Characterising a cis-regulatory element associated with Nkx2.5 in early heart development

Schatka, Magdalena (2025) Characterising a cis-regulatory element associated with Nkx2.5 in early heart development. Doctoral thesis, University of East Anglia.

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Abstract

Within an embryo, the heart is the first organ to form, as its function is essential for development and postnatal life. Past research has identified a complex network of genes controlling heart formation, with dysregulation often leading to congenital heart disease. Therefore, understanding the underlying mechanisms of cardiogenesis is crucial. The transcription factor (TF) NKX2.5 is a key regulator, yet its upstream control, especially through cis-regulatory elements (CREs) remains incompletely defined. CREs are short regions of non-coding DNA which regulate associated genes and can either enhancer or silence their expression. Here, I characterise a conserved CRE associated with Nkx2.5 and define its function in the chick. Published literature about this region focuses on mouse where the so-called activating region 2 (AR2) is mainly regulated by GATA4 and recapitulates endogenous Nkx2.5 expression in the cardiac crescent, heart tube and looping heart. This thesis demonstrates that CRISPR/dCas9-KRAB repression of the CRE reduces Nkx2.5 expression in the chick and cross-species injections show that the mouse, frog and human homologous sequences also drive cardiac expression in chick, supporting functional conservation across vertebrates. An ATAC-seq dataset generated from cardiac progenitors identified binding motifs for GATA4, NKX2.5 and PBX1. Targeted mutagenesis revealed a GATA footprint as essential for enhancer activity, with NKX2.5 and PBX1 motifs acting cooperatively or redundantly. Individual overexpression showed that GATA4, NKX2.5 and PBX1 each activate the enhancer ectopically, suggesting direct interaction between each TF and the CRE. In contrast to the mouse element, the chick CRE shows no detectable BMP/Smad responsiveness at HH8-12 despite partial motif conservation. This data supports a model in which GATA4 is the primary activator of this conserved cardiac CRE in chick, aided by NKX2.5 and PBX1. While the GATA- regulatory logic seems to be conserved across vertebrates, other regulators appear to be species-specific.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	23 Feb 2026 15:17
Last Modified:	23 Feb 2026 15:17
URI:	https://ueaeprints.uea.ac.uk/id/eprint/102013
DOI:

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