Abstract: Intellectual disability (ID) affects 1-3% of the population, resulting in cognitive and adaptive behavioral deficits. Many genes are associated with ID, including multiple mutations in KDM5C, an X-linked gene which we discovered to be a histone demethylase a decade ago. In the past funding cycle, we found that KDM5C patient mutations reduce both protein stability and catalytic activity. We showed that a mouse Kdm5c knock-out (KO) model recapitulated the cognitive and behavioral deficits seen in human patients. Kdm5c bound primarily at promoters in terminally differentiated mouse neurons to modulate methylation at lysine 4 of histone 3 (H3K4), and Kdm5c loss affected the expression of neuronal genes in the amygdala. Further studies of conditional mouse Kdm5c KOs have suggested that Kdm5c potentially plays a neurodevelopmental role. To study the function of KDM5C during neurodevelopment in a human model, we have generated patient-derived iPS cell lines bearing KDM5C mutations, and isogenic lines with the mutations corrected, both of which can undergo neuronal differentiation in culture. These cell lines provide an unprecedented opportunity to explore the effects of KDM5C in a well-defined and experimentally accessible human developmental system. The goals of this work are to obtain a comprehensive molecular and cellular understanding of how KDM5C regulates human neurodevelopment. We will conduct high-resolution time-course analyses to determine exactly which stages of neuronal differentiation, and which cell types, are compromised by KDM5C mutation. We will investigate the functionality of KDM5C-mutant neurons by interrogating the expression of synaptic markers and electrophysiology. Because some aspects of brain development (e.g. formation of multiple cell types and their organization) are not recapitulated in a 2D culture system, we will use 3D human brain organoids generated from mutant and corrected iPS cells to investigate the roles of KDM5C in promoting brain growth, generating the appropriate diversity of neural cell types, and facilitating neuronal network connectivity. Our findings will be validated in vivo during embryogenesis of WT and Kdm5c KO mice, and the critical timing and location of Kdm5c activity determined by expressing or deleting Kdm5c in specific stages/ cell types. To investigate the molecular mechanisms of the neuronal differentiation defects in KDM5C mutant cells, we will determine transcriptional profiles of mutant and corrected iPS cells during differentiation in 2D and 3D cultures. Because the critical genomic targets of KDM5C (e.g. promoters, enhancers) during neurodevelopment are unknown, we will map KDM5C binding sites genome-wide during 2D neuronal differentiation, and determine how KDM5C mutations a...