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Tahiliani, Mamta V.

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Tahiliani

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Mamta V.

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Tahiliani, Mamta V.
Author's Publications: search.filters.isAuthorOfPublication.55a8472b-2e9f-47ce-b24b-352d891eeed4

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    Conversion of 5-Methylcytosine to 5-Hydroxymethylcytosine in Mammalian DNA by the MLL Partner TET1
    (American Association for the Advancement of Science, 2009) Tahiliani, Mamta V.; Koh, Kian Peng; Shen, Yinghua; Pastor, William Abraham; Bandukwala, Hozefa S; Brudno, Yevgeny; Agarwal, Suneet; Iyer, Lakshminarayan M.; Liu, David; Aravind, L; Rao, Anjana
    DNA cytosine methylation is crucial for retrotransposon silencing and mammalian development. In a computational search for enzymes that could modify 5-methylcytosine (5mC), we identified TET proteins as mammalian homologs of the trypanosome proteins JBP1 and JBP2, which have been proposed to oxidize the 5-methyl group of thymine. We show here that TET1, a fusion partner of the MLL gene in acute myeloid leukemia, is a 2-oxoglutarate (2OG)- and Fe(II)-dependent enzyme that catalyzes conversion of 5mC to 5-hydroxymethylcytosine (hmC) in cultured cells and in vitro. hmC is present in the genome of mouse embryonic stem cells, and hmC levels decrease upon RNA interference–mediated depletion of TET1. Thus, TET proteins have potential roles in epigenetic regulation through modification of 5mC to hmC.
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    The Behaviour of 5-Hydroxymethylcytosine in Bisulfite Sequencing
    (Public Library of Science, 2010) Huang, Yun; Pastor, William Abraham; Shen, Yinghua; Tahiliani, Mamta V.; Liu, David; Rao, Anjana; Liu, Jun
    Background: We recently showed that enzymes of the TET family convert 5-mC to 5-hydroxymethylcytosine (5-hmC) in DNA. 5-hmC is present at high levels in embryonic stem cells and Purkinje neurons. The methylation status of cytosines is typically assessed by reaction with sodium bisulfite followed by PCR amplification. Reaction with sodium bisulfite promotes cytosine deamination, whereas 5-methylcytosine (5-mC) reacts poorly with bisulfite and is resistant to deamination. Since 5-hmC reacts with bisulfite to yield cytosine 5-methylenesulfonate (CMS), we asked how DNA containing 5-hmC behaves in bisulfite sequencing. Methodology/Principal Findings: We used synthetic oligonucleotides with different distributions of cytosine as templates for generation of DNAs containing C, 5-mC and 5-hmC. The resulting DNAs were subjected in parallel to bisulfite treatment, followed by exposure to conditions promoting cytosine deamination. The extent of conversion of 5-hmC to CMS was estimated to be 99.7%. Sequencing of PCR products showed that neither 5-mC nor 5-hmC undergo C-to-T transitions after bisulfite treatment, confirming that these two modified cytosine species are indistinguishable by the bisulfite technique. DNA in which CMS constituted a large fraction of all bases (28/201) was much less efficiently amplified than DNA in which those bases were 5-mC or uracil (the latter produced by cytosine deamination). Using a series of primer extension experiments, we traced the inefficient amplification of CMS-containing DNA to stalling of Taq polymerase at sites of CMS modification, especially when two CMS bases were either adjacent to one another or separated by 1–2 nucleotides.Conclusions We have confirmed that the widely used bisulfite sequencing technique does not distinguish between 5-mC and 5-hmC. Moreover, we show that CMS, the product of bisulfite conversion of 5-hmC, tends to stall DNA polymerases during PCR, suggesting that densely hydroxymethylated regions of DNA may be underrepresented in quantitative methylation analyses.