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Title: Reprogramming of DNA methylation in mouse pluripotency genes and in vivo assessment of a mechanism for demethylation
Author: Chan, C-F.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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In this study, I found that the promoters of pluripotency genes Nanog and Oct-4, and the cytidine deaminase gene Aid are hypomethylated in pluripotent embryonic stem cells compared with the majority of somatic cell types, coincident with their expression. The Nanog and Oct-4 promoters acquire DNA methylation upon ES cell differentiation, and the genes are aberrantly expressed in Dnmt1-/- embryos. A genome-wide microarray based screen for promoters that are selectively hypomethylated in pluripotent cells relative to differentiated cells reveals new candidate genes which are epigenetically regulated and connected to pluripotency in ES cells. The methylated Nanog promoter undergoes dynamic reprogramming in vivo: it is highly methylated in sperm and is actively demethylated after fertilization independent of DNA replication. When I target in ES cells the Nanog promoter (containing the Ga14 binding sequence UAS) with a Ga14 – de novo methyltransferase Dnmt3a fusion protein it becomes methylated and repressed, providing direct evidence of epigenetic regulation of its promoter. The mechanisms of active enzymatic demethylation in vivo are currently unknown, however, previous studies have shown that in vitro the cytidine deaminase Aid is capable of deaminating 5-methylcytosine to thymine. Here a nuclear form of Aid was targeted in a transgenic system to the methylated imprinting control region (DMR) of H19 in vivo (using the Ga14 fusion system). I found that Aid efficiently demethylates the DMR without causing sequence mutations.  These results show that DNA deamination coupled presumably with the base excision repair pathway can be involved in active demethylation in mammals.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available