Dr Véronique Azuara
Epigenetics and Development
Postdoctoral position available
Aims
The long-term goal of our group is to unravel the chromatin characteristics that define pluripotency in embryonic stem (ES) cells versus somatic cell commitment. We are also investigating the epigenetic relationship between in vitro maintained ES cells versus the inner cell mass from which they are derived, to define the role of epigenetic silencing in the establishment and maintenance of pluripotency both in culture and in the embryo.
Background
Stem cells are functionally characterized by their ability to both self-renew and generate progeny capable of differentiating along several defined lineage paths. Pluripotent embryonic stem (ES) cells can remarkably contribute to all tissues of the developing embryo. In the past few years, it has become clear that chromatin and epigenetic factors play a key role in maintaining gene expression programs involved in both self-renewal and cell differentiation. Deciphering how ES cell pluripotency and lineage induction is achieved at the chromatin level is likely to be very informative for understanding normal development and for successfully applying stem cell-based therapies.
Research and Progress
Profiling chromatin in a particular cell type provides a valuable ‘signature’ for cell identity and developmental stage. One approach has been to assay the timing of DNA replication across a panel of loci, as an indicator of chromatin state. This epigenetic profiling has allowed pluripotent mouse embryonic stem cells to be reliably distinguished from adult stem cells (e.g. hematopoietic stem cells) and their proliferative progeny. Indeed, these studies exemplified how DNA replication profiles may be used as a ‘predictor’ of stem cell potency. This may become increasingly useful for characterising human ES cells, where the functional verification of embryonic potential in vivo is not possible. Studies are now ongoing to further extend replication profiling in human ES cells as well as mouse ES cells to characterize the epigenetic profile that is consistent with a pluripotent state.
DNA replication timing assay. Human ES cells are pulse-labelled with 5-bromo-2-deoxyuridine (BrdU), stained with propidium iodide (PI) and separated by cell sorting into six fractions (G1, S1, S2, S3, S4 and G2/M) according to DNA content. The relative abundance of candidate sequences within newly synthesised DNA is determined using real-time PCR.
A series of reports have begun to define the chromatin profile of ES cells, revealing a critical role for the Polycomb Repressive Complexes (PRCs) in maintaining pluripotency. In undifferentiated ES cells, many silent genes that are important for lineage specification are accessible, early-replicating and carry histone marks indicative of active chromatin such as acetylated H3K9 and methylated H3K4. Their expression appears however to be restrained by PRC2-mediated H3K27 methylation. This suggests that key developmental genes are ‘poised’ for activation in ES cells yet are held in check by opposing or ‘bivalent’ (permissive/repressive) chromatin configurations. Interestingly, a subset of PRC2-target genes overlap with, and are likely to be regulated by, the pluripotency transcription factors Oct4, Sox2 and Nanog.
‘Bivalent’ chromatin profiles in ES cells. In undifferentiated ES cells, many silent, early-replicating developmental genes are marked by an unusual combination of permissive (H3K4me2 and H3K9ac) and repressive (H3K27me3) epigenetic marks. During differentiation, ‘bivalent’ chromatin domains are generally resolved while a limited number of loci (silent or weakly induced) may retain a ‘bivalent’ chromatin configuration in lineage-committed cells.
The functional importance of ‘bivalent’ chromatin signatures during development remains unclear. We focus on pre-implantation development to further explore how pluripotency is acquired and maintained while the first differentiation events (extraembryonic lineage formation) occur.
Early mouse development and embryo-derived stem cells. Progression from morula-stage embryos to early blastocysts results in differentiation into trophectoderm (TE) - an external extraembryonic layer surrounding a mosaic of cells (the inner cell mass, ICM). ICM cells further segregate into the primitive endoderm (PrE) and primitive ectoderm/epiblast (PEct). ES and EPiSC cells can be derived from the ICM and PEct, respectively. TS cells can be derived from the TE and XEN cells from the PrE. Following implantation, PEct goes on to form the three germ layers (ectoderm, mesoderm and endoderm).
We combine in vitro models of human and mouse embryo-derived stem cells with in vivo studies to identify epigenetic features that distinguish ES, TS and XEN cells, verify whether the ES cell epigenetic signature reflects the chromatin environment of the ICM and examine how such a signature may be established upon blastocyst formation. The group is also looking at dynamics of epigenetic ‘remodelling’ upon loss of pluripotency and lineage induction (neural specification) in human ES cells.
Key references
Azuara V. Profiling of DNA replication timing in unsynchronized cell populations. Nat Protoc. 2006;1(4):2171-7.
Azuara V, Perry P, Sauer S, Spivakov M, Jorgensen HF, John RM, Gouti M, Casanova M, Warnes G, Merkenschlager M, Fisher AG. Chromatin signatures of pluripotent cell lines. Nat Cell Biol. 2006 May;8(5):532-8.
Williams RR, Azuara V, Perry P, Sauer S, Dvorkina M, Jorgensen H, Roix J, McQueen P, Misteli T, Merkenschlager M, Fisher AG. Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus. J Cell Sci. 2006 Jan 1;119(Pt 1):132-40.
Perry P, Sauer S, Billon N, Richardson WD, Spivakov M, Warnes G, Livesey FJ, Merkenschlager M, Fisher AG, Azuara V. A dynamic switch in the replication timing of key regulator genes in embryonic stem cells upon neural induction. Cell Cycle. 2004 Dec;3(12):1645-50.
Azuara V, Fisher AG. Maintaining transcriptional states through DNA replication. Cell Cycle. 2003 Nov-Dec;2(6):521-4. Review.
Azuara V, Brown KE, Williams RR, Webb N, Dillon N, Festenstein R, Buckle V, Merkenschlager M, Fisher AG. Heritable gene silencing in lymphocytes delays chromatid resolution without affecting the timing of DNA replication. Nat Cell Biol. 2003 Jul;5(7):668-74.
Team members
Dr Véronique Azuara (Team Leader)
Olivia Alder (PhD student; IOG Trust funded)
Matias Autio (PhD Student; BBSRC funded)
Anne Helness (PhD student; MRC funded)
Dr Fabrice Lavial (Postdoctural Researcher; BBSRC funded)
Sandra Pinho (PhD student; FCT/Portugal funded)
