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Title: Evaluation of targeted methylation at the CDKN2B promoter
Author: Lamadema, Nermina
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2012
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DNA 5- Cytosine methylation is a modification that occurs in mammalian cells predominantly at CpG sequences, through addition of a methyl group to the C5 position of cytosine. It is a commonly held view in the current field of epigenetics that the aberrant methylation patterns observed in gene promoter regions are associated with transcriptional repression in the absence of genetic mutations. This epigenetic phenomenon is frequently reported in malignant haematopoiesis, where genes involved in cell growth and differentiation programmes are shown to be transcriptionally silenced leading to unchecked cellular proliferation. Although strongly implicated in gene shut down, it is still unclear whether promoter region methylation triggers gene repression, or if DNA methylation arises as a consequence of transcriptional gene silencing via some other mechanism. This is in part because no-one has yet managed to dissect in vivo the precise sequence of the events associated with the appearance of aberrant methylation patterns at the promoter region of actively transcribing genes. This thesis documents a research programme aiming to exogenously impose de novo cytosine methylation to specific regions of a relevant endogenous promoter in order to study spatial and temporal interactions between targeted DNA methylation and other components of the epigenetic regulatory network. The gene selected for this study was CDKN2B, a cell cycle regulating tumour suppressor gene strongly implicated in Myelodysplastic Syndrome - MDS and Acute Myeloid Leukaemia - AML. Site-biased zinc finger: DNA methyltransferase fusion proteins were specifically designed with the aim of depositing low and high density de novo methylation at different regions of the CDKN2B promoter, in an attempt to simulate methylation patterns associated with disease and to thus determine their effect on gene expression. We demonstrate here for the first time the establishment of low density de novo methylation at this endogenous locus, characterized by highly specific target site methylation downstream of zinc finger binding sites. Examination of the distribution pattern and the spread of methylation from the seeded methylation hotspots suggest that 5’ CpG positions in flanking sequences remain unmethylated, whilst 3’CpG acquire de novo methylation marks derived from the action of endogenous DNA methyltransferases. The effects of methylation spread over longer time scales on the formation of broader and more uniform methylation patterns are currently being investigated. Our data also shows that once established, the methylation pattern is inherited through successive cell divisions. Gene expression analysis indicates sustained transcriptional activity of the low density methylated promoter despite the presence of these de novo methyl marks. Overall, our results demonstrate that targeted low density methylation to the promoter region of the CDKN2B gene can be established and maintained epigenetically through subsequent cell generations, but has no effect on the transcriptional regulation of the gene. To date we have not been successful in targeting high density methylation to the same region to comprehensively evaluate the bilateral relationship between a densely methylated promoter and its transcriptional activity/regulation. The contribution of the transcription factor VEZF1 in mediating methylation protection at the CDKN2B promoter was also examined, and we showed for the first time that this transcription factor is strongly associated with this promoter in vivo. This may have important implications in our understanding of CDKN2B expression and regulation in the disease process. Related studies also revealed that TET proteins may play a significant role in cell cycle entry, which was another hitherto unknown phenomenon. In conclusion, we feel that the work in this thesis has advanced the field of targeted methylation, the further implementation of which will certainly further our understanding of key epigenetic mechanisms such as the cellular response to de novo methylation and its role in the regulation of an important cell cycle and disease associated gene.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available