Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis
Alexander Meissner,Andreas Gnirke,George W. Bell,Bernard Ramsahoye,Eric S. Lander,Rudolf Jaenisch +5 more
TLDR
Findings indicate random loss rather than specific maintenance of methylation in Dnmt[1kd,3a−/−,3b−/ −] cells, and suggest that random shotgun bisulfite sequencing can be scaled to a genome-wide approach.Abstract:
We describe a large-scale random approach termed reduced representation bisulfite sequencing (RRBS) for analyzing and comparing genomic methylation patterns. BglII restriction fragments were size-selected to 500-600 bp, equipped with adapters, treated with bisulfite, PCR amplified, cloned and sequenced. We constructed RRBS libraries from murine ES cells and from ES cells lacking DNA methyltransferases Dnmt3a and 3b and with knocked-down (kd) levels of Dnmt1 (Dnmt[1(kd),3a-/-,3b-/-]). Sequencing of 960 RRBS clones from Dnmt[1(kd),3a-/-,3b-/-] cells generated 343 kb of non-redundant bisulfite sequence covering 66212 cytosines in the genome. All but 38 cytosines had been converted to uracil indicating a conversion rate of >99.9%. Of the remaining cytosines 35 were found in CpG and 3 in CpT dinucleotides. Non-CpG methylation was >250-fold reduced compared with wild-type ES cells, consistent with a role for Dnmt3a and/or Dnmt3b in CpA and CpT methylation. Closer inspection revealed neither a consensus sequence around the methylated sites nor evidence for clustering of residual methylation in the genome. Our findings indicate random loss rather than specific maintenance of methylation in Dnmt[1(kd),3a-/-,3b-/-] cells. Near-complete bisulfite conversion and largely unbiased representation of RRBS libraries suggest that random shotgun bisulfite sequencing can be scaled to a genome-wide approach.read more
Citations
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Integrative analysis of 111 reference human epigenomes
Anshul Kundaje,Wouter Meuleman,Wouter Meuleman,Jason Ernst,Misha Bilenky,Angela Yen,Angela Yen,Alireza Heravi-Moussavi,Pouya Kheradpour,Pouya Kheradpour,Zhizhuo Zhang,Zhizhuo Zhang,Jianrong Wang,Jianrong Wang,Michael J. Ziller,Viren Amin,John W. Whitaker,Matthew D. Schultz,Lucas D. Ward,Lucas D. Ward,Abhishek Sarkar,Abhishek Sarkar,Gerald Quon,Gerald Quon,Richard Sandstrom,Matthew L. Eaton,Matthew L. Eaton,Yi-Chieh Wu,Yi-Chieh Wu,Andreas R. Pfenning,Andreas R. Pfenning,Xinchen Wang,Xinchen Wang,Melina Claussnitzer,Melina Claussnitzer,Yaping Liu,Yaping Liu,Cristian Coarfa,R. Alan Harris,Noam Shoresh,Charles B. Epstein,Elizabeta Gjoneska,Elizabeta Gjoneska,Danny Leung,Wei Xie,R. David Hawkins,Ryan Lister,Chibo Hong,Philippe Gascard,Andrew J. Mungall,Richard A. Moore,Eric Chuah,Angela Tam,Theresa K. Canfield,R. Scott Hansen,Rajinder Kaul,Peter J. Sabo,Mukul S. Bansal,Mukul S. Bansal,Mukul S. Bansal,Annaick Carles,Jesse R. Dixon,Kai How Farh,Soheil Feizi,Soheil Feizi,Rosa Karlic,Ah Ram Kim,Ah Ram Kim,Ashwinikumar Kulkarni,Daofeng Li,Rebecca F. Lowdon,Ginell Elliott,Tim R. Mercer,Shane Neph,Vitor Onuchic,Paz Polak,Paz Polak,Nisha Rajagopal,Pradipta R. Ray,Richard C Sallari,Richard C Sallari,Kyle Siebenthall,Nicholas A Sinnott-Armstrong,Nicholas A Sinnott-Armstrong,Michael Stevens,Robert E. Thurman,Jie Wu,Bo Zhang,Xin Zhou,Arthur E. Beaudet,Laurie A. Boyer,Philip L. De Jager,Philip L. De Jager,Peggy J. Farnham,Susan J. Fisher,David Haussler,Steven J.M. Jones,Steven J.M. Jones,Wei Li,Marco A. Marra,Michael T. McManus,Shamil R. Sunyaev,Shamil R. Sunyaev,James A. Thomson,Thea D. Tlsty,Li-Huei Tsai,Li-Huei Tsai,Wei Wang,Robert A. Waterland,Michael Q. Zhang,Lisa Helbling Chadwick,Bradley E. Bernstein,Bradley E. Bernstein,Bradley E. Bernstein,Joseph F. Costello,Joseph R. Ecker,Martin Hirst,Alexander Meissner,Aleksandar Milosavljevic,Bing Ren,John A. Stamatoyannopoulos,Ting Wang,Manolis Kellis,Manolis Kellis +123 more
TL;DR: It is shown that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease.
Integrative analysis of 111 reference human epigenomes
Anshul Kundaje,Wouter Meuleman,Jason Ernst,Angela Yen,Pouya Kheradpour,Zhizhuo Zhang,Jianrong Wang,Lucas D. Ward,Abhishek Sarkar,Gerald Quon,Matthew L. Eaton,Yi-Chieh Wu,Andreas R. Pfenning,Xinchen Wang,Melina Claussnitzer,Yaping Liu,Mukul S. Bansal,Soheil Feizi-Khankandi,Ah Ram Kim,Richard C Sallari,Nicholas A Sinnott-Armstrong,Laurie A. Boyer,Elizabeta Gjoneska,Li-Huei Tsai,Manolis Kellis +24 more
TL;DR: In this article, the authors describe the integrative analysis of 111 reference human epigenomes generated as part of the NIH Roadmap Epigenomics Consortium, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression.
Journal ArticleDOI
Coming of age: ten years of next-generation sequencing technologies
TL;DR: These and other strategies are providing researchers and clinicians a variety of tools to probe genomes in greater depth, leading to an enhanced understanding of how genome sequence variants underlie phenotype and disease.
Journal ArticleDOI
In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state
Marius Wernig,Alexander Meissner,Ruth K. Foreman,Tobias Brambrink,Manching Ku,Konrad Hochedlinger,Konrad Hochedlinger,Bradley E. Bernstein,Bradley E. Bernstein,Rudolf Jaenisch +9 more
TL;DR: The results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.
Journal ArticleDOI
Polycomb complexes repress developmental regulators in murine embryonic stem cells
Laurie A. Boyer,Kathrin Plath,Kathrin Plath,Julia Zeitlinger,Tobias Brambrink,Lea Ann Medeiros,Tong Ihn Lee,Stuart S. Levine,Marius Wernig,Adriana Tajonar,Mridula K. Ray,George W. Bell,Arie P. Otte,Miguel Vidal,David K. Gifford,Richard A. Young,Rudolf Jaenisch +16 more
TL;DR: It is shown that PcG proteins directly repress a large cohort of developmental regulators in murine ES cells, the expression of which would otherwise promote differentiation, and dynamic repression of developmental pathways by Polycomb complexes may be required for maintaining ES cell pluripotency and plasticity during embryonic development.
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