleation. The protein levels of c-myc (Myc) are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G1-phase and enucleation, suggesting possible roles for c-myc in either or both of these processes. Here we demonstrate that ectopic Myc expression affects terminal erythroid maturation in a dose-dependent manner. Expression of Myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown, but specifically blocked erythroid nuclear condensation and enucleation. Myc prevented deacetylation of several lysine residues in histones H3 and H4 that are normally deacetylated during erythroid maturation. When over-expressed at levels higher than the physiological range, Myc blocked erythroid differentiation and the cells continued to proliferate in cytokine-free, serum-containing culture medium with an early erythroblast morphology. These studies reveal an important dose-dependent function of Myc in regulating terminal maturation in mammalian erythroid cells. Our findings further support the emerging notion that Myc regulates chromatin structure by regulating global histone acetylation states. Five groups with three biological replicates in each.
Project description:The chromatin modifying enzymes that drive the erythroid-specific transcription program are incompletely understood. Setd8 is the sole histone methyltransferase in mammals capable of generating mono-methylated histone H4 lysine 20 (H4K20me1) and is expressed at significantly higher levels in erythroid cells than any other cell- or tissue- type, suggesting that Setd8 has an erythroid-specific function. To test this hypothesis, stable knockdown of Setd8 was established in extensively self-renewing erythroblasts (ESREs), a well-characterized, non-transformed, model of erythroid maturation. Setd8 knockdown impaired erythroid maturation, characterized by a delay in hemoglobin accumulation, larger cell area, persistent kit expression, incomplete nuclear condensation, and lower rates of enucleation than control cells. Setd8 knockdown did not alter ESRE proliferation or viability, or result in accumulation of DNA damage. Global gene expression analyses following Setd8 knockdown suggests that in erythroid cells, Setd8 functions primarily as a repressor and demonstrated high levels of Gata2 expression. Setd8 occupies critical regulatory elements in the Gata2 locus, and knockdown of Setd8 resulted in loss of H4K20me1 and gain of H4 acetylation at the Gata2 1S promoter. Taken together, these results imply that Setd8 is an important regulator of erythroid maturation that works in part through repression of Gata2. RNA-seq was performed of Setd8 knockdown and control cells, both while the cells were proliferating, and after 6 hours of maturation.
Project description:Mammals express thousands of long noncoding (lnc) RNAs, a few of which are shown to function in tissue development. However, the entire repertoire of lncRNAs and the extent to which they regulate biological processes in different tissues and species are not defined. Indeed, most lncRNAs are not conserved between species, raising questions about function. We used RNA-Seq to identify lncRNAs in primary murine fetal liver erythroblasts expressing the lineage marker TER119, megakaryocytes (CD41+) cultured from embryonic day (E) 14.5 murine fetal liver and megakaryocyte erythroid progenitors (MEPs) isolated from mouse bone marrow. We identified 683 and 594 polyadenylated lncRNAs expressed in red blood cell (erythroid) precursors of mice and humans, respectively. More than one half of erythroid lncRNAs are un-annotated, emphasizing the opportunity for new discovery through studies of specialized cell types. We analyzed the expression of these identified lncRNAs in several hematopoietic compartments using a custom microarray to identify erythroid-specific lncRNAs that were robustly expressed in both fetal liver and adult erythroid cells as targets for knockdown. Over 90% of fetal liver erythroid lncRNAs detected using RNA-seq were expressed in adult erythroblasts measured on the microarray. Analysis of the murine erythroid lncRNA transcriptome indicates that ~75% arise from promoters and 25% from enhancers, many of which are regulated by the key erythroid transcription factors GATA1 and SCL/TAL1. Erythroid lncRNA expression is largely conserved among 8 different mouse strains, yet only 15% of mouse lncRNAs are expressed in humans and vice versa, reflecting dramatically greater species-specificity than coding genes. We investigated potential functions of 21 relatively abundant erythroid-specific murine lncRNAs (both conserved and non-conserved) by RNA interference in primary mouse erythroid precursors, and identified 7 whose knockdown inhibited features of terminal erythroid maturation including cell size reduction and enucleation. Strikingly, at least 6 of the 7 lncRNAs have no detectable expression in human erythroblasts, demonstrating that lack of conservation between mammalian species does not predict lack of function. These results reflect marked evolutionary differences between protein-coding genes and lncRNAs and indicate that the latter exert tissue- and species-specific roles in development. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf A custom Agilent microarray was designed to interrogate expression levels of long noncoding RNAs identified using RNA-seq in several different hematopoietic progenitor and differentiated cell populations. LncRNA gene definitions and RNA-seq data used to identify long noncoding RNAs are deposited in GEO with the accession numbers GSE51667 and GSE40522 respectively. The Agilent eArray platform was used to customize the SurePrint G3 Mouse GE 8x60K microarray to add 3 custom-designed probes (60nt length) each against several categories of genes, namely pseudogene, genes with small RNA overlap, low stringency lncRNAs and high stringency lncRNAs . We interrogated 7 types of hematopoietic cells, HSC, CMP, MEP, GMP, early and late erythroblasts and granulocytes. Expression measurements were determined at least in duplicate for all samples and in triplicate for most samples.
Project description:The molecular mechanisms underlying erythroid-specific gene regulation remain incompletely understood. Closely spaced binding sites for GATA, NF-E2/maf and CACCC interacting transcription factors play functionally important roles in globin and other erythroid-specific gene expression. We and others recently identified the CACCC-binding transcription factor ZBP-89 as a novel GATA-1 and NF-E2/mafK interacting partner. Here, we examined the role of ZBP-89 in human globin gene regulation and erythroid maturation using a primary CD34+ cell ex vivo differentiation system. We show that ZBP-89 protein levels rise dramatically during human erythroid differentiation, and that ZBP-89 occupies key cis-regulatory elements within the globin and other erythroid gene loci. ZBP-89 binding correlates strongly with RNA Pol II occupancy, active histone marks, and high-level gene expression. ZBP-89 physically associates with the histone acetyltransferases (HATs) p300 and Gcn5/Trrap, and occupies common sites with Gcn5 within the human globin loci. Lentiviral shRNA knockdown of ZBP-89 results in reduced Gcn5 occupancy, decreased acetylated histone 3 levels, lower globin and erythroid-specific gene expression, and impaired erythroid maturation. Addition of the HDAC inhibitor valproic acid partially reverses the reduced globin gene expression. These findings reveal an activating role for ZBP-89 in human globin gene regulation and erythroid differentiation. Keywords: Human primary erythroid progenitors Expression data of human erythroid progenitors transduced with lentivirus expressing short hairpins against ZBP-89 and control empty vector
Project description:Coordination of cellular processes through the establishment of tissue-specific gene expression programmes is essential for lineage maturation. The basic helix-loop-helix haemopoietic transcriptional regulator SCL/Tal1 is required for terminal differentiation of red blood cells. To gain insight into SCL function and mechanisms of action in erythropoiesis, we performed ChIP-sequencing and gene expression analyses from primary fetal liver erythroid cells. We show that SCL coordinates expression of genes in most known red cell-specific processes. The majority of SCLâs genomic targets require direct DNA-binding activity. However, one fifth of SCLâs target sequences, mainly amongst those showing high affinity for SCL, can recruit the factor independently of its DNA binding activity. An unbiased DNA motif search of sequences bound by SCL identified CAGNTG as SCL-preferred E-box motif in erythroid cells. Novel motifs were also characterised that may help distinguish activated from repressed genes and suggest a new mechanism by which SCL may be recruited to DNA. Finally, analysis of recruitment of GATA1, a protein partner of SCL, to sequences occupied by SCL suggests that SCLâs binding is necessary prior or simultaneous to that of GATA1. This work provides the framework to study regulatory networks leading to erythroid terminal maturation and to model mechanisms of action of tissue-specific transcription factors. Total RNA extracted from wild-type (WT) day E12.5 fetal liver Ter119- erythroid progenitor cells was compared to total RNA extracted from E12.5 fetal liver Ter119- cells expressing a DNA-binding mutant form of SCL (SclRER/RER).
Project description:Expression profiling of fetal liver erythroid precursors after either Hipk1 or Hipk2 knockdown by shRNA versus control shRNA Two condition experiment, Hipk1 or Hipk2 knockdown versus control (shRNA against luciferase), two replicates each shRNA
Project description:To determine the transcriptional function (if any) of the presumed nuclear export protein Xpo7 or RanBP16 Murine fetal liver erythroid precursors (Ter119-negative cells) were isolated from C57Bl6 E14.5 embryos by magnetic depletion and infected with retroviruses containing shRNA constructs against Xpo7. They were then cultured in Epo-containing media (2U/mL) for 36hrs until they were fully differentiated and then sorted by FACS for GFP+ (infected) cells in order to isolate total RNA to be used for the profiling. Expression profiling in late cultured mouse erythroblasts before and after knockdown of gene Xpo7.