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Title: Contribution of Lsh to DNA methylation reprogramming in embryonic stem cell, epiblast stem cell and embryoid body model systems
Author: Revuelta, Ailsa Clare
ISNI:       0000 0004 7429 5868
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2018
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DNA methylation is a key epigenetic mark which undergoes global reprogramming during early mammalian embryonic development, resulting in almost complete erasure of the mark after fertilisation of the zygote. Genome-wide patterns of DNA methylation are subsequently re-established in the implanting blastocyst by de novo DNA methyltransferases Dnmt3a and Dnmt3b along with their catalytically inactive co-factor Dnmt3l, while these DNA methylation patterns are maintained through cell divisions by maintenance methyltransferase Dnmt1. The exact mechanisms by which these DNA methyltransferase enzymes are targeted to specific genomic regions remain unclear, but may involve interaction with modified histones and/or the participation of co-factors. Lsh (lymphoid specific helicase), a putative chromatin remodelling helicase, has been implicated in facilitating de novo methylation, as Lsh knockout embryos and derived somatic cell lines display substantial but specific DNA methylation losses at repetitive elements and single copy genes. This study aims to define the requirement for Lsh in establishing de novo DNA methylation and gene expression patterns during the early stages of mouse embryonic development. The '2i' culture system using two small molecule kinase inhibitors was harnessed to convert lsh-/- mouse embryonic stem cells (mESCs) to a hypomethylated 'ground state' of pluripotency. Culture conditions were then altered to transition these ground state mESCs to cells representing later, more methylated stages of development ('serum' mESCs, epiblast stem cells and embryoid bodies). Implementation of this model system suggests that Lsh does not contribute to DNA methylation establishment in a pluripotent context, but rather is important for facilitating de novo DNA methylation during differentiation to culture models representing later developmental stages. These investigations also reveal that Lsh differentially regulates DNA methylation at major and minor satellite repeats depending on cellular context, and that this regulation may involve a role for Lsh in maintenance of DNA methylation.
Supervisor: Meehan, Richard ; Jackson, Andrew Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: DNA methylation ; Lsh ; gene expression ; embryonic development ; ICF syndrome ; Immunodeficiency, Centromere instability and Facial anomalies syndrome