It is well known that DNA secondary structure and methylation are involved in regulating gene expression in mammals. Research has shown that G-quadruplex (G4) structures, which are among the most prevalent non-B DNA structures, are key players in the control of gene transcription and regulation. Methylation of cytosine in CpG dinucleotides in promoter regions is a well-characterized epigenetic modification that plays an important role in regulating numerous cellular processes, including development and tumorigenesis. CpG dinucleotides are often found within potentially G4-forming sequences in the promoter regions of numerous genes. Although early research indicated that CpGs within higher-order G4-forming DNA motifs undergo low methylation, methylation of cytosines in CpG dinucleotides within these G4 motifs nonetheless occurs in the genome. However, the interplay between DNA methylation and DNA secondary structures and the resulting effects on the regulation of gene expression have not been addressed.
Our study began from the finding that oxidative stress mediated by photodynamic therapy (PDT) and chemotherapy can down-regulate the expression of human telomerase reverse transcriptase (hTERT). hTERT is the major component of the catalytic subunit of telomerase and acts as a rate-limiting factor for telomerase activity. Telomerase activity is observed in approximately 90% of human cancers, whereas most somatic normal tissues are negative for hTERT expression. Aberrant hTERT gene expression may cause aging, cancer, and other diseases. Several transcription factors with binding sites in the promoter region have been documented to directly or indirectly regulate hTERT gene expression, including the activators MYC and SP1 and repressors such as p53 and CCCTC-binding factor (CTCF). Many recurrent mutations within the promoter region of hTERT have been identified in various cancers; such mutations correlate with elevated hTERT transcriptional activity. However, no mutation or single-nucleotide polymorphism has been identified within the CTCF binding site in the first exon of hTERT. Interestingly, methylation of the first exon of hTERT prevents CTCF binding and allows for hTERT gene expression in telomerase-positive cells. However, the underlying mechanism by which methylation regulates hTERT gene expression remains unclear.
Two findings led to our multidisciplinary research to address the interplay between DNA methylation and secondary structure. First, oxidative stress mediated by PDT and Taxol treatment altered the DNA methylation profile within the CTCF-binding region in the first exon of hTERT. Second, four CpG dinucleotides with the potential to form a G4 structure were identified in the G-rich sequence G3AGCGCACG2CTCG2CAGCG4, which is located at +13 to +37 within the hTERT CTCF-binding region (Figure 1a; this sequence has been named hT25). This sequence was identified by a group led by our collaborator Dr. Ta-Chau Chang at Academia Sinica. Without the cooperation of multidisciplinary teams, we would be unlikely to discover a possible novel mechanism for regulating gene expression.
We first conducted a 1D imino proton NMR experiment to address the possible formation of hydrogen bonds of DNA secondary structures and thereby assess the potential for G4 formation in hT25. Imino proton NMR results for hT25 showed the competitive existence of two types of secondary structures: hairpin and quadruplex structures (Figure 1b). A particularly interesting finding was that the imino proton signals indicative of the G4 structure were more pronounced after CpG methylation (Figure 1c). Further experiments demonstrated that methylation of the fourth of the four CpG dinucleotides in hT25 (C21G22) plays a key role in enhancing quadruplex formation. Furthermore, in tests with reporter constructs, we confirmed that expression was markedly higher for methylated reporter constructs than for wild-type constructs, a finding consistent with our chromatin immunoprecipitation (ChIP) results showing a significant reduction in CTCF binding after methylation. These results clearly indicate that methylation of CpG dinucleotides indeed inhibits CTCF binding and results in gene expression. However, it is unclear whether the quadruplex structure promoted by methylation has a major effect on CTCF binding and gene regulation.
Figure 1 Characterization of DNA secondary structures of a G-rich sequence in the first exon of the hTERT gene. a, Identification of the potentially G4-forming sequence hT25, d(G3AGCGCACG2CTCG2CAGCG4), in the antisense strand (from +37 to +13) in the first exon of the hTERT gene, which is located within the CTCF-binding region. b and c, Imino proton NMR spectra of hT25 (b) and hT25-Me (c) in Tris-HCl buffer without (bottom panel) and with (top panel) 150 mM KCl. In the methylated sequence hT25-Me, cytosine was synthetically modified to 5-methylcytosine at the four CpG dinucleotides of hT25.
We further designed four hT25 mutants to examine the formation of this sequence into a G4 structure and to assess its effect on CTCF binding in the regulation of hTERT gene expression. hT25-m1 and hT25-m3 were designed to disrupt quadruplex formation, and imino proton NMR results for these mutants revealed no appreciable signals of quadruplex structure, implying that hT25-m1 and hT25-m3 preferentially form hairpin structures. In contrast, hT25-m2 and hT25–m4 have imino proton NMR spectra with distinct signals of quadruplex structures. Electrophoretic mobility shift assay (EMSA) studies confirmed that CTCF binding was perturbed for hT25-m2 and hT25 structures with methylated CpG dinucleotides; in contrast, significant CTCF binding was observed for hT25-m1 and hT25-m3. The reporter expression level was much higher in A375 cells transfected with a plasmid containing hT25-m2 or hT25-m4, both of which preferentially form a quadruplex structure, than in wild-type A375 cells or A375 cells transfected with a plasmid containing hT25-m1 or hT25-m3, both of which preferentially form a hairpin structure. Furthermore, ChIP analysis revealed that the level of CTCF binding is markedly lower in cells transfected with the hT25-m2 or hT25-m4 plasmid than in WT cells or cells transfected with the hT25-m1 plasmid. These results indicated that hairpin and quadruplex structures play an important role in CTCF binding to hTERT and hTERT gene expression. Notably, DNA methylation alone is not sufficient to inhibit CTCF binding to the first exon of hTERT, suggesting that quadruplex formation promoted by CpG methylation plays a major role in preventing CTCF binding and further regulating gene expression.
In summary, the key finding of this study is that methylation of cytosine at specific CpG dinucleotides participates in quartet formation, which can shift the equilibrium from a hairpin structure to a quadruplex structure via a simple flipping process (Figure 2). CTCF prefers to bind to the hairpin structure, and this binding suppresses hTERT transcription; in contrast, quadruplex formation inhibits CTCF binding, resulting in hTERT gene expression. Our results not only identify a new example of quadruplex formation induced by CpG dinucleotide methylation but also provide mechanistic insight into the regulation of gene expression.
Figure 2. Proposed mechanism of CTCF binding to the first exon of the hTERT gene for transcriptional regulation. CTCF favors binding to the hairpin structure, whereas quadruplex formation, which is enhanced by CpG methylation, impedes CTCF binding and leads to gene expression.
Reference
Pei-Tzu Li, Zi-Fu Wang, I.-Te Chu, Yen-Min Kuan, Ming-Hao Li, Mu-Ching Huang, Pei-Chi Chiang, Ta-Chau Chang, and Chin-Tin Chen (2017). Expression of the human telomerase reverse transcriptase gene is modulated by quadruplex formation in its first exon due to DNA methylation. Journal of Biological Chemistry, 292(51), 20859-20870. DOI:10.1074/jbc.M117.808022.
Professor Chin-Tin Chen
Department of Biochemical Science and Technology
chintin@ntu.edu.tw