A method to detect DNA methyltransferase I gene transcription in vitro in aging systems

Abstract

Epigenetic alterations of DNA play key roles in determining gene structure and expression. Methylation of the 5-position of cytosine is thought to be the most common modification of the genome in mammals. Studies have generally shown that hypermethylation in gene regulatory regions is associated with inactivation and reduced transcription and that alteration in established methylation patterns during development can affect embryonic viability. Changes in methylation have also been associated with aging and cellular senescence as well as tumorogenesis. DNA methyltransferase 1 (DNMT1) is thought to play an important role in maintaining already established methylation patterns during DNA replication and catalyzes the transfer of a methyl moiety from S-adenosyl-L-methionine (SAM) to the 5-position of cytosines in the CpG dinucleotide. Several studies illustrate changes in activity and transcription of DNMT1 during aging and here we show a comprehensive method of detection of DNMT1 mRNA transcription from senescing cells in culture.

Fig. 1

Amplification of the DNMT1 cDNA. Cells were lysed and total mRNA was extracted followed by conversion to cDNA as detailed under Subheadings 3.2., 3.3., and 3.5., respectively. Amplification of cDNA was carried out by PCR and visualized by ethidium bromide staining on a 2% agarose gel.