Constitutive genomic methylation during embryonic development of Xenopus

doi:10.1016/S0167-4781(01)00280-9

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression

Volume 1521, Issues 1–3, 31 October 2001, Pages 39-44

https://doi.org/10.1016/S0167-4781(01)00280-9 Get rights and content

Abstract

Methylation of CpG dinucleotides is a predominant modification of genomic DNA in many species, especially in vertebrates. This modification, generally associated with transcriptional repression, is rapidly and globally lost during mammalian pre-implantation development. This loss of methylation is gradually reversed during subsequent stages of development. Here we show that the amphibian Xenopus laevis maintains high levels of DNA methylation during early embryonic development. The methylation status of specific loci is independent of the temporal expression profile. The observations have profound implications for the regulation of early embryonic gene regulation and genome function.

Introduction

Methylation of CpG dinucleotides, the most common modification of vertebrate genomes, is primarily associated with transcriptional repression [1]. Transcriptional repression is mediated by a family of methyl CpG-binding domain (MBD) proteins, that can interact with histone deacetylases. The extent to which genomes are methylated is vastly different, depending on the species. The genome of the mouse is highly methylated, whereas this modification is virtually absent or very infrequent in some invertebrate species, including Drosophila melanogaster and Caenorhabditis elegans. However, yet other invertebrates have significant levels of methylcytosine [2]. In one of these, in the invertebrate chordate Ciona intestinalis, genes seem to be methylated and transposable elements non-methylated [3], different from the usual situation in which methylation is thought to silence repetitive sequences and transposable elements.

Global methylation levels not only differ between species, but also between different stages of embryonic development. In the mouse, the genome-wide demethylation occurs in the fertilized egg, just before zygotic transcription starts at the two-cell stage [4], [5], [6], [7]. This hypomethylated state is gradually reversed post implantation. As the hypomethylated state is associated with a high incidence of recombination and gene loss [8], developmental demethylation comes at a cost. The function of these global changes in methylation status, however, is not known, but may be related to re-programming the epigenetic marks before embryonic development. Alternatively, global demethylation may serve to derepress the genome on a global scale at the stage at which the embryonic genome becomes transcriptionally active for the first time. Concordant with this hypothesis is the observation by Stancheva and Meehan [9] that depletion of DNA methyltransferase leads to a premature onset of embryonic gene transcription in the frog Xenopus laevis. Either explanation, however, if true, predicts developmental demethylation to occur globally in species with highly methylated genomes. In the zebrafish Danio rerio, however, no genome-wide changes of DNA methylation were observed during embryonic development [10]. Here we report that the embryonic genome of the frog X. laevis is heavily – though not uniformly – methylated during embryogenesis. This hypermethylation is observed before, during, and after activation of the embryonic genome. In contrast, mitochondrial DNA – which is very abundant relative to genomic DNA during early embryogenesis – is unmethylated, as previously reported [11]. At a global level, the frog genome is constitutively hypermethylated, as are high copy number sequences such as Satellite I and the oocyte 5S rRNA genes and a retrotransposon element. Low copy sequences are hyper- or hypomethylated, depending on the locus. The different roles methylation changes play in different species may reflect different developmental strategies regarding imprinting, uterine or extra-uterine development and may have implications for the mechanisms of developmental gene regulation.

Section snippets

Isolation of genomic DNA

Genomic DNA was isolated using a protocol adapted from Dawid [12]. Staged embryos were gently homogenized with a blue tip in 3 vols. of buffer (10 mM Tris–HCl pH 7.5, 15 mM EDTA, 1% SDS, 0.5 mg/ml proteinase K), and the homogenate was incubated for more than 2 h at 37°C. Two phenol/chloroform/isoamyl alcohol (25:24:1) extractions were performed by gently mixing on a nutator to prevent shearing of the DNA. Samples were centrifuged in phase-lock tubes (5 Prime 3 Prime) for efficient recovery of

Results

Early embryonic DNA is highly enriched in mitochondrial DNA, as the fertilized egg starts with 6 pg of genomic DNA and 4 ng of mitochondrial DNA [13]. During the initial stages of embryonic development cell division occurs without cell growth, and the amount of mitochondrial DNA initially remains the same, while the amount of genomic DNA increases exponentially. In our analysis we normalized amounts of DNA for the amount of genomic DNA present, resulting in enrichment of mitochondrial DNA in

Discussion

Our data show that the Xenopus genome is globally and constitutively hypermethylated before and after the mid-blastula transition. Recently, the methylcytosine content of frog embryonic DNA was reported using immunological detection of methylated DNA on membranes [9]. The total DNA from eggs and cleavage-stage embryos, as well as DNA from neurula or later stage embryos, was shown to be methylated. However, using this technique, DNA from blastula-stage embryos was reported to be partially

Acknowledgements

Alan Wolffe died in an accident recently. His help, encouragement and active support are remembered with gratitude. The helpful contribution of DNA constructs by the following colleagues is also gratefully acknowledged: P.A. Krieg (GS17, EF1α), R.A.W. Rupp (MyoD), P.A. Wade (MTA1L), and I.B. Dawid (mitochondrial DNA clone pXLM32, 1A11.8).

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¹: A.P.W. was at Sangamo Biosciences, Richmond, CA, USA until his death in May 2001.

View full text

Constitutive genomic methylation during embryonic development of Xenopus

Abstract

Introduction

Section snippets

Isolation of genomic DNA

Results

Discussion

Acknowledgements

Curr. Biol.

J. Mol. Biol.

Dev. Biol.

Cell

Mech. Dev.

Gene

Gene

Trends Genet.

Methyl-CpG-binding proteins: targeting specific gene repression

Eur. J. Biochem.

The role of DNA methylation in invertebrates: developmental regulation or genome defense?

Mol. Biol. Evol.

Nonmethylated transposable elements and methylated genes in a chordate genome

Science

Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development

Development

Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line

Genes Dev.

Demethylation of the zygotic paternal genome

Nature

DNA hypomethylation leads to elevated mutation rates

Nature

Transient depletion of xDnmt1 leads to premature gene activation in Xenopus embryos

Genes Dev.