Sp1 binding is inhibited by (m)Cp(m)CpG methylation

Previously it has been found that binding of the Spl transcription factor is not significantly affected by methylation of the CpG dinucleotide within its binding site, 5'-GGGCGG (lower strand, 5'-CCGCCC). Since it has been established that mammalian cells also have the capacity to methylate cytosines (C) at CpNpG sites we examined the effect of methylation of the outer C of the CpCpG on Spl binding. We find that methylation of the outer C is inhibitory and in particular methylation of both cytosines (m)Cp(m)CpG inhibits binding by 95%. Furthermore, we have identified endogenous (m)Cp(m)CpG methylation of an Spl site in the CpG island promoter of the retinoblastoma (Rb) gene by genomic sequencing. This occurs in a proportion of retinoblastoma tumors which are extensively CpG methylated in the Rb promoter. The results raise the possibility that (m)Cp(m)CpG methylation could have a biological function in preventing Spl binding, thereby contributing to the subsequent abnormal methylation of CpG islands often observed in tumor cells. (C) 1997 Elsevier Science B.V.