Role of germline transcription events in DNA methylation acquisition at imprinted genes

We inherit genes from our fathers and mothers and, for most of our genes, the copies we receive from either parent are equally active. An important exception to this general rule occurs in a process called genomic imprinting, whereby one gene copy is deliberately silenced. These imprinted genes are important in determining how the fetus grows and how infants adapt their physiology to life outside the womb. But the fact that these genes have one copy that is preselected to being silent poses a risk and makes them particularly vulnerable to mutation events, such as occurs in cancer. Imprinted genes behave in this manner because they are marked in different ways in the male and female germ cells. How these genes are so marked is not fully known, and it is important to find out, because if the marking process goes wrong problems in fertility or developmental abnormalities may arise. By analysing a single imprinted gene in some detail, we have discovered an important part of the mechanism in the germ cell marking event. In this research, we wish to understand this mechanism in more detail and we need to show that it may apply generally to imprinted genes. This work will be done in a model system, but it will provide important new insights for human studies.

Genomic imprinting refers to the differential epigenetic marking of genes in the male and female gametes, which results in the silencing of one of the parental alleles in somatic tissues. Imprinted genes perform essential roles in the placenta and fetus to control nutrient provision and growth, and after birth to coordinate physiological adaptations to feeding, independent metabolic control, and also mental function. In addition to the characterised imprinted syndromes, disruption of imprinting occurs widely in cancers, has been described in association with assisted reproductive technologies (ART), and imprinted genes are regarded as important potential targets in the developmental programming of chronic adult disease.
A fundamental but poorly understood mechanism in imprinting is the marking by CpG methylation of imprinting control regions (ICRs) in germ cells, particularly in the female germline, which is where the majority of imprinting is established. Although the protein factors responsible are known to be the de novo DNA methyltransferase Dnmt3a and its cofactor Dnmt3L, the key issue of how these factors select ICRs as targets for methylation is unclear. Recent work suggests involvement of the methylation state of lysine 4 on histone H3 and the patterning of CpG dinucleotides. Working in the mouse Gnas locus, we now have compelling evidence that a transcriptional event is also essential for establishment of germline methylation marks.
In this application, we shall investigate the mechanistic links between transcription and methylation establishment in oocytes. We shall examine their temporal relationship, including the development of a new knock-out in the Gnas locus in which we can delete the essential transcript at various times in germ cell development. We shall look at the histone properties of the sequences to be methylated and their dependence on transcriptional events. We shall investigate the generality of our proposed mechanism for imprinted loci, including the generation of a loss-of-imprinting allele, based on our model, of the Zac1 gene, which is implicated in the imprinted disorder 6q24 transient neonatal diabetes.
Insights gained from this work will be directly relevant to the causes of imprinted gene disorders and global errors in imprinting, and potentially to imprinting defects which may be associated with ART and other aberrant methylation events, such as tumour suppressor genes.