Functional annotation of orphan CpG island transcripts

CpG islands (CGIs) in the vertebrate genome are CpG-rich and GC-rich regions of approximately 1kb in size and, in contrast to the bulk genome, mostly lack DNA methylation. In mice and humans, CGIs co-localise with the majority of the transcription start sites of annotated genes. However, a significant fraction of CGIs maps to intergenic or intragenic regions and due to their lack of association with an annotated transcript are termed "orphan CGIs". Their frequent conservation indicates that they play an important role in genome regulation and they have been postulated to act as promoters for non-protein-coding RNAs (ncRNAs). With the emergence of unbiased transcriptome analysis tools such as whole transcriptome shotgun sequencing (RNA-Seq) it has been uncovered that the majority of the non-ribosomal transcriptome consists of ncRNAs. Thousands of ncRNAs have been described in the recent years, but most of them lack further experimental characterization and functional analysis. The few described functional ncRNAs act in numerous cellular pathways, with a majority playing crucial roles in regulating gene expression. In the first phase of the proposed research project I will generate a comprehensive overview of the orphan CGI transcriptome. A murine neuronal embryonic stem cell differentiation system representing various stages of development will be used to generate transcriptome sets by strand-specific RNA-Seq. The transcription start sites of novel transcripts will be compared with orphan CGIs to identify transcripts originating from an orphan CGI. Those transcripts will be further characterized, applying bioinformatic, biochemical and molecular biological tools. Functional analysis of a candidate set will involve genetic knockouts of the orphan CGI promoters and knockdowns of the novel transcripts. The effects of those genetic manipulations will be initially analysed using the neuronal embryonic stem cell differentiation system and finally in vivo by generating knockout and knockdown animals. Phenotypic analysis will involve genome-wide expression analysis and characterization of associated epigenetic modifications such as DNA methylation and chromatin modifications to elucidate the functional roles played by orphan CGI transcripts.

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