The objective of this proposal is to investigate the epigenetic mechanism of genomic imprinting of the imprinted gene Growth Factor Receptor Bound Protein-10 (GRB10) in neuronal versus non-neuronal development. Imprinted genes are unique to mammals and are expressed in a monoallelic, parent-of-origin-specific manner. Dysregulation of imprinted gene expression is associated with neurological, behavioral and growth disorders. GRB10, implicated in growth disorder Silver-Russell syndrome, is uniquely expressed from the paternal allele in neuronal cells and from the maternal allele in non-neuronal cells. Strikingly, the maternal and paternal transcripts initiate from different promoter regions and the nature of this novel switch in neurons is unclear. This proposal aims to investigate how the allelic and isoform switch occurs at the Grb10 locus to enable unique parent-of-origin expression in neurons. We and others have identified binding sites for the critical DNA architectural protein CTCF within the differentially methylated Grb10 imprinting control region (ICR). Our laboratory also described the importance of CTCF in controlling monoallelic Grb10 expression in mouse embryonic stem cells (mESCs).
Aim 1 will test the hypothesis that CTCF is required for coordinating the imprinted and molecular switch in Grb10 expression. The ability of CTCF to catalyze neuronal-specific expression will be assessed by mutating binding sites within the ICR in an in vitro neuronal differentiation model.
Aim 2 will test the hypothesis that tissue-specific enhancers regulate the Grb10 switch in neurons. A putative enhancer will be removed by CRISPR editing in mESCs, and tissue-specific Grb10 expression will be measured in derived neurons. The studies in this proposal use the imprinted gene Grb10 as a model to study the epigenetic and molecular mechanisms that govern tissue-specific gene expression. Given that many genes in the central nervous system are also tissue and allele-specific, discerning the epigenetic factors coordinating Grb10 expression in neurons will elucidate how other complex genes are differentially regulated in neurodevelopment.
The work proposed in this application aims to define the epigenetic mechanisms regulating imprinted gene expression in neurons, and how dysregulation leads to abnormal neurodevelopment. The imprinted gene Grb10 is an excellent model for studying genomic imprinting in the developing central nervous system. My findings can extend to the regulation of other complex genes in the mammalian brain, and may provide insight into the molecular basis of the imprinting disorder, Silver-Russell syndrome.
