Reprogramming a DNA methylation mutant
Hunter, Jennifer Margaret
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Chemical modification of the cytosine base via the addition of a methyl group to form 5-‐methylcytosine (5-‐mC) is a well-‐studied example of an epigenetic mark, which contributes to regulation of gene expression, chromatin organisation and other such cellular processes without affecting the underlying DNA sequence. In recent years it was shown that 5-‐mC is not the only DNA modification found within the vertebrate genome. 5-‐hydroxymethylcytosine (5-‐hmC) was first described in 1952 although it wasn’t until 2009 when it was rediscovered in mammalian tissues that it sparked intense interest in the field. Research has found that unlike the 5-‐mC base from which it is derived, 5-‐hmC displays variable levels and patterns across a multitude of tissue and cell types. As such the patterns of these DNA modifications can act as an identifier of cell state. This thesis aims to characterize the methyl and hydroxymethyl profiles of induced pluripotent stem cells (iPSCs), derived from control mouse embryonic fibroblast cell line (p53-‐/-‐) as well as and methylation hypomorphic (p53-‐/-‐, Dnmt1 -‐/-‐) mutant cell lines. As such both somatic cells were subject to reprogramming with Yamanaka factors (Oct4, cMyc, Klf4 and Sox2) via the piggyback transposition technique. Successful reprogramming was confirmed by a number of techniques and outcomes, including the de novo expression of a number of key pluripotency related factors (Nanog, Sall4 and Gdf3). Reprogrammed cells were then analysed for transcriptomic changes as well as alterations to their methyl and hydroxymethyl landscapes that accompany reprogramming. Through this work I have shown that the reprogramming of MEF derived cell lines results in a global increase in 5-‐hmC for both p53-‐/-‐ and (p53-‐/-‐, Dnmt1 -‐/-‐) hypomorphic mutant cell lines – possibly through the reactivation of an alternative form of DNMT1. I demonstrate by both antibody based dot blot assay and genome wide sequencing that the reprogramming of the (p53-‐/-‐, Dnmt1 -‐/-‐) somatic cells towards a pluripotent state brings about an increase in methylation levels within the cells. This latter observation may indicate that the reprogramming of the cells is driving them towards a more wild type phenotypic state. My studies suggest that lack of DNMT1 function is not a barrier to reprogramming of somatic cells.