Investigation of a developmentally linked transcriptional gene silencing mechanism involving an antisense RNA

An increasing number of RNA targeted gene repression mechanisms are being discovered operating in mammalian cells. Antisense-RNA mediated silencing of CpG island-associated genes is not limited to imprinting and X inactivation, but can occur at autosomal non-imprinted loci leading to disease. An antisense silencing phenomenon observed in a patient with an inherited form of anaemia was recently recreated in differentiating mouse embryonic stem cells, demonstrating that the silencing and methylation of the oppositely transcribed tissue specific alpha globin CpG island exclusively occurs in the presence of antisense RNA. The aim of this thesis is to use differentiating mouse embryonic stem cells to further characterise this silencing mechanism through analysis of the histone modifications associated with repression of the alpha globin gene upon differentiation, and to further define the developmental period in which repression is established. Also, the requirement for antisense RNA transcription through the alpha globin gene is examined through insertion of a transcriptional terminator to truncate antisense RNA transcription. The gene silencing mechanism is tested with other genes in place of the alpha globin gene to investigate whether repression is limited to the alpha globin gene or is also applicable to other tissue specific or ubiquitously expressed genes. Analysis of the tissue specific gene MYOD and the ubiquitously expressed gene UBC demonstrated that the silencing mechanism could effect other genes in a similar manner, though not all genes were susceptible to repression as the ubiquitously expressed gene ACTB remained unsilenced by antisense RNA expression. These observations establish that this silencing mechanism can play a more general role in repression, thereby suggesting its potential role as a constituent of the multivariate repressive pathways within the mammalian cell.