Regional DNA Methylation and Gene Expression

Kass, Stefan U (1993) Regional DNA Methylation and Gene Expression. PhD thesis, University of Glasgow.

Abstract

The detailed mechanisms of inhibition of transcription by DNA methylation are still unknown but it has become obvious that the formation of chromatin plays an important role in this process. Methylation of a plasmid containing the SV40 promoter linked to the chloramphenicol acetyltransferase (CAT) gene results in a significant reduction in the expression of the reporter gene after transfection into cultured cells. Transcriptional inhibition of methylated DNA is apparent only 24 to 48 hours after transfection. This coincides with a decrease in MspI accessibility of methylated DNA, in vivo, suggesting a role of chromatin formation and/or the involvement of methylated-DNA binding proteins in the inactivation process. A new method was developed to methylate, in vitro, chosen sequences of this plasmid. This localised methylation of SV40 promoter, CAT gene or vector sequences reveals a reduced CAT activity in transient transfection assays. Transcriptional inactivation appears to be proportional to the length or the CpG density of the methylated region, but independent of its localisation. Digestion with MspI of nuclei, transfected with these regionally methylated plasmids showed a reduced accessibility of MspI to both, methylated and unmethylated sequences of the plasmid. The results suggest, that upon methylation and transfection into cells, plasmid DNA forms inactive chromatin, this inactive chromatin spreads to unmethylated regions within the plasmid and can thereby inhibit gene expression. In in vitro transcription assays, no differences were observed in the level of transcription from unmethylated or methylated templates and, furthermore, histone H1 did not preferentially inhibit transcription in vitro from methylated reporter gene constructs.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Roger Adams
• Keywords: Biochemistry
• Date of Award: 1993
• Depositing User: Enlighten Team
• Unique ID: glathesis:1993-76379
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Dec 2019 09:15
• Last Modified: 19 Dec 2019 09:15
• URI: https://theses.gla.ac.uk/id/eprint/76379