A Systems Approach to Understanding Methylation Programming in Oocytes and its Consequences in Development

The genetic information we receive from our parents is not only the DNA sequence of our genes. Genes are also marked in different ways in the egg and sperm from which we arise. These marks are referred to as 'epigenetic' marks, and one of the major types of epigenetic marking is a chemical tag on certain sites in the DNA called DNA methylation. DNA methylation marks are important because they can change the activity of the genes that they tag; dense DNA methylation on genes usually causes them to be shut down. In addition, DNA methylation marks can be faithfully copied on the DNA as cells divide, so that they can provide a form of memory on the gene to reflect earlier decisions about how that gene should be active. DNA methylation is a normal mechanism by which the body controls genes throughout our development and in response to changes in our environment or diet. However, because DNA methylation marks are easier to change than the underlying DNA sequence, DNA methylation patterns may also be more vulnerable to adverse environmental conditions and abnormal DNA methylation is commonly seen in diseases such as cancer.

In this research, we aim to understand the processes that control which of our genes are marked by DNA methylation in eggs and how these marks are transmitted after the egg is fertilised by the sperm. Eggs and sperm carry different patterns of DNA methylation, so parents can transmit different epigenetic information to their offspring and these could reflect environmental factors in their own history. DNA methylation undergoes particularly dramatic changes in the early embryo, and these transitions may be important for controlling how the embryo divides and the body plan is established and different tissues arise. We are particularly interested to discover whether the normal patterns of DNA methylation in the egg and early embryo are changed as a consequence of altered diet or processes involved in assisted reproduction techniques, such as superovulation. Poor diet - which might include protein deficiency in developing countries and fat-rich diets in the Western world - in mothers before or around the time of conception is known to increase the risk of developing diseases such as diabetes and cardiovascular disorders in later life. Knowing whether DNA methylation changes occur and why would help provide informed advice about nutrition and dietary supplements to women contemplating pregnancy. Assisted reproduction techniques, such as in vitro fertilisation, often involve harvesting eggs and culturing embryos during the time at which DNA methylation patterns are undergoing most rapid change and may be most susceptible. Therefore, it is important to know whether these techniques modify DNA methylation marks to be able to develop further improved fertility treatments and minimise the risk of creating epigenetic errors.

We aim to provide an overarching account of DNA methylation in oocytes, its consequences and vulnerabilities. The work will be conducted mostly in the mouse as the model of choice for understanding epigenetic processes in gametes and preimplantation embryos. The connections we shall explore between transcription and methylation are based on known properties of Dnmt3a and Dnmt3L - which constitute the de novo methylation complex active in oocytes and which is believed to sense histone modifications - and histone modifying factors with evidence for transcription-coupled activity. Of particular interest to us is methylation of H3K4 and H3K36, which can be modified in response to transcription. We shall use conditional knock-outs for factors of the Kdm1 and Kdm5 families, which regulate H3K4 methylation, and we shall mutate Dnmt3a so that it is no longer binds the transcription-coupled mark H3K36me3. We shall map transcription units and promoter activity and how they change during oocyte growth, and correlate this with timing of DNA methylation in specific genes. We shall ablate specific transcription factors expressed in oocytes, and investigate whether this leads to predicted changes in gene body and intragenic CpG island methylation. This will be done with conditional alleles for transcription factors with known roles in oogenesis, but we shall also identify additional transcription factors with effects during the de novo methylation phase of oocyte growth by informatics analysis of expression and transcription start site datasets. We shall use our highly sensitive DNA methylation profiling (RRBS) as an endpoint in many of these mechanistic studies. We shall use RRBS (and other techniques we might develop) to profile changes in methylation in oocytes and embryos in response to manipulations in maternal diet and superovulation. We shall also profile DNA methylation in human preimplantation embryos to identify conserved sites and properties of DNA methylation.

The work proposed in this application is mostly of a fundamental nature in a model organism aimed at understanding epigenetic processes in germ cells and preimplantation embryos. It seeks to provide an overarching account of how and where DNA methylation is established in female gametes, what the consequences of this mark is for how genes are expressed in the oocyte and how gene activity is regulated in the early embryo. Correct epigenetic programming in gametes and embryos has a lifelong impact on development and health, because some epigenetic marks once established or lost cannot be corrected at later times in development. Although the work will be carried out mostly in a genetically tractable and informative model organism, we shall also conduct comparative analysis in human preimplantation embryos to demonstrate whether insights gained in the model organism are relevant to human embryogenesis. As well as providing fundamental knowledge, we expect that the research could help identify risks in assisted reproduction methods, enhance knowledge in stem cell research, and identify whether epigenetic marks can be destabilised by poor maternal nutrition and could directly contribute to adverse health outcomes in later life. Therefore, the knowledge acquired from this research will likely to have major impacts in fundamental epigenetics and germ cell biology research, but also potential translational impacts in human nutrition, assisted reproduction and stem cell research.

We also expect an impact of our research in the technical advances we shall continue to make in developing methods for epigenetic profiling in small numbers of cells. Such advances could have widespread application in biomedical research. We have already been approached by several groups (from both academic and pharmaceutical sectors) from research areas unrelated to our field with an interest in applying our methods to their own biomedical questions. Therefore, we anticipate significant impact in training and knowledge exchange also at a technical level.