title: Kidney cancer is characterized by aberrant methylation of tissue-specific enhancers that are prognostic for overall survival.
abstract: OBJECTIVE
  Even though recent studies have shown that genetic changes at enhancers can influence carcinogenesis, most methylomic studies have focused on changes at promoters. We used renal cell carcinoma (RCC), an incurable malignancy associated with mutations in epigenetic regulators, as a model to study genome-wide patterns of DNA methylation at a high resolution.
  
  
  METHODS
  Analysis of cytosine methylation status of 1.3 million CpGs was determined by the HELP assay in RCC and healthy microdissected renal tubular controls.
  
  
  RESULTS
  We observed that the RCC samples were characterized by widespread hypermethylation that preferentially affected gene bodies. Aberrant methylation was particularly enriched in kidney-specific enhancer regions associated with H3K4Me1 marks. Various important underexpressed genes, such as SMAD6, were associated with aberrantly methylated, intronic enhancers, and these changes were validated in an independent cohort. MOTIF analysis of aberrantly hypermethylated regions revealed enrichment for binding sites of AP2a, AHR, HAIRY, ARNT, and HIF1 transcription factors, reflecting contributions of dysregulated hypoxia signaling pathways in RCC. The functional importance of this aberrant hypermethylation was demonstrated by selective sensitivity of RCC cells to low levels of decitabine. Most importantly, methylation of enhancers was predictive of adverse prognosis in 405 cases of RCC in multivariate analysis. In addition, parallel copy-number analysis from MspI representations demonstrated novel copy-number variations that were validated in an independent cohort of patients.
  
  
  CONCLUSIONS
  Our study is the first high-resolution methylome analysis of RCC, demonstrates that many kidney-specific enhancers are targeted by aberrant hypermethylation, and reveals the prognostic importance of these epigenetic changes in an independent cohort.
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Caroline Y
authors.initials: CY
authors.lastname: Hu
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Davoud
authors.initials: D
authors.lastname: Mohtat
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Yiting
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Renal Electrolyte and Hypertension Division, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.
authors.forename: Yi-An
authors.initials: YA
authors.lastname: Ko
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Niraj
authors.initials: N
authors.lastname: Shenoy
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Sanchari
authors.initials: S
authors.lastname: Bhattacharya
authors.affiliation: Renal Electrolyte and Hypertension Division, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Izquierdo
authors.affiliation: Renal Electrolyte and Hypertension Division, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.
authors.forename: Ae Seo Deok
authors.initials: AS
authors.lastname: Park
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Orsolya
authors.initials: O
authors.lastname: Giricz
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Nishanth
authors.initials: N
authors.lastname: Vallumsetla
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Krishna
authors.initials: K
authors.lastname: Gundabolu
authors.affiliation: Department of Pathology, Montefiore Medical Center, New York, New York.
authors.forename: Kristin
authors.initials: K
authors.lastname: Ware
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Tushar D
authors.initials: TD
authors.lastname: Bhagat
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: Masako
authors.initials: M
authors.lastname: Suzuki
authors.affiliation: Department of Pathology, Montefiore Medical Center, New York, New York.
authors.forename: James
authors.initials: J
authors.lastname: Pullman
authors.affiliation: Dana-Farber Cancer Institute, Harvard School of Public Health, Boston, Massachusetts.
authors.forename: X Shirley
authors.initials: XS
authors.lastname: Liu
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York.
authors.forename: John M
authors.initials: JM
authors.lastname: Greally
authors.affiliation: Renal Electrolyte and Hypertension Division, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania. amit.verma@einstein.yu.edu ksusztak@mail.med.upenn.edu.
authors.forename: Katalin
authors.initials: K
authors.lastname: Susztak
authors.affiliation: Albert Einstein College of Medicine, Bronx, New York. amit.verma@einstein.yu.edu ksusztak@mail.med.upenn.edu.
authors.forename: Amit
authors.initials: A
authors.lastname: Verma
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics; D002292:Carcinoma, Renal Cell / Q000235:genetics* / Q000401:mortality*; D016022:Case-Control Studies; D002478:Cells, Cultured; D015331:Cohort Studies; D019175:DNA Methylation; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D044127:Epigenesis, Genetic / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics* / Q000401:mortality*; D009928:Organ Specificity; D011379:Prognosis; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015996:Survival Rate
chemical_list: D014408:Biomarkers, Tumor; D012333:RNA, Messenger