Doctor of Philosophy, Universiteit Utrecht (2011)
Joanna Wysocka, Postdoctoral Faculty Sponsor
During development cells change their cellular identity in response to signaling cues from their environment in a highly reproducible manner.Understanding how these cell fate decisions are driven and manifest in the cell are of key interest for developmental biology but also for cancer research and regenerative medicine. Transcriptional enhancers are the main driver of developmental cell fate transition, however we still don't understand how enhancers are driving the transcription of their target gene. In order to analyze enhancer function we have recently developed a powerful differentiation strategy that allow us now to follow enhancer activation over time in a dynamic fashion.
During early embryonic development pluripotent cell of the epiblast give rise to all cells of the embryo. We have characterized in depth the epigenetic changes that drive the transition from the early pre-implantation epiblast to the late postimplantation epiblast, we have identified key markers and have mapped changes in the enhancer landscape. In the future we can harness these results to focus on the role that single enhancer elements have the expression of their target gene(s).
We are employing
- the powerful CRISPR-Cas9 system to mutate and delete enhancers
- chromatin conformation capture to analyze changes in the 3dimensional chromatin folding
-single molecule RNA-FISH to test how changes in the population are reflected in single cells.
Naive and primed pluripotency is characterized by distinct signaling requirements, transcriptomes, and developmental properties, but both cellular states share key transcriptional regulators: Oct4, Sox2, and Nanog. Here, we demonstrate that transition between these two pluripotent states is associated with widespread Oct4 relocalization, mirrored by global rearrangement of enhancer chromatin landscapes. Our genomic and biochemical analyses identified candidate mediators of primed state-specific Oct4 binding, including Otx2 and Zic2/3. Even when differentiation cues are blocked, premature Otx2 overexpression is sufficient to exit the naive state, induce transcription of a substantial subset of primed pluripotency-associated genes, and redirect Oct4 to previously inaccessible enhancer sites. However, the ability of Otx2 to engage new enhancer regions is determined by its levels, cis-encoded properties of the sites, and the signaling environment. Our results illuminate regulatory mechanisms underlying pluripotency and suggest that the capacity of transcription factors such as Otx2 and Oct4 to pioneer new enhancer sites is highly context dependent.
View details for DOI 10.1016/j.stem.2014.04.003
View details for Web of Science ID 000341248500019
Transcriptional enhancers are the primary determinants of tissue-specific gene expression. Although the majority of our current knowledge of enhancer elements comes from detailed analyses of individual loci, recent progress in epigenomics has led to the development of methods for comprehensive and conservation-independent annotation of cell type-specific enhancers. Here, we discuss the advantages and limitations of different genomic approaches to enhancer mapping and summarize observations that have been afforded by the genome-wide views of enhancer landscapes, with a focus on development. We propose that enhancers serve as information integration hubs, at which instructions encoded by the genome are read in the context of a specific cellular state, signaling milieu and chromatin environment, allowing for exquisitely precise spatiotemporal control of gene expression during embryogenesis.
View details for DOI 10.1016/j.tig.2012.02.008
View details for Web of Science ID 000305094000004
View details for PubMedID 22487374
Murine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human iPSC reprogramming in the presence of LIF yields human stem cells that display morphological, molecular, and functional properties of murine ESCs. We termed these hLR5 iPSCs because they require the expression of five ectopic reprogramming factors, Oct4, Sox2, Klf4, cMyc, and Nanog, to maintain this more naive state. The cells are "metastable" and upon ectopic factor withdrawal they revert to standard human iPSCs. Finally, we demonstrate that the hLR5 state facilitates gene targeting, and as such provides a powerful tool for the generation of recombinant human pluripotent stem cell lines.
View details for DOI 10.1016/j.stem.2010.05.003
View details for Web of Science ID 000278840700014
View details for PubMedID 20569691
The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade.
View details for DOI 10.1016/j.stemcr.2014.09.003
View details for Web of Science ID 000345118600017
View details for PubMedID 25418731
Efficient generation of competent vasculogenic cells is a critical challenge of human induced pluripotent stem (hiPS) cell-based regenerative medicine. Biologically relevant systems to assess functionality of the engineered vessels in vivo are equally important for such development. Here, we report a unique approach for the derivation of endothelial precursor cells from hiPS cells using a triple combination of selection markers--CD34, neuropilin 1, and human kinase insert domain-containing receptor--and an efficient 2D culture system for hiPS cell-derived endothelial precursor cell expansion. With these methods, we successfully generated endothelial cells (ECs) from hiPS cells obtained from healthy donors and formed stable functional blood vessels in vivo, lasting for 280 d in mice. In addition, we developed an approach to generate mesenchymal precursor cells (MPCs) from hiPS cells in parallel. Moreover, we successfully generated functional blood vessels in vivo using these ECs and MPCs derived from the same hiPS cell line. These data provide proof of the principle that autologous hiPS cell-derived vascular precursors can be used for in vivo applications, once safety and immunological issues of hiPS-based cellular therapy have been resolved. Additionally, the durability of hiPS-derived blood vessels in vivo demonstrates a potential translation of this approach in long-term vascularization for tissue engineering and treatment of vascular diseases. Of note, we have also successfully generated ECs and MPCs from type 1 diabetic patient-derived hiPS cell lines and use them to generate blood vessels in vivo, which is an important milestone toward clinical translation of this approach.
View details for DOI 10.1073/pnas.1310675110
View details for Web of Science ID 000322441500064
View details for PubMedID 23861493
Recent data demonstrates that stem cells can exist in two morphologically, molecularly and functionally distinct pluripotent states; a naïve LIF-dependent pluripotent state which is represented by murine embryonic stem cells (mESCs) and an FGF-dependent primed pluripotent state represented by murine and rat epiblast stem cells (EpiSCs). We find that derivation of induced pluripotent stem cells (iPSCs) under EpiSC culture conditions yields FGF-dependent iPSCs from hereon called FGF-iPSCs) which, unexpectedly, display naïve ES-like/ICM properties. FGF-iPSCs display X-chromosome activation, multi-lineage differentiation, teratoma competence and chimera contribution in vivo. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions. Characterization of the key molecular signalling pathways revealed FGF-iPSCs to depend on the Activin/Nodal and FGF pathways, while signalling through the JAK-STAT pathway is not required for FGF-iPS cell maintenance. Our findings suggest that in 129 and Bl6 mouse strains, iPSCs can dominantly adopt a naive pluripotent state regardless of culture growth factor conditions.
View details for DOI 10.1371/journal.pone.0016092
View details for Web of Science ID 000285793600060
View details for PubMedID 21209851
Pluripotent stem cells (PSCs) have been classified into two distinct states: a primitive, naive LIF-dependent state represented by murine ESCs, and a primed bFGF-dependent state observed in murine and rat epiblast stem cells (EpiSCs). The vast similarities between EpiSCs and human ESCs suggest that, despite their blastocyst origin, human ESCs exist in a primed pluripotent state. Recent findings demonstrate that the naive and primed pluripotent states are interconvertible, even in human cells, and hint that growth factor-mediated Nanog expression may be an important factor regulating the balance between them.
View details for DOI 10.1016/j.stem.2010.10.007
View details for Web of Science ID 000284390500007
View details for PubMedID 21040897
The ubiquitin ligase Hul5 was recently identified as a component of the proteasome, a multisubunit protease that degrades ubiquitin-protein conjugates. We report here a proteasome-dependent conjugating activity of Hul5 that endows proteasomes with the capacity to extend ubiquitin chains. hul5 mutants show reduced degradation of multiple proteasome substrates in vivo, suggesting that the polyubiquitin signal that targets substrates to the proteasome can be productively amplified at the proteasome. However, the products of Hul5 conjugation are subject to disassembly by a proteasome-bound deubiquitinating enzyme, Ubp6. A hul5 null mutation suppresses a ubp6 null mutation, suggesting that a balance of chain-extending and chain-trimming activities is required for proper proteasome function. As the association of Hul5 with proteasomes was found to be strongly stabilized by Ubp6, these enzymes may be situated in proximity to one another. We propose that through dynamic remodeling of ubiquitin chains, proteasomes actively regulate substrate commitment to degradation.
View details for DOI 10.1016/j.cell.2006.09.051
View details for Web of Science ID 000243690000019
View details for PubMedID 17190603