Identification of Novel Regulators of Telomere Length and its Impact on Endothelial Dysfunction

Abstract

Telomere length (TL) is highly heritable and associated with a plethora of health conditions, including cancer, Alzheimer's and coronary artery disease (CAD). A number of studies have linked TL to CAD, with short TL preceding disease onset. Genetic studies support a causal link between the two, though the mechanism remains poorly understood. A model was established utilising a Cre-Lox system for reversible immortalisation of HUVECs, using TERT as an excisable transgene, and found that short TL induces markers of endothelial dysfunction, such as increased CAM expression. This work aimed to replicate previous findings to aid in our understanding of the relationship between TL and CAD risk. Unfortunately, attempts to replicate this model were unsuccessful because TL failed to stabilise. This work concludes that a more suitable model is needed to investigate the relationship between TL and CAD onset.As both longer and shorter TL influence disease risk, understanding the regulation of TL may have therapeutic benefits. Recent GWAS studies have identified 138 loci associated with leukocyte telomere length (LTL). However, many of these could not be linked to known telomere regulators, such as components of the telomerase enzyme or the shelterin complex. Bioinformatic analysis identified a number of novel candidate genes with evidence linking them to plausible roles in TL regulation: PAP-associated domain containing 4 (PAPD4), M-Phase Phosphoprotein 6 (MPHOSPH6), and Sentrin-specific protease 7 (SENP7). Previous work utilised short-term siRNA knockdowns of these genes to investigate possible roles in telomere biology. PAPD4 and MPHOSPH6 appear to regulate TL via telomerase, as the generated lines show reduced telomerase activity, likely through modulation of the TERC maturation pathway. SENP7 is hypothesised to regulate telomere-associated proteins via the SUMOylation pathway. In this work, CRISPR-Cas9 genome edited was used to generate knockout cell lines for these genes in multiple cell lines (HCT116, HeLa and HEK293A). Mutations were confirmed via gDNA sequencing, gene expression via qPCR and protein expression via Western blots. Subsequently, telomerase activity was determined using the quantitative telomerase repeat amplification protocol (Q-TRAP) and TL via qPCR. Taken together, these suggest that PAPD4 and MPSHOPH6 are regulators of TL via telomerase, while SENP7 is not.