Variable DNA methylation in the mouse genome: repetitive elements, unique regions and functional implications
Approximately 10% of the mouse genome consists of endogenous retroviruses (ERVs), a class of retrotransposon, which includes the murine-specific intracisternal A particle (IAP) elements. To prevent their mobilisation and suppress the function of the TE- encoded regulatory sequences, TEs are generally targeted by repressive histone modifications and DNA methylation. The Avy allele results from an IAP insertion into the endogenous Agouti gene locus which is variably silenced between isogenic individuals. Variable DNA methylation at the IAP inversely correlates with ectopic expression of Agouti, resulting in a spectrum of coat colours between littermates. This, and other similarly behaving loci, are termed metastable epialleles, or variably methylated IAPs (VM-IAPs), and are widely cited as the best examples of mammalian epigenetic inheritance. A recent screen in the C57BL/6J mouse strain identified over 100 candidate VM-IAPs providing novel insights into metastable epialleles. In this thesis, I revisit the phenotypic and epigenetic heritability associated with the classic metastable epiallele Avy and further characterise novel variably methylated loci in the mouse genome through transcriptional and cross-strain comparative analyses. First, breeding-intensive experiments of the Avy allele in two genetic backgrounds (C57BL/6J and C3H/HeJ) show that coat colour is weakly heritable upon maternal transmission, while DNA methylation is only weakly heritable upon paternal transmission. This implies that coat colour cannot be used as a phenotypic read-out of an epigenetic state as previously reported, since DNA methylation and coat colour appear to be uncoupled at this locus. Additionally, both coat colour and DNA methylation at Avy are subject to genetic background effects. Second, thorough experimental validations of candidate variably methylated regions result in the identification of a novel class of VM-IAP with tissue-specific variability and the potential to affect neighbouring gene expression. VM-IAPs with constitutive methylation levels across tissues (cVM-IAPs) are full-length IAP LTR1 elements or solo LTR2s and are enriched for certain sequences, although sequence alone is not predictive of variable methylation at IAP elements. The IAP elements which are variably methylated on a C57BL/6J genetic background are highly polymorphic between inbred mouse strains and are differentially epigenetically regulated between strains. Finally, I show that variable methylation exists in non-repetitive regions which can affect gene expression and is also sensitive to genetic background. Overall, this body of work indicates that: variable methylation outside of evolutionarily young ERVs is rare; variable methylation can, albeit rarely, affect neighbouring gene expression and, in all contexts studied so far, is subject to strain-specific regulation suggesting that genetic background-specific modifiers contribute to the establishment of metastable epialleles within and across generations. Furthermore, my data indicates that our previous understanding of Avy is open to alternative interpretations.
metastable epiallele, endogenous retrovirus, intracisternal A particle, Agouti viable yellow, epigenetic inheritance, DNA methylation
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