Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine

Booth, Michael J.; Ost, Tobias W B; Beraldi, Dario; Bell, Neil M.; Branco, Miguel R.; Reik, Wolf; Balasubramanian, Shankar

doi:10.1038/nprot.2013.115

Cited by 310 publications

(217 citation statements)

References 28 publications

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“…We note that the bisulphite data do not rule out the presence of hm 5 C, which is also resistant to bisulphite conversion. Oxidative bisulphite sequencing, which can distinguish m 5 C and hm 5 C in DNA (Booth et al , 2013), resulted in degradation of the RNA. However, hm 5 C was not observed upon ABH1 oxidation in vitro and had no beneficial effect in ribosome binding assays, suggesting that this modification might not play a major role for mt‐tRNA Met .…”

Section: Resultsmentioning

confidence: 99%

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

et al. 2016

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Mitochondrial gene expression uses a non‐universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt‐)tRNAM et mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt‐tRNAM et to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt‐tRNAM et to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m5C34 mt‐tRNA Met in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt‐tRNAM et function. Together, our data reveal how modifications in mt‐tRNAM et are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAM et to recognise the different codons encoding methionine.

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Section: Resultsmentioning

confidence: 99%

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

et al. 2016

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“…However, the recent discovery of endogeneous TET-mediated conversion of 5-methylcytosine (5mC) back to the unmethylated state through 5hmC, 5fC, and 5caC intermediates has raised some concerns over the interpretation of the past methylation data produced by bisulfite modification technique, since this technique is unable to distinguish between 5mC and 5hmC, leaving them both as cytosines [133]. Although some new techniques such as oxidative bisulfite sequencing have been developed to make this distinction possible, the number of studies using such methods yet remains limited [134,135]. Taken together, although the exact cause and effect relationship between epigenetics and male infertility has not been elucidated, further investigation of this area holds a significant potential and great promise for understanding the molecular mechanisms of infertility.…”

Section: Resultsmentioning

confidence: 99%

The role of epigenetics in idiopathic male infertility

Güneş

Arslan

Hekim

et al. 2016

J Assist Reprod Genet

109

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Infertility is a complex disorder with multiple genetic and environmental causes. Although some specific mutations have been identified, other factors responsible for sperm defects remain largely unknown. Despite considerable efforts to identify the pathophysiology of the disease, we cannot explain the underlying mechanisms of approximately half of infertility cases. This study reviews current data on epigenetic regulation and idiopathic male infertility. Recent data have shown an association between epigenetic modifications and idiopathic infertility. In this regard, epigenetics has emerged as one of the promising research areas in understanding male infertility. Many studies have indicated that epigenetic modifications, including DNA methylation in imprinted and developmental genes, histone tail modifications and short non-coding RNAs in spermatozoa may have a role in idiopathic male infertility.

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“…41 To examine this possibility, we determined the 5hmC content of the hypermethylated ERV-9 LTR by oxBS-Sseq. 53 The results showed that 5hmCs comprised a very minor portion of total 5mCs (< 5%; Fig. 2e).…”

Section: Hypermethylated Erv-9 Ltr Possesses In Vivo Enhancer Activitymentioning

confidence: 96%

Hypermethylated LTR retrotransposon exhibits enhancer activity

Zhu

et al. 2017

Epigenetics

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LTR retrotransposons are repetitive DNA elements comprising »10% of the human genome. They are silenced by hypermethylation of cytosines in CpG dinucleotides and are considered parasitic DNA serving no useful function for the host genome. However, hypermethylated LTRs contain enhancer and promoter sequences and can promote tissue-specific transcription of cis-linked genes. To resolve the apparent paradox of hypermethylated LTRs possessing transcriptional activities, we studied the ERV-9 LTR retrotransposon located at the 5 0 border of the transcriptionally active b-globin gene locus in human erythroid progenitor and erythroleukemia K562 cells. We found that the ERV-9 LTR, containing 65 CpGs in 1.7 kb DNA, was hypermethylated (with > 90% methylated CpGs). Hypermethylated LTR possessed transcriptional enhancer activity, since in vivo deletion of the LTR by CRISPR-cas9 suppressed transcription of the globin genes by > 50%. ChIP-qPCR and ChIP-seq studies showed that the hypermethylated LTR enhancer spanning recurrent CCAATCG and GATA motifs associated respectively with key transcription factors (TFs) NF-Y and GATA-1 and -2 at reduced levels, compared with the unmethylated LTR in transfected LTR-reporter gene plasmids. Electrophoretic mobility shift assays with methylated LTR enhancer probe showed that the methylated probe bound both NF-Y and GATA-1 and -2 with lower affinities than the unmethylated enhancer probe. Thus, hypermethylation drastically reduced, but did not totally abolish, the binding affinities of the enhancer motifs to the key TFs to assemble the LTR-pol II transcription complex that activated transcription of cis-linked genes at reduced efficiency.

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Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine

Cited by 310 publications

References 28 publications

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

The role of epigenetics in idiopathic male infertility

Hypermethylated LTR retrotransposon exhibits enhancer activity

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Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine

Cited by 310 publications

References 28 publications

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

The role of epigenetics in idiopathic male infertility

Hypermethylated LTR retrotransposon exhibits enhancer activity

Contact Info

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NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation