Effects of 5-Hydroxymethylcytosine Epigenetic Modification on the Stability and Molecular Recognition of VEGF i-Motif and G-Quadruplex Structures.

Rhianna K Morgan,Michael M Molnar,Tracy A Brooks,Harshul Batra,Bethany Summerford,Randy M Wadkins

doi:10.1155/2018/9281286

Rhianna K Morgan, Michael M Molnar + Show 4 more

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https://doi.org/10.1155/2018/9281286

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Abstract

Promoters often contain asymmetric G- and C-rich strands, in which the cytosines are prone to epigenetic modification via methylation (5-mC) and 5-hydroxymethylation (5-hmC). These sequences can also form four-stranded G-quadruplex (G4) or i-motif (iM) secondary structures. Although the requisite sequences for epigenetic modulation and iM/G4 formation are similar and can overlap, they are unlikely to coexist. Despite 5-hmC being an oxidization product of 5-mC, the two modified bases cluster at distinct loci. This study focuses on the intersection of G4/iM formation and 5-hmC modification using the vascular endothelial growth factor (VEGF) gene promoter's CpG sites and examines whether incorporation of 5-hmC into iM/G4 structures had any physicochemical effect on formation, stability, or recognition by nucleolin or the cationic porphyrin, TMPyP4. No marked changes were found in the formation or stability of iM and G4 structures; however, changes in recognition by nucleolin or TMPyP4 occurred with 5-hmC modification wherein protein and compound binding to 5-hmC modified G4s was notably reduced. G4/iM structures in the VEGF promoter are promising therapeutic targets for antiangiogenic therapy, and this work contributes to a comprehensive understanding of their governing principles related to potential transcriptional control and targeting.

Highlights

Guanine- and cytosine-rich DNAs are capable of forming higher order, noncanonical, intramolecular structures known as G-quadruplexes (G4s) and i-motifs, respectively. iM/G4 forming sequences are common throughout the genome with high abundances in promoters and 5󸀠 untranslated regions (UTRs) of both oncogenes and tumor suppressor genes [1, 2]
Endothelial stem cells play key roles in vasculogenesis and angiogenesis, as they form the lining of blood vessels
vascular endothelial growth factor (VEGF) is overexpressed by tumors, secreted into the local milieu, and acts upon nearby endothelial cells to trigger angiogenesis in order to increase oxygen levels and provide essential nutrients, mediating tumor growth, invasion, and metastasis [43,44,45]

Summary

Introduction

Guanine- and cytosine-rich DNAs are capable of forming higher order, noncanonical, intramolecular structures known as G-quadruplexes (G4s) and i-motifs (iMs), respectively. iM/G4 forming sequences are common throughout the genome with high abundances in promoters and 5󸀠 untranslated regions (UTRs) of both oncogenes and tumor suppressor genes [1, 2]. IM/G4 forming sequences are common throughout the genome with high abundances in promoters and 5󸀠 untranslated regions (UTRs) of both oncogenes and tumor suppressor genes [1, 2]. These structures are associated with an array of human diseases, including cancer and neurodegeneration [3, 4]. Most iMs and G4s identified to date act as transcriptional suppressors by hindering RNA polymerase activity and/or transcription factor binding Such structures span the hallmarks of cancer; most research focuses on utilizing small iM/G4-stabilizing molecules in order to decrease tumor activity [3,4,5,6,7]

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