Project description:Purpose:The purpose of this study is to create unbiased, stage-specific transcriptomes by RNA-seq analyses of pure populations of both murine and human erythroblasts at distinct developmental stages. Methods: Recently developed FACS-based methods (Chen et al, PNAS, Liu et al, Blood, Hu et al Blood) were employed to purify morphologically and functionally discrete populations of cells, each representing specific stages of terminal erythroid differentiation. RNA was prepared from these cells and subjected to RNA-seq analyses. Results: There were vast temporal changes in gene expression across the differentiation stages, with each stage exhibiting unique transcriptomes.Clustering and network analyses revealed that differing stage-specific patterns of expression across differentiation were transcriptionally enriched for genes of differing function. Numerous differences were present between human and murine transcriptomes, with significant variation in the global patterns of gene expression. Conclusions: These data provide a significant resource for studies of normal and perturbed erythropoiesis, allowing a deeper understanding of mechanisms of erythroid development, differentiation, and inherited and acquired disease. Both murine and human erythroblasts at distinct developmental stage mRNA profiles were generated by deep sequencing, in triplicate, using IlluminaHiSeq 2000.
Project description:Purpose:The purpose of this study is to create unbiased, stage-specific transcriptomes by RNA-seq analyses of pure populations of both murine and human erythroblasts at distinct developmental stages. Methods: Recently developed FACS-based methods (Chen et al, PNAS, Liu et al, Blood, Hu et al Blood) were employed to purify morphologically and functionally discrete populations of cells, each representing specific stages of terminal erythroid differentiation. RNA was prepared from these cells and subjected to RNA-seq analyses. Results: There were vast temporal changes in gene expression across the differentiation stages, with each stage exhibiting unique transcriptomes.Clustering and network analyses revealed that differing stage-specific patterns of expression across differentiation were transcriptionally enriched for genes of differing function. Numerous differences were present between human and murine transcriptomes, with significant variation in the global patterns of gene expression. Conclusions: These data provide a significant resource for studies of normal and perturbed erythropoiesis, allowing a deeper understanding of mechanisms of erythroid development, differentiation, and inherited and acquired disease.
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: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: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.
Project description: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.
Project description:Histone deacetylases (HDACs) are a group of enzymes catalyzing the removal of acetyl groups from histone and non-histone proteins. HDACs have been shown to play diverse functions in a wide range of biological processes. However, their roles in mammalian erythropoiesis remain to be fully defined. We show here that of the eleven classic HDAC family members, six of them (HDAC 1,2,3 and HDAC 5,6,7) are expressed in human erythroid cells with HDAC5 significantly up regulated during terminal erythroid differentiation. Knockdown of HDAC5 in human CD34+ cells followed by erythroid cell culture led to increased apoptosis, decreased chromatin condensation, and impaired enucleation of erythroblasts.
Project description:Primary murine fetal liver cells were freshly isolated from day e14.5 livers and then sorted for successive differentiation stages by Ter119 and CD71 surface expression (ranging from double-negative CFU-Es to Ter-119 positive enucleated erythrocytes) [Zhang, et al. Blood. 2003 Dec 1; 102(12):3938-46]. RNA isolated from each freshly isolated, stage-sorted population was reverse-transcribed, labelled, and then hybridized onto 3' oligo Affymetrix arrays. Important erythroid specific genes as well as the proteins that regulate them were elucidated through this profiling based on coexpression and differential expression patterns as well as by extracting specific GO categories of genes (such as DNA-binding proteins). Abstract (submitted paper): rationale for expression profiling Gene-targeting experiments report that the homeodomain-interacting protein kinases 1 and 2, Hipk1 and Hipk2, are essential but redundant in hematopoietic developmentâbecause Hipk1/Hipk2 double-deficient animals exhibit severe defects in hematopoiesis and vasculogenesis while the single knockouts do not. These serine-threonine kinases phosphorylate, and consequently modify the functions of, several important hematopoietic transcription factors and cofactors. Here we show that Hipk2 knockdown alone plays a significant role in terminal fetal liver erythroid differentiation. Hipk1 and Hipk2 are highly induced during primary mouse fetal liver erythropoiesis. Specific knockdown of Hipk2 inhibits terminal erythroid cell proliferationâexplained in part by impaired cell cycle progression as well as increased apoptosisâand terminal enucleation as well as the accumulation of hemoglobin. Hipk2 knockdown also reduces the transcription of many genes involved in proliferation and apoptosis as well as important, erythroid-specific genes involved in hemoglobin biosynthesisâsuch as alpha-globin and mitoferrin 1âdemonstrating that Hipk2 plays an important role in some but not all aspects of normal terminal erythroid differentiation.
Project description:Terminal differentiation is characterized by a respecification of the global protein complement, as epitomized by erythrocytes, whose cytosol is ~98% globin. Remodeling of the proteome involves programmed elimination of generic cellular constituents in parallel with synthesis of cell-type-specific proteins. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving elimination of preexisting cytosolic proteins are unidentified. UBE2O is a ubiquitin-conjugating enzyme expressed at elevated levels during late stage erythropoiesis. A Ube2o null mutation in mice results in anemia. Proteomic analysis of this mutant suggests that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, a hallmark of the reticulocyte to mature erythrocyte transition—ribosome elimination—is defective in Ube2o-/- mutants. UBE2O recognizes ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, and perhaps other highly differentiated cells, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.