KLF1 mutation E325K induces cell cycle arrest in erythroid cells differentiated from congenital dyserythropoietic anemia patient-specific induced pluripotent stem cells.

Hideki Muramatsu, Kenzaburo Tani, Hitoshi Kanno, Seiji Kojima, Etsuro Ito, Seishi Ogawa, Kenichi Yoshida, Shouichi Ohga, Takashi Yamamoto, Masayuki Shiba, Tetsushi Sakuma, Shohei Miyamoto, Ryo Kurita, Takaaki Abe, Hiroshi Kohara, Yusuke Okuno, Sayoko Doisaki, Takayoshi Asai, Takuya Iwasaki, Takako Aoki, Jiyuan Liao, Ai Sugawara, Hiromi Ogura, Yoshie Ogawa, Lisa Hirose, Chika Sakamoto, Taiju Utsugisawa

Journal: Experimental hematology 2020;73():25-37.e8

PMID: 30876823

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Abstract

Krüppel-like factor 1 (KLF1), a transcription factor controlling definitive erythropoiesis, is involved in sequential control of terminal cell division and enucleation via fine regulation of key cell cycle regulator gene expression in erythroid lineage cells. Type IV congenital dyserythropoietic anemia (CDA) is caused by a monoallelic mutation at the second zinc finger of KLF1 (c.973G>A, p.E325K). We recently diagnosed a female patient with type IV CDA with the identical missense mutation. To understand the mechanism underlying the dyserythropoiesis caused by the mutation, we generated induced pluripotent stem cells (iPSCs) from the CDA patient (CDA-iPSCs). The erythroid cells that differentiated from CDA-iPSCs (CDA-erythroid cells) displayed multinucleated morphology, absence of CD44, and dysregulation of the KLF1 target gene expression. In addition, uptake of bromodeoxyuridine by CDA-erythroid cells was significantly decreased at the CD235a/CD71 stage, and microarray analysis revealed that cell cycle regulator genes were dysregulated, with increased expression of negative regulators such as CDKN2C and CDKN2A. Furthermore, inducible expression of the KLF1 E325K, but not the wild-type KLF1, caused a cell cycle arrest at the G1 phase in CDA-erythroid cells. Microarray analysis of CDA-erythroid cells and real-time polymerase chain reaction analysis of the KLF1 E325K inducible expression system also revealed altered expression of several KLF1 target genes including erythrocyte membrane protein band 4.1 (EPB41), EPB42, glutathione disulfide reductase (GSR), glucose phosphate isomerase (GPI), and ATPase phospholipid transporting 8A1 (ATP8A1). Our data indicate that the E325K mutation in KLF1 is associated with disruption of transcriptional control of cell cycle regulators in association with erythroid membrane or enzyme abnormalities, leading to dyserythropoiesis.

Address: Project Division of ALA Advanced Medical Research, The Institute of Medical Science, University of Tokyo, Tokyo, Japan.; Department of Transfusion Medicine and Cell Processing, Tokyo Women's Medical University, Tokyo, Japan.; Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.; Japanese Red Cross Chiba Blood Center, Chiba, Japan.; Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.; Department of Research and Development, Central Blood Institute, Japanese Red Cross Society, Tokyo, Japan.; Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan.; Department of Pediatrics, Yamaguchi University Graduate School of Medicine, Ube, Japan.; Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.; Department of Pediatrics, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.; Department of Transfusion Medicine and Cell Processing, Tokyo Women's Medical University, Tokyo, Japan. Electronic address: [email protected].; Project Division of ALA Advanced Medical Research, The Institute of Medical Science, University of Tokyo, Tokyo, Japan; Division of Molecular and Clinical Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; Department of Advanced Molecular and Cell Therapy, Kyushu University Hospital, Fukuoka, Japan. Electronic address: [email protected].

DOI: 10.1016/j.exphem.2019.03.001