Understanding X-chromosome regulation and early development

In this PhD thesis, I aimed to investigate how chromatin mechanisms contribute to transcriptional processes and cell fate decisions in mammals. I used X-chromosome dosage compensation in reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) as a paradigm to study chromatin organization and gene regulation. In addition, my goal was to understand how chromatin-based processes regulate naive human pluripotency and their developmental potential. This work has led to exciting scientific discoveries and advances in our understanding of cell reprogramming, X-chromosome regulation and pluripotency. The main publications I largely worked on were used to write this thesis. In article 1, I aimed to unravel the transcriptional kinetics and mechanisms of reprogramming of mouse somatic cells to iPSCs and concomitant X-chromosome reactivation (XCR). Using allele-specific transcriptomic analyses, we defined the dynamics of XCR during reprogramming of somatic cells into iPSCs. We found that XCR is a gradual process with different genes reactivating at different times during reprogramming. Interestingly, we observed that a subset of early reactivating genes have reduced genomic distance to genes that escape inactivation (escapee genes) compared with late reactivating genes. This result suggests that the localization of a gene may be related to gene silencing reversibility. In addition, our analyses revealed that early genes on the active X chromosome show increased pluripotency transcription factor (TF) binding compared to late genes. Last, we also showed that histone deacetylases restrict XCR in reprogramming. In article 2, I studied the chromatin accessibility and transcriptional state of the X-chromosomes as a paradigm to understand how gene dosage is regulated. First, using allele-specific analyses on chromatin accessibility and transcriptomics, I revealed that transcriptional upregulation on the sole active X in mouse female and male somatic cells is accompanied by an increase in chromatin accessibility compared to autosomal levels. Moreover, we confirmed that X-chromosome upregulation (XCU) is driven by increased burst frequency. Next, our results showed that, XCU, increased chromatin accessibility and increased transcriptional burst frequency are erased by reprogramming, concomitant with XCR in female cells. Moreover, upon loss of one X chromosome (XO cells), XCU is reacquired, indicating that X-chromosome aneuploidies are dosage compensated. Our results reveal dosage compensatory mechanisms that involve modulation of chromatin accessibility to balance X-to-Autosome gene dosage in mammals. Furthermore, we characterized the gene regulatory networks (GRNs) during reprogramming to iPSCs and identified candidate TFs that might have a role in XCR. In article 3, I explored chromatin mechanisms that play a role in the developmental plasticity of naive human pluripotent stem cells (hPSCs). First, we performed a multi-omics study to define the chromatin-associated proteome, histone post-translational modifications and transcriptome of naive and primed hPSCs. Surprisingly, we found that the repressive histone mark histone H3 lysine 27 trimethylation (H3K27me3) is strongly enriched in naive compared to primed hPSCs. Specifically, we determined that H3K27me3 marks lineage-determining genes. Moreover, I revealed that H3K27me3 acts as a chromatin barrier that oppose the induction of the trophoblast fate in naive to trophoblast conversion and during human blastoid formation.

Jaar van publicatie:2022

Toegankelijkheid:Closed