The obvious question concerns the functional importance of such differences in methylation. DNA methylation affects gene expression either by attracting methylated DNA-binding proteins to a densely methylated region of a gene or by site-specific interference with the binding of a transcription factor to its recognition element.91,112 Our data showing site-specific differences in methylation of the cytosine within the 5' CpG dinucleotide of the NGFIA response element suggests alterations in the ability of the NGFIA protein to bind to its response element. We118 determined the in vitro binding of increasing concentrations of purified recombinant NGFIA protein119 to its response element under different states of methylation using the electrophilic mobility shift assay (EMSA) technique with four 32P-labelled synthetic oligonucleotide sequences bearing the NGFIA binding site that was either (i) nonmethylated; (ii) methylated in the 3' CpG site; (hi) methylated in the 5' CpG site; (iv) methylated in both sites; or (v) mutated at the two CpGs with an adenosine replacing the cytosines. NGFIA formed a protein-DNA complex with the nonmethylated oligonucleotide, while the protein is unable to form a complex with either
