5-hydroxymethylcytosine in cancer: significance in diagnosis and therapy

Vasanthakumar, Aparna; Godley, Lucy A.

doi:10.1016/j.cancergen.2015.02.009

Cited by 81 publications

(75 citation statements)

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“…In particular, it seems important for pluripotence of mouse embryonic stem cells (Tahiliani et al ., 2009; Ito et al ., 2010; Koh et al ., 2011; Dawlaty et al ., 2014) and is preferentially associated with transcriptionally active genes (Ficz et al ., 2011; Pastor et al ., 2011; Wu et al ., 2011; Xu et al ., 2011; Kubiura et al ., 2012; Mellén et al ., 2012). Studies reporting a link between 5hmeC and many diseases, in particular cancers (review (Vasanthakumar and Godley, 2015)) and neurologic disorders such as autism (Papale et al ., 2015), have also greatly increased over the last few years. Due to its link to DNA demethylation and to these functions, 5hmeC appears as a potential crucial mark during early development.…”

Section: Introductionmentioning

confidence: 99%

Assessment of ‘one-step’ versus ‘sequential’ embryo culture conditions through embryonic genome methylation and hydroxymethylation changes

et al. 2016

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STUDY QUESTIONIn comparison to in vivo development, how do different conditions of in vitro culture (‘one step’ versus ‘sequential medium’) impact DNA methylation and hydroxymethylation in preimplantation embryos?SUMMARY ANSWERUsing rabbit as a model, we show that DNA methylation and hydroxymethylation are both affected by in vitro culture of preimplantation embryos and the effect observed depends on the culture medium used.WHAT IS KNOWN ALREADYCorrect regulation of DNA methylation is essential for embryonic development and DNA hydroxymethylation appears more and more to be a key player. Modifications of the environment of early embryos are known to have long term effects on adult phenotypes and health; these probably rely on epigenetic alterations.STUDY DESIGN SIZE, DURATIONThe study design we used is both cross sectional (control versus treatment) and longitudinal (time-course). Each individual in vivo experiment used embryos flushed from the donor at the 2-, 4-, 8-, 16- or morula stage. Each stage was analyzed in at least two independent experiments. Each individual in vitro experiment used embryos flushed from donors at the 1-cell stage (19 h post-coïtum) which were then cultured in parallel in the two tested media until the 2-, 4-, 8- 16-cell or morula stages. Each stage was analyzed in at least three independent experiments. In both the in vivo and in vitro experiments, 4-cell stage embryos were always included as an internal reference.PARTICIPANTS/MATERIALS, SETTING, METHODSImmunofluorescence with antibodies specific for 5-methylcytosine (5meC) and 5-hydroxymethylcytosine (5hmeC) was used to quantify DNA methylation and hydroxymethylation levels in preimplantation embryos. We assessed the expression of DNA methyltransferases (DNMT), of ten eleven translocation (TET) dioxigenases and of two endogenous retroviral sequences (ERV) using RT-qPCR, since the expression of endogenous retroviral sequences is known to be regulated by DNA methylation. Three repeats were first done for all stages; then three additional repetitions were performed for those stages showing differences or tendencies toward differences between the different conditions in the first round of quantification.MAIN RESULTS AND THE ROLE OF CHANCEThe kinetics of DNA methylation and hydroxymethylation were modified in in vitro cultured embryos, and the observed differences depended on the type of medium used. These differences were statistically significant. In addition, the expression of TET1 and TET2 was significantly reduced in post-embryonic genome activation (EGA) embryos after in vitro culture in both tested conditions. Finally, the expression of two retroviral sequences was analyzed and found to be significantly affected by in vitro culture.LIMITATIONS REASONS FOR CAUTIONOur study remains mostly descriptive as no direct link can be established between the epigenetic changes observed and the expression changes in both effectors and targets of the studied epigenetic modifications. The results we obtained suggest that gene expression could be a...

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Section: Introductionmentioning

confidence: 99%

Assessment of ‘one-step’ versus ‘sequential’ embryo culture conditions through embryonic genome methylation and hydroxymethylation changes

et al. 2016

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“…Indeed, the ten-eleven translocation family of proteins has been shown to function as enzymes capable of altering the methylation status of DNA by oxidizing 5mC to 5-hydroxymethylcytosine (5hmC). Emerging evidence has suggested that 5hmC may act both as a transient intermediate in the process of 5mC demethylation and a functional epigenetic mark that regulates transcription (Bachman et al, 2014;Vasanthakumar and Godley, 2015).…”

Section: Introductionmentioning

confidence: 99%

OxyBS: estimation of 5-methylcytosine and 5-hydroxymethylcytosine from tandem-treated oxidative bisulfite and bisulfite DNA

2016

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Summary: The use of sodium bisulfite (BS) treatment followed by hybridization to an Illumina Infinium BeadChip (HumanMethylation450 and MethylationEPIC) is a common method for interrogating 5-methylcytosine (5mC) at single nucleotide resolution. However, standard treatment of DNA with BS does not allow disambiguation of 5mC from an additional cytosine modification, 5-hydroxymethylcytosine (5hmC). Recently, it has been demonstrated that paired BS and oxidative bisulfite (oxBS) treatment on the same sample followed by hybridization to an Infinium microarray permits the differentiation of 5hmC from 5mC. Nevertheless, estimation of 5hmC and 5mC from tandem-treated arrays has been shown to produce irregular estimates of cytosine modifications. Results: We present a novel method using maximum likelihood estimation to accurately estimate the parameters of unmethylated cytosine (5C), 5mC and 5hmC from Infinium microarray data given the signal intensities from the oxBS and BS replicates. Availability and Implementation: OxyBS is an R package available on CRAN.

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“…5-hmC is also likely to have its own epigenetic function beyond simply being a demethylating base (3). The highest levels of 5-hmC are found in stem cells of various origins and in neural tissues (6,7).…”

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confidence: 99%

“…The TET enzymes convert the 5-methylcytosine (5-mC) in DNA sequentially to 5-hydroxymethylcytosine (5-hmC), 5-formylcytocine, and 5-carboxylcytocine, leading to DNA demethylation (3,(5)(6)(7). 5-hmC is also likely to have its own epigenetic function beyond simply being a demethylating base (3).…”

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confidence: 99%

“…The substrates for 2-OGDDs vary from proteins to DNA, RNA, and fatty acids (1). Interestingly, a large number of 2-OGDDs act on the chromatin structure, most notably the ten-eleven-translocation 5-methylcytosine dioxygenases (TETs) and the Jumonji domain-containing histone demethylases (1)(2)(3). The stability of the ␣ subunit of the key regulator of the hypoxia response, the hypoxia-inducible factor (HIF), is also regulated by 2-OGDDs, namely the HIF-prolyl 4-hydroxylases (HIF-P4Hs), also known as PHDs and EglNs (1,2,4).…”

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confidence: 99%

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Fumarate and Succinate Regulate Expression of Hypoxia-inducible Genes via TET Enzymes

Laukka

Mariani

Ihantola

et al. 2016

Journal of Biological Chemistry

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253

204

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The TET enzymes are members of the 2-oxoglutarate-dependent dioxygenase family and comprise three isoenzymes in humans: TETs 1-3. These TETs convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA, and high 5-hmC levels are associated with active transcription. The importance of the balance in these modified cytosines is emphasized by the fact that TET2 is mutated in several human cancers, including myeloid malignancies such as acute myeloid leukemia (AML). We characterize here the kinetic and inhibitory properties of Tets and show that the K m value of Tets 1 and 2 for O 2 is 30 M, indicating that they retain high activity even under hypoxic conditions. The AML-associated mutations in the Fe 2؉ and 2-oxoglutaratebinding residues increased the K m values for these factors 30 -80-fold and reduced the V max values. Fumarate and succinate, which can accumulate to millimolar levels in succinate dehydrogenase and fumarate hydratase-mutant tumors, were identified as potent Tet inhibitors in vitro, with IC 50 values ϳ400 -500 M. Fumarate and succinate also down-regulated global 5-hmC levels in neuroblastoma cells and the expression levels of some hypoxia-inducible factor (HIF) target genes via TET inhibition, despite simultaneous HIF␣ stabilization. The combination of fumarate or succinate treatment with TET1 or TET3 silencing caused differential effects on the expression of specific HIF target genes. Altogether these data show that hypoxia-inducible genes are regulated in a multilayered manner that includes epigenetic regulation via TETs and 5-hmC levels in addition to HIF stabilization.The 2-oxoglutarate-dependent dioxygenases (2-OGDDs) 2 comprise an enzyme family of about 70 members in humans (1, 2). These enzymes all share the same basic reaction mechanism, in which the substrate is hydroxylated by molecular oxygen in the presence of a divalent metal cofactor (most commonly Fe 2ϩ ) and the 2-oxoglutarate cosubstrate is decarboxylated to succinate and CO 2 (1). The substrates for 2-OGDDs vary from proteins to DNA, RNA, and fatty acids (1). Interestingly, a large number of 2-OGDDs act on the chromatin structure, most notably the ten-eleven-translocation 5-methylcytosine dioxygenases (TETs) and the Jumonji domain-containing histone demethylases (1-3). The stability of the ␣ subunit of the key regulator of the hypoxia response, the hypoxia-inducible factor (HIF), is also regulated by 2-OGDDs, namely the HIF-prolyl 4-hydroxylases (HIF-P4Hs), also known as PHDs and EglNs (1, 2, 4).The TET enzymes convert the 5-methylcytosine (5-mC) in DNA sequentially to 5-hydroxymethylcytosine (5-hmC), 5-formylcytocine, and 5-carboxylcytocine, leading to DNA demethylation (3, 5-7). 5-hmC is also likely to have its own epigenetic function beyond simply being a demethylating base (3). The highest levels of 5-hmC are found in stem cells of various origins and in neural tissues (6, 7). There are three human TET isoenzymes. TET1 is highly expressed in embryonic stem cells, whereas TETs 2 and 3 are required for normal hematopoiesis...

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5-hydroxymethylcytosine in cancer: significance in diagnosis and therapy

Cited by 81 publications

References 158 publications

Assessment of ‘one-step’ versus ‘sequential’ embryo culture conditions through embryonic genome methylation and hydroxymethylation changes

Assessment of ‘one-step’ versus ‘sequential’ embryo culture conditions through embryonic genome methylation and hydroxymethylation changes

OxyBS: estimation of 5-methylcytosine and 5-hydroxymethylcytosine from tandem-treated oxidative bisulfite and bisulfite DNA

Fumarate and Succinate Regulate Expression of Hypoxia-inducible Genes via TET Enzymes

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