Investigating the Role of MEF2C Isoforms in Human Erythroid Cell Differentiation.

Abstract

Transcription factors and cofactors play a major role in cell proliferation and differentiation during development. They achieve this by executing specific programs of gene expression. Myocyte enhancer factor (MEF) 2C is from the MEF2 family of transcription factors which regulate various cellular processes, in many different cell types. Alternative splicing of the last coding exon of the MEF2C gene results in two isoforms: MEF2Cγ+ (gamma isoform) and MEF2Cγ- (regular isoform). MEF2Cγ- was demonstrated to have a higher transcriptional activity while the γ domain is thought to recruit repressors of transcription via phosphorylation action.
In a previous study, MEF2C was identified as one of the highly expressed transcription factors at the early stages of adult primary erythroid culture differentiation compared to late-stage erythroid cells by transcriptomics, suggesting it may be involved in expansion. Sequencing of transcript from primary erythroid cells identified the two aforementioned isoforms, which both lacked the β exon and contained the α1 exon.
In this study, detection and distinction between the two MEF2C isoforms in erythroid cells was optimised for protein and transcript. Knockdown of gamma MEF2C isoform in erythroleukemic K562 cells during expansion also led to some reduction of the regular MEF2C isoform. It also led to a reduction in cellular proliferation and an induction of erythroid differentiation, with increased haemoglobinization observed.
Immunoprecipitations of both isoforms, at endogenous and ectopic overexpression levels, using a total-MEF2C antibody and gamma MEF2C isoform specific antibodies revealed that isoforms do not heterodimerise in erythroid cells, though may still homodimerize.
Optimisation of co-immunoprecipitations of both isoforms, at endogenous and ectopic overexpression levels, using all aforementioned MEF2C antibodies, followed by tandem-mass-tag liquid-chromatography mass-spectrometry (MS1, MS2) allowed for an extensive study of MEF2C interactome,
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with quantitative comparison. Revealing clear distinctions among the commonalities of isoform interactomes, with functional complexes identified with both isoforms.

Date of Award	10 Dec 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Jan Frayne (Supervisor) & Belinda Singleton (Supervisor)