Project Summary Precise control of gene expression in response to external signals and cues is essential for organismal development, growth, and homeostasis. A critical step in metazoan gene regulation involves pausing RNA polymerase II (RNAPII) in early elongation and its controlled release into productive RNA synthesis. Pausing of RNAPII consists of the association of pause-inducing factor NELF and the central scaffolding protein SPT5, which maintain RNAPII stably within the promoter region while awaiting the signal for pause release. Pause release is triggered by the kinase P-TEFb, which phosphorylates RNAPII and SPT5 to cause the dissociation of NELF and promote productive elongation. However, our recent work demonstrated that paused RNAPII can experience a different fate: it can be targeted by the Integrator complex (INT), a multi-subunit transcription termination machine. INT association with paused RNAPII drives promoter-proximal termination, with RNAPII releasing a short, non-productive RNA. In this way, INT potently attenuates messenger RNA (mRNA) expression. Importantly, INT antagonizes RNAPII pause release using two distinct activities: endonuclease-mediated cleavage of the nascent RNA and phosphatase-dependent removal of stimulatory phosphorylation. Indeed, our recent work revealed that INT recruits the protein phosphatase 2A (PP2A), directing its activity toward the elongation complex. Studies in multiple model systems have demonstrated that mutation or depletion of INT subunits activates stress-responsive genes, indicating a conserved role for INT in suppressing these pathways in normal, healthy cells. Accordingly, mutations in INT have been associated with a large number of diseases, with every one of the subunits in the INT complex being implicated in one or more pathophysiological state(s). Improving our understanding of INT activity will thus provide invaluable insights into mechanisms of diseases associated with INT mutations, such as blood, hepatic and gastric cancers, and developmental disorders, including ciliopathies and aberrant neuronal migration. The proposed research will address central questions that remain about INT function and regulation. Specifically, Aim 1 will dissect the roles of the INT endonuclease and phosphatase modules in gene regulation, assessing coordination between these modules and the requirement for each catalytic activity across the repertoire of target genes. Given the established role of INT in attenuating stress- responsive genes during normal cell growth, Aim 2 will determine how stress signaling can activate these genes. In particular, we will investigate several potential models for INT inactivation by cellular stressors. Our work shows the ‘Z3’ complex is an uncharacterized INT module, and, given its previous connections to DNA/chromatin binding and transcriptional repression, Aim 3 will probe the role of Z3 in INT function.