Lennox-Gastaut syndrome (LGS) is an early-onset epileptic encephalopathy with seizures that are often refractory to treatment. In recent years, next generation sequencing has revealed several novel genes associated with LGS, however little is known about how pathogenic variation in these genes leads to disease. Improved treatment options are necessary to improve the outcome and quality of life of LGS patients and the first step in finding drug targets will be to better understand the molecular biology of these disrupted genes. Pathogenic variants in the CHD2 gene are associated with epileptic encephalopathy, including several LGS cases. CHD2 is a chromatin remodeling protein associated with sites of active transcription. Our hypothesis is that disruption of CHD2 will lead to decreased and/or aberrant binding of CHD2 to its genomic targets, resulting in misregulation of gene expression. In order to study the function and molecular mechanisms of CHD2 we have disrupted one copy of CHD2 in induced pluripotent stem cells (iPSCs) using the CRISPR/Cas9 system. We differentiated CHD2 heterozygous cell lines (CHD2+/-) into neuronal progenitor cells (NPCs) and cortical excitatory neurons to study the effect of CHD2 disruption in human cell models. To identify genes misregulated upon CHD2 disruption, we will perform mRNA sequencing in CHD2+/- NPCs and wildtype (WT) controls at different points in differentiation from iPSCs to NPCs. To determine genome-wide CHD2 occupancy, we will perform chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) in WT NPCs. We will then correlate the results of CHD2 occupancy with the data on which genes are misregulated upon CHD2 disruption to identify a set of candidate genes that are regulated by CHD2. Secondly, we plan to identify binding partners of CHD2 in NPCs and cortical neurons by performing co-immunoprecipitation of CHD2 followed by mass spectrometry. Our hypothesis is that CHD2 will bind master regulators of neurodevelopment, along with novel proteins, which will illuminate new and possibly unexpected pathways in which CHD2 is involved. Combined, the identification of CHD2 genomic targets, regulated genes, and binding partners in human neuronal cell types will improve our understanding of the biological mechanisms behind CHD2-associated epilepsies and point toward novel drug targets for LGS.
|Effective start/end date||9/1/18 → 8/31/20|
- LGS Foundation (Agmt 11/8/18)
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