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Title: Regulation of nuclear receptor Esrrb function by Ncoa3 in mouse embryonic stem cell pluripotency
Author: Percharde, Michelle
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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The mouse embryonic stem cell (ESC) state depends upon a gene regulatory network centred on Oct4, Sox2 and Nanog (OSN). Notably, these and other key transcription factors (TFs) assemble in large, multi-protein complexes at enhancer loci to drive the high expression of target genes. It remains unclear, however, how each specific TF functions in transcription, and whether each depends on protein-protein interactions with other complexes. Furthermore, little is known about potential coregulators in ESCs that may regulate TF activity. To study the functional importance of TF-cofactor interactions in ESCs, work here was focused on the nuclear receptor Estrogen-related receptor beta (Esrrb). As with the core OSN triad, Esrrb is vital for the maintenance of ESC identity and self-renewal, as well as playing important roles in somatic cell reprogramming. Moreover, Esrrb was recently found to associate with components of the basal transcription machinery, hinting at a specific role for Esrrb in ESC transcription that remains to be characterised. Here, it was investigated how Esrrb function is conferred in ESCs, drawing on established knowledge of the regulation of estrogen-related receptors (ERRs) in somatic contexts. Both on somatic cell targets as well as in ESCs, Esrrb transcriptional activity is found to be absolutely dependent upon its ligand-binding domain (LBD) and AF-2 region. Functionally, inactivating the Esrrb AF-2 region mirrors Esrrb depletion itself, triggering differentiation and preventing Esrrb-dependent enhancement of self-renewal. Critically, Ncoa3 is discovered to be the key coactivator recruited to the Esrrb AF-2 region at ESC target genes. Ncoa3, like Esrrb, is essential for the maintenance of self-renewal and also enhances LIF-independence when stably overexpressed. Furthermore, Ncoa3 is highly upregulated in induced pluripotent stem cells (iPSCs), and its depletion inhibits somatic cell reprogramming. Genome-wide Ncoa3 ChIP-sequencing and DNA microarray analysis further demonstrate that Ncoa3 co-operates with Esrrb and the OSN circuitry at active enhancers, driving the expression of self-renewal and germ cell related genes. Finally, Ncoa3 is shown to bind RNA polymerase II in ESCs, and bridges Esrrb via its AF-2 region to the general transcription machinery. This work reveals an integrated model of transcriptional and coactivator control, mediated by Ncoa3, for the maintenance of ESC self-renewal and reprogramming.
Supervisor: Parker, Malcolm ; Azuara, Veronique Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available