Molecular and in silico analysis of chd2 and chd8 de novo mutations in neurodevelopmental disorders

Neurodevelopmental disorders (NDDs) such as intellectual disability and autism spectrum disorder are associated with impaired intellectual and adaptive functioning, deficits in social communication accompanied by the presence of limited interests and repetitive behaviors. A number of genes that have been identified to regulate chromatin remodeling-an active and key epigenetic mechanism of altering the structure and function of chromatin- have been implicated in both disorders. Chromodomain helicase DNA-binding (CHD) proteins are important factors for remodeling chromatin and for gene regulation. CHD2 and CHD8 pathogenic variants have been extensively reported for ID and ASD. The initial goals of this study are to evaluate the effects of CHD2 and CHD8 loss of function and putative pathogenic missense mutations on DNA/chromatin binding dynamics, on protein stability and gene expression, and to assess the effects of mutations in these genes on neuronal morphology and differentiation. CHD2 and CHD8 are critical for methylation and acetylation of chromatin, important for chromatin conformation and resultant gene expression. We performed a review of the literature to identify de novo missense mutations, and checked for prediction of pathogenicity using SIFT, PROVEAN, Mutation taster, and Polyphen. Next, we cloned full length CHD2 and CHD8 cDNAs into expression vectors, and introduced missense mutations by site-directed mutagenesis, followed by transfection into cell lines (HeLa and HEK293). We examined protein expression levels of methyltransferases (SET8, SET7/9, RBBP5, WDR5, ESET) as indicators of chromatin conformation or state) via immunoblotting. This was followed by RT-qPCR and ChIP experiments to evaluate important genes involved in development (including HDAC1/2, histones H3.3A, H3.3B, H3C1). We also generated in silico prediction data for the effects of SNVs on the CHD2/CHD8 protein surface energy, stability, flexibility and binding dynamics using docking and MD simulations analysis. Preliminary findings revealed that these mutations affect the protein levels of active histones H3.3, key methyltransferases such as RBBP5, SET8, SET7/9, ESET, that are required for regulation of numerous developmentally related genes. Preliminary ChIP data suggests that these mutations affect the binding dynamics of the CHD2 and CHD8 proteins. Furthermore, the docking and MD simulations predict changes in binding with their interacting proteins partners, protein stability and flexibility as compared to the control CHD2 and CHD8 proteins. These pathogenic CHD2 and CHD8 mutations affect the expression of active histone H3.3, dysregulate various methyltransferases, and affect binding dynamics of CHD2 and CHD8 proteins. We are now utilizing the CRISPR/Cas9 system to knockout CHD2 and CHD8, and introduce point mutations, in P19 embryonic cells and NPC (Neuronal Progenitor Cells) to get isogenic cell lines for detailed mechanistic studies.