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Title: Identification of a silencing element at the imprinted H19 locus using mouse and Drosophila transgenes
Author: Brenton, J. D.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2001
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Imprinting of H19 transgenes and the wild type locus is dependent on a CpG-rich differentially methylated region (DMR) that lies between -2 to -4kb upstream of the promoter. Embryos deficient for the paternal DMR, or that have an unmethylated DMR secondary to targeted mutation of DNA (cytosine-5-)-methyltransferase 1, show reactivation of the paternal H19 allele. I have used mini transgenes as a stringent test for sufficiency of imprinting elements. I demonstrate here that H19-lacZ and H19-PLAP reporter transgenes that do not contain the H19 coding region but have -3.8 or -10.5 kb of upstream sequence undergo imprinting. These transgenes were repressed and hypermethylated after paternal inheritance at multiple integration sites. In order to test whether the upstream region might have a conserved epigenetic function, an H19-lacZ transgene with -3.8 kb of sequence was introduced into Drosophila. This demonstrated strong bidirectional silencing activity despite the absence of methylation. Using a p-element deletion screen the silencer was localized to a 1.1 kb region within the 3' region of the DMR. As this suggested a mechanistic link between silencing in Drosophila and imprinting in mice, I deleted the same region in H19-PLAP transgenes with -10.5 kb of upstream sequence. In contrast to the original lines, transgenes that lacked the 1.1 kb silencer element were not repressed on paternal transmission. Strikingly however, the paternal methylation of the remaining upstream region remained and was appropriately methylated and demethylated on successive passage through the paternal and maternal germ line. This study demonstrated that the 1.1 kb control element identified in Drosophila is required to silence H19 transgenes. This suggests that a conserved silencing mechanism may suppress transcription at the H19 locus. Sequence within the DMR that are upstream of the silencer may be responsible for other functions which involve differential methylation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available