Developmental Biology - Genome|
Kick-Starting Cell Division
Developing a picture of what happens in earliest stages of life in mammals...
After the fertilisation of an egg cell, now two sets of genetic information from dad and mom combine to form a new individual's genome - that individual's entire genetic identity. The egg and sperm act as information capsules with stored instructions for starting this new life. But what kick starts interpretation of these instruction packages?
Researchers at the Babraham Institute are providing some of the pieces in this puzzle through use of a rare sub-group of mouse embryonic stem cells — '2C-like' cells. These cells act as a 'mirror' for observing the two celled embryo (now called a zygote) in pregnant mice. All of this occurs 24 hours after fertilisation at the stage when a zygote's genome normally activates.
Using these '2C-like' cells, researchers can now probe the hierarchy of events on the molecular level which initiates formation of the zygote's genome. Their findings are published in the journal Genes & Development.
Waking up the genome occurs through events called the minor wave and the major wave. Researchers knew the protein that binds to DNA and promotes its expression i.e. function is a transcription factor called Dux. Dux activates a range of genes in the second major wave but not in the first wave.
"Genome activation is the first thing the embryo has to do. Despite being crucial to formation of the embryo, we know surprisingly little about it. The 2C-like system provides an experimental approximation of these very early embryo cells, allowing us to use the full arsenal of research techniques to tease apart what is happening."
Melanie Eckersley-Maslin PhD, Marie Curie/EMBO Research Fellow, The Babraham Institute and Christ's College, The University of Cambridge, Cambridge, United Kingdom.
Researchers began by screening for factors to increase the number of rare 2C-like cells in a population of mouse embryonic stem cells — factors able to promote genome activation. They identified two: Development Pluripotency Associated 2 (Dppa2) and Development Pluripotency Associated 4 (Dppa4).
"Looking at what we have discovered about Dppa2 and Dppa4 they fit the profile of being responsible for kick-starting expression of the new genome. They are already present in the egg before the new embryo is formed; and if we delete Dppa2 and Dppa4 genes from the genome, we see a loss of 2C-like cells and loss of the Dux-initiated wave of genome expression."
Melanie Eckersley-Maslin PhD
The model proposed by this research connects (1) genome activation with (2) epigenetic reprogramming of cells to eventually form the sperm and eggs. This chain of events secures that Dppa2 and Dppa4 expression in egg cells will initiate genome activation at the right time.
"Looking forward, it will be interesting to uncover the details of genome activation in human development as this is an unexplored area. Understanding more about regulators of the genome in early life is also relevant to situations where we wish to revert cells to their early-life state, such as in generating induced pluripotent stem cells for future therapeutic uses."
Wolf Reik MD, University of Hamburg, Head of the Epigenetics Research Programme - Babraham Institute, Honorary Professor of Epigenetics at the University of Cambridge and Associate Faculty Member, Wellcome Sanger Institute.
The molecular regulation of zygotic genome activation (ZGA) in mammals remains an exciting area of research. Primed mouse embryonic stem cells contain a rare subset of “2C-like” cells that are epigenetically and transcriptionally similar to the two-cell embryo and thus represent an in vitro approximation for studying ZGA transcription regulation. Recently, the transcription factor Dux, expressed in the minor wave of ZGA, was described to activate many downstream ZGA transcripts. However, it remains unknown what upstream maternal factors initiate ZGA in either a Dux-dependent or Dux-independent manner. Here we performed a candidate-based overexpression screen, identifying, among others, developmental pluripotency-associated 2 (Dppa2) and Dppa4 as positive regulators of 2C-like cells and transcription of ZGA genes. In the germline, promoter DNA demethylation coincides with expression of Dppa2 and Dppa4, which remain expressed until embryonic day 7.5 (E7.5), when their promoters are remethylated. Furthermore, Dppa2 and Dppa4 are also expressed during induced pluripotent stem cell (iPSC) reprogramming at the time that 2C-like transcription transiently peaks. Through a combination of overexpression, knockdown, knockout, and rescue experiments together with transcriptional analyses, we show that Dppa2 and Dppa4 directly regulate the 2C-like cell population and associated transcripts, including Dux and the Zscan4 cluster. Importantly, we teased apart the molecular hierarchy in which the 2C-like transcriptional program is initiated and stabilized. Dppa2 and Dppa4 require Dux to initiate 2C-like transcription, suggesting that they act upstream by directly regulating Dux. Supporting this, ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) analysis revealed that Dppa2 and Dppa4 bind to the Dux promoter and gene body and drive its expression. Zscan4c is also able to induce 2C-like cells in wild-type cells but, in contrast to Dux, can no longer do so in Dppa2/4 double-knockout cells, suggesting that it may act to stabilize rather than drive the transcriptional network. Our findings suggest a model in which Dppa2/4 binding to the Dux promoter leads to Dux up-regulation and activation of the 2C-like transcriptional program, which is subsequently reinforced by Zscan4c.
Mélanie Eckersley-Maslin, Celia Alda-Catalinas, Marloes Blotenburg, Elisa Kreibich1, Christel Krueger and Wolf Reik.
© 2019 Eckersley-Maslin et al.; Published by Cold Spring Harbor Laboratory Press
This article, published in Genes & Development, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
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Jan 31, 2018 Fetal Timeline Maternal Timeline News News Archive
Mouse colony of embryonic stem cells fluorescently stained for Dppa2 (RED) - Dppa4 (GREEN)
and DNA (BLUE). Image: Dr Melanie Eckersley-Maslin, Babraham Institute