Using synthetic biology to investigate human gene body DNA methylation

DNA methylation is an epigenetic modification important in development, genomic imprinting and gene regulation. Classically, methylation at promoters is associated with transcriptional repression, whereas methylation within gene bodies has been correlated with highly expressed genes. This has led to the hypothesis that gene body methylation may enhance transcription. However, most of these conclusions have been drawn from correlative whole- genome analyses. To directly test the effect of gene body methylation on gene expression, I utilised a synthetic system to integrate in-vitro methylated gene bodies into the genome of human cell lines. Utilising the Bxb1-Recombinase landing-pad system, I integrated methylated gene bodies at a single fixed genomic locus expressed by an unmethylated, active promoter. I observed that gene body methylation of eGFP and mCherry reduced gene expression by ~2- fold relative to unmethylated controls. This reduction of gene expression was restored upon removal of gene body methylation through inhibition of the major maintenance methyltransferase DNMT1 by GSK-3484862 treatment. To understand how gene body methylation functions at endogenous loci, I analysed data from HCT116 cells which had been treated with the DNMT1 inhibitor. I found that most genes were unaffected by loss of gene body methylation, although subsets were up- or down-regulated. This suggested that gene body methylation does not exert uniform effects across the genome, but instead influences specific genes in a context-dependent manner. To test whether sequence-specific effects explained these differences, I used my targeted platform to examine splicing, intragenic promoter activity, and CpG density, and discovered that increased CpG density correlated with increased gene body-mediated repression. Together, my findings demonstrate that gene body methylation can directly modulate gene expression, causing repression and in some cases upregulation. Its effects are not uniform across the genome but depend on sequence context, with CpG density emerging as an important factor. This work refines our understanding of gene body methylation and introduces av novel application of a targeted integration system for locus-specific investigation of gene body methylation, enabling future mechanistic studies of its role in transcriptional regulation and disease.

Author dc:creator

• Rodger, Christine

Advisors dc:contributor.advisor

• Sproul, Duncan
• Kudla, Grzegorz

• DNA methylation
• Transcription
• gene expression
• Epigenetics
• Cell biology
• Gene regulation
• synthetic biology

Language dc:language.iso

en

Identifier URI

https://doi.org/10.7488/era/7031

OAI identifier oai:identifier

oai:era.ed.ac.uk:1842/44514