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Epigenetic reprogramming and pronuclear asymmetry in early mouse development

Reference	BB/E023355/1
Principal Investigator / Supervisor	Dr Sari Pennings
Co-Investigators / Co-Supervisors	Professor Richard Meehan, Dr Jane Taylor, Professor Ian Wilmut
Institution	University of Edinburgh
Department	College of Medicine and Veterinary Medic
Funding type	Research
Value (£)	466,042
Status	Completed
Type	Research Grant
Start date	01/12/2007
End date	30/11/2010
Duration	36 months

Abstract

CpG methylation is a post-transcriptional DNA modification that is associated with gene silencing, while histone modifications such as lysine acetylation and methylation can induce either gene activation or repression. Together, DNA and histone marks form an epigenetic code that holds the clues to understanding cell differentiation and lineage specification in normal development, as well as the disease states of genomic imprinting, Rett syndrome and cancer. Somatic cell nuclear transfer experiments have demonstrated that the epigenetic memory of heritable DNA methylation and chromatin states in differentiated cells is not easily erased in order to restore developmental potential, and that incorrect nuclear reprogramming leads to developmental failure. This has refocused attention on understanding the natural nuclear reprogramming process that with each new generation reinstates totipotency in the fertilised zygote. Demethylation of DNA is thought to be necessary for the activation of essential developmental genes. Rapid DNA demethylation of the mouse male pronucleus with passive DNA demethylation of the female genome in subsequent cell divisions was taken to indicate that nuclear reprogramming constitutes the global removal of epigenetic marks. This striking pronuclear asymmetry and demethylation dynamics is not conserved in other mammals, however, an observation at odds with the evolutionary conserved developmental context. Hypothesising that sperm chromatin variability in protamines, histones, variants and epigenetic modifications can explain these differences, we will investigate elements of somatic and zygotic chromatin structure that delimit the nuclear reprogramming process and determine its epigenetic outcome.

Summary

A detailed picture is emerging of the changes in cell nuclear architecture and epigenetic marks that accompany fertilisation and are necessary for subsequent early embryo development (Morgan et al., 2005). The molecular mechanisms underlying these events are nevertheless still poorly understood. A key question is how the zygote is reprogrammed by these mechanisms to initiate its full developmental potential. The importance of the nuclear remodelling process is highlighted by somatic cell nuclear transfer (SCNT) experiments into enucleated oocytes. In spite of well publicised successes in recreating zygotic development and even cloned mammals (Wilmut et al., 1997), high rates of early developmental arrest attributed to incorrect nuclear reprogramming are encountered in every species tested (Hochedlinger and Jaenisch, 2006). These experiments, which exploit the remodelling activity of oocytes in order to restore zygotic transcription potential to somatic cell nuclei, have identified an 'epigenetic memory' of tissue-specific cell characteristics that cannot easily be erased (Ng and Gurdon, 2005). Developmental potential is gradually lost in the transitions from zygote to somatic tissues. The specification of cell lineages involves the programmed activation and silencing of genes. DNA and chromatin modifications that are heritable over many cell cycles can act in concert to define the transcription profile associated with particular cell lineages. These epigenetic patterns that maintain cell differentiation in normal development can only be reversed with low frequency by SCNT, and they are disturbed upon malignant transformation in cancer. Natural reprogramming occurs solely in the germ line and upon fertilisation (Morgan et al., 2005), where it is necessary to reverse the differentiated state of the gametes. The preimplantation development period after fertilisation presents a unique window during which a dynamic behaviour of epigenetic markers can be observed while patterns are being reset. Mechanistic clues may be gleaned more readily from this dynamic sequence than from a steady state. Observations in the mouse have led to a model of nuclear reprogramming in which the epigenetic marks are globally erased upon fertilisation and preimplantation development. This model has been challenged by the observations from a number of other mammals showing varying degrees of epigenetic remodelling. In particular, the striking asymmetry between male and female mouse pronuclei resulting from the rapid DNA demethylation of the male pronucleus appears not conserved amongst mammals, which requires further explanation. Our earlier work suggested the existence of mammalian variation in oocyte remodelling activity, as well as in sperm remodelling susceptibility (Beaujean et al., 2004c). The species specific variations between male and female pronuclei on one hand and the poor outcome with somatic nuclei on the other hand suggest that the chromatin substrate is a determinant of the epigenetic outcome of nuclear reprogramming. In view of the special chromatin packaging of the male genome, this may depend on initial DNA methylation levels, the protamine composition, or the residual histones and their genomic distribution, variants and modifications. A complete investigation of the molecular mechanisms involved in nuclear programming needs to include the chromatin substrate perspective, to complement ongoing efforts in characterising the oocyte remodelling activity. Building on earlier work, as well as a combined expertise in reproductive biology, epigenetics of normal and cloned preimplantation embryos, DNA methylation, histone modification and chromatin assembly, the proposed research aims to fill this gap by employing physiological and biochemical substrates of known composition to pinpoint the elements of somatic and zygotic chromatin structure that delimit the nuclear reprogramming process and determine its epigenetic outcome.

Committee	Closed Committee - Genes & Developmental Biology (GDB)
Research Topics	Stem Cells
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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