Mechanisms of epigenetic gene silencing caused by the long non-coding Kcnq1ot1 RNA - a key regulator of BWS

All the genes in the human genome are now known, but it is not known in detail how they are regulated in normal development and misregulated in diseases. The fact that the number of genes a fruitfly has is quite similar to the number of genes of a human, has given rise to the idea that vast areas in the genome that do not contain genes have this regulatory function. In particular so called non-coding RNAs outside of genes have been implicated in genome regulation and disease. We study a particular non-coding RNA which when it is aberrantly made leads to the Beckwith-Wiedemann syndrome in which babies are born that are very large and can develop cancer. Our studies will help to understand better how the disease is caused, and will therefore help the families with children who have the disease.

Noncoding RNAs are increasingly implicated in key regulatory functions in mammalian genome biology and disease. Many ncRNAs are involved in gene silencing, but the precise mechanisms of silencing and the links to human diseases are not well understood. We are working on the long, nuclear, and imprinted ncRNA Kcnq1ot1 which silences in cis a large cluster of imprinted genes amongst which are key regulators of placental function and fetal growth. Loss of imprinting of Kcnq1ot1 is a frequent cause of the fetal overgrowth and cancer syndrome, Beckwith-Wiedemann syndrome. We have recently found that gene silencing mediated by Kcnq1ot1 shares remarkable parallels with X chromosome inactivation mediated by the ncRNA Xist. This includes the fact that the Kcnq1ot1 RNA appears to establish a physical structure in the nucleus which may be a silencing compartment, and the targeting by the ncRNA of repressive histone modifications to the cluster. We have also begun to establish a Kcnq1ot1 alelle which can be activated or silenced in mice with the Tet system, and fully quantitative conditions for 3C (chromosome conformation capture) of the locus using the Sequenom masspec system. The overall aim of this proposal is to understand how expression and physical organisation of the Kcnq1ot1 RNA together with its target locus results in epigenetic gene silencing in a large gene cluster. The specific objectives are 1.to determine in preimplantation embryos the precise epigenetic dynamics leading from Kcnq1ot1 expression and coating to gene silencing, 2. to construct in mice a tetracycline inducible Kcnq1ot1 allele and to test at which stages of development and in which lineages expression of the Kcnq1ot1 RNA can induce and maintain epigenetic gene silencing in cis, 3. to analyse higher order chromatin structure of the Kcnq1ot1 cluster by quantitative allele-specific 3C with the Sequenom system, and to determine its dependence on the ncRNA using the tetracycline regulatable Kcnq1ot1 allele and, 4. to analyse imprinted gene expression and epigenetic marks in the cluster in a conditional Dicer knockout deleted in embryonic or extraembryonic tissues, respectively.
This work will establish comprehensively the physical organisation of the Kcnq1ot1 RNA ?silencing compartment? and how this, together with developmental factors, may lead to epigenetic gene silencing. This may result in novel biomarkers which we will translate to BWS patients through our established clinical collaboration. Significant new insights may also emerge into why the risk of BWS (and other epigenetic disorders) is increased by ART in humans.