title: Epigenetic regulation of CDH1 exon 8 alternative splicing in gastric cancer.
abstract: BACKGROUND
  The tumor suppressor gene CDH1 is critical for intercellular adhesion. In our previous work, we reported a nonfunctional CDH1 transcript that lacks the final 83 base pairs of exon 8 (1054del83). In this work, we probed the role of histone epigenetic modifications as well as DNA methylation in selection of this isoform.
  
  
  METHODS
  RT-qPCR was used to detect CDH1 RNA expression. Methylation of CDH1 was analyzed by bisulphite sequencing PCR. ChIP assay was performed to show histones level. Cell lines were treated with DNA methyltransferase inhibitor AZA, HDAC inhibitor TSA, or siRNA oligonucleotides to test regulation of CDH1 splicing.
  
  
  RESULTS
  Greater CDH1 1054del83 transcripts were observed in gastric cancer (GC) cell lines than human gastric mucosal epithelial cell line GES-1. All the cell lines showed significant methylation pattern at the CpG sites of CDH1 exon 8. AZA treatment did not influence selection of 1054del83 transcripts. A significant decrease in acetylation for histones H3 and H4K16Ac in an internal region of the CDH1 gene surrounding the alternative exon 8 were detected in GC cell lines. Treatment with TSA preferentially expressed the correctly spliced transcript and not the exon 8 skipped aberrant transcripts, showing that histone acetylation was involved in the splicing regulation. SiRNA-mediated knockdown of SETD2 (The specific methyltransferase of H3K36) decreased exclusion of exon 8, suggesting that the presence of this mark correlates with increased skipping of the final 83 base pairs of CDH1 exon 8. However, CDH1 splicing was not affected by SRSF2 knockdown.
  
  
  CONCLUSIONS
  H3K36me3 correlates with increased skipping of the final 83 base pairs of CDH1 exon 8. Histone acetylation was involved in the splicing regulation as well.
authors.affiliation: Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. lixiaowei0825@126.com.
authors.forename: Xiao-Wei
authors.initials: XW
authors.lastname: Li
authors.affiliation: Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. yellowistone@126.com.
authors.forename: Bing-Yu
authors.initials: BY
authors.lastname: Shi
authors.affiliation: Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. 348609649@qq.com.
authors.forename: Qing-Lan
authors.initials: QL
authors.lastname: Yang
authors.affiliation: Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. airlamb@foxmail.com.
authors.forename: Jie
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. b101230079@smail.nju.edu.cn.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Wu
authors.affiliation: Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. b101230076@smail.nju.edu.cn.
authors.forename: Yu-Feng
authors.initials: YF
authors.lastname: Wang
authors.affiliation: Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. wzjlufy@sina.com.
authors.forename: Zhi-Jiao
authors.initials: ZJ
authors.lastname: Wu
authors.affiliation: Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. ymfan@nju.edu.cn.
authors.forename: Yi-Mei
authors.initials: YM
authors.lastname: Fan
authors.affiliation: Department of Medical Genetics, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Hankou Road 22, Nanjing, 210093, China. wangyap@nju.edu.cn.
authors.forename: Ya-Ping
authors.initials: YP
authors.lastname: Wang
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D015703:Antigens, CD; D001483:Base Sequence; D015820:Cadherins / Q000235:genetics*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D019175:DNA Methylation / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000235:genetics*; D005091:Exons / Q000235:genetics; D006657:Histones / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics*
chemical_list: D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; D006657:Histones