COMPLEX DEMETHYLATION PATTERNS AT SP1 BINDING-SITES IN F9 EMBRYONAL CARCINOMA-CELLS

The ubiquitous transcription factor Sp1 has been implicated in the mec hanism which maintains CpG islands methylation-free. Plasmids containi ng GC boxes (Sp1 sites) were in vitro methylated at every CpG dinucleo tide. After stable introduction into F9 embryonal carcinoma cells, we analysed the methylation of the sequence around the GC boxes with bisu lphite sequencing. In agreement with restriction site analysis by othe r labs, we found preferential demethylation at GC box DNA versus contr ol 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 patte rn became simpler, with most sites demethylated but certain sites bein g consistently methylated.