Edited by Joel M. Gottesfeld This work was supported by Natural Sciences and Engineering Research Council of Canada (NSERC) Grant RPGIN-2016 -04297 (to M. S. K.), a PDF fellowship (to M. J. A.), and a USRA studentship (to M. S.). The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S6. Microarray data were submitted to the Gene Expression Omnibus under accession number GSE128936.

Cdk8 of the RNA Polymerase II mediator kinase complex regulates gene expression by phosphorylating sequence-specific transcription factors. This function is conserved amongst eukaryotes, but the signals and mechanisms regulating Cdk8 activity and phosphorylation of its substrates are unknown. Full induction of the GAL genes in yeast requires phosphorylation of the transcriptional activator Gal4 by Cdk8. We used a screen to identify regulators of the Cdk8-dependent phosphorylation on Gal4, from which we identified multiple mutants with defects in TORC1 signaling. One mutant, designated gal four throttle 1 (gft1) was identified as a recessive allele of hom3, encoding aspartokinase, and mutations in hom3 caused effects typical of inhibition of TORC1, including rapamycin sensitivity and enhanced nuclear localization of the TORC1-responsive transcription factor Gat1. Mutations in hom3 also inhibit phosphorylation of Gal4 in vivo at the Cdk8-dependent site on Gal4, as did mutations of tor1, but these mutations did not affect activity of Cdk8 assayed in vitro. Disruption of cdc55, encoding a regulatory subunit of the TORC1-regulated protein phosphatase PP2A, suppressed the effect of hom3 and tor1 mutations on GAL expression, and also restored phosphorylation of Gal4 at the Cdk8-dependent site in vivo. These observations demonstrate that TORC1 signaling regulates GAL induction through the activity of PP2A/Cdc55, and suggest that Cdk8-dependent phosphorylation of Gal4 is opposed by PP2A/Cdc55 dephosphorylation. These results provide insight into how induction of transcription by a specific inducer can be modulated by global nutritional signals through regulation of Cdk8-dependent phosphorylation.

CDK8 encodes an evolutionarily conserved Mediator complex kinase subunit that functions in general and context-specific transcription regulation by phosphorylating core components of the transcription machinery and gene-specific transcription factors. To better understand the role Cdk8 in transcription regulation, we performed high-resolution gene expression time course analysis following nuclear depletion of Cdk8. Focusing on the earliest gene expression alterations revealed dysregulation of genes encoding glycolysis enzymes, suggesting a functional link to Gcr1 and Gcr2, key transcriptional activators of these genes. Consistently, we found that nuclear depletion of Cdk8 altered the mRNA levels of glycolysis genes as well as the promoter occupancy of Gcr2, but not Gcr1. Examination of the Gcr2 protein sequence revealed a putative Cdk8 phosphorylation site at serine 365, which we confirmed using in vitro and in vivo assays. Importantly, phospho-mutant GCR2 recapitulated the growth and gene expression defects of the GCR2 deletion mutant, effects not observed with a phospho mimetic mutant. As such, our work highlights Gcr2 as a new Cdk8 substrate, revealing that its phosphorylation is critical for the activation of genes encoding glycolysis enzymes.

1Cdk8 of the RNA Polymerase II mediator complex regulates genes by 2 phosphorylating sequence specific transcription factors. Despite conserved importance 3 for eukaryotic transcriptional regulation, the signals regulating Cdk8 are unknown. Full 4 induction of the yeast GAL genes requires phosphorylation of Gal4 by Cdk8, and we 5 exploited this requirement for growth of gal3 yeast on galactose to identify mutants 6 affecting Cdk8 activity. Several mutants from the screen produced defects in TOR 7 signaling. A mutant designated gal four throttle (gft) 1, gft1, was identified as an allele of 8 hom3, encoding aspartokinase. Defects in gft1/ hom3 caused hypersensitivity to 9 rapamycin, and constitutive nuclear localization of Gat1. Furthermore, mutations of tor1 10 or tco89, encoding TORC1 components, also prevented GAL expression in gal3 yeast, 11and tco89 was determined to be allelic to gft7. Disruption of cdc55, encoding a subunit 12 of PP2A regulated by TOR signaling, suppressed the effect of gft1/ hom3, gft7/ tco89, 13 and tor1 mutations on GAL expression in gal3 yeast, but not of cdk8/ srb10 disruptions or 14 Gal4 S699A mutation. Mutations of gft1/ hom3 and tor1 did not affect kinase activity of 15Cdk8 in vitro, but caused loss of Gal4 phosphorylation in vivo. These observations 16 demonstrate that TOR signaling regulates GAL induction through the activity of PP2A/ 17 Cdc55, and are consistent with the contention that Cdk8-dependent phosphorylation of 18 Gal4 S699 is opposed by PP2A/ Cdc55 dephosphorylation. These results provide insight 19 into how induction of transcription by a specific inducer can be modulated by global 20 nutritional signals through regulation of Cdk8-dependent phosphorylation. 21 on EB-gal. Because less than 10% of otherwise wild type gal3 yeast produce a colony on 128 EB-gal (Figure 2), we were unable to implement a conventional synthetic genetic array 129 (SGA) screen using the non-essential gene deletion collection for this purpose. 130Consequently, we employed u.v.-irradiation of a gal3 W303 strain and replica plating, to 131 identify mutants that were incapable of growth on EB-gal, but which produced robust 132 colonies on media containing 3-carbon molecules as the sole carbon source. Initial 133 mutants recovered from this process were transformed with a plasmid bearing genomic 134 GAL3 and re-assayed for growth on EB-gal; mutants capable of growth when expressing 135Gal3 were designated the gal four throttle (gft) mutants, a collection which is comprised 136 of several groups with distinct phenotypes that will be detailed in a separate report. The 137

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