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Title: Dissecting the upstream regulation of the GATA genes during heart development
Author: Wennington, Caroline
ISNI:       0000 0004 8507 3638
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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The cardiac GATA transcription factors (TFs) play vital roles in the developing myocardium and potentially during regeneration. While their function as cardiac effectors has been well studied, the precise molecular mechanisms driving their activity have yet to be parsed. Here, I have investigated the upstream regulation of the cardiac GATA factors during cardiogenesis in zebrafish by interrogating their cis-regulatory genomic landscapes. Using cardiomyocyte-specific ATAC-Seq datasets, I have identified putative enhancers around the gata4, 5 and 6 loci that are accessible during cardiac development and regeneration and tested their activity in vivo using a novel, highly efficient reporter system in zebrafish. I observed heart-specific reporter activity driven by the majority of the newly identified putative gata5 and gata6 enhancers, within particular subpopulations of these TFs domains of expression. The reported activity of Tg(Gata5-E5-mCherry) was of particular interest. I characterised Gata5-E5-mCherry positive populations using a combination of microscopic and transcriptomic analyses. I then tested the requirement of overrepresented TF binding site motifs for the activity of Gata5-E5 by assessing changes in enhancer-driven reporter expression after binding site perturbation. Gata5-E5 was active in a population of extracardiac cells adjacent to the inflow tract that expressed epithelial markers, hypothesised to act as support cells for second heart field progenitor cells. Gata5-E5 was also active in a population of CMs in the inflow tract region that expressed the hallmarks of cardiac conduction system cells. Binding site studies revealed a potential upstream regulation by Gata5, Nkx2.5/Nkx2.7 and Tbx2a/2b/5a to drive the activity of gata5 in these cells. Gata5-E5 was also active in CMs throughout the atrium and ventricle and binding site studies suggested an upstream role for Gata5, Meis1b, Nkx2.5/Nkx2.7 and Tbx20 in regulating Gata5-E5 activity in this subpopulation of CMs. Future experiments will involve ChIP-Seq to test the direct interactions suggested by binding site perturbation studies, and Capture-C to investigate topologically associating domains, ultimately revealing the functional interaction between these enhancers and their regulated GATA genes. Taken together, my work has begun to uncover the exact upstream spatiotemporal regulation of the cardiac GATA factors in various subpopulations of the heart during early cardiogenesis at high resolution. Comprehension of these complex networks is critical to understand the molecular basis of GATA-related congenital heart defects.
Supervisor: Patient, Roger ; Simoes, Filipa ; Riley, Paul Sponsor: British Heart Foundation
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available