May 2009

Document Type


Degree Name



Dept. of Molecular and Medical Genetics


Oregon Health & Science University


Aberrant epigenetic silencing is a prevalent mechanism of tumor suppressor gene inactivation that promotes cancer initiation and progression. Although repressive epigenetic modifications have been identified that are specific to tumor suppressor genes after aberrant silencing occurs, the factors that initiate these changes are not clearly understood. The aim of this thesis research was to examine epigenetic changes during the dynamic transition from active to inactive transcription states during epigenetic silencing; this strategy is in contrast to measuring epigenetic modifications after transcriptional silencing has stabilized. To achieve this goal I designed a system to directly test the hypothesis that transient reductions in gene activation are an initiating factor of aberrant epigenetic silencing. Aberrant silencing of an experimental transgene was induced by reversibly inhibiting transcriptional activation, and the frequency of silencing increased with longer durations of reduced transcriptional activation. Thus, these observations confirmed the hypothesis, and epigenetic modification associated with experimentally induced silencing were the same as those measured at silenced tumor suppressor genes in cancer cells. These data demonstrated that results from the experimentally induced silencing are relevant to epigenetic processes that contribute to tumorigenesis. In the experimental system, the initiation of silencing was dependent on the activity of class I/II histone deacetylases, but not DNA methylation. This demonstrated the value of this system of induced silencing as a way to examine specific molecular requirements for initiation and stabilization of aberrant epigenetic silencing. Aberrant silencing of endogenous Aprt in mouse cells was also examined to identify characteristics of distinct silencing pathways and requirements for reactivation. In D7 cells, Aprt silencing was correlated with a bivalent histone modification pattern, enrichment of methylation at H3K4 and H3K9, which did not require DNA methylation of the Aprt promoter. In contrast, Aprt silencing in D3 and D3S1 cells correlated with hypermethylation of the promoter region DNA, loss of methyl-H3K4, and increased methyl-H3K9. Comparison between D3 and D3S1 cells demonstrated that increased DNA methylation of the promoter stabilized transcriptional silencing and required demethylation of promoter DNA for reactivation. However, the silenced Aprt promoter in D3 cells that was only partially methylated, did not require a loss of DNA methylation for reactivation. Maintaining reactivated expression by selection did promote eventual removal of DNA methylation from Aprt, but re-silencing of Aprt still occurred at a high frequency. Therefore, some epigenetic modification other than DNA methylation is responsible for the instability of reactivated expression of silenced alleles. In summary, these results identify transient reduction of gene activation as an early initiator of aberrant epigenetic silencing. This has allowed us to distinguish between early and late events during the silencing process and assign specific functions to individual repressive modifications. Additionally, these results demonstrate the existence of a stably inherited epigenetic state that does not require DNA methylation and prevents stable reactivation of previously silenced alleles. Finally, the experimental system designed for these experiments provides the means to examine steps in the silencing process upstream or independent of DNA methylation. This is a critical experimental tool for the study of both normal and aberrant epigenetic processes.




School of Medicine



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