title: Sequencing of the rat light neurofilament promoter reveals differences in methylation between expressing and non-expressing cell lines, but not tissues.
abstract: 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 DNA. We analysed twenty-five potential CpG methylation sites between nucleotide (nt) positions -311 and +103 of pNF-L, containing Sp1- and AP-2-binding sites, a CGCCCCCGC box and a cAMP-responsive element. Six out of 25 possible CpG methylation sites are within these elements. The pNF-L promoter was unmethylated in NF-L-expressing rat brain, as well as in liver not expressing NF-L. In NF-L-expressing PC12 cells, the promoter was unmethylated, whereas in non-expressing glioma C6 cells intensive methylation occurred. A cluster of methylated CpG dinucleotides spanned the region from nt -176 to -67 bp. Thus, methylation of this 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 sequence, in the CpApG trinucleotide at nt position -114, cytosine was found to be partially methylated. It is thus possible to describe the methylation state of each cytosine present in the area of genomic DNA of interest.
authors.affiliation: Institute of Biotechnology, University of Helsinki, Finland.
authors.forename: M
authors.initials: M
authors.lastname: Reeben
authors.forename: S
authors.initials: S
authors.lastname: Myöhänen
authors.forename: M
authors.initials: M
authors.lastname: Saarma
authors.forename: H
authors.initials: H
authors.lastname: Prydz
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008745:Methylation; D008969:Molecular Sequence Data; D016900:Neurofilament Proteins / Q000096:biosynthesis* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013447:Sulfites; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D016900:Neurofilament Proteins; D012333:RNA, Messenger; D013447:Sulfites; D014157:Transcription Factors; C044882:neurofilament protein L; D004247:DNA; C042345:hydrogen sulfite