Theme leaders: Marc Timmers (UMCU) & Albert Heck (UU)

Background

Stem cells are receiving considerable interest because of their ability to self renew as well as to form one or more differentiated cell types. Embryonic stem cells are unique in their ability to self renew indefinitely whilst maintaining pluripotency, the ability to differentiate to a wide variety of specialized cells. Stem cells promise a range of clinical applications in the area of regenerative medicine and a detailed molecular understanding provides clues to both their unique self renewal properties and their ability to make different developmental decisions. A number of studies postulated the presence of stem cell-specific proteins and histone modifications that impose ‘stemness’ properties on undifferentiated cells. Indeed, a number of proteins have been identified which are decreased or lost upon the onset of differentiation (Oct4, Nanog, etc). Most of these proteins are chromatin remodeling proteins and transcription factors that regulate subsets of genes. Conversely, it has been shown that forced (ectopic) expression of a limited number of these stem-cell specific genes is sufficient to reprogram adult cells into pluripotent stem cells, inducing the phenotypic properties characteristic of embryonic stem cells (pluripotency, differentiation behavior, expression of stem cell markers). An important aspect of this is the unique pattern of activating and repressive histone modifications on the gene regulatory sequences of developmentally important genes. This research program will focus on important aspects of stem cell biology, induced pluripotency and epigenetic regulation, employing proteomic approaches.

The generation of cells with induced pluripotency (iPS cells) hallmarks an important breakthrough in the development of stem cell therapy and platforms for disease research. Importantly, iPS could solve both technical and ethical issues in the clinical use of embryonic stem cells since patient-derived cells could replace non-autologous cells and prevent complications arising from immunological rejection. Although early experiments have shown the similarity between pluripotent SC and iPS, there is an obvious need to classify iPS molecularly, and to establish whether they have re-acquired the full potential to differentiate and correct epigenetic hallmarks. An important question is whether molecular pathways and networks are re-established in iPS cells to control gene expression in a fashion similar to that in embryonic stem cells (ESCs) previously. In this program we will investigate to what extent the characteristic molecular properties of human and mouse ESCs that have emerged over the past few years can be extrapolated to iPS. We will study how both cell types respond to external stimuli and inhibitors in terms of loss of pluripotency. Early events in the loss of pluripotency are mainly propagated by activation of signaling pathways, and therefore identification of these pathways and the kinases involved in this process will shed light on similarities and differences between ESC and iPS. Furthermore gene expression in ESC has been shown to be regulated by epigenetic mechanisms. We will therefore focus on the enzymes involved in chromatin remodeling and on histone modifications associated with transcriptional activation and silencing to establish whether iPS are reprogrammed in this aspect as well.

Approach

Here we propose to establish a program in stem cell proteomics focusing on the pluripotent nature of ESC and cross compare this with that of iPS. This will be done using mouse as well as human cell lines, and will be approached from various angles. We will especially focus on signaling cascades and epigenetic mechanisms invoked before and after induction of differentiation. Signalling cascades₂ chromatography. Quantitative analysis of these data will provide insight into the phosphorylation events over time. At the same time, this will be an excellent resource for defining profiling protein networks and defining kinase motifs that are enriched upon stem cell stimulation. will be studied by quantitative phospho-proteomics. Specifically, cells will be stimulated with various growth factors (BMP, activin, LIF, FGF), either alone or in combination with inhibitors or the factors will be removed. These cells will be mixed with SILAC-labeled untreated control cells and subjected to enrichment for phosphopeptides by TiO

Pluripotency and the loss thereof will be studied in mouse ESC and iPS focusing on epigenetic mechanisms involving polycomb(Pc)- and Trx-class of proteins. We will be especially interested in proteins interacting with these crucial chromatin regulatory proteins, the dynamics of these interactions, and their dependence on posttranslational modifications (e.g. methylation, phosphorylation, acetylation, ubiquitination, sumoylation). A similar approach will be used for TFIID complexes. Likewise, there is preliminary evidence that epigenetic regulators such as polycomb factors and chromatin remodeling complexes are regulated both on the level of expression and through post-translational modifications. In addition, we will employ affinity purification and advanced MS methods to determine the co-occurrence of histone modification marks.

All of these studies will be accompanied by functional analyses to corroborate proteomic data and to cross correlate results obtained in human and mouse cells, both ESC and iPS.

This program brings together partners with interests in stem cell pluripotency and differentiation (Mummery, Van Lohuizen, Verrijzer), signal transduction (Krijgsveld, Mummery, Verrijzer), protein interactions (Verrijzer, Van Lohuizen, Timmers, Krijgsveld), epigenetic gene regulation (Verrijzer, Van Lohuizen, Timmers). At the same time, they bring in complementary expertise in cell biology (Mummery, Van Lohuizen), quantitative mass spectrometry (Krijgsveld, Demmers), protein tagging approaches (Timmers, Verrijzer).

Deliverables

• Characterization of phosphorylation pathways in ESC and iPS before/after induction of differentiation
• Kinase profiling in ESC and iPS
• Functional analysis of phosphorylated proteins and kinases in ESC and iPS
• Purification of H3K4me-marked nucleosomes from ES and iPS cells
• Tagging/purification of TFIID from mouse ES cells
• Tagging/purification of histone H3 modifying complexes from mouse ES cells
• Purification and characterization of antagonistic epigenetic regulators from ESC, neural stem cells and differentiated neural cells
• Characterization of post-translational modifications of epigenetic regulatory complexes during ESC differentiation
• Identification of targeting mechanisms of epigenetic regulators during differentiation
• Profiling of Polycomb protein complexes in iPS and ES cells
• Profiling of changes in Polycomb complexes upon differentiation of ES and iPS cells