Dr Richard Meehan: Chromosomes & Gene Expression
Epigenetic Mechanisms in Development and Disease
Our Work
Xenopus Laevis System
Over the past decade, my research interests have become centred on the role of DNA methyltransferases and methylated DNA binding proteins in development and latterly in disease. As an independent investigator, I decided to use Xenopus laevis as a model organism.
Mammalian System’s
In addition, I have had collaborations determining (1) the global pattern of DNA methylation in normal and cloned sheep embryos (L. Young, A. Bird and I. Wilmut) and (2) The basis of HP1alpha binding to chromatin (S. Pennings). We currently use mouse and human cell line models in our analysis of epigenetic pathways and components.
Conclusion
In general, we have been able to show that the molecular mechanisms of repression by DNA methylation are highly conserved in vertebrates, but that the application of epigenetic mechanisms is 'plastic'. Mice utilize DNA methylation for regulatory pathways (imprinting and X-inactivation) that are not present in frogs. Equally, frogs utilize xDnmt1 for transcription silencing prior to MBT, a phenomenon not observed in mice. However, the phenotypes of resulting from Dnmt1 inactivation are very similar in both systems (an embryonic lethal phenotype that includes activation of p53 dependent apoptosis). What is required in the future is the identification of how epigenetic regulatory mechanisms can be translated in different developmental contexts and systems.
Current Research
Our current research program is focused on determining if the regulatory pathways (transcriptional regulation and activation of apoptosis) that we have identified for xDnmt and MeCPs in Xenopus laevis are applicable in mammalian systems. We primarily use somatic cells and embryonic stem cells (wild type and mutant) in conjunction with genome wide arraying approaches to study these processes in the context of development and cancer.
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Our Work (this page)
An advantage of this system is that it permits both developmental and biochemical analysis of whole embryos that can develop to tadpole stage in less than 3 days in tap water. It is comparatively easy and rapid to generate embryo mutants that are deficient in epigenetic components during development through the use of morpholino (MO) injection. Each MO sequence (~25 nt) is designed to block the transcriptional start site of the targeted gene. In our hands there is a strong correlation between the ability of a MO to block translation in vitro and reduction of protein expression in vivo. If elimination of specific functions by MO microinjection into 1 and 2 cell stage embryos results in perturbation of the developmental program, then this is reflected in the phenotype of a 3-day old embryo.
The specificity of the resulting phenotype can be assessed by micro-injection of mRNA (cross-species or modified) that does not bind the MO and rescues the mutant phenotype. In addition, there are a number of developmental and molecular landmarks that we have developed and these can be assessed to determine if specific molecular pathways are altered as a result of MO mediated depletion xenbase. A disadvantage is that it is difficult to do genetics and that it does not have such a well developed genome annotation or reagents. From the prospective of DNA methylation, Xenopus laevis (unlike mammals) does not have imprinted genes or morphologically distinguishable sex chromosomes. So it was of interest to determine the phenotype of embryos depleted of the maintenance methyltransferase xDnmt1.