A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription

with Ribosomal RNA Synthesis

Benjamin Albert,¹Britta Knight,¹Jason Merwin,¹Victoria Martin,¹Diana Ottoz,¹Yvonne Gloor,¹Maria Jessica Bruzzone,¹ Adam Rudner,^2,3and David Shore^1,4,*

1Department of Molecular Biology and Institute for Genetics and Genomics in Geneva (iGE3), 30 quai Ernest-Ansermet, 1211 Geneva, Switzerland

2Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA

3Present address: Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, 451 Smyth Road, Ottawa, ON KIH 8M5, Canada

4Lead Contact

*Correspondence:david.shore@unige.ch http://dx.doi.org/10.1016/j.molcel.2016.10.003

SUMMARY

Cell growth potential is determined by the rate of ribosome biogenesis, a complex process that requires massive and coordinated transcriptional output. In the yeastSaccharomyces cerevisiae, ribo-some biogenesis is highly regulated at the transcrip-tional level. Although evidence for a system that coor-dinates ribosomal RNA (rRNA) and ribosomal protein gene (RPG) transcription has been described, the molecular mechanisms remain poorly understood.

Here we show that an interaction between the RPG transcriptional activator Ifh1 and the rRNA process-ing factor Utp22 serves to coordinate RPG transcrip-tion with that of rRNA. We demonstrate that Ifh1 is rapidly released from RPG promoters by a Utp22-in-dependent mechanism following growth inhibition, but that its long-term dissociation requires Utp22.

We present evidence that RNA polymerase I activity inhibits the ability of Utp22 to titrate Ifh1 from RPG promoters and propose that a dynamic Ifh1-Utp22 interaction fine-tunes RPG expression to coordinate RPG and rRNA transcription.

INTRODUCTION

Ribosomes provide the anabolic capacity for mass accumula-tion, making the production of adequate numbers of ribosomes a prerequisite for cell growth. A significant fraction of cellular en-ergy reserves is devoted to this complex process. In the budding yeast,Saccharomyces cerevisiaeRNA polymerase I (RNAPI) ac-tivity accounts for approximately 60% of total cellular RNA, and in rapidly growing cells nearly half of all RNA polymerase II (RNAPII) initiation events occur on ribosomal protein genes (RPGs) (Warner, 1999). Both ribosomal RNA (rRNA) and RPG transcrip-tion are tightly regulated in yeast, where competitranscrip-tion for limiting

resources may be important for survival. Regulated production of roughly equimolar amounts of ribosomal proteins (RPs) and rRNAs may be important in all organisms, as evidenced by the fact that RPGs are often haplo-insufficient, both in yeast and in humans, where heterozygosity can lead to a number of diseases referred to as ribosomopathies (Teng et al., 2013).

Several studies in yeast, Drosophila, and mammalian cells demonstrate coordinated transcriptional regulation of ribosome components, though the underlying mechanisms are not well un-derstood (Calo et al., 2015; Grewal et al., 2007; Jouffe et al., 2013; Laferte´ et al., 2006). Under optimal growth conditions inS. cerevisiae, the growth-promoting TORC1 (target of rapamy-cin 1) kinase is active and stimulates both RPG and rRNA tran-scription (Lempi€ainen and Shore, 2009). A key factor activating RNAPI is the Rrn3 protein (TIF1-A in vertebrates). Rrn3 associa-tion with RNAPI allows its recruitment to 35S rDNA gene pro-moters, whereas inhibition of growth, for example by TORC1 inactivation with rapamycin, triggers decreased Rrn3 associa-tion with RNAPI and a rapid decline of rRNA producassocia-tion. In a strain where Rrn3 is covalently linked to its binding partner Rpa43, and thus constitutively bound to RNAPI, there is a concomitant failure to downregulate both rRNA and RPG tran-scription following rapamycin treatment (Laferte´ et al., 2006).

Thistranseffect on RNAPII is specific to RPGs, providing strong evidence for crosstalk between RNAPI and RNAPII that helps to coordinate rRNA and RPG transcription.

Transcription of RPGs is thought to be regulated in large part by the controlled association of the Ifh1 activator protein with its constitutively promoter-bound partner protein Fhl1, a forkhead-like transcription factor (Martin et al., 2004; Rudra et al., 2005;

Schawalder et al., 2004; Wade et al., 2004). This interaction is dependent on the forkhead-associated (FHA) domain of Fhl1 and the forkhead binding (FHB) domain of Ifh1 (Martin et al., 2004; Schawalder et al., 2004). Although signaling mechanisms upstream of Fhl1/Ifh1 remain to be clarified, one salient observa-tion is that Ifh1 is rapidly removed from RPG promoters following inhibition of TORC1 kinase by rapamycin (Schawalder et al., 2004). This effect might be mediated by the AGC kinase Sch9, a major TORC1 downstream effector. Sch9 regulates binding

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of Stb3 to RPG promoters, which in turn promotes the recruit-ment of the Rpd3 deacetylase complex to repress transcription (Huber et al., 2011).

In addition to its association with Fhl1 and Rap1 at RPG pro-moters, Ifh1 is also present in a complex called CURI, which in-cludes casein kinase 2 (CK2), Utp22, Rrp7, and Ifh1 (Rudra et al., 2007). Utp22, Rrp7, and CK2 can also be found together in the SSU processome, where they form the UTP-C sub-complex (Krogan et al., 2004). The fact that CURI contains Utp22 and Rrp7, two essential participants in the processing of pre-rRNA, suggests that it may play a role in coupling RPG transcription and pre-rRNA processing. However, the function of the CURI complex is not well understood, and its study is complicated by the fact that many of its components are required for viability.

Here we present evidence for a mechanism by which Utp22 ti-trates Ifh1, thereby regulating its release from RPG promoters following growth inhibition. We show that the ability of Utp22 to sequester Ifh1 and inhibit its binding at RPG promoters is inversely proportional to RNAPI activity, thus allowing for a link-age between rRNA production and RPG transcription. Our data thus illuminate a pathway that can act in parallel with TORC1 to coordinate RPG transcription and rRNA production.

RESULTS

Utp22 Is Physically and Genetically Linked to Ifh1 Ifh1 is an essential RPG activator whose promoter binding is positively correlated with transcription, unlike that of its binding partner Fhl1 or the general regulatory factor (GRF) Rap1 (Jorgen-sen et al., 2004; Knight et al., 2014; Rudra et al., 2005; Scha-walder et al., 2004; Wade et al., 2004). Thus, in cultures exiting exponential growth phase, or in cells where the growth-promot-ing TORC1 kinase is inactivated by rapamycin, Ifh1 is rapidly released from RPG promoters concomitant with a strong decrease in RNAPII binding and transcription. Although post-translational modification (both phosphorylation and acetylation) of Ifh1 has been extensively studied (Cai et al., 2013; Downey et al., 2013), none of the modifications characterized to date have been shown to affect Ifh1 promoter release. However, Ifh1 has also been shown to exist in a complex called CURI, together with casein kinase 2 (CK2) and two proteins (Utp22 and Rrp7) involved in rRNA biogenesis, that has been proposed to act in some way as an interface between pre-rRNA processing and RP production (Rudra et al., 2007). We performed a purifica-tion of endogenously TAP-tagged Ifh1, which showed that a sig-nificant fraction of soluble Ifh1 is found together in a complex with Utp22, Rrp7, and CK2 (Figure 1A), consistent with previous results (Rudra et al., 2007). We thus decided to investigate the possibility that the CURI complex plays a direct role in controlling Ifh1 promoter binding.

We began with an extensive yeast two-hybrid (Y2H) analysis to determine whether Ifh1 displays a particularly strong interaction with one specific CURI protein, which might be suggestive of a direct interaction. In a commonly used hybrid/reporter system (James et al., 1996), we examined all possible combinations of Gal4 activation domain (GAD) and Gal4 DNA binding domain (GBD) hybrids, initially using the pGAL1-HIS3 reporter, and growth on media lacking histidine and containing increasing

concentrations of the His3 inhibitor 3-amino-1,2,4-triazole (3AT), as a readout for the strength of the interactions. Based on the resulting growth profiles, evidence for two distinct groups emerged: one composed of Ifh1, Utp22, and Rrp7, and another comprising the CK2 subunits (Figure 1B). We consistently found that Ifh1 interacts more strongly with Utp22 than with Rrp7, regardless of whether the corresponding proteins are expressed as GAD or GBD hybrids (Figure 1B). An independent test using selection for the more stringent pGAL2-ADE2 reporter gene further supported this conclusion (Figure 1C). Unfortunately, the fact that bothUTP22andRRP7are essential for viability pre-cludes a more straightforward epistasis analysis of their involve-ment in Ifh1 association with the CURI complex.

We previously observed that overexpression of Ifh1, in addi-tion to increasing expression of RPGs, also upregulates a small subset of other genes, including UTP22 (Schawalder et al., 2004). Consistent with the idea that Ifh1 directly regulates UTP22expression, ChIP-seq analysis revealed theUTP22 pro-moter as one of the few non-RPG targets of Ifh1 (Knight et al., 2014) (Figure 1D). Rapid nuclear depletion of Ifh1 by the an-chor-away method (Haruki et al., 2008) results in a concomitant decrease inUTP22mRNA levels, indicating that Ifh1 is indeed a direct regulator ofUTP22(Figure 1E). No Ifh1 binding was de-tected at the promoters of any other CURI complex gene.

Utp22 Levels Regulate Growth and Ifh1 Promoter Binding at RPGs

This specific regulatory link between Ifh1 andUTP22transcription prompted us to explore its possible biological significance. We began by expressingUTP22under the control of the galactose-inducibleGAL1promoter in both a wild-typeIFH1strain and a strain expressing a hypomorphic allele ofIFH1(ifh1-AA), which contains alanine substitutions in the Fhl1-binding domain at Ser680 and Thr681. These residues are phosphorylated by CK2, and the double alanine mutation causes a slight growth defect (Figure S1A) and compromises Ifh1’s interaction with Fhl1 (unpub-lished data;Kim and Hahn, 2016), but not with Utp22 (Figure S1B).

Utp22 overexpression (growth in galactose medium) has only a minor effect on growth in theIFH1strain but triggers a severe growth defect in theifh1-AAbackground (Figures 2A andS1C).

To confirm this genetic link with glucose as the carbon source, we used a pTet promoter to drive overexpression ofUTP22in a doxycycline-repressible system. As was the case for galactose-inducedUTP22overexpression, pTet-drivenUTP22 overexpres-sion only leads to a growth defect in anifh1-AAstrain (Figure 2B).

Further consistent with a requirement of balanced Utp22/Ifh1 levels for normal growth, partial depletion of wild-type Ifh1 protein by the Tet-OFF system (pTet-IFH1), combined with overexpres-sion of Utp22 (endogenous pGAL1-UTP22), creates a growth defect (Figure 2C; right panel, Utp22 OE). Importantly, overex-pression of Rrp7 from either the pTet or pGAL1promoter had no effect on growth in either theIFH1orifh1-AAbackgrounds, consistent with a more direct role for Utp22 in the functional inter-action with Ifh1 (Figures S1D and S1E).

We next assessed by ChIP whether this growth phenotype conferred by Utp22 overexpression could be explained by a direct effect on Ifh1 promoter binding. Utp22 overexpression in anIFH1wild-type strain had no significant effect on Ifh1 binding

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at the two different RPG promoters tested, whereas in the ifh1-AAstrain binding was reduced to a very low level by Utp22 over-expression (Figure 2D). These results suggest a direct ability of Utp22 to regulate growth by modulating Ifh1 promoter binding.

In order to determine if lower than normal Utp22 levels can influ-ence binding of wild-type Ifh1, we took advantage of the pTet-UTP22 construct to reduce Utp22 levels. Because Utp22 is essential, we identified a doxycycline concentration (0.5mg/ml) that significantly changes Utp22 levels but has only a minor ef-fect on cell growth (Figures 2E and 2F). Ifh1 binding at the RPL30 and RPL37 promoters showed an inverse correlation with total Utp22 protein, suggesting that Utp22 levels near phys-iological can directly influence promoter association of wild-type Ifh1 (Figure 2G) without changing RNAPI occupancy on rDNA

B Figure 1. Ifh1 Binds to Utp22 in the CURI

Complex and Targets theUTP22Promoter (A)IFH1-TAPpurification. E1 is eluate following TEV protease cleavage (step 1), and S3 is unbound material from step 2 (calmodulin affinity resin). E2 is the final eluate (step 2). Samples from control (un-tagged) and IFH1-TAP strains, as indicated, were silver stained. Ifh1 and Utp22 migrate at >220 kDa (top arrow) and140 kDa (bottom arrow), respec-tively, in SDS-PAGE. Rrp7 and all four CK2 subunits were identified at20–45 kDa, as marked.

(B) The indicated GBD fusions (columns) were expressed together with the corresponding set of GAD fusions (rows) in the Y2H reporter strain PJ69-4A. In the last column and the last row of each group of eight strains, cells bear empty GBD or GAD expressing plasmids, respectively. In the upper left panel, cells from overnight liquid cul-tures were spotted on a synthetic medium that selects only for the presence of the two plasmids (-Ura-Leu). In the upper right panel, cells were spotted onto selective medium forHIS3in addi-tion (-Ura-Leu-His). In the bottom panels, cells were spotted in -Ura-Leu-His medium containing, in addition, the His3 inhibitor 3-amino-triazole (3AT) at the indicated concentrations.

(C) The same experiment as in (B), except that cells were spotted onto selective medium for the pGAL2-ADE2reporter (-Ura-Leu-Ade).

(D) ChIP-seq profile of Ifh1 and Fhl1 in a 17 kb region centered on theUTP22gene promoter.

(E) Quantitation of cDNA fromUTP22relative to a control gene (ACT1) at 0, 15, or 30 min following treatment with rapamycin (rap) or vehicle (veh).

Data are plotted relative to the values at t = 0, which are set to 1.

genes or Ifh1 partner binding (Fhl1 or Rap1) at RPG promoters (Figures S1F and S1G).

Utp22 Is Required for Stable Release of Ifh1 and RNAPII from RPG Promoters following TORC1 Inhibition

The results described above suggest that Utp22 could play a critical role in regu-lating Ifh1 binding to RPG promoters following TORC1 inhibition.

To more precisely define the function of Utp22 during rapamycin stress, we depleted Utp22 from cells by placing the endogenous UTP22gene under control of theGAL1promoter and shifting cells from galactose to glucose medium 3 hr prior to rapamycin treatment (Figures 3A and 3B). Notably, we found that Utp22 depletion did not alter Ifh1 release at 5 min after addition of rapamycin, but instead alloweded a rapid return of Ifh1 to RPG promoters by 10 min that was complete at 20 min (Figure 3C).

Depletion of Rrp7 or CKB2deletion also triggered a return of Ifh1 at 20 min, but to a lesser extent than Utp22 depletion (Figure S2).

To test the role of Utp22 for Ifh1 release under more physiolog-ical conditions, we examined Ifh1 promoter association following

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both glucose and glucose plus amino acid starvation, in cells with normal levels of Utp22 and in cells where Utp22 was depleted (Figure 3D). Ifh1 promoter association dropped rapidly following glucose deprivation and slightly more when both glucose and amino acids were removed from the medium, as expected, but it rapidly returned to the promoters when Utp22 was depleted. Importantly, we observed that re-binding of Ifh1 following rapamycin treatment was mirrored by a return of RNAPII with similar kinetics, whereas two Ribi genes (NOP1 and NSR1), whose RNAPII association was also strongly reduced by rapamycin, did not recover RNAPII binding

(Fig-ure 3E). These observations support the hypothesis that Utp22 specifically regulates Ifh1 and RNAPII binding to RPG pro-moters on a long timescale following TORC1 inhibition. They also suggest that Ifh1 promoter association immediately following TORC1 inhibition is regulated by a distinct, Utp22-independent mechanism.

Separate Ifh1 Domains Implicated in RPG Promoter Release

The finding that Ifh1 promoter release involves two temporally and mechanistically distinct processes prompted us to test the A

D E

B C

Figure 2. Utp22 Levels Regulate Growth and Ifh1 Promoter Binding

(A) Growth curves on liquid medium with galactose as the sole carbon source of either wild-typeIFH1(blue, green) orifh1-S680A/S681A(ifh1-AA) (red, purple) strains, expressing the endogenous chromosomalUTP22gene under the control of the native promoter or theGAL1promoter (pGAL1-UTP22). Doubling times (minutes) during exponential phase growth are noted.

(B) 10-fold serial dilution ofIFH1orifh1-AAcells transformed with either a plasmid containing theUTP22gene under the control of a doxycycline-repressible promoter (pTet-UTP22) or an empty pTet vector ( ). Cells were spotted onto rich medium lacking (–Dox; left panels) or containing (+Dox; right panels) doxycycline and grown at 30C for 2 days.

(C) 10-fold serial dilution spotting assays of strains expressingIFH1from the doxycycline-repressible pTet promoter (pTet-IFH1) at increasing doxycycline concentrations (top to bottom, as indicated). The strain in the left panels contained, in addition, an empty pPGK1plasmid ( ), whereas the strain on the right overexpressed Utp22 from a pPGK1-UTP22plasmid (Utp22 OE).

(D) Ifh1 orifh1-AAoccupancy (qPCR-ChIP) at theRPL30andRPL37Apromoters (fold enrichment relative toACT1control), in strains with normalUTP22 expression (UTP22) orUTP22overexpression (pGAL1-UTP22). In these and all subsequent qPCR-ChIP assays, bars represent SD from at least three inde-pendent experiments (*p < 0.05).

(E) Serial dilution of yeast expressingUTP22under the control of the doxycycline-repressible pTet promoter (pTet-UTP22) or carrying the empty pTet vector (UTP22). Cells were grown in different concentrations of doxycycline, as indicated.

(F) Utp22 was detected by western blot using FLAG antibody in a whole-cell extract from wild-type (UTP22) cells or a strain expressingUTP22under the control of pTet promoter (pTet-UTP22), at the indicated doxycycline concentrations, as in (E) (top panel). Ponceau red stain served as a loading control (bottom panel).

(G) Ifh1 promoter occupancy (qPCR-ChIP) on theRPL30andRPL37Apromoters, measured as in (D), in a strain carrying pTet-UTP22and grown at the indicated doxycycline concentrations. See alsoFigure S1.

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A C

Figure 3. Utp22 Protein Is Essential for Ifh1 Release after Rapamycin Treatment

(A) Schematic of protocol for Utp22 depletion followed by rapamycin treatment in a strain containing the endoge-nousUTP22gene under control of theGAL1promoter.

(B) Utp22 was detected by western blot using an HA antibody in a whole-cell extract from a strain expressing UTP22under the control ofGAL1promoter, grown in liquid galactose medium (Gal) then shifted in glucose (Glu) for the indicated times (top panel). Ponceau red stain served as a loading control (bottom panel).

(C) Ifh1 occupancy at theRPL30andRPL37Apromoters at the indicated times following rapamycin treatment in apGAL1-UTP22strain grown for 3 hr on glucose, as described in (A) and (B). qPCR-ChIP; fold enrichment relative toACT1was normalized to values at t = 0, which were set to 1.

(D) Ifh1 occupancy at theRPL30andRPL37Apromoters, measured and plotted as in (C), at the indicated times following glucose or glucose plus amino acid depletion in a pGAL1-UTP22strain ( Utp22) or in wild-type strain (+Utp22) grown for 3 hr on glucose, as described in (A).

(E) Rpb3 promoter occupancy at the indicated genes in apGAL1-UTP22( Utp22) or wild-type strain (+Utp22) grown for 3 hr on glucose before rapamycin treatment (t = 0), as described in (A). Aliquots taken at 0, 5, and 20 min after rapamycin addition were analyzed by qPCR-ChIP (as inFigure 2D). Results in this case are presented as fold enrichment relative to theSNR52control locus.