Context-dependent and shared features of CHD8 activity reveal the mechanism of developmental brain disorder

ABSTRACT Molecular regulation of the genome is the primary mechanism of developmental constraints and disease phenotype. Gene disruptions sculpt human developmental diseases and provide an opportunity to hypothesize a functional role for molecular regulation of genome in cell and tissue. For example, an important paradigm in understanding nervous system development is the mechanism of neurodevelopmental disorders and analyzing the role of risk genes in disease etiology. In this work, I decided to tackle genetic instability in autism spectrum disorder (ASD), employing embryonic stem cell and human neural cell system, previously established at Wernig laboratory. I studied transcription, chromatin structure, and cellular signaling, employing a conditional knockout (KO) model of the causative gene in autism. To model the pathophysiology of autism, I choose to study CHD8, a gene with the highest number of discovered mutations in trio-families (parents and the affected child). My initial characterization of CHD8 knockout revealed a stark difference in apoptosis induction between embryonic stem cells and neurons. Similarly, the analysis of genomic bindings suggested that the interaction of CHD8 with the genome is not conserved between the cell types. In neurons, activating bindings of CHD8 seem to be near the promoters, but in embryonic stem cells, the binding shifts to distal-promoter regions, potentially to the elements of silent enhancers. Analysis of chromatin structure in knockout cells similarly revealed cell-type-specific changes in CHD8 knockout, even at the common targets. In neurons, CHD8 is a strong chromatin activator, but in embryonic stem cells regulates the distant upstream of promoters. A critical observation in embryonic stem cells revealed a group of genes with CHD8 regulated distal promoters distinctly relate to neuronal cell function (e.g., GO:0045211; postsynaptic membrane); however, I did not observe a term for another specialized cell type. These findings proved to me that CHD8 distinctly regulates neuronal genes from the early stages of development. Another insightful result obtained from the chromatin interaction experiment revealed the genomic bindings of CHD8, enriched for the motif of a MAPK/ERK effector molecular-ELK1. The motif is overrepresented at the strong binding sites, but the composite motif for ELK1 and SRF is presented at the weak bindings. This finding shows the relevance of ELK1 to play a biological role at CHD8 binding sites, as the composite motif is a regulatory element by which the serum response factor regulates target genes in partnership with ELK1. The ELK1 binding at these sites is transient and signal-dependent; thus, CHD8 binding should also reflect low affinity and transient interactions at ELK1-SRF motif sites. That is indeed what we observed: the composite motif of SRF-ELK1 enriches at the 'weak' binding sites of CHD8. Finally, the examination of ELK1 knockdown in the context of CHD8 knockout out showed a remarkable cellular phenotype. First, I observed that ELK1 knockdown rescues CHD8 mediated apoptosis in embryonic stem cells. Similar but slightly indirect results revealed a neurogenesis role, as I observed the pro-neural function of ELK1 in wild-type ES cells diminishes in CHD8 knockout cells. This finding shows that cooperativity between ELK1 and CHD8 is molecularly conserved, but it is also cell-type dependent. Altogether, my observations suggest kinase signaling not only activates effector molecules for chromatin binding but also regulates the co-partner chromatin network. Since chromatin factors generally do not have sequence specificity on binding sites, the kinase effector molecular plays a role in guiding the chromatin factor's specificity on the genome. In conclusion, my thesis provides insights into cell type and cell-state-dependent activities of CHD8 within a kinase pathway for gene and chromatin regulation. Additionally, I found that ASD genes, within a functionally related module, significantly overlap with targets of CHD8

Keywords: Stem Cell Biology and Regenerative Medicine.
Advisor: Wernig Marius
Palmer Theo
Wysocka Joanna