1. Volume 64, Issue 4, Pages 720-733 (November 2016)
A Molecular Titration System Coordinates Ribosomal Protein Gene Transcription with Ribosomal RNA Synthesis 
Benjamin Albert, Britta Knight, Jason Merwin, Victoria Martin, Diana Ottoz, Yvonne Gloor, Maria Jessica Bruzzone, Adam Rudner, David Shore 
Molecular Cell 
Volume 64, Issue 4, Pages (November 2016)
DOI: /j.molcel
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
Ifh1 Binds to Utp22 in the CURI Complex and Targets the UTP22 Promoter
(A) IFH1-TAP purification. 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 (untagged) and IFH1-TAP strains, as indicated, were silver stained. Ifh1 and Utp22 migrate at >220 kDa (top arrow) and ∼140 kDa (bottom arrow), respectively, in SDS-PAGE. Rrp7 and all four CK2 subunits were identified at ∼20–45 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 cultures 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 for HIS3 in addition (-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-ADE2 reporter (-Ura-Leu-Ade).
(D) ChIP-seq profile of Ifh1 and Fhl1 in a 17 kb region centered on the UTP22 gene promoter.
(E) Quantitation of cDNA from UTP22 relative 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.
Molecular Cell  , DOI: ( /j.molcel )
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3. 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-type IFH1 (blue, green) or ifh1-S680A/S681A (ifh1-AA) (red, purple) strains, expressing the endogenous chromosomal UTP22 gene under the control of the native promoter or the GAL1 promoter (pGAL1-UTP22). Doubling times (minutes) during exponential phase growth are noted.
(B) 10-fold serial dilution of IFH1 or ifh1-AA cells transformed with either a plasmid containing the UTP22 gene 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 30°C for 2 days.
(C) 10-fold serial dilution spotting assays of strains expressing IFH1 from 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 pPGK1 plasmid (−), whereas the strain on the right overexpressed Utp22 from a pPGK1-UTP22 plasmid (Utp22 OE).
(D) Ifh1 or ifh1-AA occupancy (qPCR-ChIP) at the RPL30 and RPL37A promoters (fold enrichment relative to ACT1 control), in strains with normal UTP22 expression (UTP22) or UTP22 overexpression (pGAL1-UTP22). In these and all subsequent qPCR-ChIP assays, bars represent SD from at least three independent experiments (∗p < 0.05).
(E) Serial dilution of yeast expressing UTP22 under 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 expressing UTP22 under 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 the RPL30 and RPL37A promoters, measured as in (D), in a strain carrying pTet-UTP22 and grown at the indicated doxycycline concentrations. See also Figure S1.
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4. 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 endogenous UTP22 gene under control of the GAL1 promoter.
(B) Utp22 was detected by western blot using an HA antibody in a whole-cell extract from a strain expressing UTP22 under the control of GAL1 promoter, 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 the RPL30 and RPL37A promoters at the indicated times following rapamycin treatment in a pGAL1-UTP22 strain grown for 3 hr on glucose, as described in (A) and (B). qPCR-ChIP; fold enrichment relative to ACT1 was normalized to values at t = 0, which were set to 1.
(D) Ifh1 occupancy at the RPL30 and RPL37A promoters, measured and plotted as in (C), at the indicated times following glucose or glucose plus amino acid depletion in a pGAL1-UTP22 strain (−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 a pGAL1-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 in Figure 2D). Results in this case are presented as fold enrichment relative to the SNR52 control locus. See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
N- and C-Terminal Regions of Ifh1 Are Not Essential for Growth but Regulate Short and Long Timescale Promoter Release after TORC1 Inhibition
(A) Schematic representation of a series of Ifh1 deletion mutants. A cross (to the right) indicates those that fail to support growth.
(B) Y2H interaction of Ifh1 mutants with Utp22. Each row contains PJ69-4A cells expressing GBD-Utp22 (or the empty GBD vector) and the indicated GAD-Ifh1 mutant. 10-fold serial dilutions were spotted onto the indicated selective media, as described in Figure 1B.
(C) Promoter occupancy at RPL30 of Ifh1, ifh1-1, and ifh1-6 at the indicated times following rapamycin treatment, analyzed by qPCR-ChIP and normalized as above to values at t = 0.
(D) 10-fold serial dilutions of overnight cultures (right) from strains carrying the indicated alleles of IFH1 (left) were spotted onto rich medium and grown at 30°C for 40 hr.
(E) Ifh1 s promoter occupancy (qPCR-ChIP; fold enrichment relative to ACT1) on RPL30 and RPL37A at the indicated times following rapamycin treatment. See also Figure S3.
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6. Figure 5
Sch9 Inactivation Triggers Ifh1 Promoter Release and Enhances Titration of Ifh1 by Utp22
(A) Ifh1 promoter occupancy on RPL30 (qPCR-ChIP; fold enrichment relative to ACT1) at the indicated times following 1NM-PP1 treatment of wild-type (white), pka-as (gray), or sch9-as (black) strains.
(B) Stb3 promoter occupancy on RPL30 (qPCR-ChIP; fold enrichment relative to ADH4) at the indicated times following rapamycin treatment.
(C) Ifh1 promoter occupancy on RPL30 and RPL37A (qPCR-ChIP; fold enrichment relative to ACT1) at the indicated times following rapamycin treatment in wild-type (STB3) or stb3Δ cells (∗p < 0.05).
(D) 10-fold serial dilution of wild-type (WT), sch9-as, pka-as strains transformed with a doxycycline-repressible pTet-UTP22 plasmid (pTet-Utp22) or with the empty pTet vector (−). Cells were spotted in the presence of increasing concentrations of 1NM-PP1, and in the presence or absence of doxycycline, as indicated. Plates were incubated at 30°C for 48 hr.
(E) Wild-type (WT) or rpd3Δ strains were transformed as indicated at the top with an empty vector (−), or plasmids overexpressing either Stb3 (pADH1-STB3) or a phospho mutant of Stb3 (pADH1-stb3-3A). These different strains were transformed with a second vector allowing overexpression of UTP22 under control of the PGK1 promoter (Utp22 OE) or with an empty vector (−). 10-fold serial dilutions of overnight cultures were grown at 30°C for 48 hr.
(F) Ifh1 promoter occupancy on RPL30 and RPL37A (qPCR-ChIP; fold enrichment relative to ACT1) in rpd3Δ cells transformed with a pTet-stb3-3A plasmid. Cells were treated (+Dox) or not (−Dox) with doxycycline. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Utp22-Ifh1 Controls RNAPI-RNAPII Crosstalk Independently of the TORC1 Pathway
(A) Utp22, Rrp7, Ifh1 occupancy (qPCR-ChIP; fold enrichment relative to 35S promoter, P) at the indicated times following rapamycin treatment on different sites within the rDNA repeat locus, as indicated.
(B and C) Ifh1 (B) and Rpb3 (C) promoter occupancy at RPL30 and RPL37A (qPCR-ChIP; fold enrichment relative to SNR52) at the indicated times following rapamycin treatment of wild-type (WT) and CARA cells.
(D) Rpa190 occupancy (qPCR-ChIP; fold enrichment relative to ACT1) on the indicated rDNA regions in an RPA135-FRB “anchor away” strain at the indicated times (min) following addition of rapamycin or vehicle alone.
(E) Ifh1 promoter occupancy at RPL30 and RPL37A (qPCR-ChIP; fold enrichment relative to ACT1) following Rpa135 anchor away, as in (D).
(F) Rpb3 promoter occupancy on the indicated genes (qPCR-ChIP; fold enrichment relative to SNR52, normalized to value at t = 0 min) at times indicated following Rpa135 anchor away, as in (D).
(G) Ifh1 or ifh1-6 promoter occupancy at RPL30 and RPL37A (qPCR-ChIP; fold enrichment relative to ACT1) at 60 min following rapamycin treatment in the presence (+) or absence (−) of Utp22 (vehicle or auxin treatment, respectively, in a strain carrying UTP22-AID). Data are plotted relative to t = 60 value for an untreated culture (vehicle alone) whose Ifh1 enrichment was normalized to 1. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
A Schematic Representation of the Proposed Mechanism of RNAPI-RNAPII Crosstalk through a Utp22-Ifh1 Interaction
During exponential growth (black arrows), Sch9 kinase activity stimulates RNAPI, prevents the repressor protein Stb3 from entering the nucleus, and promotes Ifh1 binding on RPG promoters to stimulate transcription. Upon growth inhibition (colored arrows) TORC1 and Sch9 kinases are inactivated, leading to a reduction in RNAPI transcription, and rapid release of Ifh1 and Sfp1 from RPG promoters concomitant with Stb3 binding (short timescale, ∼5 min; blue arrows). Under persistent stress conditions, Ifh1 promoter release is sustained through Utp22 binding, which is facilitated by reduced RNAPI activity (long timescale, ∼20–30 min; red arrows). See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Molecular Cell 2016 64, 720-733DOI: (10.1016/j.molcel.2016.10.003)
Copyright © 2016 Elsevier Inc. Terms and Conditions