The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance.
Base-Resolution Analysis of 5-Hydroxymethylcytosine in the Mammalian Genome.
Miao Yu, Gary C. Hon, Keith E. Szulwach, Chun-Xiao Song, Liang Zhang, Audrey Kim, Xuekun Li, Qing Dai, Yin Shen, Beomseok Park, Jung-Hyun Min, Peng Jinsend email, Bing Rensend email, Chuan He. Cell, Volume 149, Issue 6, 1368-1380, 17 May 2012
The image shows the concept of a new sequencing system capable of differentiating between cytosine, methylcytosine and 5-hydroxymethyl cytosine in the mammalian genome. This method will allow us to read the epigenome with resolution up to now unimaginable. The University of Chicago.