Investigating the molecular dynamics of gene re-silencing following treatment with epigenetic therapies

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Copyright: Patil, Vibha
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Abstract
Aberrant transcriptional silencing of genes by promoter hypermethylation is an important mechanism of tumour suppressor gene (TSG) inactivation in cancer. DNA hypomethylating agents such as 5-Aza-2’deoxycytidine (5-Aza-dC) are approved for the treatment of myelodysplastic syndrome and show promise in the treatment of solid tumours. These therapies are thought to exert their anti-tumour effects by reactivating hypermethylated genes. However, resistance to these drugs inevitably develops and this is associated with the silencing of genes initially re-expressed by therapy, termed gene re-silencing. The molecular basis of this is unknown, however understanding how it occurs may aid in the development of strategies to prevent or overcome resistance. We hypothesised that other chromatin modifications may silence gene expression in the absence of methylation. To study gene re-silencing following 5-Aza-dC exposure, a model gene (MLH1) and cell line (the colorectal carcinoma cell line RKO) were identified and validated. Gene reactivation was dependent on DNMT1 depletion and hypomethylation, nucleosome eviction and the acquisition of active histone marks at the MLH1 promoter. However, close inspection of the early stages of gene re-silencing showed that loss of expression occurred despite persistent hypomethylation. Instead, gene re-silencing coincided with the reassembly of nucleosomes at the MLH1 transcription start site, which preceded DNA re-methylation. Unexpectedly, changes in non-CpG methylation (methylation at CpT, CpA and CpC dinucleotides) were also observed, which we find may be attributable to alterations in the expression of the DNMT3A, DNMT3B or DNMT3L genes. Finally, despite the persistence of the repressive histone mark H3K9me3 following 5-Aza-dC treatment we find that, in this model, inhibitors of H3K9 methyltransferases do not delay gene re-silencing. These findings show that gene re-silencing is not driven by DNA re-methylation but involves other mechanisms, namely the regulation of gene expression by nucleosome positioning. This has important implications for resistance to DNA hypomethylating agents and suggests that strategies to overcome this will require targeting of nucleosomes.
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Author(s)
Patil, Vibha
Supervisor(s)
Hesson, Luke
Sloane, Mathew
Hawkins, Nicholas
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Publication Year
2015
Resource Type
Thesis
Degree Type
PhD Doctorate
UNSW Faculty
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