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.