Biology subjects

Lister, R.

Publications and source records attributed to Lister, R..

4 recordsLinked to original sources

bioRxiv2018

A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs

AbstractDNA methylation is a covalent modification of the genome that plays important roles in genome regulation and vertebrate development. Although detection of this modification in the genome has been possible for several decades, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded the direct determination of the consequence of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this outstanding question. Recent adaptations of genome editing technologies, such as the fusion of the DNMT3A methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to provide new tools for altering DNA methylation at desired loci. Here, we performed a deeper analysis of the performance of these tools, revealing consistent off-target binding events and DNA methylation deposition in the genome, limiting the capacity of these tools to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA-methylation. dC9Sun-D3A is tunable, specific and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target binding and methylation by the dC9-D3A direct fusion construct. Furthermore, we used dC9Sun-D3A to test the impact of DNA methylation upon the DNA binding of CTCF and NRF1 upon targeted methylation of their core binding sites, demonstrating the binding sensitivity of these proteins to DNA methylation in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest amount of off-target DNA methylation reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.

molecular biology↗

bioRxiv2017

HOME: A histogram based machine learning approach for effective identification of differentially methylated regions

BackgroundThe development of whole genome bisulfite sequencing has made it possible to identify methylation differences at single base resolution throughout an entire genome. However, a persistent challenge in DNA methylome analysis is the accurate identification of differentially methylated regions (DMRs) between samples. Sensitive and specific identification of DMRs among different conditions requires accurate and efficient algorithms, and while various tools have been developed to tackle this problem, they frequently suffer from inaccurate DMR boundary identification and high false positive rate.\n\nResultsWe present a novel Histogram Of MEthylation (HOME) based method that takes into account the inherent difference in the distribution of methylation levels between DMRs and non-DMRs to discriminate between the two using a Support Vector Machine. We show that generated features used by HOME are dataset-independent such that a classifier trained on, for example, a mouse methylome training set of regions of differentially accessible chromatin, can be applied to any other organisms dataset and identify accurate DMRs. We demonstrate that DMRs identified by HOME exhibit higher association with biologically relevant genes, processes, and regulatory events compared to the existing methods. Moreover, HOME provides additional functionalities lacking in most of the current DMR finders such as DMR identification in non-CG context and time series analysis. HOME is freely available at https://github.com/ListerLab/HOME.\n\nConclusionHOME produces more accurate DMRs than the current state-of-the-art methods on both simulated and biological datasets. The broad applicability of HOME to identify accurate DMRs in genomic data from any organism will have a significant impact upon expanding our knowledge of how DNA methylation dynamics affect cell development and differentiation.

bioinformatics↗

bioRxiv2017

Frequent lack of repressive capacity of promoter DNA methylation identified through genome-wide epigenomic manipulation

It is widely assumed that the addition of DNA methylation at CpG rich gene promoters silences gene transcription. However, this conclusion is largely drawn from the observation that promoter DNA methylation inversely correlates with gene expression. The effect of forced DNA methylation on endogenous promoters has yet to be comprehensively assessed. Here, we conducted artificial methylation of thousands of promoters in human cells using an artificial zinc finger-DNMT3A fusion protein, enabling assessment of the effect of forced DNA methylation upon transcription and histone modifications, and the durability of DNA methylation after the removal of the fusion protein. We find that DNA methylation is frequently insufficient to transcriptionally repress promoters. Furthermore, DNA methylation deposited at promoter regions associated with H3K4me3 is rapidly erased after removal of the zinc finger-DNMT3A fusion protein. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3. These findings suggest that promoter DNA methylation is not generally sufficient for transcriptional inactivation, with implications for the emerging field of epigenome engineering.

genomics↗

bioRxiv2017

Regulatory remodeling in the allo-tetraploid frog Xenopus laevis

BackgroundGenome duplication has played a pivotal role in the evolution of many eukaryotic lineages, including the vertebrates. The most recent vertebrate genome duplication is that in Xenopus laevis, resulting from the hybridization of two closely related species about 17 million years ago [1]. However, little is known about the consequences of this duplication at the level of the genome, the epigenome and gene expression.\n\nResultsOf the parental subgenomes, S chromosomes have degraded faster than L chromosomes ever since the genome duplication and until the present day. Deletions appear to have the largest effect on pseudogene formation and loss of regulatory regions. Deleted regions are enriched for long DNA repeats and the flanking regions have high alignment scores, suggesting that non-allelic homologous recombination (NAHR) has played a significant role in the loss of DNA. To assess innovations in the X. laevis subgenomes we examined p300 (Ep300)-bound enhancer peaks that are unique to one subgenome and absent from X. tropicalis. A large majority of new enhancers are comprised of transposable elements. Finally, to dissect early and late events following interspecific hybridization, we examined the epigenome and the enhancer landscape in X. tropicalis x X. laevis hybrid embryos. Strikingly, young X. tropicalis DNA transposons are derepressed and recruit p300 in hybrid embryos.\n\nConclusionsThe results show that erosion of X. laevis genes and functional regulatory elements is associated with repeats and NAHR, and furthermore that young repeats have also contributed to the p300-bound regulatory landscape following hybridization and whole genome duplication.

evolutionary biology↗