Investigating the role of the ETS transcription factor ELK1 in stem cell transcription

Abstract

ELK1 is a member of the E-twenty-six transcription factor family which is known to be both an activator and a repressor of transcription and is able to toggle between the two modes. Even in a single cell line, ELK1 can act as both an activator and repressor in response to a single stimulus, such as epidermal growth factor stimulation. To answer this apparent paradox, ELK1 binding can be parsed into distinct binding modules, with a distinct biological function, and these binding modules may explain ELK1’s dual roles. A recent study performed in human embryonic stem cells (hESC) suggested that ELK1 exists in an active module which co-localises with SRF, and a repressive module co-localizing with members of Polycomb repressive complex 2 (PRC2) on loci near to developmental genes. The PRC2 complex has shown to have repressive roles in pluripotent stem cells, however, the regulation and recruitment of PRC2 remains a mystery. Elucidating the possible relationship between ELK1 and the PRC2 complex may further explain the pathways that govern PRC2 recruitment and shed light on the role of ELK1 as a repressor. To investigate an ELK1-PRC2 interaction, we first scanned the ELK1 protein interactome using the rapid immunoprecipitation and mass spectrometry of endogenous proteins (RIME) protocol. Subsequently, we investigated the co-binding of ELK1 and PRC2, and its effect on the regulation of gene transcription during ELK1 depletion and differentiation. We did not detect any interaction between ELK1 and PRC2 using these protocols, although we saw an increase in transcription of the developmental gene SIX1 upon ELK1 depletion, hinting at a PRC2-independent repressive role for ELK1. We next investigated the role of ELK1 in pluripotency and both early mesoderm and neural differentiation. Although we did not uncover a central role for ELK1 in these processes, concurring with the in vitro studies, we find that ELK1 depletion increased the expression of genes in both mesoderm and neural differentiation, pointing to the role of ELK1 as a repressor. We finally revisit the interaction between ELK1 and SRF in the context of mesoderm differentiation. We uncovered ELK1 and SRF co-binding near many genes. We see a decrease in ELK1 binding in both hESC and mesoderm upon SRF depletion, affirming the role of SRF as a recruiter of ELK1.

Date of Award	1 Aug 2018
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Andrew Sharrocks (Supervisor)