CTCF and Epigenetic Regulation of the H19/Igf2 Locus
Sammanfattning: An overall coordination between the expressions of genes is required for the proper development of an individual. Although most genes are expressed from both the constituent alleles of the genome, a small subset of autosomal genes are preferentially expressed from only one of the parental alleles, a phenomenon known as genomic imprinting. The imprinted H19 and Igf2 genes are considered paradigms of genomic imprinting as their monoallelic expression pattern is coordinated by a short stretch of sequence located upstream of H19, known as the imprinting control region (ICR). This region shows differential methylation, with hypermethylation specifically on the paternal allele. On the maternal allele this region acts as an insulator and harbours maternal specific hypersensitive sites. The hypersensitive sites were identified as the result of association of the vertebrate insulator protein CTCF with the region. This association was investigated in both an in vitro episomal system and in an in vivo mouse model system by mutating the CTCF target sites at the H19 ICR. The importance of CTCF for the insulator property of the region was confirmed in both instances. In the mouse model, the disruption of the binding was also observed to affect the methylation profile of the ICR, which ultimately resulted in the de-repression of the maternal Igf2 allele.The relevance of multiple CTCF target sites in higher vertebrates for the proper insulator function was investigated using another knock-in mouse model with mutation at a single CTCF target site in the H19 ICR. The investigation confirmed the cooperation between the target sites for the establishment of a functional insulator on the maternal allele. Target sites in the ICR were also analysed for their differential binding affinity for the CTCF protein.The utilisation of the CTCF target sites was examined in different human tumours and cell lines. Methylation analysis conveyed a lack of correlation between the loss of insulator function and methylation status of the ICR with the loss of imprinting (LOI) of IGF2. Investigations also identified a novel mechanism, which neutralised the chromatin insulator function of the H19 ICR without affecting its chromatin conformation. This principle might also help in explaining the loss of IGF2 imprinting observed in some instances.In conclusion, this thesis confirms the importance of CTCF in the formation of an epigenetically regulated chromatin insulator at the ICR, which in turn controls the expression pattern of H19 and Igf2. The studies also confirm the role of CTCF in the maintenance of the methylation profile of the region. Investigations into the loss of IGF2 imprinting in human cancer indicate the involvement of other novel mechanisms besides CTCF in the regulation of insulator function.
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