Abstract
Both 5-methylcytosine (5mC) and its oxidized form 5-hydroxymethylcytosine (5hmC) have been proposed to be involved in tumorigenesis. Because the readout of the broadly used 5mC mapping method, bisulfite sequencing (BS-seq), is the sum of 5mC and 5hmC levels, the 5mC/5hmC patterns and relationship of these two modifications remain poorly understood. By profiling real 5mC (BS-seq corrected by Tet-assisted BS-seq, TAB-seq) and 5hmC (TAB-seq) levels simultaneously at single-nucleotide resolution, we here demonstrate that there is no global loss of 5mC in kidney tumors compared with matched normal tissues. Conversely, 5hmC was globally lost in virtually all kidney tumor tissues. The 5hmC level in tumor tissues is an independent prognostic marker for kidney cancer, with lower levels of 5hmC associated with shorter overall survival. Furthermore, we demonstrated that loss of 5hmC is linked to hypermethylation in tumors compared with matched normal tissues, particularly in gene body regions. Strikingly, gene body hypermethylation was significantly associated with silencing of the tumor-related genes. Downregulation of IDH1 was identified as a mechanism underlying 5hmC loss in kidney cancer. Restoring 5hmC levels attenuated the invasion capacity of tumor cells and suppressed tumor growth in a xenograft model. Collectively, our results demonstrate that loss of 5hmC is both a prognostic marker and an oncogenic event in kidney cancer by remodeling the DNA methylation pattern.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Accession codes
References
Jones PA, Baylin SB . The fundamental role of epigenetic events in cancer. Nat Rev Genet 2002; 3:415–428.
Eden A, Gaudet F, Waghmare A, Jaenisch R . Chromosomal instability and tumors promoted by DNA hypomethylation. Science 2003; 300:455.
Herman JG, Baylin SB . Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003; 349:2042–2054.
Ehrlich M, Gama-Sosa MA, Huang LH, et al. Amount and distribution of 5-methylcytosine in human DNA from different types of tissues of cells. Nucleic Acids Res 1982; 10:2709–2721.
Gama-Sosa MA, Slagel VA, Trewyn RW, et al. The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res 1983; 11:6883–6894.
Ziller MJ, Gu H, Müller F, et al. Charting a dynamic DNA methylation landscape of the human genome. Nature 2013; 500:477–481.
Koh KP, Yabuuchi A, Rao S, et al. Tet1 and Tet2 regulate 5-hydroxymethylcytosine production and cell lineage specification in mouse embryonic stem cells. Cell Stem Cell 2011; 8:200–213.
Tahiliani M, Koh KP, Shen Y, et al. Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science 2009; 324:930–935.
Ko M, Huang Y, Jankowska AM, et al. Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2. Nature 2010; 468(7325):839–843.
Figueroa ME, Abdel-Wahab O, Lu C, et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 2010; 18:553–567.
Yamazaki J, Taby R, Vasanthakumar A, et al. Effects of TET2 mutations on DNA methylation in chronic myelomonocytic leukemia. Epigenetics 2012; 7:201–207.
Huang Y, Pastor WA, Shen Y, Tahiliani M, Liu DR, Rao A . The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One 2010; 5:e8888.
Jin SG, Kadam S, Pfeifer GP . Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res 2010; 38:e125.
Varela I, Tarpey P, Raine K, et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 2011; 469:539–542.
Network TCGAR . Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature 2013; 499:43–49.
Sato Y, Yoshizato T, Shiraishi Y, et al. Integrated molecular analysis of clear-cell renal cell carcinoma. Nat Genet 2013; 45:860–867.
Yu M, Hon GC, Szulwach KE, et al. Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 2012; 149:1368–1380.
Lian CG, Xu Y, Ceol C, et al. Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma. Cell 2012; 150:1135–1146.
Haffner MC, Chaux A, Meeker AK, et al. Global 5-hydroxymethylcytosine content is significantly reduced in tissue stem/progenitor cell compartments and in human cancers. Oncotarget 2011; 2:627–637.
Chen ML, Shen F, Huang W, et al. Quantification of 5-methylcytosine and 5-hydroxymethylcytosine in genomic DNA from hepatocellular carcinoma tissues by capillary hydrophilic-interaction liquid chromatography/quadrupole TOF mass spectrometry. Clin Chem 2013; 59:824–832.
Liu J, Ghanim M, Xue L, et al. Analysis of Drosophila segmentation network identifies a JNK pathway factor overexpressed in kidney cancer. Science 2009; 323:1218–1222.
Loenarz C, Schofield CJ . Expanding chemical biology of 2-oxoglutarate oxygenases. Nat Chem Biol 2008; 4:152–156.
Mellen M, Ayata P, Dewell S, Kriaucionis S, Heintz N . MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. Cell 2012; 151:1417–1430.
Xu Y, Wu F, Tan L, et al. Genome-wide regulation of 5hmC, 5mC, and gene expression by Tet1 hydroxylase in mouse embryonic stem cells. Mol Cell 2011; 42:451–464.
Guelen L, Pagie L, Brasset E, et al. Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions. Nature 2008; 453:948–951.
Turcan S, Rohle D, Goenka A, et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature 2012; 483(7390):479–483.
Quinlan AR, Hall IM . BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 2010; 26:841–842.
Berman BP, Weisenberger DJ, Aman JF, et al. Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat Genet 2012; 44:40–46.
Lister R, Weisenberger DJ, Aman JF, et al. Human DNA methylomes at base resolution show widespread epigenomic differences. Nature 2009; 462:315–322.
Feng S, Cokus SJ, Zhang X, et al. Conservation and divergence of methylation patterning in plants and animals. Proc Natl Acad Sci USA 2010; 107:8689–8694.
Zemach A, McDaniel IE, Silva P, Zilberman D . Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science 2010; 328:916–919.
Kaelin WG Jr. Molecular basis of the VHL hereditary cancer syndrome. Nat Rev Cancer 2002; 2:673–682.
Peric-Hupkes D, Meuleman W, Pagie L, et al. Molecular maps of the reorganization of genome-nuclear lamina interactions during differentiation. Mol Cell 2010; 38:603–613.
Jones PA . The DNA methylation paradox. Trends Genet 1999; 15:34–37.
Ward PS, Patel J, Wise DR, et al. The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate. Cancer Cell 2010; 17:225–234.
Shim EH, Livi CB, Rakheja D, et al. L-2-Hydroxyglutarate:an epigenetic modifier and putative oncometabolite in renal cancer. Cancer Discov 2014; 4:1290–1298.
Wang F, Travins J, DeLaBarre B, et al. Targeted inhibition of mutant IDH2 in leukemia cells induces cellular differentiation. Science 2013; 340:622–626.
Iyer LM, Tahiliani M, Rao A, Aravind L . Prediction of novel families of enzymes involved in oxidative and other complex modifications of bases in nucleic acids. Cell Cycle 2009; 8:1698–1710.
Jiang L, Zhang J, Wang JJ, et al. Sperm, but not oocyte, DNA methylome is inherited by zebrafish early embryos. Cell 2013; 153:773–784.
Krueger F, Andrews SR . Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 2011; 27:1571–1572.
Trapnell C, Pachter L, Salzberg SL . TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 2009; 25:1105–1111.
Trapnell C, Williams BA, Pertea G, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 2010; 28:511–555.
Anders S, Pyl PT, Huber W . HTSeq — a Python framework to work with high-throughput sequencing data. Bioinformatics 2015; 31:166–169.
Love MI, Huber W, Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014; 15:550.
Acknowledgements
We thank Dr Kevin P White for providing the TMA slides. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB13040000 to WC and JL), the National Basic Research Program of China (973 Program; 2015CB856200 to JL), the 863 High-Tech Foundation (2014AA020608 to YK), the National Natural Science Foundation of China (91231112, 81422035 and 91519307 to WC, 81372746 to LZ, 81101940 to KC, and 31200958 to JZ), the Youth Innovation Promotion Association of Chinese Academy of Sciences (to KC and JZ), the KC Wong Education Foundation (to JZ), and the Chinese Academy of Sciences Funds for distinguished young scientists (to JZ).
Author information
Authors and Affiliations
Corresponding authors
Additional information
( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1 (download PDF )
Global dynamics of 5mC and 5hmC levels in kidney cancer. (PDF 173 kb)
Supplementary information, Figure S2 (download PDF )
Genome-wide dynamic patterns of 5hmC during kidney tumorigenesis. (PDF 286 kb)
Supplementary information, Figure S3 (download PDF )
Genome-wide single nucleotide resolution mapping of 5mC by combing BS-seq and TAB-seq method. (PDF 434 kb)
Supplementary information, Figure S4 (download PDF )
Loss of 5hmC facilitates the gene body hypermethylation in tumor tissue and promotes tumorigenesis. (PDF 473 kb)
Supplementary information, Figure S5 (download PDF )
Manipulating 5hmC generating pathway has potential therapeutical effect. (PDF 188 kb)
Supplementary information, Table S1 (download PDF )
Summary of single-base 5hmC sequencing using TAB-seq. (PDF 15 kb)
Supplementary information, Table S2 (download PDF )
Summary of RNA-seq. (PDF 34 kb)
Supplementary information, Table S3 (download PDF )
Summary of shotgun bisulfite sequencing (BS-seq). (PDF 12 kb)
Supplementary information, Table S4 (download XLSX )
The consistency between TCGA KIRC 450K data and our BS-seq data. (XLSX 17 kb)
Supplementary information, Table S5 (download PDF )
Summary of DMS (differentially methylated sites). (PDF 48 kb)
Supplementary information, Table S6 (download PDF )
The overlaps between aberrant 5hmC and 5mC changes at single nucleotide resolution during tumorigenesis. (PDF 12 kb)
Supplementary information, Table S7 (download XLSX )
The GO terms of genes with 5mC/5hmC changes in gene body regions. (XLSX 17 kb)
Rights and permissions
About this article
Cite this article
Chen, K., Zhang, J., Guo, Z. et al. Loss of 5-hydroxymethylcytosine is linked to gene body hypermethylation in kidney cancer. Cell Res 26, 103–118 (2016). https://doi.org/10.1038/cr.2015.150
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/cr.2015.150
Keywords
This article is cited by
-
EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution
Clinical Epigenetics (2023)
-
Dissection of transcriptomic and epigenetic heterogeneity of grade 4 gliomas: implications for prognosis
Acta Neuropathologica Communications (2023)
-
Analysis of myosin genes in HNSCC and identify MYL1 as a specific poor prognostic biomarker, promotes tumor metastasis and correlates with tumor immune infiltration in HNSCC
BMC Cancer (2023)
-
Regional gain and global loss of 5-hydroxymethylcytosine coexist in genitourinary cancers and regulate different oncogenic pathways
Clinical Epigenetics (2022)
-
5-Hydroxymethylcytosine (5hmC) at or near cancer mutation hot spots as potential targets for early cancer detection
BMC Research Notes (2022)
