title: Possible role of intragenic DNA hypermethylation in gene silencing of the tumor suppressor gene NR4A3 in acute myeloid leukemia.
abstract: Expression of the tumor suppressor gene NR4A3 is silenced in the blasts of acute myeloid leukemia (AML), irrespective of the karyotype. Although the transcriptional reactivation of NR4A3 is considered to have a broad-spectrum anti-leukemic effect, the therapeutic modalities targeting this gene have been hindered by our minimal understanding of the transcriptional mechanisms regulating its expression, particularly in human AML. Here we show the role of intragenic DNA hypermethylation in reducing the expression of NR4A3 in AML. Bisulfite sequencing analysis revealed that CpG sites at the intragenic region encompassing exon 3 of NR4A3, but not the promoter region, are hypermethylated in AML cell lines and primary AML cells. A DNA methyltransferase inhibitor restored the expression of NR4A3 following a reduction in DNA methylation levels at intragenic CpG sites. The in silico data revealed an enrichment of H3K4me1 and H2A.Z at exon 3 of NR4A3 in human non-malignant cells but that was excluded specifically in leukemia cells with CpG hypermethylation. This suggests that exon 3 represents a functional regulatory element involved in the transcriptional regulation of NR4A3. Our findings improve the current understanding of the mechanism underlying NR4A3 silencing and facilitate the development of NR4A3-targeted therapy.
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan; Department of Clinical Cell Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Ryo
authors.initials: R
authors.lastname: Shimizu
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan. Electronic address: tomoya.muto@gmail.com.
authors.forename: Tomoya
authors.initials: T
authors.lastname: Muto
authors.affiliation: Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Kazumasa
authors.initials: K
authors.lastname: Aoyama
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
authors.forename: Kwangmin
authors.initials: K
authors.lastname: Choi
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takeuchi
authors.affiliation: Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Shuhei
authors.initials: S
authors.lastname: Koide
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan; Department of Clinical Cell Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Nagisa
authors.initials: N
authors.lastname: Hasegawa
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan; Department of Clinical Cell Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Isshiki
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan; Department of Clinical Cell Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Emi
authors.initials: E
authors.lastname: Togasaki
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan; Department of Clinical Cell Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Chika
authors.initials: C
authors.lastname: Kawajiri-Manako
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan; Department of Clinical Cell Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Yuhei
authors.initials: Y
authors.lastname: Nagao
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan.
authors.forename: Shokichi
authors.initials: S
authors.lastname: Tsukamoto
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan; Department of Transfusion Medicine and Cell Therapy, Chiba University Hospital, Chiba, Japan.
authors.forename: Shio
authors.initials: S
authors.lastname: Sakai
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Takeda
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan; Department of Transfusion Medicine and Cell Therapy, Chiba University Hospital, Chiba, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Mimura
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan.
authors.forename: Chikako
authors.initials: C
authors.lastname: Ohwada
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan.
authors.forename: Emiko
authors.initials: E
authors.lastname: Sakaida
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan; Department of Transfusion Medicine and Cell Therapy, Chiba University Hospital, Chiba, Japan.
authors.forename: Tohru
authors.initials: T
authors.lastname: Iseki
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Starczynowski
authors.affiliation: Department of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Iwama
authors.affiliation: Department of Clinical Cell Biology and Medicine, Graduate School of Medicine, Chiba University, Chiba, Japan.
authors.forename: Koutaro
authors.initials: K
authors.lastname: Yokote
authors.affiliation: Department of Hematology, Chiba University Hospital, Chiba, Japan.
authors.forename: Chiaki
authors.initials: C
authors.lastname: Nakaseko
mesh_terms: D000328:Adult; D000368:Aged; D000964:Antimetabolites, Antineoplastic; D001374:Azacitidine / Q000031:analogs & derivatives / Q000494:pharmacology; D001752:Blast Crisis / Q000235:genetics / Q000473:pathology; D016022:Case-Control Studies; D002478:Cells, Cultured; D018899:CpG Islands; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics*; D000077209:Decitabine; D005091:Exons / Q000235:genetics; D005260:Female; D015973:Gene Expression Regulation, Leukemic; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D011987:Receptors, Steroid / Q000235:genetics*; D011988:Receptors, Thyroid Hormone / Q000235:genetics*
chemical_list: D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; C490412:NR4A3 protein, human; D011987:Receptors, Steroid; D011988:Receptors, Thyroid Hormone; D000077209:Decitabine; D001374:Azacitidine
keywords: Acute myeloid leukemia; DNA hypermethylation; NR4A3; Tumor suppressor gene