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Title: DNA and chromatin binding by the methyl-CpG-binding protein, MeCP2
Author: Goddard, C.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2003
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Abstract:
In eukaryotes, methylation at CpG dinucleotides causes transcriptional repression. MeCP2 is a good candidate for a transducer of this methylation signal as it is capable of binding to methylated DNA and can repress transcription in vitro. This thesis describes experiments to further investigate the DNA and chromatin binding properties of MeCP2. Chapter 2 describes the subcloning, overexpression and purification of recombinant rat MeCP2 and also the purification of native MeCP2 from rat brain. The similarity in purification protocols indicates that the recombinant protein is a good model for the native protein. Chapter 3 describes in vitro assays for DNA and chromatin binding. The DNA binding assays confirm that MeCP2 binds to both methylated and unmethylated DNA but has a preference for methylated DNA. The chromatin binding assays show that MeCP2 binds chromatin of all lengths in vitro and most evidence indicates that it is capable of displacing histone H1. Crosslinking experiments showed that MeCP2 crosslinks to core histones and maybe also to itself. The electrophoretic mobility shift assay and also the arrangement of endogenous MeCP2 in native chromatin indicate that MeCP2 may exhibit co-operativity. The crosslinking data suggested that co-operativity may be mediated by protein-protein interactions but this was shown not to be the case by gel filtration. Another possibility was that MeCP2 arrays could be protected by higher order folding. Analytical ultracentrifugation and electron microscopic analysis show that MeCP2 does not induce higher order folding itself. It may be that other factors cause folding of inactive chromatin and protect MeCP2 arrays. The in vitro data indicates that MeCP2 acts non-specifically. In the work described in Chapter 4, a chromatin immunoprecipitation (ChIP) assay was used to investigate whether MeCP2 displays specificity in vivo. It was found that MeCP2 binds NSE, Mash2, H19 and HPRT, but not GAPDH in both female rat brain and female rat liver nuclei, and to ADH in female rat brain but not female rat liver nuclei.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.599452  DOI: Not available
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