Dissecting molecular mechanisms mediating ERK1/2 regulation of neural differentiation

Many signals directing cell differentiation have been identified, but how they control chromatin organisation and coordinate the initiation of differentiation gene expression in lineage-specific progenitor cells is poorly understood. Bipotent neuromesodermal progenitors (NMPs) in the caudal lateral epiblast of the developing vertebrate embryo can contribute to neural or mesodermal tissues during body axis elongation. Previous work by the Storey laboratory demonstrated that NMP neural differentiation involves a reduction in FGF-ERK1/2 signalling: ERK1/2 inhibition rapidly de-compacts chromatin at neural differentiation gene loci and accelerates their transcriptional onset. However, the mechanism(s) by which ERK1/2 signalling regulates chromatin accessibility have yet to be determined. To address this, I took an unbiased approach to capture phospho-proteomic changes following brief inhibition of MEK1/2 (the ERK1/2 upstream kinase) in human ES cell derived NMP cells. After differential analysis, the phospho-proteomic screen was validated, focusing on RSK1 kinase phosphorylation, a downstream target of ERK1/2. Functional classification of altered proteins revealed five major protein classes: MAPK-related proteins, chromatin regulators, cytoskeleton, metabolism, and E3-ligase activity. Moreover, these peptides predominantly exhibited phosphorylation motifs for ERK1/2 (S/T-P) and RSK (R-X-X-S/T), aligning with MEK1/2 pathway inhibition. Focussing on chromatin regulators I selected two proteins for further characterisation and functional analyses. Capicua (CIC) is a transcriptional repressor and tumour suppressor known in other contexts to be regulated by ERK1/2 and RSK1 phosphorylation, which inactivates CIC. Preliminary data revealed reduced expression of neural marker progenitor gene PAX6 following knock down of CIC in NMPs, suggesting it is involved in the initiation of neural differentiation. PHF2 is a histone lysine demethylase widely expressed during embryogenesis. I established PHF2 as a novel ERK1/2 and RSK1 target and current experiments address how these phospho-sites may mediate neural differentiation.