Exploring functional significance of asymmetric DNA methylation in early mammalian development

DNA methylation is an epigenetic modification important for the regulation of transcriptional activity in processes like genomic imprinting, retrotransposon silencing and centromeric stabilisation. It is also crucial for correct embryonic development and the differentiation of embryonic stem cells (ESCs) into diverse cell types. Although in mammals DNA methylation occurs predominantly in the symmetric CG context, it has been shown that certain cell types and tissues (ESCs, oocytes, primordial germ cells) have substantial amounts of methylation outside of the CG dinucleotide, which is asymmetric. Its presence in early developmental stages related to toti- or pluri-potency raises the intriguing possibility that non-CG context methylation may have a role in differential gene expression during those stages, contributing to their transcriptional plasticity. I investigated the distribution, dynamics and functional significance of non-CG methylation in early mouse development. Most methods for DNA methylation analysis are either targeted for the analysis of CG methylation or, in the case of bisulfite sequencing, suffer from potentially confounding issues. I have compared existing approaches to detect methylation outside of CG context and developed novel tools, namely the use of antibodies against non-CG methylation, to either analyse global levels of non-CG methylation, or validate its genomic distribution. With these, I have evaluated the role of components of the DNA methylation machinery and have followed the dynamic changes of non-CG context methylation throughout development. My analysis reveals that the highest levels of non-CG methylation in the mouse are present in the mature oocyte and the zygote. The enzymes responsible for establishing and maintaining its levels are the de novo Dnmts (3a and 3b), among which the activity of Dnmt3a2 towards CH seems regulated, suggesting a specific rather than an unspecific role. In ES cells, CH methylation correlates with active histone marks e.g. H3K4me3, and inversely with H3K27me3. The distribution of mCH, both in ESC naïve and primed pluripotency states, is very heterogeneous, while its nuclear distribution is very homogenous. mCH is physically recognised by a number of pluripotency factors such as Oct4 and Sox2, as well as by other DNA modification-sensitive proteins like MeCP2, Foxk1 and Foxk2. Moreover, mCH cannot be hydroxylated by the Tet family of enzymes, and repels proteins involved in the initiation of base-excision repair (AID and RPA), thus potentially escaping active demethylation in the zygote. In summary, my results show that mCH is a valid methylation mark, with a functional significance in early mouse development

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

BACKGROUND: Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. RESULTS: We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies. CONCLUSIONS: We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets

An endosiRNA-Based Repression Mechanism Counteracts Transposon Activation during Global DNA Demethylation in Embryonic Stem Cells

Erasure of DNA methylation and repressive chromatin marks in the mammalian germline leads to risk of transcriptional activation of transposable elements (TEs). Here, we used mouse embryonic stem cells (ESCs) to identify an endosiRNA-based mechanism involved in suppression of TE transcription. In ESCs with DNA demethylation induced by acute deletion of Dnmt1, we saw an increase in sense transcription at TEs, resulting in an abundance of sense/antisense transcripts leading to high levels of ARGONAUTE2 (AGO2)-bound small RNAs. Inhibition of Dicer or Ago2 expression revealed that small RNAs are involved in an immediate response to demethylation-induced transposon activation, while the deposition of repressive histone marks follows as a chronic response. In vivo, we also found TE-specific endosiRNAs present during primordial germ cell development. Our results suggest that antisense TE transcription is a “trap” that elicits an endosiRNA response to restrain acute transposon activity during epigenetic reprogramming in the mammalian germline.ISSN:1934-5909ISSN:1875-977

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

BACKGROUND: Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. RESULTS: We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies. CONCLUSIONS: We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets

Additional file 3: of Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

An Excel spreadsheet with reference genome compositions. (XLSX 269 kb

Additional file 1: of Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

An Excel spreadsheet with three tabs listing 1) datasets generated in this study, 2) datasets used from the publically domain, together with key parameters, and 3) number of datasets used per analysis. (XLSX 21 kb

Additional file 2: of Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

A PDF file with supplementary Tables S1–S3 and supplementary Figures S1–S10. (PDF 11655 kb