Project Summary RNA polymerase II (RNAPII), which catalyzes mRNA synthesis, is co-regulated by cyclin-dependent kinases (CDKs) and phosphatases to control transcription cycle progression and ensure coupling of elongation and processing. Derangements of the RNAPII cycle underlie cancer and other diseases; CDKs and phosphatases are potential drug targets, but how they collaborate to regulate gene expression is largely unknown. We seek to determine how specific phosphorylations are placed and removed in precise order to execute transitions between phases of transcription. In the past five years we uncovered two CDK-phosphatase switches that regulate RNAPII elongation. Our goals for the next five years are to understand how these circuits work to coordinate the transcription cycle, and how they might be disrupted with small molecules. Dissecting CDK-PPP switches: We identified two members of the phosphoprotein phosphatase (PPP) family as targets of inhibitory phosphorylation by Cdk9 (P-TEFb). PP4 and PP1 dephosphorylate the Cdk9 substrate Spt5 to regulate the onset and end of rapid elongation, respectively. To define roles of Cdk9-PPP crosstalk in transcription, we will introduce mutations that lock PP4 or PP1 in constitutively active (unphosphorylated) or inactive (phosphorylated) states and analyze genome-wide effects on RNAPII dynamics and gene expression. Unraveling control of RNAPII elongation by PPP family phosphatases: PP4 and the related PP2A, another phosphatase recruited to transcription complexes, dephosphorylate Spt5 with similar site specificity, and may cooperate to regulate RNAPII release from the promoter-proximal pause. We plan integrated genomic analyses to ask whether PP4 and PP2A work independently, in combination, or in response to different signals. Understanding how phosphorylation events are ordered in the RNAPII cycle: By analogy with the cell division cycle—also regulated by CDKs—the order of phosphorylations during transcription is likely determined by substrate specificities of CDKs and phosphatases, and by structural features of the substrates themselves. We will test this hypothesis for Cdk9, which has scores of substrates and is active throughout elongation. Functional dissection of a conserved elongation regulator: Spt5, large subunit of the conserved elongation factor DSIF, is the linchpin of elongation control by Cdk9-PPP switches; phosphorylations placed by Cdk9 at different sites in Spt5 are removed at different times by PP4 or PP1 and are likely to perform different functions in the transcription cycle. We will take a genome editing approach in human cells to dissect the functions of distinct Spt5 phosphorylations, and to elucidate their differential regulation by CDKs and phosphatases. Illuminating a link between transcription and cell cycle control: One CDK is a core component of both cell-division and transcriptional machineries: the TFIIH-associated Cdk7. As human cells re-enter the division cycle from quiesce...