Detection of DNA Methylation of G-Quadruplex and i-Motif-Forming Sequences by Measuring the Initial Elongation Efficiency of Polymerase Chain Reaction

Yoshida, Wataru; Yoshioka, Hitomi; Bay, Daniyah Habiballah; Iida, Keisuke; Nagasawa, Kazuo; Karube, Isao

doi:10.1021/acs.analchem.6b00982

Cited by 31 publications

(19 citation statements)

References 38 publications

(47 reference statements)

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“…The formation of i‐motif structures can lead to arrest of cellular processes that require transient opening of the double helix, such as transcription and DNA replication . To determine the effect of i‐motif structures in ACC1 promoters on DNA replication, a polymerase stop assay was performed.…”

Section: Resultsmentioning

confidence: 99%

Structural Characterization of i‐Motif Structure in the Human Acetyl‐CoA Carboxylase 1 Gene Promoters and Their Role in the Regulation of Gene Expression

2018

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The polypurine/polypyrimidine-rich sequences within the promoters (PI and PII) of human acetyl coenzyme A (CoA) carboxylase 1 (ACC1) gene play a vital role in determining hormone- or diet-inducible expression of ACC1. PI and PII contain consecutive runs of three and three to five G/C base pairs, respectively. In a previous study, G-rich DNA sequences of human ACC1 PI and PII were found to fold into G-quadruplex structures; these consequently acted as strong barriers to transcription and DNA replication. Typically, stretches of C-rich sequences that coexist with stretches of guanines have the capacity to form another four-stranded secondary structure known as an i-motif. However, studies on the i-motif structure are limited and its functional significance is unclear. In the current study, through the use of a combination of different techniques, it is demonstrated that C-rich single-stranded DNA derived from ACC1 PI and PII form intramolecular i-motif structures and affect normal DNA metabolic processes. Additionally, the C-rich strands of PI and PII in duplex DNA adopt the i-motif conformation in crowded solution environments at neutral pH. Notably, the i-motif-forming sequences of PI and PII suppressed luciferase gene transcription in HeLa cells. Furthermore, substitution of a nucleotide sequence that has no potential to form the i-motif structure increases luciferase gene expression in HeLa cells. These results support the idea that C-rich sequences within ACC1 PI and PII can form intramolecular i-motif structures, cause suppression of transcription, and thus reveal the functional significance of C-rich sequences in the regulation of ACC1 gene expression.

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Section: Resultsmentioning

confidence: 99%

Structural Characterization of i‐Motif Structure in the Human Acetyl‐CoA Carboxylase 1 Gene Promoters and Their Role in the Regulation of Gene Expression

2018

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“…It has been reported that the Bcl-2 G4 structure and quadruplex structure of C9orf72 repeat were stabilised by DNA methylation 36 , 37 . We reported that the initial elongation efficiency of PCR decreased with increasing DNA methylation levels in VEGFA and RET G4-forming sequences 38 , indicating that the G4 structures are also stabilised by DNA methylation. These reports suggest that our method could be applied to detect epigenetic modification by identifying G4 clusters stabilised by DNA methylation.…”

Section: Discussionmentioning

confidence: 89%

Identification of G-quadruplex clusters by high-throughput sequencing of whole-genome amplified products with a G-quadruplex ligand

Yoshida

Saikyo

Nakabayashi

et al. 2018

Sci Rep

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G-quadruplex (G4) is a DNA secondary structure that has been found to play regulatory roles in the genome. The identification of G4-forming sequences is important to study the specific structure-function relationships of such regions. In the present study, we developed a method for identification of G4 clusters on genomic DNA by high-throughput sequencing of genomic DNA amplified via whole-genome amplification (WGA) in the presence of a G4 ligand. The G4 ligand specifically bound to G4 structures on genomic DNA; thus, DNA polymerase was arrested on the G4 structures stabilised by G4 ligand. We utilised the telomestatin derivative L1H1-7OTD as a G4 ligand and demonstrated that the efficiency of amplification of the G4 cluster regions was lower than that of the non-G4-forming regions. By high-throughput sequencing of the WGA products, 9,651 G4 clusters were identified on human genomic DNA. Among these clusters, 3,766 G4 clusters contained at least one transcriptional start site, suggesting that genes are regulated by G4 clusters rather than by one G4 structure.

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“…Given the exclusivity of 5-mC with both iM/G4-forming regions and with 5-hmC, the overall purpose of this study was to examine the effects of 5-hmC modification on the formation, stability, and recognition of secondary DNA structures. As a physiologically relevant model, we focused on the structures in the promoter of the VEGF gene, which have been well-described for both iM and G4 formation [ 12 , 14 , 29 – 31 ] and containing natural CpG islands [ 40 ]. Cytosine modification of this promoter has been shown to vary with pathophysiological states [ 41 , 42 ].…”

Section: Discussionmentioning

confidence: 99%

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

Morgan

Molnar

Batra

et al. 2018

Journal of Nucleic Acids

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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.

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Detection of DNA Methylation of G-Quadruplex and i-Motif-Forming Sequences by Measuring the Initial Elongation Efficiency of Polymerase Chain Reaction

Cited by 31 publications

References 38 publications

Structural Characterization of i‐Motif Structure in the Human Acetyl‐CoA Carboxylase 1 Gene Promoters and Their Role in the Regulation of Gene Expression

Structural Characterization of i‐Motif Structure in the Human Acetyl‐CoA Carboxylase 1 Gene Promoters and Their Role in the Regulation of Gene Expression

Identification of G-quadruplex clusters by high-throughput sequencing of whole-genome amplified products with a G-quadruplex ligand

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

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