< Terug naar vorige pagina

Publicatie

Uncovering the epigenetic landscape of cell fate reprogramming and X chromosome reactivation

Boek - Dissertatie

In this thesis, I aimed to define the dynamics and the mechanisms that orchestrate the reversal of stable gene silencing during reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). I used X chromosome reactivation as a paradigm to better understand the reversal of silent chromatin. In addition, my goal was also to define the gene regulatory logic of mammalian pre- implantation development and reprogramming, as well as to evaluate totipotency in extended pluripotent stem cells. The work has led to exciting scientific advances in our understanding of reprogramming and early development. In article 1, I used allele-specific transcriptomics to define the timing of X Chromosome Reactivation (XCR) during mouse iPSC reprogramming at a chromosome-wide scale. This enabled us to find that transcriptional activation of genes from the inactive X chromosome takes place in several steps. We found that several genes initiate reactivation earlier than others. Moreover, we thoroughly studied the relationship between the reactivation kinetics and a broad range of genetic and epigenetic features. These analyses revealed that genes that initiate reactivation early reside closer to genes that escape inactivation (termed escapee genes) than those that reactivate later. Moreover, we found that early reactivating genes are more enriched in the binding of pluripotency transcription factors (TF) on the active X chromosome (Xa) than late genes in embryonic stem cells (ESCs). We also identified histone deacetylases as barriers to XCR during reprogramming to iPSCs. In article 2, I aimed to extend our understanding of how gene transcription is reacquired during XCR with a particular focus on the role of transcription factors. First, we employed single-cell transcriptomics to define the dynamics of gene reactivation while resolving heterogeneity during reprogramming as well as to define gene regulatory networks of reprogramming to iPSCs. In line with previous bulk studies, we found that several genes initiate reactivation early, however most genes reactivate to full levels later during reprogramming. Moreover, our analyses revealed that the upregulation of the active X chromosome in starting cells is erased during reprogramming to pluripotency and that the rate of erasure is proportional to that of XCR. We also described the dynamic remodelling of gene regulatory networks during reprogramming. Second, we set out to define the chromatin accessibility landscape of the reactivating X chromosome. We identified the region-specific timing of chromatin accessibility acquisition on the reactivating X chromosome. Our results also revealed that the erasure of X chromosome upregulation (XCU) is manifested by a decrease of chromatin accessibility. Third, we integrated single-cell RNAseq data and gene regulatory networks with chromatin accessibility information. This allowed us to identify TFs that might directly target the inactive X chromosome (Xi) for transcriptional reactivation. In article 3, we addressed the need in the developmental and stem cell biology fields for standardized criteria to assess the developmental potential of stem cells. Specifically, we defined the molecular and cellular criteria of various levels of stringency that allow distinguishing totipotent stem cells using in vitro and in vivo assays. In this work, we evaluated most recently established mouse in vitro models of cells with extended plasticity against those criteria and established a rigorous benchmark for defining totipotency.
Jaar van publicatie:2021
Toegankelijkheid:Closed