Genome-wide high throughput analysis of DNA methylation in eukaryotes

Pomraning, Kyle R.; Smith, Kristina M.; Freitag, Michael

doi:10.1016/j.ymeth.2008.09.022

Cited by 185 publications

(133 citation statements)

References 67 publications

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“…Methylated fractions of genomes can also be sequenced using a combination of NGS and methyl-DNA immunoprecipitation (MeDIP). By using this approach, detailed methylation maps of the genome will become available (Pomraning et al, 2009), and by comparing these for different samples we will be able to address the importance of methylation for a large range of ecologically and evolutionary important questions, like the genetic architecture behind differential gene expression because of natural selection.…”

Section: Epigeneticsmentioning

confidence: 99%

Applications of next generation sequencing in molecular ecology of non-model organisms

2010

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As most biologists are probably aware, technological advances in molecular biology during the last few years have opened up possibilities to rapidly generate large-scale sequencing data from non-model organisms at a reasonable cost. In an era when virtually any study organism can 'go genomic', it is worthwhile to review how this may impact molecular ecology. The first studies to put the next generation sequencing (NGS) to the test in ecologically well-characterized species without previous genome information were published in 2007 and the beginning of 2008. Since then several studies have followed in their footsteps, and a large number are undoubtedly under way. This review focuses on how NGS has been, and can be, applied to ecological, population genetic and conservation genetic studies of non-model species, in which there is no (or very limited) genomic resources. Our aim is to draw attention to the various possibilities that are opening up using the new technologies, but we also highlight some of the pitfalls and drawbacks with these methods. We will try to provide a snapshot of the current state of the art for this rapidly advancing and expanding field of research and give some likely directions for future developments.

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Section: Epigeneticsmentioning

confidence: 99%

Applications of next generation sequencing in molecular ecology of non-model organisms

2010

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“…For MeDIP assays, 5-10 mg of genomic DNA was sonicated into small fragments, and 1 mg of the 5-methylcytosine monoclonal antibody (Active Motif, catalog no. 39649) was used to perform the MeDIP assay as previously described (Pomraning et al 2009). MeDIP samples were analyzed with qPCR.…”

Section: Small Rna Sequencingmentioning

confidence: 99%

Mechanism of siRNA production from repetitive DNA

Yang

Liu

2015

Genes Dev.

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RNAi is a conserved genome defense mechanism in eukaryotes that protects against deleterious effects of transposons and viral invasion. Repetitive DNA loci are a major source for the production of eukaryotic small RNAs, but how these small RNAs are produced is not clear. Quelling in Neurospora is one of the first known RNAi-related phenomena and is triggered by the presence of multiple copies of transgenes. Here we showed that DNA tandem repeats and double-strand breaks are necessary and, when both are present, sufficient to trigger gene silencing and siRNA production. Introduction of a site-specific double-strand break or DNA fragile site resulted in homologous recombination of repetitive sequences, which is required for gene silencing. In addition to siRNA production, the quelling pathway also maintains tandem repeats by regulating homologous recombination. Our study identified the mechanistic trigger for siRNA production from repetitive DNA and established a role for siRNA in maintaining genome stability.

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“…MeDIP-seq analyses were performed as described previously (Pomraning et al 2009) by using the NEBNext DNA library preparation kit (New England Biolabs, catalog no. E6040).…”

Section: Dna Methylation Analysesmentioning

confidence: 99%

Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing

et al. 2016

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In eukaryotes, antisense transcription can regulate sense transcription by induction of epigenetic modifications. We showed previously that antisense transcription triggers Dicer-independent siRNA (disiRNA) production and disiRNA locus DNA methylation (DLDM) in Neurospora crassa. Here we show that the conserved exonuclease ERI-1 (enhanced RNAi-1) is a critical component in this process. Antisense transcription and ERI-1 binding to target RNAs are necessary and sufficient to trigger DLDM. Convergent transcription causes stalling of RNA polymerase II during transcription, which permits ERI-1 to bind nascent RNAs in the nucleus and recruit a histone methyltransferase complex that catalyzes chromatin modifications. Furthermore, we show that, in the cytoplasm, ERI-1 targets hundreds of transcripts from loci without antisense transcription to regulate RNA stability. Together, our results demonstrate a critical role for transcription kinetics in long noncoding RNA-mediated epigenetic modifications and identify ERI-1 as an important regulator of cotranscriptional gene silencing and post-transcriptional RNA metabolism.

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Genome-wide high throughput analysis of DNA methylation in eukaryotes

Cited by 185 publications

References 67 publications

Applications of next generation sequencing in molecular ecology of non-model organisms

Applications of next generation sequencing in molecular ecology of non-model organisms

Mechanism of siRNA production from repetitive DNA

Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing

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