Effects of CpG methylation on the thermal stability of c-kit2, c-kit*, and c-kit1 G-quadruplex structures.

In genomic DNA, G-quadruplex (G4)-forming DNA can form either a duplex or G4 structure, suggesting that understanding the factors regulating G4 formation is important for revealing the cellular functions controlled by G4 formation. Cytosine DNA methylation in the CpG islands is known to play an important role in transcriptional regulation. Additionally, CpG methylation increases the thermal stability of G4 structures such as BCL2 and VEGF G4. In this study, we evaluated the effects of CpG methylation in three G4 structures (c-kit2, c-kit*, and c-kit1) produced by the c-KIT promoter. Each was analyzed using circular dichroism (CD) melting analysis. The results demonstrate that CpG methylation does not alter the thermal stability of c-kit2 G4 structure when formed in the presence of K+; a single-CpG methylation at C1 or C11 decreases the thermal stability of any c-kit2 G4 structure formed in the presence of Na+ and Mg2+ while methylation at C5 increases the thermal stability; CpG methylation does not alter the thermal stability of c-kit1 or c-kit* G4 structures formed in the presence of K+; and the c-kit1 and c-kit* G4-forming oligonucleotides do not form G4 structures in the presence of Na+ and Mg2+. These results provide important clues for understanding the regulatory mechanisms underlying the formation of CpG methylation-induced G4 structures.

Destabilisation of the c-kit1 G-quadruplex structure by N6-methyladenosine modification.

N6-methyladenine (m6dA) has been recently discovered in eukaryotic genomic DNA. However, there have been few reports on its biological roles. G-quadruplex (G4) is a non-canonical nucleic acid structure formed by the stacking of G-tetrads. G4-forming sequences are enriched with cis-regulatory elements in genomic DNA and the G4 structures have important roles in various cellular functions. We previously reported that CpG methylation stabilized vascular endothelial growth factor (VEGF) G4 structure. Here we report that m6dA modification destabilizes the human c-kit1 G4 structure. These results suggest that epigenetic modifications may affect G4 formation in order to regulate the biological functions.

Stabilization of G-quadruplex structure on vascular endothelial growth factor gene promoter depends on CpG methylation site and cation type.

BACKGROUND: DNA methylation at the 5-position of cytosine is an epigenetic modification of CpG dinucleotides. In addition to CpG methylation, the G-quadruplex (G4) structure has been reported as a regulator of gene expression. The identification of G4 forming sequences in CpG islands suggests an involvement of CpG-methylated G4 structures in biological processes; however, few reports have addressed the effects of CpG methylation on G4 structure. METHODS: The thermostability of a methylated, 21-mer G4 structure located on the vascular endothelial growth factor (VEGF) gene promoter containing four CpG sites (C1, C6, C11, and C17) were investigated using circular dichroism (CD) spectral analysis. RESULTS: CD melting analysis revealed that VEGF G4 was stabilized by a single CpG methylation on C11 in the presence of Na+ and Mg2+. However, either C1 or C11 methylation enhanced VEGF G4 thermal stability in the presence of K+. CONCLUSIONS: Single CpG methylation appears to enhance VEGF G4 thermostability in a manner dependent on both the CpG methylation site and cation type. GENERAL SIGNIFICANCE: These results are expected to contribute to the elucidation of the roles of CpG methylation-stabilized G4 structures in biological processes.