Voigt, Phillipp

Allshire, Robin

Amendola, Roberta Ilaria

2019-08-12T12:02:22Z

2019-08-12T12:02:22Z

2019-07-06

DNA methylation at cytosine (5meC) is essential for central cellular mechanisms, such as genomic imprinting, tissue-specific gene expression, silencing of retroviral elements and inactivation of the X chromosome in female. Impairment of proteins that either deposit or bind the 5meC leads to human disease, such as imprinting disorders, Rett Syndrome, Immunodeficiency Centromeric instability and facial abnormalities (ICF) syndrome and cancer. DNA methyl transferases (DNMTs) are directly responsible for the deposition of methyl groups on the DNA and can be divided into de novo and maintenance DNMTs, accordingly to structural and functional features. The de novo DNMTs are responsible for depositing methyl groups on non-methylated DNA, mostly in the form of heterochromatin. The maintenance DNMTs methylate hemimethylated DNA at the replication fork, and are thereby responsible for preventing the loss of 5meC during cell division. These proteins are crucial during embryonic development, since DNA methylation goes through severe reprogramming events. In fact, 5meC is almost completely lost after 3.5 days from the fertilisation and is re-established by day 6.5 of the embryonic development, when the epiblast is formed. From day 3.5 the de novo DNMTs, DNMT3B and DNMT3A, are highly expressed and methylate the genome. The maintenance DNMT1 is then expressed and maintains the newly methylated genomic loci. Mutations of these proteins determine diseases in humans, such as the ICF syndrome. The majority of ICF patients is characterised by mutations in DNMT3B and hypomethylation of DNA. However, a small percentage of patients is characterised by mutations in the Lsh gene. Lymphoid Specific Helicase (LSH) is an ATP-dependent chromatin remodeler, whose remodeler activity has not been proven in vitro. It is essential for DNA methylation throughout the genome, in mammals and plants and it has been linked to developmentally programmed de novo methylation at unique and repetitive sequences. However, there is still debate on whether LSH has a role in maintenance of DNA methylation as well. Knockout of Lsh gene in embryos results in ~50% reduction of 5meC in the genome. It is hypothesized that LSH remodels chromatin to enable the access of DNA methyltransferases to DNA during development, when DNA methylation patterns undergo dramatic reprogramming. In this work, by using an in vitro culturing system, we mimicked the reprogramming of the DNA methylation typical of embryonic development and analysed changes occurring in 5meC deposition and localisation in absence of LSH. To do so, we used mouse ESCs maintained in 2i or serum-containing culturing media. The 2i-containing medium was used to deplete 5meC and ensure a naïve state of pluripotency, resembling the methylation level in the blastocyst state; the serum-containing medium determined an accumulation of DNA methylation, which took the cells to a primed pluripotent state, resembling the epiblast. Using this system, we could analyse how cells lacking LSH responded to DNA methylation reprogramming. We showed that the role of LSH is concomitant with the de novo DNA methylation timing. Furthermore, analyses of DNA demethylation suggested that LSH was not involved in maintenance of DNA methylation. Interestingly, we found that in absence of LSH; loss and gain of methylation were faster. This suggested that the chromatin in cells lacking LSH was more easily accessible to methyltransferases and demethylases, supporting the hypothesis of an involvement of LSH in DNA methylation via its remodelling activity. The same system was advantageous to investigate on a whole genome base the genomic regions requiring LSH activity. Sequencing of the unmethylated fraction of the genome was carried out on cells before and after reprogramming of the DNA methylation and on cells in the early differentiation stage. This experiment confirmed the previous findings that LSH is required both at unique and repetitive regions of the genome, such as IAPs, and gave further insights into the regions that require LSH to be de novo methylated. In conclusion, this study has demonstrated that LSH is required only concomitantly to the reprogramming of DNA methylation, that occurs during early phases of embryonic development. Furthermore, it has provided with further evidence supporting LSH activity as chromatin remodeler and facilitator of de novo methylation, but not maintenance. Finally, the high throughput sequencing analysis has deepened the understanding of the loci-specific activity of LSH.

http://hdl.handle.net/1842/36027

en

The University of Edinburgh

DNA methylation

LSH

DNMTs

Lymphoid Specific Helicase

Establishment and maintenance of DNA methylation in absence of the chromatin remodeller LSH

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy