Role of chromatin in resilencing dynamics after drug-induced DNA demethylation Público
Kagey, Jacob Daniel (2010)
Abstract
In cancer, large-scale epigenetic alterations are observed including the aberrant hypermethylation and silencing of a subset of CpG island containing tumor suppressor genes. The causes underlying the establishment and maintenance of this silencing remain incompletely understood. To study these processes, we have utilized Target of Methylation-induced Silencing 1 (TMS1), a gene frequently methylated and silenced during carcinogenesis, to investigate the relationship between the epigenetic regulation of TMS1 and the DNA methylation status at the CpG island. We found that in normal cells the unmethylated CpG island is enriched for a subset of active histone modifications Conversely, in cancer cells, the methylated CpG island associated the repressive histone modifications. To investigate the role of DNA methylation in the establishment and maintenance of TMS1 gene silencing we treated cells, methylated for TMS1, with 5-aza- 2'deoxycytidine (5-azaCdR). TMS1 demethylation, induced by 5-azaCdR, was accompanied by gene re-expression, the loss of repressive histone modifications, and the re-acquisition of active marks. We kept these cells in culture following 5-azaCdR treatment and found that a sub-population of unmethylated TMS1 alleles is stably maintained in the absence of drug which does not fully recapitulate the chromatin structure of the unmethylated TMS1 locus, providing evidence for two independent repressive pathways negatively regulating TMS1. We next examined kinetics of DNA demethylation and remethylation following a transient treatment with 5-azaCdR on a genome-wide scale. CpGs specifically methylated in cancer demonstrated distinct remethylation kinetics. One group was significantly enriched for genes occupied by SUZ12 in embryonic stem cells, suggesting these loci may be marked by histone H3 lysine 27 tri-methylation in normal tissues. This finding suggests that the mechanisms underlying de novo aberrant methylation differ between individual CpGs. Together, these data provide insight into how cancer specific methylation is established and may help to improve existing therapeutic strategies in cancer.
Table of Contents
Contents
Chapter 1: Introduction ............................................... 1
Figures ...................................................................... 26
Chapter 2: Role of hMOF-dependent H4 Lysine 16 acetylation in the maintenance of
TMS1/ASC gene activity ............................................... 34
Figures ...................................................................... 56
Chapter 3: The 5' and 3' boundaries of the unmethylated TMS1 CpG island function as enhancer blockers...................................................................... 72
Figures ....................................................................... 81
Chapter 4: Long-term stability of maintenance of a poised epigenetic state after transient exposure to 5-aza-2'-deoxycytidine correlates with occupancy by RNA polymerase II …………..……82
Figures ....................................................................... 107
Chapter 5: Determinants of genome-wide remethylation kinetics after transient exposure to 5-azaCdR .................................................................................. 125
Figures ....................................................................... 144
Chapter 6: Discussion .................................................... 152
Figures ........................................................................ 177
References.................................................................... 182
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