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The CHD8 regulatory network in the developing brain is enriched for ASD risk genes
Gene discovery in autism spectrum disorders by exome sequencing coupled with co-expression analyses have revealed chromatin modification as a significant point of biological convergence. ASD-associated chromatin modifiers and other regulatory genes may directly control other ASD-associated genes and thereby organize pathways and networks that, when perturbed, lead to ASD. To investigate this mechanism, we have undertaken studies to map the regulatory networks of ASD genes during brain development. Here we focus on Chromodomain Helicase DNA binding protein 8 (CHD8), an ATP-dependent chromatin remodeler that exhibits a high number of de novo loss of function mutations in unrelated individuals with ASD, strongly supporting a role for this gene in autism pathology. The loss of CHD8 function may result in aberrant expression of downstream target genes required for proper brain patterning, cell fate determination, or other critical neurodevelopmental processes. Characterizing the CHD8 regulatory network in human brain will therefore provide biological insights into the developmental etiology of autism.
Objectives:
To elucidate the CHD8 regulatory network via a) chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) to identify CHD8 target genes in developing brain b) generation of CHD8 regulatory maps by the co-localization of CHD8 binding with active or repressive chromatin marks; c) identification of global changes in gene expression and chromatin marks due to CHD8 depletion using shRNA; and d) examination of the CHD8 target gene networks to determine if previously identified ASD genes are significantly enriched.
Methods:
We have mapped CHD8 binding sites in developing brain and human cell lines using ChIP-seq, and have cross-referenced these sites with maps of specific active or repressive histone modifications to identify CHD8-targeted genes and regulatory elements. The genes targeted by CHD8 have been assessed for enrichment of ASD genes by permutation testing. We will evaluate the biological relevance of these binding events using CHD8-specific knockdown using shRNAs followed by global analysis of the transcriptome and histone regulome.
Results:
We found that CHD8 gene targets are significantly enriched in ASD genes bearing one or more de novo loss of function mutations, and in genes found in ASD-associated spatio-temporal co-expression networks during human brain development. In developing brain and human cell lines, CHD8 is targeted to promoters and regulatory elements that bear active chromatin signatures, with a strong predilection for active promoters.
Conclusions:
Identification and analysis of biologically relevant CHD8 binding sites allows for the generation of CHD8 (and therefore likely ASD-associated) regulatory networks, and provides a general framework for characterizing other chromatin and transcription regulating ASD genes. By generating and integrating regulatory networks for multiple ASD genes, we will uncover molecular and biological points of convergence in pathways that contribute to ASD pathophysiology.