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Dahhan, Rania (2024) Identification of genes and pathways regulated by the PRH/HHEX transcription factor in endothelial and lymphatic cells. PhD thesis, University of Nottingham.

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Abstract

The Proline-Rich Homeodomain protein (PRH/HHEX) is recognized as an essential transcription factor in development and in the adult. PRH regulates transcription by binding directly to DNA at specific regions of target genes, often containing specific DNA sequence motifs to which it has a high affinity. Moreover, PRH is also known to interact with many other transcription factors, including activator protein 1 (AP-1) to modulate complex gene regulatory networks either directly or via indirect mechanisms. As a transcriptional regulator, PRH can act either as a transcriptional repressor or as an activator, depending on the context and its interactions with other cofactors. This study aims to better understand the role of PRH in normal vascular homeostasis through the manipulation of PRH expression in Human Umbilical Vein Endothelial Cells (HUVECs) and Human Dermal Lymphatic Endothelial Cells (HDLECs). Endothelial cells line blood and lymphatic vessels, orchestrating processes including tube formation, cell extravasation, and vessel permeability which are essential for maintaining the integrity and functionality of the vascular system. This thesis demonstrates that PRH regulates angiogenic tube formation as well as the proliferation, and migratory behaviour of blood and lymphatic endothelial cells. Adenoviral overexpression of PRH results in decreased proliferation, decreased migration in wound closure assays, and decreased angiogenic tube formation in HUVECs and HDLECs. Conversely, RNA interference (RNAi) knockdown of PRH in HUVECs and HDLECs results in increased cell proliferation, increased migration in wound closure assays, and increased angiogenic tube formation. Interestingly, PRH protein expression varies in correlation with endothelial cell density, where confluent HUVECs exhibit higher PRH levels than sub-confluent HUVECs indicating a role for PRH in modulating cellular behaviour during proliferation and quiescence. Further exploration through whole transcriptome analysis using RNA sequencing analysis post-PRH manipulation indicates that blood and vascular related genes are over-represented in the Differentially Expressed Genes (DEGs); genes that show a significant change in expression levels between normal and altered PRH conditions. This identified wound healing, cell adhesion, cell motility, and circulatory system development as significantly enriched biological processes regulated by PRH.

Additionally, chromatin Immunoprecipitation followed by sequencing of exogenously expressed Myc tagged PRH in HUVEC cells identified 28494 PRH ChIP peaks. Analysis of ChIP sequencing data binding motifs present within these peaks showed the enrichment of the AP-1 motif which is bound by jun-fos transcription factor heterodimers. Overlap of the RNA sequencing DEGs with AP-1-regulated genes (target genes regulated by AP-1 based on experimental evidence as well as predicted AP-1 target genes identified using MotifMap, a database that identifies and annotates transcription factor binding sites in the human genome, suggests many PRH-regulated genes that are involved in tube development and wound healing are also AP-1-regulated genes. Moreover, overlap of PRH binding peaks with c-Jun binding peaks in HUVECs (CistromeDB: 45848) reveals that many genes involved in 'cell motility', 'regulation of cell motility', 'blood vessel development' and 'cell adhesion' have regulatory regions that are bound both by PRH and AP-1. Co-immunoprecipitation experiments reveal that PRH interacts directly with the AP-1 subunit FOSL2 in HUVECs while PRH interacts directly with the AP-1 subunits FOSL2, JunB and c-Jun in HDLECs reflecting that PRH can directly interact with different components of the AP-1 transcription factor complex in a cell-type specific manner. Together, this research advances our understanding of vascular biology and provides insights into the molecular regulation of angiogenesis and lymphangiogenesis. These data also indicate that PRH may be a potential therapeutic target in diseases characterized by dysfunctional angiogenesis. The findings suggest that manipulation of PRH activity could be a novel strategy for treating vascular disorders.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Gaston, Kevin Jayaraman, Sheela Benest, Andrew
Keywords:	Transcription factors; Vascular homeostasis; Endothelial cells; Angiogenic tube formation; Cell proliferation; Vascular biology; Angiogenesis; Lymphangiogenesis
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QU Biochemistry
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID:	79962
Depositing User:	Dahhan, Rania
Date Deposited:	11 Dec 2024 04:40
Last Modified:	11 Dec 2024 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/79962

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