KRAB/KAP1 Regulation of Embryonic Stem Cell Pluripotent Self-renewal

KRAB-ZFPs constitute the largest family of transcription factors (TFs) encoded by the mouse genome and are known to interact with the co-repressor KAP1. KRAB/KAP1-mediated regulation is essential for several development- and ESC- specific functions, in particular regulation of pluripotent self-renewal. We performed an expression analysis in mouse pluripotent ESCs and differentiated cells. A large number of KRAB-ZFPs was expressed in pluripotent cells and many of them were differentially regulated in other cell types, suggesting that a set of KRAB-ZFPs can be involved in ESC-specific functions. We identified a set of candidate genes, including Zfp459, which show a pluripotency-specific expression pattern. Zfp459 depletion in ESCs induced transcriptional perturbation of pluripotency- and differentiation-associated genes, even if their ability to self-renew or to differentiate was not impaired, and its expression was not required for the formation of blastocysts ex vivo. To understand the precise role of Zfp459 in ESCs further experiments for the identification of its targets are still required. We used a lentiviral vector (LV) to over-express in ESCs ZFP57, which had been previously demonstrated to play a role in regulating genomic imprinting, in fusion with HA-tags. ChIP-seq experiments using a HA antibody demonstrated that ZFP57-HA binds all the known and predicted imprinted loci and identified a hexanucleotide consensus in most of the ZFP57 binding sites co-occupied also by KAP1 and its associated factor SetDB1. We also demonstrated that our LV based expression system could be exploited to perform ChIP-seq analyses in ESCs of TFs, such as Zfp459, for which specific antibodies are not available. Finally, when we cultured ESCs in different conditions, we observed that KAP1 recruitment upstream of Nanog, which had been previously observed, was dependent on the MAPK and GSK3 signaling pathways. We hypothesized that this could be due to the presence in these conditions of ESCs expressing heterogeneous Nanog levels. However, when Nanoghigh and Nanoglow cells were sorted, both the populations shown a comparable enrichment for KAP1 binding upstream of Nanog. The precise role for the KAP1-mediated regulation of Nanog in ESCs and its dependence on extrinsic signaling pathways remains unclear, even if we hypothesize that it can depend on the mono-/bi-allelic expression of Nanog in ESCs and during early development.

Quantitative analysis of SOX2 and OCT4 expression in pluripotency and differentiation

Daniel Strebinger

Embryonic stem (ES) cells have the capacity to give rise to all cell types of the adult organism and can be used as a model to study early cell fate decisions as they closely recapitulate in vivo events. The M and G1 phases have been suggested to be the key time windows for pluripotent cells to engage toward differentiation and SOX2 and OCT4 have been shown to orchestrate the dissolution of pluripotency and induction of cell fate commitment. Furthermore, it is known that the levels of some pluripotency transcription factors fluctuate over time, leading to the notion that ES cells experience dynamic heterogeneity, where for example low levels of NANOG can be found in cells prone to differentiation, whereas cells with high NANOG expression are in a robust pluripotent state. However, it remains unclear if and how (i) the action of transcription factors in specific cell cycle phases and (ii) protein level variations of factors that show mild differences between single cells impact cell fate decisions. This is in part due to the lack of quantitative single-cell meth-ods allowing dissecting the origin and functional consequences of gene expression heterogeneity. In this work, we established a method based on internal tagging of endogenous proteins with an excisable reporter to perform absolute molecule quantification in single living cells. This enabled us to investigate fluctuations of endogenous protein expression levels, the heterogeneity of degradation rates between individual cells and determine absolute amounts of proteins synthesized over the cell cycle. Additionally, inducible excision of the tag enabled us to estimate endogenous mRNA half-lives. Next, by using live-cell microscopy of fusion proteins, we show that SOX2 and OCT4 stay bound to mitotic chromosomes through their DNA binding domains. We then characterized the function of SOX2 at the M-G1 transition, showing that its absence specifically in this phase impairs pluripotency maintenance and abolishes its ability to induce neuroectodermal commitment. This suggests, that transcription factor activity at the M-G1 transition is essential for cell fate maintenance and differentiation. Lastly, we show that mild endogenous fluctuations in the levels of key transcription factors in ES cells bias cell fate decisions. We found that high OCT4 levels at the onset of differentiation increase the probability of single cells to commit to both neuroectoderm and mesendoderm. Further, we showed that high SOX2 levels result in a higher number of cells acquiring a neuroectodermal cell fate. This not only shows that the differentiation potential of embryonic stem cells is highly sensitive to small variations in intracellular concentrations of pluripotency transcription factors but further suggests that endogenous fluctuations of these are a major source of heterogeneity in cell fate decisions. In conclusion, the presented work identifies that the SOX2 activity in the M-G1 transition is important for cell fate maintenance and differentiation. Furthermore, the heterogeneity of protein levels governs cell fate decisions, suggesting the existence of different short-lived cell states in populations of presumed homogenous cells. We think that the minor differences in the levels of transcription factors and the cell cycle specific sensitivity of cells to transcription factor activity could be important mechanisms guiding maintenance and differentiation potential in various stem cell types.

EPFL2018

RhoV, a Potential Notch Target Gene in Murine Skin, Does not Play an Essential Role in Epidermis Development and Homeostasis

April Bezdek Pomey

The evolutionarily conserved Notch signaling cascade plays a pivotal role in the regulation of many fundamental processes such as stem cell maintenance, proliferation, and differentiation. Recently, we aimed to identify downstream target genes regulated by Notch1 during epidermal differentiation. For this purpose an Affymetrix mouse gene chip array of 14.000 genes was performed in both Notch1 gain and loss of function experiments using murine primary keratinocytes. Thirteen genes positively or negatively regulated, based on expression levels and relevance of the involved signaling pathways, were further validated by real-time PCR. Among these, we decided to focus on the Rho GTPase RhoV because it showed robust up- and down-regulation under both gain- and loss-of-function conditions. Therefore, we generated different transgenic mice expressing either a dominant active or a dominant negative form of RhoV specifically in the skin. Morphological and histological analyses did not reveal any overt skin phenotype in transgenic mice. In addition, challenging the system by performing wound healing assays demonstrated that the healing process in transgenic mice occurred normally and that the migration and/or differentiation of keratinocytes was unimpaired. Taken together, our data suggest that gain or loss of RhoV function does not perturb skin development and homeostasis, and does not affect the wound healing process. In addition to the investigation of the potential Notch target gene RhoV, we sought to generate both a floxed Jag2 gene targeted mouse line and a Notch2-specifc reporter mouse. The former was intended to enable conditional deletion of the Notch ligand Jagged2 and therefore allow its role to be studied in a loss-of-function context. The Notch2 reporter mouse is a tool enabling the in vivo study of cells that receive a Notch2-mediated signal. The strategy employs elements of a Tet-Off system and allows cells receiving a Notch2 specific signal to be identified in any tissue at any given time point.

EPFL2010

Differentiation of human embryonic stem cells to hepatocytes

Noam Beckmann

Many liver diseases involve hepatocyte malfunction. In most cases, transplantation of corrected hepatocytes would correct for disease symptoms. Liver donor shortage and hepatocyte inability to proliferate in vitro accentuate this problem. Embryonic stem and induced pluripotent stem cells proliferate indefinitely and have the capacity to differentiate into every cell type of the body. They could thus represent a solution to this organ donor shortage. In this project, we aim to differentiate ES and iPS cells to hepatocytes. First, we consider an approach to select the ES/iPS cell line showing the best potential to generate liver lineage using teratomas as a selection tool. Then we use lentiviral vectors to transduce this selected cell line with essential liver-‐specific development factors to help differentiation efficiency. Next, we developed an in vitro differentiation protocol based on existing protocols and developmental clues of liver development to generate a pool of hepatocyte precursors. Later on, we injected these differentiated cells in mice liver of Fah-‐, Rag2-‐ and Il-‐2rγ-‐deficient mice to check for engraftment and liver marker expression. Our findings show, after checking for hepatocyte-‐ specific markers in the differentiated cells, that our previous selection for the potentially best cell line seems correct. We also find that the transcription factors involved in liver development appear to have a negative effect in vitro on liver differentiation. Applying our protocol to regular ES cells results on the production of late maturity hepatocyte markers, showing correct differentiation and maturation of newly formed hepatocytes. Finally, mouse in vivo liver transplantation of these differentiated cells gives us human albumin expression detectable in blood serum of said mice