This proposal dissects the roles of long non-coding RNAs (lncRNAs) in the epigenetic phenomenon of genomic imprinting. Imprinted gene expression is enriched in the brain and ultimately affects neuron signaling, differentiation, and survival. Notably, lncRNAs originating from differentially methylated regions are functionally required for proper monoallelic expression of several imprinted genes. Detailed investigation on lncRNAs in the establishment and maintenance of genomic imprinting will have significant implications for imprinted disorders, such as Angelman syndrome, which results from loss of maternal Ube3a. The paternal copy of Ube3a is intact in patients, but silenced by a neuron-specific repressive lncRNA called Ube3a-ATS. While the mechanism of action is unknown, it has been demonstrated that inhibition of Ube3a-ATS partially restores Ube3a in Angelman syndrome. With more than 100 imprinted protein-coding genes identified to date, a comprehensive study of lncRNAs at imprinted loci will increase our mechanistic understanding of lncRNAs-mediated gene regulation. Here, in Specific Aim 1, an in vitro neuron differentiation system will be established that permits allele-specific transcriptome characterization. Embryonic stem cells from reciprocal crosses of divergent mouse subspecies will be harvested and differentiated to the neuronal lineage.
In Specific Aim 2, this unique model system will be used to identify novel lncRNAs at imprinted loci. Transcriptome sequencing and alignment of allele-specific single nucleotide polymorphisms will be used to assign allelic biases in expression.
In Specific Aim 3, the molecular mechanisms by which Ube3a-ATS regulates imprinted expression of Ube3a will be determined. CRISPR/Cas9-targeting strategies will be used to functionally dissect the activity of Ube3a-ATS in the epigenetic and transcriptional control of Ube3a in neurons. The long-term goals of this research are to leverage the fundamental activities of imprinted lncRNAs to therapeutically modulate imprinted genes in neurodevelopmental disorders.
This proposal seeks to characterize the molecular mechanisms by which long non-coding RNAs regulate imprinted gene expression, including detailed studies on how the causative gene in Angelman syndrome is controlled in neurons. Many genes are imprinted in the brain, and a complete understanding of the fundamental process by which genomic imprinting is established will have implications for neurobiology and application to neurological diseases.