Epigenetic modifiers of transgene silencing in the mouse

Daniel Morgan (2008). Epigenetic modifiers of transgene silencing in the mouse PhD Thesis, School of Medicine, The University of Queensland.

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Author Daniel Morgan
Thesis Title Epigenetic modifiers of transgene silencing in the mouse
School, Centre or Institute School of Medicine
Institution The University of Queensland
Publication date 2008-11
Thesis type PhD Thesis
Supervisor Professor Emma Whitelaw
Dr Suyinn Chong
Total pages 176
Total colour pages 27
Total black and white pages 149
Subjects 11 Medical and Health Sciences
Abstract/Summary It is well established that epigenetic modifications to the genome are crucial for the exquisite control of gene expression required for an organism to develop and differentiate. These modifications are maintained through mitotic rounds of cell division, but must be cleared and reset through meiosis in order for the cells of the early embryo to achieve totipotency. Although we know these mechanisms exist, the rules determining which modifications are established where on the genome and the genes involved in these processes remain poorly characterised. Much of what is known about epigenetic processes has come from studies in non-mammalian organisms, such as Drosophila. However, in our laboratory we have developed a mammalian system for identifying modifiers of epigenetic gene silencing. An ENU mutagenesis screen is being carried out using an inbred mouse line carrying a GFP transgene, with an erythroid-specific promoter, that is particularly sensitive to changes in epigenetic modifications. Currently, 14 mutant lines that display a heritable shift in GFP expression have been recovered. These have been termed Modifiers of Murine Metastable Epialleles (Mommes). When I began my PhD in 2005, we had not identified any of the mutations underlying the phenotypes observed. To confirm the efficacy of the screen, I have tested the effect of heterozygosity for null alleles of two known epigenetic modifiers, Dnmt3a and Dnmt3b, on expression of the GFP transgene. Heterozygosity for the Dnmt3b knockout allele does shift expression while heterozygosity for the Dnmt3a knockout allele does not. This highlights the limitations of the screen. With this particular screen we will only detect modifiers that are expressed during haematopoiesis in the bone marrow. I have also worked on MommeD5. MommeD5 is a semi-dominant, homozygous embryonic lethal mutation that acts as an enhancer of variegation. I have found that the MommeD5 allele carries a 7 bp deletion in the major histone deacetylase, Histone deacetylase 1 (Hdac1), and this significantly alters the C-terminus of the mutant protein. The finding of Hdac1 attests to the screen design. The MommeD5 homozygous mutants die at approximately the same time as the published knockout of Hdac1 and the heterozygous mutants show increased levels of Hdac2 and acetylated histone H3, as reported in Hdac1-deficient embryonic stem cells. In addition, I have studied the effect of heterozygosity for each of the mutations on the phenotype of the mouse. In general, heterozygous Momme mutants are viable and fertile, but show subtle abnormal phenotypes. However, in the case of MommeD5 none were observed and this may relate to the compensatory upregulation of other histone deacetylases. In the case of Dnmt3a and Dnmt3b a sex ratio distortion is seen in the colonies, with less males seen than expected. Also, Dnmt3a heterozygous mutant males that inherited the mutant allele from the dam are smaller and show an increased range of body weights compared to their wild-type male littermates. This may be an example of intangible variation, i.e. phenotypic variation observed in isogenic individuals raised in standardised environments. These results suggest that epigenetic mechanisms have a role in intangible variation, also known as developmental noise. Despite the fact that it is now acknowledged by many that stochastic events occur at the level of the cell, the idea that it can happen at the level of the whole organism is rarely considered.
Keyword DNA methyltransferase, epigenetic gene silencing, epigenetic modifiers, heterozygosity, histone deacetylase, intangible variation, sex ratio distortion
Additional Notes * The page numbers that need to be printed in COLOUR, according to the pagination in Adobe Reader, are: 1, 40, 46, 49, 51, 52, 80, 81, 91, 94, 98, 100, 102, 105, 113, 115, 116, 118, 119, 121, 122, 123, 125, 127, 129, 139, 140 (27 colour pages total). * The page number that needs to be printed in LANDSCAPE, according to the pagination in Adobe Reader, is: 174 (1 landscape page total)

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Created: Thu, 10 Sep 2009, 23:51:33 EST by Mr Daniel Morgan on behalf of Library - Information Access Service