Epigenetics

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Lab Head: Professor Emma Whitelaw

Welcome to the Epigenetics Laboratory. We are a molecular genetics lab, interested in mammalian genes where expression is regulated by epigenetic modification. Characteristics like physical appearance and personality traits are commonly considered to be the result of interactions between genetic and environmental factors alone. However genetically identical individuals raised in similar environments, for example identical twins, show wide variation in phenotype. Some of these variations may be the result of epigenetic differences between these individuals. Epigenetics is the study of mechanisms which modify DNA structure, and thus change gene expression, without influencing the DNA base sequence. Epigenetic modifications such as cytosine methylation and chromatin condensation have been implicated in many important physiological phenomena, from control of cell differentiation to schizophrenia. Our lab is particularly interested in the molecular mechanisms of epigenetic modification and how these mechanisms influence phenotype.

PROJECTS

Epigenetic modification and inheritance of endogenous genes in mice
Coat colour in mice is a characteristic which can be influenced by epigenetic effects. The agouti viable yellow (Avy) allele contains a section of inserted retroviral sequence which usually results in constitutive expression of the agouti protein, producing mice which are butter yellow. However this inserted sequence can be subject to DNA methylation, silencing the allele to varying extents and producing widely varyiable coat colour among genetically identical (isogenic) mice. Surprisingly, this epigenetic modification in Avy can be inherited through the germline, so that a yellow female produces more yellow offspring than a mottled female, even though the parents are genetically identical (Morgan et al, 1999). This is the first convincing evidence that epigenetic modifications can be inherited in mammals. The work has stimulated much interest from both the scientific and lay communities and has been reviewed in Science, Nature Genetics and New Scientist, as well as being the subject of a number of newspaper articles.

These mice are all genetically identical and raised in very similar environments, yet varying levels of epigenetic modification of their Avy allele produces this striking range in coat colour

We have now made similar observations at another endogenous gene, Axin fused , which also appears to be controlled by epigenetic modification (Rakyan et al, 2003). We are investigating the role of co-suppression and RNA interference (RNAi) in the initial establishment of these epigenetic states.

A mutation in Axin, called Axin-fused, produces mice with kinky tails! The degree of kinkiness however, varies wildly among isogenic, Axin-fused littermates. This is another gene which is controlled epigentically

Epigenetic regulation of transgene expression in mice
Transgenes are usually inserted into host DNA in large, head-to-tail arrays rather than as single copies. One might expect that higher copy number would result in greater transgene expression, but often the opposite is true. At high copy numbers the cell recognises the gene as 'foreign' and goes about silencing its expression. In some transgenic lines, transgene expression varies between isogenic individuals as a result of varying extents of epigenetic modification. We have observed this variable expressivity in mice carrying a LacZ transgene, and a similar phenomenon using a GFP transgene (Garrick et al, 1998).

These 'red' blood cells all carry a Green Flourescent Protein (GFP) transgene and so they flouresce green. In some cells, the transgene is epigenetically silenced and no GFP expression is observed

The presence of widespread methylation and varying chromatin structure in mammalian cells, as well as the high frequency of retroviral sequence elements, suggests there may be many genes where expression is subject to epigenetic regulation. We have used differential display screening techniques and microarray technology to look for other endogenous genes which are variably expressed in isogenic mice. We have identified one gene which behaves like this in the C57 inbred mouse (Druker et al, 2004). We now call these genes "metastable epialleles" (Rakyan et al, 2002)

Ultimately, of course, we would like to extend this analysis to look for similar phenomena in humans, where epigenetic regulation may contribute to the incomplete penetrance of certain phenotypes. Since humans are essentially an outbred population, it is difficult to study this at the population level. However, monozygotic twins provide an ideal opportunity to search for metastable epialleles. We have recently reported increased methylation at the AXIN1 gene in an MZ twin from a pair discordant for a caudal duplication anomaly (Oates et al, 2006) This case may be paradigmatic for some MZ discordance. Epigenetic control of gene expression could potentially be responsible for much of the complex variation observed in human characteristics.

In collaboration with Professor Nick Martin (QIMR), we will continue to look for transcripts that are differentially expressed between monozygotic twins. We are developing genome-wide methods to look for novel transcripts that are variably expressed between genetically identical mice (littermates of inbred strains) eg differential display, microarrays etc. We will then use these methods with human twin samples. One experiment of this type has been completed (Oates et al, 2006)

Finding the genes involved in establishing and maintaining the epigenetic marks
We have also initiated two related projects, both of which are aimed at determining the molecular mechanisms involved in establishing epigenetic states in gametogenesis and early development in mammals. The first approach is to use random ENU mutagenesis in mice to find modifiers (suppressors and enhancers) of variegation. We have carried out our mutagenesis on a mouse line carrying a GFP transgene which is expressed in erythroid cells. The transgene is expressed in a variegated manner, approximately 50% cells express the transgene. Flow cytometric analysis provides us with a fast and accurate measure of transgene expression and enables us to screen a large numbers of descendents of ENU treated males. We have identified twelve dominant mutations and three recessive mutations which we know are stably inherited. Our initial results from this screen are promising (Blewitt et al, 2005, Chong et al, 2007, Blewitt et al, 2008). In this way we hope to identify novel genes involved in epigenetic modifications. We are now extending this screen to saturation. These mice are available upon request.

We are also using the more traditional approach of determining whether null mutants of candidate genes can alter the establishment of the epigenetic state at epigenetically-sensitive alleles. To this end, we have imported the Dnmt1 (DNA methyltransferase 1) knockout , the mel-18 knockout (from Japan), the Dnmt 3a and 3b knockouts from Professor En Li (Harvard Medical School, Boston) and the Dnmt3L knockout (Dr Hamish Scott,Walter and Eliza Hall Institute, Melbourne). The effects of absence (seen in homozygous KOs) and reduction (seen in heterozygotes) of these proteins on the establishment of epigenetic state at these and other alleles is being studied.

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Staff

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Funding

NHMRC (National Health and Medical Research Foundation)
ARC (Australian Research Council)
ACRF (Australian Cancer Research Foundation)
NCRIS (National Collaboration Research Infrastructure Scheme)

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Key Publications

Garrick, D., Fiering, S., Martin, D. I. & Whitelaw, E. (1998) Repeat-induced gene silencing in mammals. Nature Genetics, 18, 56-59.

Morgan, H. D., Sutherland, H. G., Martin, D. I. & Whitelaw, E. (1999) Epigenetic inheritance at the agouti locus in the mouse. Nature Genetics, 23, 314-8

Whitelaw, E. & Martin, D. I. (2001) Retrotransposons as epigenetic mediators of phenotypic variation in mammals. Nature Genetics, 27(4), 361-5

Rakyan, V. K., Blewitt, M. E., Druker, R., Preis, J. I. & Whitelaw, E. (2002) Metastable epialleles in mammals. Trends in Genetics, 18, 348-51

Rakyan, V., Chong, S., Champ, M.E., Cuthbert, P.C., Morgan, H.D., Luu, K.V.K. and Whitelaw, E. (2003) Transgenerational inheritance of epigenetic state at the murine AxinFu allele occurs following maternal and paternal transmission. Proceedings of the National Academy of Science (USA), 100(5), 2538-4.

Blewitt M, Vickaryous N, Hemley S, Preis J, Ashe A, Bruxner T, Arkell R and Whitelaw E (2005) An ENU screen for genes involved in variegation in the mouse, Proceedings of the National Academy of Science (USA), 102, 7629-34.

Blewitt M, Vickaryous N, Paldi A, Koseki H and Whitelaw E (2006) Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice PLoS. Genetics, 2,e49

Oates NA, van Vliet J, Duffy D, Kroes HY, Martin N, Boomsma DI, Campbell M, Coulthard MG, Whitelaw E, Chong, S (2006) Increased DNA methylation at the AXIN1 gene in an MZ twin from a pair discordant for a caudal duplication anomaly. American Journal of Human Genetics, 79, 155-162

Chong S, Vickaryous N, Ashe A, Zamudio N, Youngson N, Hemley S, Stopka T, Skoultchi A, Matthews J, Scott H, de Kretser D, O'Bryan M, Blewitt M and Whitelaw E (2007). Modifiers of epigenetic reprogramming display paternal effects in the mouse. Nature Genetics, 39, 614-622

Blewitt M, Gendrel A, Pang Z, Sparrow D, Whitelaw N, Craig J, Apedaile A, Hilton D, Dunwoodie S, Brockdorff N, Kay G and Whitelaw E. SmcHD1, a protein containing a structural maintenance of chromosome hinge domain, has a critical role in X inactivation , Nature Genetics, in press

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