Complex demethylation patterns at Sp1 binding sites in F9 embryonal carcinoma cells.
The ubiquitous transcription factor Sp1 has been implicated in the mechanism which maintains CpG islands methylation-free. Plasmids containing GC boxes (Sp1 sites) were in vitro methylated at every CpG dinucleotide. After stable introduction into F9 embryonal carcinoma cells, we analysed the methylation of the sequence around the GC boxes with bisulphite sequencing. In agreement with restriction site analysis by other labs, we found preferential demethylation at GC box DNA versus control DNA. However, the bisulphite sequencing which permits the analysis of every CpG site on a given DNA molecule, revealed a complex pattern of methylated and unmethylated sites. Upon prolonged culture the pattern became simpler, with most sites demethylated but certain sites being consistently methylated.
Silke
J
J
Institut für Molekularbiologie II, Universität Zürich, Switzerland.
Rother
K I
KI
Georgiev
O
O
Schaffner
W
W
Matsuo
K
K
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015139:Blotting, Southern; D018236:Carcinoma, Embryonal / Q000737:chemistry* / Q000473:pathology; D004247:DNA / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D008745:Methylation; D051379:Mice; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D017422:Sequence Analysis, DNA; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D013379:Substrate Specificity; D013447:Sulfites / Q000737:chemistry; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009711:Nucleotides; D016329:Sp1 Transcription Factor; D013447:Sulfites; D014157:Transcription Factors; D004247:DNA; D015257:DNA-Cytosine Methylases