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Title: Chromatin organisation at differentially methylated regions of imprinted mouse loci
Author: Gregory, R. I.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2001
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Genomic imprinting is an epigenetic process by which certain genes are expressed in a parental-origin dependent manner. Most imprinted loci have regulatory sequence elements referred to as 'differentially methylated regions' (DMRs) that are methylated only on one of the two parental chromosomes. Methylation differences at certain DMRs are established in the germline and persist throughout development. Allele-specific constitutive nuclease hypersensitive sites were detected at the constitutive DMRs of the imprinted H19 and U2af1-rs1 genes suggesting that non-histone proteins are associated with the unmethylated allele. It was found that the parental alleles of U2af1-rs1 display differential sensitivity to nucleases throughout the DMR suggesting the involvement of nucleosomal modifications. To address the latter, I developed a novel allele-specific chromatin immunoprecipitation (ChIP) assay to investigate the involvement of histone acetylation. Antibodies to specific acetylated lysine residues on core histones H3 and H4 revealed complex, allele-specific, patterns of acetylation throughout U2af1-rs1 and also at the constitutive DMR of Snrpn. At both DMRs, the parental alleles had high levels of H4 acetylation except at lysine 5, which was underacetylated when inherited from the mother. H3 was underacetylated on the maternal allele at all lysines analysed. A genetic approach was used to demonstrate that CpG methylation is linked to deacetylation of H3, but not of H4, at U2af1-rs1. This deacetylation of H3 may involve 'methyl-CpG-binding-domain' proteins, such as MeCP2, which was found to be associated with the methylated maternal allele. To investigate the regulation of H3 and H4 acetylation, ChIP was performed on control and Trichostatin-A (TSA)-treated cells. Chromatin compaction at U2af1-rs1 may be dependent on the acetylation status of individual lysine residues of histones H3 and H4.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available