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Epigenetic regulation of pluripotency and lineage commitment in the early mouse embryo

Reference	BB/G011117/1
Principal Investigator / Supervisor	Dr Veronique Azuara
Co-Investigators / Co-Supervisors
Institution	Imperial College London
Department	Surgery and Cancer
Funding type	Research
Value (£)	382,111
Status	Completed
Type	Research Grant
Start date	01/04/2009
End date	30/09/2012
Duration	42 months

Abstract

Pluripotent cells develop within the inner cell mass (ICM) of early-stage blastocyst embryos, a cell population surrounded by an extraembryonic layer, the trophectoderm (TE). Here, we use both embryo-derived stem cells and in vivo studies to investigate the epigenetic mechanisms leading to blastocyst lineage segregation and pluripotency safeguarding. Recent studies in ES cells showed that many silent genes that are required later on during development are simultaneously marked with Polycomb repressor (PRC)-mediated H3K27 methylation, and marks normally associated with gene activity. Moreover, these so-called bivalent genes assemble RNA Polymerase (RNAP) complexes and are transcribed at low level, specifying a 'primed' state in pluripotent cells. We have begun to compare the chromatin profiles of ES and TE-derived trophoblast stem (TS) cells. Though many genes are bivalently marked in TS cells as in ES cells, they are not normally expressed in this lineage. To explore this further, we will examine the chromatin environment of ES and TS cells at bivalent promoters by assessing PRC1 and PRC2 binding, RNAP occupancy and conformation and, additional 'lock in' mechanisms in TS cells that may operate to avoid reprogramming. We also address the role of key stem cell transcription factors in targeting chromatin changes using ES cell de-differentiation models upon Oct4 loss or gain of Cdx2. In order to confirm the biological significance of 'primed' chromatin in the embryo we will use carrier ChIP assay to investigate the epigenetic relationship between ES and TS cells and in vivo ICM and TE cells. In particular, we intend to explore how and when specific differences in epigenetic programming are specified, at the locus level, between embryonic and extraembryonic tissues. This will lead to in vivo lineage tracing studies where single blastomere knock-down (RNA interference), jointly labelled with a dye, can reveal the effects of epigenetic program failure on cell allocation.

Summary

In the last few years the genetic code or 'blueprint' for man has become available. Although this information is nearly complete, we still do not know how to translate the code, how to use it to rebuild the cells of damaged tissues or how to understand the remarkable development of an individual from a single fertilised egg. What we do know now is that this code is interpreted in each cell in the body by a group of agents that are collectively called 'chromatin-modifiers'. Our laboratory looks at the chromatin structure of embryonic stem (ES) cells. These remarkable cells are pluripotent, meaning they have the potential to differentiate into any cell type found in the body. Twenty-five years ago the first ES cell lines were derived from early mouse embryos into culture. The regulatory mechanisms, which define ES cell 'pluripotency' or differentiation, were not understood, although potential application for regenerative medicine was immediately foreseen. Deciphering how pluripotency is achieved and how it can be harnessed and maintained in culture are key questions for understanding normal development and successfully applying the knowledge to stem cell-based therapies. We intend to unravel the unique chromatin 'make-up' of pluripotent ES cells and their counterparts in the embryo that confers them flexibility and unlimited cell fate options. We also explore how this can be dynamically changed in a manner that is predictable when a stem cell decides to become a specific cell type (e.g. nerve, muscle or blood cell). This information will help us to understand how the genetic code is used, and help us to design better strategies for turning stem cells into cells that are useful for treating degenerative diseases and other human diseases.

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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