Capturing The Dynamics Of Transcription Regulation During Neuromesodermal Progenitor Fate Specification

Abstract

During embryogenesis, cells undergo a series of critical fate decisions where lineage competence reflects the cell’s unique environment. External signals and internal regulatory mechanisms converge in the nucleus to control gene expression and ultimately determine cell fates. Mammalian genes are regulated by the complex interplay between millions of cis- regulatory elements (CREs), including enhancers and promoters, and transcription regulators (TRs), including transcription factors (TFs) and chromatin regulators. However, much remains unknown about the spatiotemporal interplay of these elements, particularly within the context of 3D chromatin organisation, and how it modulates context-dependent gene expression, fate competence, and fate specification. Moreover, how these dynamics are controlled at the molecular level during a cell fate choice remains underexplored. My PhD project therefore aimed to capture how the spatiotemporal interplay of TFs and the chromatin landscape modulates transcription regulation in development by integrating 1) live-cell single-molecule dynamics of chromatin and TFs with 2) genome-wide changes in the chromatin landscape and transcription during neuromesodermal fate specification and differentiation. To explore whether chromatin mobility changes in development, I used live-cell single- molecule localisation microscopy (SMLM) to characterise the biophysical properties of single nucleosomes from pluripotency exit to progenitor specification, and finally neuromesodermal differentiation (Chapter 3). Interestingly, I observed striking differences in the diffusive properties of chromatin during caudalisation. I further uncovered region-specific differences in the chromatin mobility along the anterior-posterior axis. To address the functional relevance of such changes, we next integrated population-wide sequencing modalities (Micro-C, CUT&Tag, CUT&RUN, RNA-seq), showing caudalisation is accompanied by reorganisation of the chromatin landscape, including chromatin compartments, enhancer-promoter loops, histone post-translational modifications, and chromatin-bound proteins (Chapter 4). I next explored the drivers of chromatin reorganisation, showing cell type-specific sensitivity to transcription regulation and acetylation inhibitors. Instead, I showed chromatin mobility is driven by modulation of the signalling environment, most notably WNT/β-catenin, a major determinant of caudal fate specification (Chapter 5). Interestingly, changes in chromatin mobility precede genome-wide chromatin reorganisation, transcriptional and proteomic changes. I further identified the TF CDX2 as a central chromatin regulator in caudal progenitors (Chapter 4). To address the role of CDX2 in chromatin regulation, I captured single-nucleosome dynamics in the absence of CDX factors, showing no changes in chromatin mobility during caudal fate acquisition and WNT/β-catenin modulation (Chapter 6). Moreover, CDX KO cells fail to establish caudal-specific chromatin loops and acquire appropriate region- specific cell identities in the presence of caudalising factors. Considering the TFs SOX2 and CDX2 are central regulators of the caudal progenitor gene regulatory network, but also other fates, I hypothesised context-specific transcription activation may be achieved through different nuclear dynamics, including nuclear diffusion and DNA binding (Chapter 7). We therefore captured live-cell single-molecule dynamics of SOX2 and CDX2 during differentiation, demonstrating cell-type specific signatures. Together, these findings provide a unique perspective into the spatiotemporal changes of chromatin and transcription factors during a cell fate choice, suggesting modulation of the signalling environment stirs the chromatin, potentially facilitating reorganisation of the chromatin landscape, and hence affecting TF nuclear dynamics and transcriptional activation.