Weeks, Samuel Luke; (2022) Characterisation of the roles of Stag paralogs in transitions of pluripotency. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Specification of cell identity requires spatio-temporal gene expression changes, which are broadly controlled by epigenetic and post-transcriptional mechanisms. De-regulation of these processes leads to a plethora of loss of cell identity-associated phenotypes, including cancer and developmental disorders. Stag proteins are established regulators of the cohesin complex, which dynamically structures 3D genome topology during development to influence gene expression. Despite their homology, Stag1, Stag2 and Stag3 paralogs display non-redundancy in maintenance of cell identity through mechanisms that remain poorly understood. Here, I utilise an in vitro murine Embryonic Stem (ES) cell commitment model to characterise Stags roles in cell fate decisions during transitions of pluripotency. First, I find exit of naïve ES cell identity is concomitant with Stag1 down-regulation and Stag2 up-regulation. Stag1 protein variants containing different intrinsically disordered regions (IDRs) were identified in ES cells and their selective knockdown resulted in heterogeneous cell fate decisions, indicating fine-tuning of genome topology. Through generation of a CRISPR/Cas9-tagged Stag1 ES cell line, I further uncover Stag1 co-localisation at heterochromatin domains. Degradation of Stag1 increases compaction of these condensates and disrupts nucleolar structure. Further, assessment of chromatin topology using SPRITE methodologies reveals Stag1 degradation promotes increased chromatin contacts at both Topologically Associated Domain (TAD) and compartment levels of genome organisation. In this project, I also identify expression of the germ cell-specific Stag3 paralog in pluripotent states. Stag3 knockdown decreased mRNA expression of the cell fate markers, including Dppa3, and resulted in a reduced commitment potential that was maintained through differentiation into Embryoid Bodies (EBs). Unexpectedly Stag3 knockdown also correlated with up-regulated Dppa3 protein levels. Contrasting to nuclear-localised Stag1 and Stag2, Stag3 exists as discrete cytoplasmic foci at established sites of post-transcriptional regulation, notably at the centrosome, around the nuclear envelope and along the cytoskeleton. Co-Immunofluorescence (co-IF) assays under Stag3 knockdown conditions suggests a role for Stag3 in structuring these sites. Thus, Stag3 is proposed to mediate ES cell fate decisions by post-transcriptional regulation of Dppa3. Overall, this project provides evidence for mechanisms in which Stags co-ordinate cell fate decisions and, for the first time, has reported on an extra-nuclear post-transcriptional function for Stag3.

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