SEQUENCING OF THE RAT LIGHT NEUROFILAMENT PROMOTER REVEALS DIFFERENCES IN METHYLATION BETWEEN EXPRESSING AND NON-EXPRESSING CELL-LINES, BUTNOT TISSUES

The DNA methylation pattern of the promoter (pNF-L) region of the rat light-neurofilament-encoding gene (NF-L), a neuron-specific gene, was assessed in NF-L expressing and non-expressing cell lines and tissues by genomic sequencing using PCR amplification of bisulfite-modified DN A. We analysed twenty-five potential CpG methylation sites between nuc leotide (nt) positions - 311 and + 103 of pNF-L, containing Sp1- and A P-2-binding sites, a CGCCCCCGC box and a cAMP-responsive element. Six out of 25 possible CpG methylation sites are within these elements. Th e pNF-L promoter was unmethylated in NF-L-expressing rat brain, as wel l as in liver not expressing NF-L. In NF-L-expressing PC12 cells, the promoter was unmethylated, whereas in non-expressing glioma C6 cells i ntensive methylation occurred. A cluster of methylated CpG dinucleotid es spanned the region from nt - 176 to - 67 bp. Thus, methylation of t his promoter region could play a role in silencing NF-L in the glioma cell line in vitro, but not in liver tissue in vivo. In a non-CpG sequ ence, in the CpApG trinucleotide at nt position - 114, cytosine was fo und to be partially methylated. It is thus possible to describe the me thylation state of each cytosine present in the area of genomic DNA of interest.