title: Epigenetic Silencing of Eyes Absent 4 Gene by Acute Myeloid Leukemia 1-Eight-twenty-one Oncoprotein Contributes to Leukemogenesis in t(8;21) Acute Myeloid Leukemia.
abstract: BACKGROUND
  The acute myeloid leukemia 1 (AML1)-eight-twenty-one (ETO) fusion protein generated by the t(8;21)(q22;q22) translocation is considered to display a crucial role in leukemogenesis in AML. By focusing on the anti-leukemia effects of eyes absent 4 (EYA4) gene on AML cells, we investigated the biologic and molecular mechanism associated with AML1-ETO expressed in t(8;21) AML.
  
  
  METHODS
  Qualitative polymerase chain reaction (PCR), quantitative reverse transcription PCR (RT-PCR), and Western blotting analysis were used to observe the mRNA and protein expression levels of EYA4 in cell lines. Different plasmids (including mutant plasmids) of dual luciferase reporter vector were built to study the binding status of AML1-ETO to the promoter region of EYA4. Chromatin immunoprecipitation assay was used to study the epigenetic silencing mechanism of EYA4. Bisulfite sequencing was applied to detect the methylation status in EYA4 promoter region. The influence of EYA4 gene in the cell proliferation, apoptosis, and cell clone-forming ability was detected by the technique of Cell Counting Kit-8, flow cytometry, and clonogenic assay.
  
  
  RESULTS
  EYA4 gene was hypermethylated in AML1-ETO+ patients and its expression was down-regulated by 6-fold in Kasumi-1 and SKNO-1 cells, compared to HL-60 and SKNO-1-siA/E cells, respectively. We demonstrated that AML1-ETO triggered the epigenetic silencing of EYA4 gene by binding at AML1-binding sites and recruiting histone deacetylase 1 and DNA methyltransferases. Enhanced EYA4 expression levels inhibited cellular proliferation and suppressed cell colony formation in AML1-ETO+ cell lines. We also found EYA4 transfection increased apoptosis of Kasumi-1 and SKNO-1 cells by 1.6-fold and 1.4-fold compared to negative control, respectively.
  
  
  CONCLUSIONS
  Our study identified EYA4 gene as targets for AML1-ETO and indicated it as a novel tumor suppressor gene. In addition, we provided evidence that EYA4 gene might be a novel therapeutic target and a potential candidate for treating AML1-ETO+ t (8;21) AML.
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: Sai
authors.initials: S
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Cancer Biology, First Hospital Affiliated to The Fourth Military Medical University, Xi'an, Shaanxi 710032, China.
authors.forename: Meng-Meng
authors.initials: MM
authors.lastname: Jiang
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: Guo-Feng
authors.initials: GF
authors.lastname: Chen
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Qian
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: Hong-Hao
authors.initials: HH
authors.lastname: Gao
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Guan
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: Jin-Long
authors.initials: JL
authors.lastname: Shi
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: An-Qi
authors.initials: AQ
authors.lastname: Liu
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: Bian-Hong
authors.initials: BH
authors.lastname: Wang
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: Yong-Hui
authors.initials: YH
authors.lastname: Li
authors.affiliation: Department of Hematology, Chinese PLA General Hospital, Beijing 100853, China.
authors.forename: Li
authors.initials: L
authors.lastname: Yu
mesh_terms: D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics / Q000502:physiology; D047369:Chromatin Immunoprecipitation; D050676:Core Binding Factor Alpha 2 Subunit / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation / Q000235:genetics; D044127:Epigenesis, Genetic / Q000235:genetics*; D020868:Gene Silencing; D018922:HL-60 Cells; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D000075142:RUNX1 Translocation Partner 1 Protein; D015531:Radioimmunoprecipitation Assay; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*
chemical_list: C107844:AML1-ETO fusion protein, human; D050676:Core Binding Factor Alpha 2 Subunit; C117736:EYA4 protein, human; D015514:Oncogene Proteins, Fusion; D034741:RNA, Small Interfering; D000075142:RUNX1 Translocation Partner 1 Protein; D015534:Trans-Activators