Characterization of the Integrator-Z3 module as a regulator of neuronal differentiation

Abstract Transcriptional regulation is an essential process for proper development that a host of cellular machinery orchestrates. Among these cellular factors is the Integrator complex (INT), a 17-subunit complex that associates with paused RNA polymerase II and is responsible for the 3'-end processing of non-coding RNAs and premature termination of promoter-proximally paused RNAPII. Consistent with a fundamental role of Integrator function, mutations within INT subunits cause neuronal dysfunction, including complex neurological syndromes marked by cerebellar ataxia, intellectual defects, and seizures. Similarly, three zinc finger proteins comprising the ‘Z3 Complex’ (ZNF592, ZNF687, and ZMYND8) thought to be associated with INT also have diverse neurological phenotypes when genetically perturbed. Despite a fundamental requirement of Integrator for gene expression and apparent overlap of neurological symptoms in patients with mutations in INT and Z3 subunits, the molecular basis of this profound connection between Z3-INT and brain disorders is unknown. My preliminary biochemical purifications and proteomics described below indicate that the Z3 complex is strongly bound to INT. The formation of a Z3-INT complex indicates that specific DNA binding proteins can potentially influence INT recruitment to promoter-proximal regions in the genome. Consistently, my RNA-sequencing analyses from cells depleted of Z3 subunits reveal broad transcriptional changes that significantly overlap with changes observed upon depletion of INT subunits. Nothing is known about Z3-INT subunit occupancy during neurogenesis nor how perturbation of Z3-INT expression would affect neural development. This is despite the compelling human patient phenotypes caused by their mutation revealing their importance to brain development. Based on these data, I hypothesize that Z3 interacts with INT to modulate its recruitment and occupancy to promoters, and perturbation of this process leads to disrupted neuronal differentiation. To address this, I will first determine expression and occupancy of Z3-INT during neuronal differentiation. Second, I will elucidate the functional role of Z3-INT neuronal fitness. Third, I will uncover biochemical interactions between Z3 and INT. Completion of these aims will converge to uncover the link between INT and associated transcription factors, Z3.