Mef2 in muscle: A CRISPR-based approach to investigate the regulation of a master transcription factor

Hubbert, Sean 2023. Mef2 in muscle: A CRISPR-based approach to investigate the regulation of a master transcription factor. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Myocyte enhancer factor-2 (Mef2) is a highly conserved transcription factor required for muscle differentiation in Drosophila, and functions by activating the expression of hundreds of genes. By using CRISPR-Cas9 genome engineering, not previously used for the study Mef2 function in vivo, I have generated a novel suite of valuable tools which can be used to probe endogenous Mef2 function in vivo. Interestingly, Mef2 function is also highly expressed in undifferentiated myoblast populations, many hours before these cells differentiate and express many Mef2 target genes. Therefore, Mef2 activity must be regulated in space and time to enable to proper coordination of muscle development in vivo. The Class IIa histone deacetylases (HDACs) have been shown to physically interact with, and negatively regulate Mef2 in vitro. However, before this project, there had been relatively little research as to their function in the context of muscle differentiation in vivo. In this project, I further our understanding of the role of Class IIa HDACs during muscle differentiation using Drosophila melanogaster as a model. Firstly, I use the Gal4-UAS system to probe the functional consequences of overexpressing HDAC4 in vivo, as well as to identify important residues for HDAC4 function in vivo. Secondly, I utilise CRISPR-Cas9 genome engineering to generate a novel, rescuable HDAC4 null allele and show that HDAC4 is essential for viability, while its loss also disrupts normal muscle patterning in vivo. Importantly, this allele will be a critical tool to further define the role of HDAC4 during muscle differentiation in vivo, as well for investigating Class IIa HDAC function more broadly. Finally, I also identify critical residues required for Mef2 transcriptional activity in vivo. I link these observations to, and investigate the function of the Drosophila orthologue of the p300/CBP Histone acetyltransferase (HAT), Nejire, during muscle differentiation in vivo. In this work, I utilised CRISPR-Cas9 genome engineering to develop valuable tools to facilitate the understanding of Mef2 function and regulation, while I have also investigated the role of HDAC4 and Nejire in the regulation of muscle differentiation in vivo.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	8 August 2024
Last Modified:	08 Aug 2024 13:19
URI:	https://orca.cardiff.ac.uk/id/eprint/171253

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