Co-Localization of DNA i-Motif-Forming Sequences and 5-Hydroxymethyl-cytosines in Human Embryonic Stem Cells

Abstract

G-quadruplexes (G4s) and i-motifs (iMs) are tetraplex DNA structures. Sequences capable of forming G4/iMs are abundant near the transcription start sites (TSS) of several genes. G4/iMs affect gene expression in vitro. Depending on the gene, the presence of G4/iMs can enhance or suppress expression, making it challenging to discern the underlying mechanism by which they operate. Factors affecting G4/iM structures can provide additional insight into their mechanism of regulation. One such factor is epigenetic modification. The 5-hydroxymethylated cytosines (5hmCs) are epigenetic modifications that occur abundantly in human embryonic stem cells (hESC). The 5hmCs, like G4/iMs, are known to participate in gene regulation and are also enriched near the TSS. We investigated genomic co-localization to assess the possibility that these two elements may play an interdependent role in regulating genes in hESC. Our results indicate that amongst 15,760 G4/iM-forming locations, only 15% have 5hmCs associated with them. A detailed analysis of G4/iM-forming locations enriched in 5hmC indicates that most of these locations are in genes that are associated with cell differentiation, proliferation, apoptosis and embryogenesis. The library generated from our analysis is an important resource for investigators exploring the interdependence of these DNA features in regulating expression of selected genes in hESC.

The i-Motif as a Molecular Target: More Than a Complementary DNA Secondary Structure

“…The Waller laboratory examined a range of cytosine modifications and determined that i-motifs stable at physiological pH were more likely to consist of methylated cytosines [37]. The presence of i-motif sequences within CpG islands, which are well-known sites of methylation, supports this finding and indicates that methylation may contribute to i-motif formation within living cells [37][38][39].…”

Stability and context of intercalated motifs (i-motifs) for biological applications

“…Interrogation of 5hmC using TET-assisted bisulfite sequencing, compared with putative i-motif structures using the Quadfinder computational prediction tool, revealed a weak negative correlation of 5hmC density with potential i-motif formation [83]. Furthermore, only a small fraction (15%) of putative i-motif regions contained more than one 5hmC modification.…”

“…While multiple studies have analysed the effects of 5mC and 5hmC in vitro using synthetic oligonucleotides [42,70,71], it is important to consider biologically relevant epigenetic modification patterns in the human genome. The prevalence of i-motif-forming sequences 1 kb upstream and downstream of global transcription start sites (TSS) coincide with CpG islands [42,82,83].…”

“…Furthermore, only a small fraction (15%) of putative i-motif regions contained more than one 5hmC modification. Further analysis revealed that within human embryonic stem cells, sequences that possess three or more 5hmC and colocalise with i-motif-forming sequences are predominantly involved in ligand binding and enzyme regulatory activities [83].…”

“…Bhavsar-Jog et al also highlighted the high abundance of putative i-motif-forming sequences within 100 bp of promoters of 15,760 reference genes from the human GRCh37.p10 primary assembly [83].…”