Transient triple TET (tet1/2/3/) knockouts in zebrafish

In our latest work, Sam Ross provides a detailed protocol for the generation of triple TET zebrafish CRISPants (F0 CRISPR/Cas9 KOs) and their subsequent molecular analyses by reduced representation bisulfite sequencing (RRBS). The full protocol can be accessed here, while the entire book (TET proteins and DNA demethylation, Methods and Protocols) can be obtained through the Springer website.

TET proteins and DNA Demethylation

Check out the latest book on protocols in the field of TET proteins and DNA demethylation coedited by Michiel Vermeulen (Radboud University) and myself. The book is divided in five parts. Part One describes technologies aimed at detecting and quantifying DNA methylation turnover using massively parallel sequencing, ELISA, and mass spectrometry approaches. Part Two looks at data analyses protocols for distinguishing acting versus passive DNA demethylation and estimation of 5mC and 5hmC levels. Part Three deals with a new topic that takes advantage of modified CRISPR/Cas9 genome editing systems to target DNA demethylation activity to genomic loci of interest. Part Four discusses protocols that detail how to purify TET proteins and unravel their protein interactions, and Part Five looks at the assessment of TET protein function and activity in vivo and in vitro. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. 

Developmental Accumulation of Gene Body and Transposon Non-CpG Methylation in the Zebrafish Brain

Our latest is out in Frontiers in Cell and Developmental Biology. Here we demonstrate that zebrafish, just like mammals, accumulate non-canonical DNA methylation (mCH) during brain development. Moreover, we identify accumulation of mCH at transposons from the Tc1-mariner superfamily.  Overall, this work demonstrates the evolutionary conservation of developmental mCH dynamics and highlights the potential of zebrafish as a model to study mCH regulation and function during normal and perturbed development (i.e Rett syndrome).

Developmental remodelling of non-CG methylation at satellite DNA repeats

The latest from our lab is out in Nucleic Acids Research. This exciting project was led by the very talented Sam Ross who uncovered a novel non-CG DNA (mCH) methylation signature that is highly abundant at zebrafish MOsaic SATellite repeats (MOSAT). MOSAT mCH is high in gonads, decreases during cleavage stages, and reaches its lowest point at ZGA. Unlike mammalian mCH – that is mostly present in oocytes and in the nervous system – MOSAT mCH increases during gastrulation and is deposited in tissues originating from all embryonic layers. Also, in contrast to mammals, this mCH type positively correlates with heterochromatin (H3K9me3). We uncovered the actinopterygian-specific Dnmt3ba as the principal MOSAT mCH DNA methyltransferase and fully resolved its phylogenetic origin. This work thus provides proof for yet another “love story” between repetitive elements and DNMTs in the animal kingdom.

Evolution of DNA methylome diversity in eukaryotes

Our lab has participated in the special issue of the Journal of Molecular Biology: “Reading DNA modifications”. We contributed with a review: “Evolution of DNA methylome diversity in eukaryotes”. In this piece, we summarise a decade worth of insights obtained from whole genome bisulfite sequencing studies across eukaryotes. We speculate on the origins and functions of diverse DNA methylation patterns observed in different organisms and propose how such patterns might have evolved. This work expands our knowledge on the function and evolutionary origins of DNA methylation in plants, animals and fungi. The bonus was that our artwork (designed by Scot Nicholls) was selected for the cover of this special issue.

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Evolutionary conservation of DNA methylation loss during chordate development. Hourglass represents the developmental morphological divergence across species, where the mid-developmental phylotypic stage shows similar morphologies across distant taxa whereas early and late development are more prone to lineage specific forms.

Retention of paternal DNA methylome in the developing zebrafish germline

In mammals, DNA methylation undergoes two rounds of developmental remodelling; in primordial germ cells and in the pre-implantation embryo. However, it is currently unclear whether such DNA methylome reprogramming is more broadly conserved among vertebrates. This work led by Ksenia Skvortsova from our lab, published today in Nature Communications, demonstrates inheritance of the paternal methylation pattern in developing germ cells and absence of genome-wide reprogramming events. This study was published back to back with a similar study from the Hore lab (University of Otago).

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Functions and mechanisms of epigenetic inheritance in animals

Our review on inter- and transgenerational epigenetic inheritance in animals is just out in Nature Reviews Molecular Cell Biology. This is our take on trying to summarise a highly complex and polemic field on a few pages of text. This effort was led by Ksenia Skvortsova and our long term collaborator Nicola Iovino from the Max Planck Institute of Immunobiology and Epigenetics. For those who don’t feel like reading the whole piece, this is a two-sentence summary of the review: Briefly, epigenetic inheritance (EI) across generations is widespread in animals. There are excellent examples in flies and worms. In mammals EI that spans multiple generations is a rare event. Anamniotes that lack embryonic epigenome reprogramming might be useful models for EI.

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Amphioxus functional genomics and the origins of vertebrate gene regulation

This long-awaited study was published in Nature this week. As a part of a large international consortium, we generated a functional genomics resource for amphioxus (B. lanceolatum) comprising 96 datasets of RNA-seq, MethylC-seq, RRBS, CAGE-seq, ChIP-seq and ATAC-seq. Amphioxus (also known as “the lancelet”), is an invertebrate chordate that can help us resolve some of the earliest steps of vertebrate evolution. We uncovered that DNA methylation is removed from Amphioxus enhancers in a similar fashion as previously described in vertebrates. More about the study can be found at here.

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Germ line–inherited H3K27me3 restricts enhancer function during maternal-to-zygotic transition

Great to see this exciting collaboration led by the Iovino lab finally out in Science! Besides DNA itself, the parents provide a multitude of epigenetic information to the embryo. Here we show that the Polycomb repressive histone mark H3K27me3 is contributed maternally, to regulate the timing of zygotic genome activation in Drosophila. Preventing the propagation of maternally inherited H3K27me3 leads to precocious gene activation and, ultimately, embryo lethality.

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