title: Elucidation of the role of LMO2 in human erythroid cells.
abstract: LIM-only protein 2 (LMO2) is a non-DNA-binding component of a protein complex containing master regulators of hematopoiesis, including GATA-1, SCL/TAL1, and LDB1. However, the role of LMO2 in human erythroid differentiation is unclear. LMO2 knockdown in hemin-treated K562 cells reduced the benzidine-positive cell ratio, suggesting that LMO2 retards hemin-mediated K562 cell differentiation. Microarray analysis using K562 cells after siRNA-mediated LMO2 knockdown indicated that 177 and 78 genes were upregulated and downregulated (>1.5-fold), respectively. The downregulated gene ensemble contained prototypical erythroid genes (HBB, SLC4A1). Whereas LMO2 knockdown did not affect GATA-1 or SCL/TAL1 expression, it resulted in significantly reduced chromatin occupancy of GATA-1, SCL/TAL1, and LDB1 at the β-globin locus control region and SLC4A1 locus in both K562 cells and human induced pluripotent stem cell-derived erythroid cells. Introduction of GATA-1 mutations, shown to impair direct interaction with LMO2, significantly diminished chromatin occupancy. On the other hand, knockdown of either SCL/TAL1 or LDB1 also resulted in significantly reduced chromatin occupancy of GATA-1 at endogenous loci, suggesting that impaired assembly of these components also affects GATA-1 chromatin occupancy. In an ex vivo model of erythroid differentiation from CD34(+) cells, LMO2 protein level peaked on day 5 and decreased at later stages of differentiation. The LMO2 expression pattern was similar to those of GATA-1 and SCL/TAL1. Furthermore, shRNA-mediated LMO2 knockdown in primary erythroblasts suggested that LMO2 regulates HBB, HBA, and SLC4A1 expression. LMO2 contributes to GATA-1 target gene expression by affecting assembly of the GATA-SCL/TAL1 complex components at endogenous loci.
authors.affiliation: Department of Hematology and Rheumatology, Tohoku University Graduate School of Medicine, Sendai, Japan.
authors.forename: Ai
authors.initials: A
authors.lastname: Inoue
authors.forename: Tohru
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Yoko
authors.initials: Y
authors.lastname: Okitsu
authors.forename: Yuna
authors.initials: Y
authors.lastname: Katsuoka
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: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002454:Cell Differentiation / Q000235:genetics; D045744:Cell Line, Tumor; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism; D041905:Erythroid Cells / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006427:Hemin / Q000378:metabolism; D006801:Humans; D060588:LIM Domain Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D060588:LIM Domain Proteins; C085117:LMO2 protein, human; D011518:Proto-Oncogene Proteins; D006427:Hemin