Article

Identification of gene networks associated with erythroid differentiation.

The Laboratory of Hematology, Assaf-Harofeh Medical Center, Zerifin, Israel.
Blood Cells Molecules and Diseases (Impact Factor: 2.26). 04/2009; 43(1):74-80. DOI: 10.1016/j.bcmd.2009.01.020
Source: PubMed

ABSTRACT Erythropoiesis is a multistep process involving a large number of genes, which balance between proliferation, differentiation and survival of the erythroid cells. To understand the molecular mechanisms of erythropoiesis and related pathological aberrations, we analyzed three stages of in vitro differentiating human erythroid cells by expression profiling. We identified distinct clusters of genes, each with a unique expression pattern during differentiation. As JAK2 was shown to play a central role in myeloproliferative disorders, we focused on one cluster which includes JAK2 and other genes with high correlation to JAK2 expression. These genes had a low expression at the early erythroblast which increased in the intermediate stage and further slightly increased in the last stage of differentiation. Our results indicate that gene networks may associate with JAK2 expression in erythroid differentiation. It is intriguing to determine whether the pathogenesis of polycythemia vera (PV), harboring a common or uncommon JAK2 mutation, involves alterations in independent gene pathways that underlie the normal erythropoietic process.

0 Bookmarks
 · 
82 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: We recently developed FACS-based methods to purify morphologically and functionally discrete populations of cells, each representing specific stages of terminal erythroid differentiation. We utilized these techniques to obtain pure populations of both human and murine erythroblasts at distinct developmental stages. RNA was prepared from these cells and subjected to RNA-seq analyses, creating unbiased, stage-specific transcriptomes. Tight clustering of transcriptomes from differing stages, even between biologically different replicates, validated the utility of the FACS-based assays. Bioinformatic analyses revealed that there were marked differences between differentiation stages, with both shared and dissimilar gene expression profiles defining each stage within transcriptional space. There were vast temporal changes in gene expression across the differentiation stages, with each stage exhibiting unique transcriptomes. Clustering and network analyses revealed that varying stage-specific patterns of expression observed across differentiation were enriched for genes of differing function. Importantly, numerous differences were present between human and murine transcriptomes, with significant variation in the global patterns of gene expression. These data provide a significant resource for studies of normal and perturbed erythropoiesis, allowing a deeper understanding of mechanisms of erythroid development in various inherited and acquired erythroid disorders.
    Blood 03/2014; · 9.78 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fetal stem cells isolated from umbilical cord blood (UCB) possess a great capacity for proliferation and differentiation and serve as a valuable model system to study gene regulation. Expanded knowledge of the molecular control of hemoglobin synthesis will provide a basis for rational design of therapies for β-hemoglobinopathies. Transcriptome data are available for erythroid progenitors derived from adult stem cells, however studies to define molecular mechanisms controlling globin gene regulation during fetal erythropoiesis are limited. Here, we utilize UCB-CD34+ stem cells induced to undergo erythroid differentiation to characterize the transcriptome and transcription factor networks (TFNs) associated with the γ/β-globin switch during fetal erythropoiesis. UCB-CD34+ stem cells grown in the one-phase liquid culture system displayed a higher proliferative capacity than adult CD34+ stem cells. The γ/β-globin switch was observed after day 42 during fetal erythropoiesis in contrast to adult progenitors where the switch occurred around day 21. To gain insights into transcription factors involved in globin gene regulation, microarray analysis was performed on RNA isolated from UCB-CD34+ cell-derived erythroid progenitors harvested on day 21, 42, 49 and 56 using the HumanHT-12 Expression BeadChip. After data normalization, Gene Set Enrichment Analysis identified transcription factors (TFs) with significant changes in expression during the γ/β-globin switch. Forty-five TFs were silenced by day 56 (Profile-1) and 30 TFs were activated by day 56 (Profile-2). Both GSEA datasets were analyzed using the MIMI Cytoscape platform, which discovered TFNs centered on KLF4 and GATA2 (Profile-1) and KLF1 and GATA1 for Profile-2 genes. Subsequent shRNA studies in KU812 leukemia cells and human erythroid progenitors generated from UCB-CD34+ cells supported a negative role of MAFB in γ-globin regulation. The characteristics of erythroblasts derived from UCB-CD34+ stem cells including prolonged γ-globin expression combined with unique TFNs support novel mechanisms controlling the γ/β-globin switch during UCB-derived erythropoiesis.
    PLoS ONE 09/2014; 9(9):e107133. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: To globally survey the changes in transcriptional landscape during terminal erythroid differentiation, we performed RNA sequencing (RNA-seq) on primary human CD34(+) cells after ex vivo differentiation from the earliest into the most mature erythroid cell stages. This analysis identified thousands of novel intergenic and intronic transcripts as well as novel alternative transcript isoforms. After rigorous data filtering, 51 (presumptive) novel protein-coding transcripts, 5,326 long and 679 small non-coding RNA candidates remained. The analysis also revealed two clear transcriptional trends during terminal erythroid differentiation: first, that the complexity of transcript diversity was predominantly achieved by alternative splicing, and second, that splicing junctional diversity diminished during erythroid differentiation. Finally, 404 genes that were not known previously to be differentially expressed in erythroid cells were annotated. Analysis of the most extremely differentially expressed transcripts revealed that these gene products were all closely associated with hematopoietic lineage differentiation. Taken together, this study will serve as a comprehensive platform for future in-depth investigation of human erythroid development that, in turn, may reveal new insights into multiple layers of the transcriptional regulatory hierarchy that controls erythropoiesis.
    Human Molecular Genetics 04/2014; · 7.69 Impact Factor