The epigenetic reprogramming in DNA methylation that occurs in the preimplantation embryo appears vulnerable to disruption when in vitro embryo production technologies are applied. Thus we reasoned that blastocyst-derived, human embryonic stem cells isolated and cultured through a diverse range of protocols in different laboratories (hESC), may also be subject to epigenetic instability and variation (Allegrucci et al., 2004. Lancet 364:206-20). In order to define the degree of epigenetic variation between independently-derived hESC lines we have employed Restriction Landmark Genome Scanning (RLGS) to examine the genome-wide methylation profiles of gene-rich CpG islands in hESC. Our studies to date have revealed significant epigenetic variation between lines that cannot be accounted for by inherent genetic variability. Studies on the effect of a range of culture conditions have also revealed epigenetic instability over time in culture, with evidence of stable inheritance of changes occurring at lower passage number. The majority of loci which changed over time within a line were not in common between lines, suggesting that passage–associated changes are stochastic and unpredictable. Our data suggest that further optimisation and standardization of hESC culture conditions may be required to develop a safe therapeutic product to which tissuespecific differentiation protocols can be generically applied. Current work from our laboratory focussing on developing culture conditions that minimise epigenetic instability will be presented.

Cell fate decisions in the hematopoietic system

Constanze Bonifer¹, Nicola Wilson¹, Hanna Krysinska¹, Maarten Hoogenkamp¹, Richard Ingram¹, Georgia Salvagiotto, Meinrad Busslinger², and Hiromi Tagoh¹

1 Section of Experimental Haematology, Leeds Institute for Molecular Medicine, University of Leeds, UK.
2 Institute for Molecular Pathology, Vienna, Austria.

Epigenetic processes involve the establishment of patterns of gene expression via heritable alterations in the chromatin structure of genes, however, the molecular details how these processes are regulated in development are poorly understood. We therefore asked the questions: (i) How is active chromatin established at specific genes in stem cells and early precursor cells? (ii) How is chromatin altered during the stages when cell fates are decided, and can we draw general conclusions about how such decisions are made? (iii) How is gene expression in individual cell lineages specified and restricted, and last, but not least: which transcription factors regulate these processes and how do they cooperate with chromatin templates? One of the genes we study is the gene for the macrophage-colony-stimulating factor receptor (csf1R or c-fms). This gene is expressed in hematopoietic stem cells (HSCs), up-regulated in macrophages and silenced in lymphoid cells. To identify the molecular events taking place during developmentally controlled gene activation and silencing, we have established methods allowing to study the development of an active chromatin structure in hematopoietic precursor cells at high resolution and we use knock-out mice to examine the impact of specific factors on chromatin structure development.
We have previously shown that the c-fms locus is organized into active chromatin in HSCs and committed myeloid precursors (1, 2). Here we show that transcription factor complex assembly and active chromatin formation at the c-fms locus are critically dependent on the presence of the transcription factor PU.1. Using precursor cells from PU.1 knock-out mice carrying an inducible PU.1 gene, we examined how active chromatin structure in precursor cells is established and defined a precise order of assembly events from the silent state (3).
To study lineage specification at the chromatin level we examined silencing of c-fms during B cell development (1). We showed that epigenetic silencing of the c-fms locus during B lymphopoiesis occurs in a distinct order and that even mature B cells display a poised chromatin structure. The transcription factor Pax5 (BSAP) is required for the expression of a B cell specific genetic program and for B cell differentiation, but is also required to suppress genes of alternative lineages and silencing of c-fms is crucially dependent on the presence of this factor. Pax5 has to be present throughout B lymphopoiesis to counteract active chromatin formation and to maintain myeloidspecific genes in a silent state. To gain insight into the molecular mechanism by which Pax5 represses c-fms expression we have examined how Pax5 alters c-fms chromatin modification and chromatin accessibility (4). We defined the c-fms promoter as a direct target of Pax5 and show that Pax5 targets the basal transcription machinery of c-fms by binding to a low-affinity binding site. Our results point to a 5 model by which Pax5 blocks recruitment of the basal transcription machinery when the levels of myeloid transcription factors are low and chromatin is in a neutral state.
Taken together, our experiments support a model by which gene expression is primed in HSCs by the selective activation of specific cis-elements. These elements are subject to regulation by the balanced expression of cross-regulatory transcription factors leading to either lineage-specific activation or silencing.

(1) Tagoh, H., Himes, R., Clarke, D.,Leenen, P., Riggs, A.D., Hume, D. and Bonifer, C. (2002). Transcription factor complex formation and chromatin fine structure alterations at the murine c-fms (CSF-1 receptor) locus early myeloid precursor maturation. Genes & Development 16, 1721 – 1737
(2) Tagoh H, Schebesta A, Wilson N, Hume, DA, Busslinger M, and Bonifer C. Epigenetic silencing of the c-fms locus during B-lymphopoiesis occurs in discrete steps and is reversible. EMBO J. 2004; 23: 4275-4285
(3) Krisinska,H. Hoogenkamp, M., Tagoh, H.,Laslo, P., Singh, and Bonifer, C. Transcription factor at assembly of the c-fms locus in development begins at the promoter and is crucially dependent on the transcription factor PU.1. Submitted.
(4) H. Tagoh, R. Ingram, N.Wilson, G. Salvagiotto, A. Warren, M. Busslinger and C. Bonifer (2006). The mechanism of repression of the myeloid-specific c-fms gene by Pax5 during B lineage restriction. EMBO Journal. Feb. 16.

Epigenetic memory of donor cell differentiation status in Xenopus nuclear transplant embryos

Ray Ng and John Gurdon

Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, United Kingdom.

Different cell types have characteristic patterns of gene expression. Once a cell has differentiated, its daughter cells nearly always differentiate in the same way. The maintenance of cell lineage involves either instructions from a cell's surroundings or the inheritance of memory from a parent cell. In normal development, the differentiation state of a cell is remarkably stable and irreversible. However, we have demonstrated that in Xenopus nuclear transfer experiments using both endoderm and neuroectoderm donor cells, there is a substantial overexpression of donor celltype specific genes in the donor irrelevant cell-type in some nuclear transplant embryos. This refers to the epigenetic memory of donor nuclei. This actively transcribed state of a gene can be propagated through many mitotic cell divisions in the absence of the stimulus that first induced the activity of this gene. To understand the basis of epigenetic memory, we studied the Xenopus MyoD promoter and demonstrated that its transcriptional activity is not regulated by DNA methylation; nevertheless, the memory of MyoD is seen in some somite-derived nuclear transplant embryos. This implies that DNA methylation is not responsible for the maintenance of epigenetic memory of active gene state in nuclear transplant embryos. Furthermore, chromatin immunoprecipitation analysis demonstrated that there is enrichment of histone variant H3.3 in the MyoD promoter region in cells of nuclear transplant embryos with active memory. This suggests that H3.3 incorporation took part in the maintenance of epigenetic memory of donor active gene state in nuclear transplant embryos. These results may provide new insights into the stability and maintenance of cell lineage in normal development.

Embryo technology: inheritance and implications for stem cells

Prof. Miodrag Stojkovic

Centre de Investigación, Príncipe Felipe, C/E.P. Avda. Autopista del Saler, 16-3 (junto Oceanográfico) 46013 Valencia, Spain

Human embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development. To data, all hESC lines has been derived from viable developing human embryos at morula or blastocyst stage obtained after in vitro fertilisation (IVF) or intracytoplasmic sperm injection (ICSI). The most obvious difference between the two procedures is the level of selection of sperm for assisted reproduction technology (ART). Some of the published work concluded that inappropriate maternal methylation is the most common epimutation that arises, resulting in imprinting disorders including Beckwith- Wiedemann Syndrome (BWS) and that there is some relation between ART and BWS (Olivennes et al., 2001; Maher et al., 2003). The nature of the oocyte derived factors responsible for reprogramming is largely unknown although it is clear from activation studies that their existence is transitory (Boiani et al., 2005). From the point of view of the normally fertilised oocyte, their limited persistence is undoubtedly sufficient for the task of rapidly demethylating the incoming paternal DNA, but the highly differentiated state of a transplanted adult somatic cell in the case of nuclear transfer (NT) may be more problematic. Therefore, other types of cells which are not terminally differentiated may behave different and make better donors than somatic cells. Donor cells from early preimplantation stage embryos or the use of undifferentiated embryonic stem cells (ESC) as nuclear donors gives rise to viable offspring with greater efficiency than many somatic cell types since they may be more easily reprogrammed as they have a lower level of genomic DNA methylation per blastomere/cell. Many of the epigenetic errors which result from NT affect imprinted genes typically involved in extra embryonic development, they would be less likely to affect the embryos from which ESC are derived. Therefore, gene expression aberrations observed in tissues of NT animals have also raised concerns regarding the medical application of stem cells derived from human NT embryos. On the contrary, previous experiments in mice have demonstrated that the developmental and differentiation potential of nuclear transfer stem cells (NTSC) is identical to that of cells derived from fertilised blastocysts (Brambrink et al., 2006). Based on their transcriptional profiles, NTSC lines derived from NT and fertilised mouse blastocysts were indistinguishable which supports the notion that stem cells derived from fertilised or NT embryos at least in mouse have an identical potential.

Nanog in self-renewal of mouse embryonic stem cells

Ian Chambers, Jose da Silva, Jennifer Nichols & Austin Smith

Centre Development in Stem Cell Biology, Institute for Stem Cell Research, University of Edinburgh, Scotland

Nanog overexpression enhances the efficiency of self-renewal of mouse embryonic stem (ES) cells during culture in the presence of LIF and can allow propagation of pluripotency in the absence of LIF. The phenotypic similarity between differentiated cells formed following LIF withdrawal and those formed in response to elevated Oct4 levels suggests that Nanog can modulate effects of Oct4. Data on this point will be presented together with a continuing assessment of the requirement of Nanog for pluripotency.

Specification of neural fate by coiled-coil and chromatin remodelling proteins.

Costis Papanayotou¹, Anne Mey², Anne-Marie Birot², Yasushi Saka³, Sharon Boast¹, Jim C Smith³, Jacques Samarut² and Claudio D Stern¹

1 Department of Anatomy & Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
2 Laboratoire de Biologie Moléculaire de la Cellule, Ecole Normale Supérieure de Lyon, UMR CNRS 5161, INRA 1237, 46 allée d'Italie, F-69364 Lyon Cedex 07, France
3 Wellcome/CR-UK Gurdon Institute for Cancer and Developmental Biology, Tennis Court Road, Cambridge CB2 1QN, UK

Early embryonic cells are multipotent, their fates gradually being allocated by sequential intercellular signalling events, many of which are now known. However we know little about the downstream intracellular mechanisms of cell commitment. Here we study the regulation of a definitive neural plate marker, Sox2. We show that competitive interactions between three coiled-coil domain proteins, ERNI, BERT and Geminin, regulate Sox2 expression. ERNI functions as an antagonist of Sox2 expression in early development. Later, BERT binds to ERNI and blocks it. We provide evidence that these proteins modulate the recruitment of different HP1 transcriptional repressors to Brahma-containing chromatin remodelling complexes, which otherwise activate Sox2 expression. These results provide a mechanism regulating multipotency and commitment during gastrulation and early neural development.