Project Summary Nuclear condensate formation by chromatin regulatory proteins and transcription factors is a new paradigm for explaining genome partitioning and gene regulation mechanisms. Recent studies showed that disease- associated mutations of DNA-binding protein, such as MeCP2, are associated with aberrant condensate formation and disrupting the heterochromatin domains. However, the most current studies of phase-separation by nuclear proteins are limited to in vitro biochemical studies and overexpression studies in cells showing the formation of puncta or nuclear bodies, which are insufficient to dissect the molecular function of endogenous protein condensates in disease. To address these issues, the objective of this R21 application is to develop novel chemogenetic tools for manipulating, monitoring, and testing endogenous MeCP2 condensates in neurons. Specifically, we will develop SPARK-OFF to dissolve MeCP2 condensates and SPARK-ON to restore condensation for MeCP2 mutants from Rett Syndrome patients in differentiated neurons. Through sequencing analysis, we will further investigate the roles of MeCP2 condensates in chromatin regulation, chromatin structure, and gene regulation. Our study will provide new technologies for studying the biological function of nuclear condensates in the nervous system and help demystify the causal relationship between MeCP2 condensates and Rett Syndrome etiology.