title: Role of transcriptional corepressor ETO2 in erythroid cells.
abstract: Transcriptional corepressor ETO2 is a component of a protein complex containing master regulators of hematopoiesis, including GATA-1, SCL/TAL1, LMO2, and LDB1. To elucidate the role of ETO2 during erythroid differentiation, including the effects of ETO2 on GATA-1 targets, we performed gene expression profiling using K562 cells overexpressed with ETO2. The analysis demonstrated that 667 and 598 genes were upregulated and downregulated (more than twofold), respectively, in ETO2-overexpressing cells. ETO2-repressed genes included those encoding prototypical erythroid proteins. To test what percentages of ETO2-repressed genes could be direct target genes of GATA-1 in K562 cells, we merged the microarray results with ChIP-seq profile (n = 5,749), demonstrating that 23.1% of ETO2-repressed genes contained significant GATA-1 in their loci. However, there was no significant enrichment of PU.1 target genes among ETO2-repressed genes. Gene ontology analysis among ETO2-repressed genes revealed significant enrichment of genes related to "oxygen transporter," corresponding to globin genes. Quantitative chromatin immunoprecipitation and ETO2 knockdown analyses confirmed that ETO2 directly regulates globin genes in K562 cells. Next, we evaluated the role of ETO2 in human primary erythroblasts, derived from cord blood CD34-positive cells. In an ex vivo model of erythroid differentiation from CD34-positive cells, ETO2 protein level peaked at day 2-4 and almost diminished at the later stage of differentiation. Furthermore, short hairpin RNA-mediated knockdown and retroviral vector-mediated overexpression of ETO2 in primary erythroblasts suggested that ETO2 significantly represses HBB, HBA, and ALAS2 expression. In summary, ETO2 regulates GATA-1 target genes critical for erythroid differentiation, and the decrease of ETO2 levels during erythroid differentiation would contribute to the activation of these targets.
authors.affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan. fujiwara-to@apple.email.ne.jp
authors.forename: Tohru
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Yarob Wael
authors.initials: YW
authors.lastname: Alqadi
authors.forename: Yoko
authors.initials: Y
authors.lastname: Okitsu
authors.forename: Noriko
authors.initials: N
authors.lastname: Fukuhara
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Onishi
authors.forename: Kenichi
authors.initials: K
authors.lastname: Ishizawa
authors.forename: Hideo
authors.initials: H
authors.lastname: Harigae
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation / Q000235:genetics; D047369:Chromatin Immunoprecipitation / Q000379:methods; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism; D041905:Erythroid Cells / Q000378:metabolism*; D050982:GATA1 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling; D015973:Gene Expression Regulation, Leukemic; D005914:Globins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020014:K562 Cells; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D034622:RNA Interference; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; C115744:CBFA2T3 protein, human; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D012097:Repressor Proteins; D025521:Tumor Suppressor Proteins; D005914:Globins