CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation.

By Sanae M. M. Iguchi-Ariga, W. Schaffner. 1989

In mammals and other vertebrates, cytosine methylation in CpG sites is often negatively correlated with gene activity. Because methylation of the promoter region is most crucial for this effect, the simplest hypothesis is that CpG methylation interferes with the binding of specific transcription factors. We have examined this hypothesis with two different transcription factor-binding sites that contain a CpG dinucleotide, namely the cAMP-responsive element (CRE; 5'-TGACGTCA) and the Sp1-binding site (5'-GTGAGGCGGTGAGACT). We have reported previously that CpG methylation of the Sp1-binding site affected neither factor binding nor transcription in HeLa cells, which may be related to the fact that Sp1 is typically associated with promoters of housekeeping genes. In contrast, CREs are often associated with promoters of cell type-specific genes. A synthetic oligonucleotide containing two tandem CREs derived from the gene encoding the human glycoprotein hormone alpha-subunit was cloned upstream of a reporter gene. Transcription of this gene was dependent on the CRE sequences in both PC12 and HeLa cells. Bandshift and methylation interference assays show that similar, if not the same, factor(s) bind to the CRE in both cell lines, even though induction by cAMP was only observed in PC12 cells. CpG methylation of the CRE consensus sequences (TGACGTCA) resulted in loss of specific factor binding, as well as loss of transcriptional activity in vitro and in vivo, in both cell types. This suggests that the inactivity of methylated promoters can, at least in some cases, be explained by their inability to bind specific transcription factors.

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Citations in the corpus

• Impact of cytosine methylation on DNA binding specificities of human transcription factors (2017, background): “In contrast to most previously reported cases [for example, (9, 10, 12, 17, 18)], we found that methylation can affect TF binding positively in many cases [see also (16, 62)].”
• CG methylated microarrays identify a novel methylated sequence bound by the CEBPB|ATF4 heterodimer that is active in vivo (2013, background): “Both the CEBP and CRE motifs have a CG dinucleotide at the center of the transcription factor binding site (TFBS), and methylation has the opposite effect on the DNA binding: It enhances CEBPA binding and inhibits CREB binding (Iguchi-Ariga and Schaffner 1989; Rishi et al. 2010).”
• CREB and the CRTC co-activators: sensors for hormonal and metabolic signals (2011, background): “Most of the 750,000 palindromic and half-site CREs in the human genome, a number of which are near silenced genes, are unoccupied in cells owing to disruptive cytosine methylation within the CREB-binding sit”
• Evolution of dioecy and sex chromosomes via methylation driving Muller's ratchet (2003, background): “Methylation blocks binding sites for enzymes that mediate transcription (Iguchi-Ariga & Schaffner, 1989; Tate & Bird, 1993; Henry et al ., 1999; Yung et al ., 2001) (Fig.”
• CpG methylation of half-CRE sequences creates C/EBPα binding sites that activate some tissue-specific genes (2010, background): “This was surprising because methylation of the CRE has been reported to inhibit DNA binding of CREB, the protein known to bind the CRE (13).”
• Transcriptional regulation by the phosphorylation-dependent factor CREB (2001, background): “The CRE palindrome (TGACGTCA) contains a central CpG dinucleotide, and methylation of this site blocks CREB binding in vitro”