|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Ectopic expression of Hmgn2 antagonizes mouse erythroid differentiation in vitro
Kasem Kulkeaw*, Tomoko Inoue*†, Chiyo Mizuochi*, Yuka Horio*, Yasushi Ishihama‡ and Daisuke Sugiyama*1
*Advanced Medical Initiatives, Division of Hematopoietic Stem Cells, Department of Advanced Medical Initiatives, Faculty of Medical Sciences, Kyushu University, Fukuoka, 8128582, Japan, †Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, 8128582, Japan, and ‡Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, 6068501, Japan
Hmgn2 (high mobility group nucleosomal 2), a ubiquitous nucleosome-binding protein that unfolds chromatin fibres and enhances DNA replication, reportedly regulates differentiation of epithelial and mesenchymal cells. To investigate how Hmgn2 regulates HC (haemopoietic cell) differentiation, we quantified Hmgn2 expression in HCs of mouse FL (fetal liver) during erythroid differentiation. Hmgn2 expression levels were >10-fold higher in immature erythroid progenitors than in mature erythroid cells, suggesting that Hmgn2 antagonizes erythroid differentiation. To address this issue, Hmgn2 were transfected into both Friend erythroleukaemia cells and FL HCs. There was a 3.3-fold decrease in relatively mature c-Kit+/CD71+ erythroid cells, a 2.9-fold increase in immature c-Kit+/CD71− erythroid cells in transfected Friend cells, a 1.1-fold decrease in relatively mature CD71+/Ter119+ erythroid cells, and a 1.7-fold increase in relatively immature c-Kit+/CD71+ erythroid cells in FL HCs accompanied by down-regulation of genes encoding the erythroid transcription factors, Gata1 and Klf1. Two days after Hmgn2 transfection of Friend erythroleukaemia cells, the number of S-phase cells increased, whereas the number of cells in G1 decreased, while that of mitotic cells remained unchanged. We conclude that ectopic expression of Hmgn2 antagonizes mouse erythroid differentiation in vitro, which may be due to enhancement of DNA replication and/or blocking entry of mitosis at S-phase.
Key words: erythroid differentiation, erythroleukaemia cell, fetal liver, Hmgn2
Abbreviations: AcGFP, Aequorea coerulescens green fluorescent protein, APC, allophycocyanin, BFU-E, burst-forming unit-erythroid, CFU-E, colony-forming unit-erythroid, CHUD, chromatin-unfolding domain, dpc, day post coitum, EPO, erythropoietin, FBS, fetal bovine serum, FL, fetal liver, GFP, green fluorescent protein, HC, haemopoietic cell, Hmgn, high mobility group nucleosomal, HSC, haemopoietic stem cell, IL-3, interleukin 3, IRES, internal ribosome entry site, α-MEM, α-minimum essential medium, MNC, mononuclear cell, NBD, nucleosome-binding domain, NLS, nuclear localization signal, PE, phycoerythrin, PI, propidium iodide, PBSBA, BSA in PBS, RQ, relative quantity, RT–PCR, reverse transcription–PCR, Sca-1, stem cell antigen-1, SCF, stem cell factor, TBS-T, TBS containing 0.1% Tween-20, TPO, thrombopoietin, WB, Western blot
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Received 18 March 2011/25 July 2011; accepted 12 October 2011
Published as Cell Biology International Immediate Publication 12 October 2011, doi:10.1042/CBI20110169
© The Author(s) Journal compilation © 2012 Portland Press Limited