Investigating the role of human-specific 3D genome conformation in the context of brain development and disease - PROJECT SUMMARY The human brain has undergone many specializations (e.g., increased synapse density) resulting in unmatched cognitive complexity, as well as increased vulnerability to neurodevelopmental disorders, such as autism. Differences in gene expression are responsible for most human-specific brain specializations, and comparative (epi)genomics strategies are remarkably powerful in identifying the mechanisms underlying these differences. Spatial genome conformation, particularly organization of topologically associated domains (TADs), is a very promising candidate mechanism for driving human-specific gene expression patterns in the brain but remains understudied in this context. In our preliminary comparative investigation, we found that human-specific TAD organization in lymphoblastoid cells are associated with neurodevelopmental genes, and are recurrently disrupted in patients with developmental delay. However, genome conformation differs drastically across tissues, thus accurate identification of human-specific TAD organization and understanding its contributions to neuronal development and pathology can only be achieved by investigating genome conformation in brain cells. To this end, here we will analyze well-validated cortical neuroprogenitor cells (NPCs) and glutamatergic neurons in human and other primates to identify human-specific TAD boundaries, predict their functionality through in silico mutagenesis, and validate their contribution human neurodevelopmental specialization and disease by using targeted genome editing. In Aim 1, we will obtain Hi- C data from cortical NPCs and mature neurons derived from induced pluripotent stem cells (iPSC) of human, chimpanzee, orangutan, and rhesus macaque using well-validated differentiation protocols. Human-specific TAD boundaries identified in these cells will also be compared against primary NPC and neuron data to validate findings in-vivo. We will complement our findings with epigenetic and transcriptomics data from the same cells and species, to identify differential gene expression and genomic and epigenetic signatures associated with human-specific TAD organization. In Aim 2, we will use novel computation tools to predict effects of disrupting each human-specific boundary and prioritize 15 candidates whose disruption is most likely to cause neuropathogenicity. We will use CRISPR-Cas9 to delete these candidates in human iPSC-derived NPCs and neurons. The molecular consequences of each deletion on chromatin interactions/structure and gene expression will be assessed, along with electrophysiology (for neurons) or proliferation assays (for NPCs) to study the physiological consequences. We will then select a subset of 5 most effective candidates and knock them into chimpanzee iPSC, followed by differentiation to NPC and neurons. “Humanized” chimpanzee cells will be characterized at the molecular and physiological level like the human knockout lines, to further shed light on the function of human-specific TAD boundaries to human neurodevelopment. Overall, this study will provide new insights into mechanisms underlying human-specific brain development and disease.
