Regulators of DNA methylation in mammalian cells
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Date
29/06/2013Author
Termanis, Ausma
Metadata
Abstract
Although the many cells within a mammal share the same DNA sequence,
their gene expression programmes are highly heterogeneous, and their functions
correspondingly diverse. This heterogeneity within an isogenic population of cells
arises in part from the ability of each cell to respond to its immediate surroundings
via a network of signalling pathways. However, this is not sufficient to explain many
of the transcriptional and functional differences between cells, particularly those that
are more stable, or, indeed, differences in expression between parental alleles within
the same cell. This conundrum lead to the emergence of the field of epigenetics - the
study of heritable changes in gene expression independent of DNA sequence. Such
changes are dependent on “epigenetic modifications”, of which DNA methylation is
one of the best characterised, and is associated with gene silencing. The
establishment of correct DNA methylation patterns is particularly important during
early development, leading to cell type specific and parental allele specific gene
regulation. Besides DNA methyltransferases, various other proteins have recently
been implicated in DNA methylation. The absence of these proteins leads to defects
in DNA methylation and development that can be even more severe than those in
DNA methyltransferase knockouts themselves. In this study I focus on three such
accessory proteins: LSH (a putative chromatin remodelling ATPase), G9a (a histone
lysine methyltransferase) and SmcHD1 (a structural maintenance of chromosomes
protein). To compare DNA methylation between WT cells and cells knocked out for
each of these proteins, I used whole genome methylated DNA affinity purification
and subsequent hybridization to promoter microarrays. This enabled me to compare
the requirement for each protein in DNA methylation at specific genomic regions.
The absence of LSH in mouse embryonic fibroblasts (MEFs) resulted in the
loss of DNA methylation at 20% of usually methylated promoters, and the
misregulation of associated protein coding genes. This revealed a requirement for
LSH in the establishment of DNA methylation at promoters normally methylated
during pre-implantation as well as post-implantation development.
Secondly, I identified hypomethylation at 26% of normally methylated
promoters in G9a-/- compared to WT ES cells. Strikingly, this revealed that G9a is
required for maintenance of DNA methylation at maternal as well as paternal
imprinting control regions (ICRs). This is accompanied by expression defects of
imprinted genes regulated by these ICRs.
Finally, in collaboration with the Brockdorff lab at the University of Oxford I
identified a role for SmcHD1 in establishing DNA methylation at promoters on the X
chromosome normally methylated slowly during X chromosome inactivation.
Interestingly, SmcHD1 was also required for DNA methylation at autosomal gene
promoters, contrary to previous reports that it is mainly involved in X chromosome
methylation.
I conclude that different accessory proteins are required to facilitate correct
DNA methylation and gene repression at distinct regions of the genome, as well as at
different times during development. This function of accessory proteins may be in
part dependent on the prior establishment of specific chromatin signatures and
developmental signals, together comprising a precisely regulated system to establish
and maintain appropriate DNA methylation throughout development.