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Unit Programme: Epigenetic Mechanisms of Toxicity

Dr Mathew Van de Pette

Geno-toxicity, whereby a toxic compound causes changes to the genetic code through mutation, has received widespread attention from the research community. Effects of toxic insults on the epigenome, where the genetic code is not directly affected, instead the ability to correctly read this code, remain poorly understood. The programme focusses on developing our understanding of the action and mechanism of chronic sub-toxic insults on the epigenome. These occur when a substances dose is insufficient to cause acute toxicity, but instead alters the environment of the DNA. The programme will have a particular emphasis on the lasting impact of these exposures during pregnancy. These toxic events can be environmental or pharmaceutical, and we aim to identify similarities and differences in such diverse challenges. We employ standard genomics and molecular biology techniques, in addition to using a new approach to studying epigenetics. We have generated a series of transgenic mouse lines, which can “see” the epigenetic status of a gene, through imaging of a mouse in vivo. Using these approaches, we can track epigenetic change through life-course and across generations, thereby “seeing” the toxic insult in its entirety.



Figure (a) Imprinted genes display parent-of-origin specific mono-allelic expression. Around 100 imprinted genes have so far been found, representing around 0.1% of the genome. Their allelic expression is established through differential methylation in the germline, and further bolstered by an array of epigenetic marks

(b) We have targeted a number of imprinted genes, in this case the Cdkn1c gene, a maternally expressed imprinted gene. This targeting allows for firefly luciferase (FLuc) and LacZ reporters to be transcriptionally controlled by the endogenous Cdkn1c loci, without disrupting the natural expression of the target gene. T2A sites allow for cleavage of the translated proteins, meaning the reporter is linked to endogenous expression through transcription and translation.

The FLuc reporter allows bioluminescent imaging of Cdkn1c expression. Here mouse embryonic stem cells (mESCs) are imaged. Clones with a maternal knock-in (KImat) of the reporter display bioluminescent signal (blue-green). Clones with a paternal knock-in (KIpat) display no signal, as the reporter is epigenetically silenced. Genetically identical, Epigenetically different.

(c) This method allows reporting from the whole body to single cell. Here, immunostaining of Cdkn1c+ somites (red nuclei) overlaps with luciferase staining in KImat cells, with no staining observed in KIpat cells.