Reiterative de novo methylation maintainsmethylation levels in somatic cells
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Date
28/09/2022Item status
Restricted AccessEmbargo end date
28/09/2023Author
Kafetzopoulos, Ioannis
Metadata
Abstract
DNA methylation is a pervasive epigenetic mark in normal cells. DNA
methylation abnormalities are a fundamental hallmark of cancer that can
promote carcinogenesis. DNA methylation is lost specifically in
heterochromatic regions in tumours. These hypomethylated regions are
termed partially methylated domains (PMDs) and replicate during late S-phase. The late replication of PMDs has been proposed to play a key role in
their hypomethylation. Specifically, it has been suggested that PMDs passively
lose methylation due to incomplete maintenance of methylation after
consecutive cell divisions. This model directly implicates DNMT1 as the
maintenance methyltransferase and suggests that it does not have enough
time to fully methylate late replicating regions. I aimed to elucidate how PMDs
become hypomethylated during tumorigenesis and address this ‘passive loss’
model. I investigated the levels and patterns of DNA methylation in HCT116
colorectal cancer cells and their DNMT1 Knock-Out (DNMT1KO) derivatives.
I identified that PMDs show distinct hypomethylation in HCT116 cells,
depending on their heterochromatic state. Constitutive heterochromatic PMDs,
marked by H3K9me3, showed more pronounced hypomethylation than
facultative ones, marked by H3K27me3. In DNMT1KO cells, I observed global
loss of methylation levels. However, hypomethylation was particularly
prominent within PMDs, suggesting that hindering DNMT1 activity led to
poorer maintenance of methylation, in agreement with the model. I also
observed a subgroup of PMDs that were predominantly marked by H3K9me3
and bordered by H3K27me3 in HCT116 cells, which unexpectedly showed
increased methylation levels in DNMT1KO cells. These hypermethylated
PMDs remain late replicating in DNMT1KO cells despite their high methylation.
However, these regions were no longer marked by H3K9me3 and H3K27me3
in DNMT1KO cells, indicating the loss of their heterochromatic state. Finally,
using ChIP, I identified that DNMT3A and DNMT3B were not recruited in
constitutive and facultative heterochromatic regions. DNMT3A, but not
DNMT3B, recruitment was detected in these hypermethylated PMDs in
DNMT1KO but not HCT116 cells, aligning with the loss of the heterochromatic
marks in the hypermethylated PMDs. Taken together, my results suggested
that hypermethylated PMDs in DNMT1KO cells could maintain high
methylation levels, despite their late replication timing, due to the recruitment
of DNMT3A. More generally, this suggested that de novo DNMTs play an
important role in maintenance of methylation levels via reiterative de novo
methylation, while highlighting that chromatin environment and its role in
DNMT recruitment might play a more important role than replication timing in
the hypomethylation observed in cancers.