|dc.description.abstract||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
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.||en