Abstract
Embryonic Stem Cells (ESCs) can self-renew or differentiate into all specialised cell types in the adult body, a phenomenon known as pluripotency. Distinct pluripotent states have emerged as a key concept in ESC biology; the naïve state safeguards selfrenewal whilst primed pluripotency provides a window for differentiation. However, the molecular mechanisms that control transition between naïve and primed pluripotent states are poorly understood.Kinases and protein phosphorylation control all aspects of cellular decisionmaking, including ESC pluripotency and differentiation. I therefore performed a targeted screen to identify kinase inhibitors that modulate the naïve-primed transition in mouse ESCs. I show that selective compounds which inhibit both ERK5 and BET family bromodomains drive ESCs from the naïve state towards primed pluripotency. Using compound selectivity engineering and CRISPR/Cas9 genome editing, I deconvolve distinct functions for ERK5 and BRD4 in pluripotency regulation. Furthermore, I show that ERK5 signalling, in a manner dependent upon ERK5 kinase activity, upstream activation by MEK5 and a C-terminal transcriptional domain, maintain mESCs in the naïve state, and activation of the ERK5 pathway suppresses transition to primed pluripotency.
I employ quantitative phosphoproteomics to elucidate molecular mechanisms by which ERK5 functions in mESCs. Within a cohort of high confidence ERK5responsive phosphopeptides, I identify a dual phosphosite on the core naïve pluripotency transcription factor KLF2. ERK5 directly phosphorylates KLF2 at tandem Thr-Pro/Ser-Pro motifs, which form consensus binding sites for the Peptidyl-Prolyl Isomerase PIN1. KLF2 phosphorylation at these sites promotes ubiquitylation and degradation. These results identify a novel function for ERK5 in maintaining expression of naïve pluripotency factors and directly regulating KLF2 stability. These findings have exciting potential applications in deriving the naïve pluripotent state and therefore in regenerative therapeutics and disease modelling.
| Date of Award | 2017 |
|---|---|
| Original language | English |
| Sponsors | Medical Research Council |
| Supervisor | Greg Findlay (Supervisor) |