Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I–Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.DOI:http://dx.doi.org/10.7554/eLife.20832.001 The nucleolus constitutes a cellular hub dedicated to ribosome biogenesis, which starts with the transcription of ribosomal RNA (rRNA) precursor genes by RNA polymerase I (Pol I). Ribosomes are completed by the action of Pol II, synthesizing messenger RNA, and Pol III, involved in 5S rRNA and transfer RNA production. The critical requirement for ribosomes in actively growing cells causes that Pol I retains up to 60% of the total transcriptional activity within the eukaryotic nucleus . HoweverInitiation of rRNA synthesis in yeast is a sequential process that involves four components: the upstream activating factor (UAF) complex, the TATA box-binding protein (TBP), the core factor (CF) heterotrimer and the Rrn3 protein . While tYeast Pol I is a 590 kDa enzyme composed of 14 subunits, whose atomic architecture was recently revealed . The twoIn the Pol I crystal structure, the enzyme forms homodimers that exhibit an unexpectedly open cleft occupied by a DNA-mimicking loop, which is incompatible with transcription. The structure suggests that the interaction between the A43 C-terminal tail and the opposite monomer’s clamp is important to maintain Pol I dimers. Interestingly, Pol I dimers in solution present the same structural arrangement, as shown by cryo-EM . While hHere, we have investigated the molecular mechanisms underlying Pol I activation using a holistic approach. Live-cell imaging shows that nutrient depletion causes rapid clearance of Pol I–Rrn3 complexes, followed by the formation of Pol I homodimers, while nutrient addition reverts both repressive conditions following a quasi-symmetric pattern. Additionally, we report the cryo-EM structures of monomeric Pol I alone and in complex with Rrn3 at 4.9 and 7.7 Å resolution, respectively, representing the two steps in Pol I activation. Finally, we designed stalk mutants affecting Pol I dimerization and/or Rrn3 binding, and used them to explore the influence of these complexes on rDNA association.To investigate the formation and disruption of Pol I transcription complexes in vivo, we used PICT (Protein interactions from Imaging Complexes after Translocation), a fluorescence microscopy technique to analyse protein interactions in living cells . This tetor1-1 mutation  and the second to FRB (A190-FRB). The presence of these tags did not alter the doubling times of the cells . When rapamycin is added, GFP-labelled Pol I will only co-translocate to the anchor-RFP-FKBP if it interacts with FRB-tagged Pol I . Cells iThe crystal structure of inactive Pol I identified the A43 C-terminal tail, encompassing residues 260–326, as a key element to form Pol I homodimers , inset. To evaluate whether RNA polymerase dimerization is a more general regulatory mechanism, we applied PICT analysis to the other nuclear RNA polymerases. In the case of Pol III, no oligomerization was observed on the anchor in either growing or starving medium, indicating that Pol III does not dimerize in these conditions . SimilarTo rule out the possibility that Pol I homodimerization could involve the synthesis of new proteins, we followed the formation of Pol I dimers in the presence of cycloheximide, which targets ribosomes and inhibits protein synthesis. Starved cells exposed to this compound did not show any defect in Pol I homodimerization. Instead, we detect a 40% increase in the homodimers levels with respect to untreated cells. This indicates that no additional protein synthesis is required to induce this cellular event and that ribosome inhibition could reinforce Pol I dimerization . InteresThe Pol I–Rrn3 complex represents the activated form of the enzyme . We usedTo further investigate the effect of starvation on Pol I transcription, we used our A43 C-terminal truncation abolishing Pol I dimerization. For that, we constructed A43ΔCt haploid cells that, while expressing similar levels of Rrn3 and A190 to wild-type cells, present reduced growth . StructuWe also used a strain, termed CARA for Constitutive Association of Rrn3 and A43, expressing a Pol I–Rrn3 chimera that cannot form Pol I homodimers . ChIP exWe then quantified the temporal progression in the assembly and disassembly of Pol I inactive dimers and Pol I–Rrn3 active complexes in response to nutrient availability. When growing cells are transferred to starving medium, detected levels of Pol I–Rrn3 are rapidly depleted by about 30%, in a process that follows an exponential decay , left. AChIP experiments performed at an equivalent regime show that the promoter association of both A190 and Rrn3 drops by two thirds within the first 10 min from starvation but requires about 2 hr to reach completion , left. WTo unveil the molecular details of Pol I activation, we aimed to characterize this process structurally. Negatively-stained 2D averages of the Pol I–Rrn3 complex showed a portion of additional density next to the stalk . AntibodOur monomeric Pol I structure provides a detailed picture of the structural transition from homodimers to monomers, a critical step in Pol I activation . The PolOur cryo-EM map of the Pol I–Rrn3 complex shows the precise location of the activating factor on the enzyme . The eloOur Pol I–Rrn3 structure underscores Pol I regions that are critical to bind the activating factor . The A43While the relevance of A43 in Rrn3 binding has been demonstrated , no evidFinally, we engineered a strain lacking an internal loop in the TA domain that appears disordered in the crystal structure of dimeric Pol I . Strikingly, this mutant behaves opposite to ΔA14 and helix α2 mutants, as it shows a 2-fold increase in detected Pol I–Rrn3 complexes . In accoIn this study, which includes a wealth of techniques ranging from structural analysis to in vivo interaction experiments, we shed light on the regulation of Pol I activation, the first step in ribosome biogenesis. An improved PICT assay allowed us to investigate the levels of Pol I homodimers and Pol I–Rrn3 complexes in response to nutrient availability. We also provide a detailed picture of the conformational rearrangements taking place in the transition between the inactive and activated states of Pol I, and identify distinct stalk regions as central structural elements in this process.Cellular polymerases are recruited to the promoter in an active, monomeric conformation . HoweverWhile only a minor fraction of Pol I is bound to Rrn3 in growing cells , the forIn contrast to eukaryotes, bacteria use a single form of RNA polymerase to transcribe their entire genome. In response to stress, bacteria induce the production of the ppGpp alarmone, which targets RNA polymerase at an allosteric site . This seS. pombe and A. thaliana, where likely no Pol I dimerization will take place in nutrient-arrested cells. Additionally, our cryo-EM structure shows that the main Pol I element involved in Rrn3 interaction is the OB domain in the A43 elongated core. This is consistent with reported data in yeast  falls in the region where Rrn3 is located and contacts equivalent domains of the RNA polymerase core  the stalk, (ii) the dock domain, and (iii) the AC40/AC19 heterodimer. Contact of the activating factor with these three regions is therefore required for activation and subsequent binding to promoter-attached initiation factors. In the Pol II system, the Mediator complex acts as an activating cofactor required for association of the enzyme to promoter-bound initiation factors . Interesase core . Moreoveoter DNA  and mammoter DNA . These aRPA14  and the C-terminal regions of RPA14 (A14ΔCt) and RPA43 (A43ΔCt). The remaining A14 mutants were obtained by directed mutagenesis of the wild type RPA14 gene cloned in a centromeric plasmid, which was used to transform the rpa14Δ strain, followed by selection in appropriate medium.The strains used are listed in http://rsb.info.nih.gov/ij/) was used to analyze the images and a custom image analysis workflow was developed and implemented in ImageJ macro language to enable the automatic processing of complete data sets. This workflow independently processes RFP (red) and GFP (green) channels to segment spot like structures of a specific size in the images; the functional steps  was used to analyze 10 cells for each strain. As the nucleus is found between two Tub4-RFP-FKBP anchoring platforms of yeast cells with two spindle pole bodies, recruited preys-GFP that specifically accumulate between the two anchoring platforms were scored as nuclear  slurry overnight at 4°C, and then, after extensive washing and decrosslinking at 65°C for several hours, analysed by western-blot using a monoclonal antibody against the Myc epitope.ChIP and purification, quantitative real-time PCR (qPCR) amplification and data analysis were performed described . For staE. coli Rosetta Cells  in TB autoinducing medium overnight at 24°C. Cells were harvested by centrifugation and resuspended in L Buffer  supplemented with protease inhibitors . Cells were sonicated and the lysate was centrifuged at 20,000 g for 40 min. The supernatant was loaded on HisTrap  equilibrated in L Buffer with 0.5 mM Phenylmethylsulfonyl fluoride and eluted in a linear gradient to 400 mM imidazole. Rrn3-containing fractions were pooled, loaded on Mono Q  equilibrated in MQ Buffer , and eluted in a linear gradient to 750 mM NaCl. Pooled fractions were concentrated and loaded on a Superdex 200  equilibrated in GF Buffer . Eluted fractions were concentrated to 20 mg/mL, frozen in liquid N2 and stored at −80°C.Rrn3 was amplified by PCR from yeast genomic DNA and cloned into pETM11 between NcoI and Acc65I restriction sites, resulting in an N-terminal hexahistidine tag followed by a tobacco etch virus (TEV) protease target sequence. His-TEV-Rrn3 was expressed in 2SO4, 3 mM DTT) to isolate the complex of interest from the free antibody and crosslinking aggregates.Pol I was obtained according to published protocols . A Pol IFor negative-staining EM, crosslinked Pol I–Rrn3 (20 ng/μl) was adsorbed on a glow-discharged carbon-coated copper grid and stained with 2% uranyl formate. Observations were performed in a JEOL-1230 electron microscope operated at 100 kV and micrographs were recorded under low-dose conditions (~10 e–/Å2) using a 4k × 4k TemCam-F416 camera (TVIPS) at 2.28 Å/pixel. The same procedure was used for the Pol I–Rrn3–antibody complex, except that micrographs were collected at 2.84 Å/pix.For cryo-EM, 4 µl of crosslinked Pol I–Rrn3 (60 ng/µl) were placed onto glow-discharged Quantifoil R2/2 grids and incubated in the chamber of a FEI Vitrobot at 4°C and 95% humidity for 30 s before blotting for 2 s at an offset of –2 mm. Data were collected on a FEI Titan Krios electron microscope operated at 300 kV, using FEI automated single particle acquisition software (EPU) on a back-thinned FEI Falcon II detector at a calibrated magnification of 79,096 (pixel size of 1.77 Å). Defocus values ranged from 1.9 to 4.2 µm. Videos were intercepted at a rate of 68 frames for 4 s exposures.For negative-staining, around 63,500 images of Pol I and 42,400 images of Pol I–Rrn3 were extracted using EMAN  and binnFor cryo-EM, 1288 movies were averaged using optical flow correction  and theiwww.pymol.org).The available crystal structure of dimeric Pol I (PDB entry 4C3H) was fitted into the cryo-EM map of monomeric Pol I at 4.9 Å resolution, using UCSF Chimera . RegionsThe Pol I–Rrn3 and Pol I monomer cryo-EM maps were deposited under accession numbers EMD-4086 and EMD-4087. The cryo-EM map of the Pol I monomer at 4.9 Å resolution and its corresponding pseudo-atomic model were deposited under accession codes EMD-4088 and PDB-5LMX. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Kevin Struhl (Senior Editor) and three reviewers, one of whom, Alan G Hinnebusch (Reviewer #1), is a member of our Board of Reviewing Editors. The following individuals involved in review of your submission have agreed to reveal their identity:.Thank you for submitting your article "The dynamic balance between RNA polymerase I complexes regulates rDNA transcriptional activation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this paper, the authors study the down-regulation of Pol I activity by nutrient starvation, focusing on the roles of Pol I homodimerization and dissociation of the activated complex formed with cofactor Rrn3 as possible mechanisms that diminish recruitment of the Pol I:Rrn3 complex to the RDN promoter. Introducing an improvement in the PICT assay, they provide the first evidence that Pol I forms homodimers in cells, in a manner that depends on nutrient starvation or inhibition of either translation or ribosome biogenesis using drugs. The dimerization requires the C-terminal domain of subunit A43, implicated in dimerization by the previous crystal structure of a Pol I dimer. They could also use the PICT assay to monitor Pol I:Rrn3 complex levels and show that these are down-regulated in starved cells, although not to the same extent as promoter occupancy of these proteins measured by ChIP. They examine the kinetics of formation/dissociation of the Pol I dimer and Pol I:Rrn3 complexes by PICT in the transition from growth to starvation and back to growth, which are complex and multiphasic and show that at least to some extent the two processes are not tightly coupled in time. They then switch gears and present two cryo-EM structures, a Pol I::Rrn3 complex at 7.7 Å resolution, which appears similar in most important respects to a higher resolution structure (4.8 Å) of the same complex recently published by another group; and a Pol I monomer at 4.9 Å, of higher resolution than one also published this year. Comparison of their Pol I monomer to the crystallized dimer gives some insights into the transition from the presumably inactive dimer to active monomer, but these transitions were either not dramatic or were not well explained in the text/figures. The Pol I:Rrn3 complex is highly relevant to Pol I activation and they go on to perform a limited structure-function analysis of this interaction, showing that it is impaired if the A14 subunit (which interacts extensively with Rrn3) is simply deleted from Pol I. The Pol I:Rrn3 complex assembly and recruitment are either diminished or enhanced if the unstructured C-terminal tail of A43 is removed or the internal unstructured "TA-loop" of A14 is removed, respectively. The effect of deleting A14 entirely could be indirect, as it also interacts extensively with A43; and the opposing effects of deleting A43-CTT or A14 TA-loop, while interesting, are difficult to rationalize because they are unstructured segments that don't make known contacts with Rrn3.Essential revisions:The consensus of the reviewers is that the structural information in the second half of the paper is valuable, even if mostly confirmatory of other recently published structures, as are the structure-function studies, even if somewhat limited in scope. However, the structural insights into the transition from presumably inactive dimer to active monomer gleaned from the structures were either not dramatic or not well explained in the text/figures and should be highlighted better. In addition, the possible effects of the A14 subunit deletion on Pol I integrity and abundance would have to be examined.Regarding the results obtained using the PICT assay in the first part of the paper, there are important concerns about whether the results are sufficient to conclude, as the title of the paper implies, that formation/disassembly of the homodimer versus Pol I-Rrn3 complexes are important events regulating rDNA transcription in response to nutrient availability. All three reviewers had difficulty understanding how the PICT results have been quantified; Rev. #2 had the most serious concerns, and has explained the difficulties thoroughly in his/her review. The large halo of background GFP signal compared to the small amount associated with the tethered RFP makes quantification of the co-localizing PICT signal highly questionable; and the images in Specific recommendations:1) It would be very helpful if they had an independent assay(s) for dimerization. Two choices. First, they could formaldehyde crosslink under the various in vivo situation and do a co-IP with essentially the same constructs they already have. Second, they could replace the targeting tags with Gal4, which would allow Gal4-Pol I fusions to targeted to Gal4 binding sites (assayed by ChIP). If the model is correct, one should see the non-Gal4 version of Pol I associating with Gal4 binding sites under starvation. Both of these approaches might be better for kinetics and quantification, but the real virtue of them is that they are independent assays for dimerization.2) The authors haven't demonstrated "regulation", so their title needs to be softened. What they have "shown" is that the dimerization and change in complexes is associated with regulation.Reviewer #1:Summary of work:In this paper, the authors study the down-regulation of Pol I activity by nutrient starvation, focusing on the roles of Pol I homodimerization and dissociation of the activated complex formed with cofactor Rrn3 as possible mechanisms that diminish recruitment of the Pol I:Rrn3 complex to the RDN promoter. Introducing an improvement in the PICT assay, they provide the first evidence that Pol I forms homodimers in cells, in a manner that depends on nutrient starvation or inhibition of either translation or ribosome biogenesis using drugs. The dimerization requires the C-terminal domain of subunit A43, implicated in dimerization by the previous crystal structure of a Pol I dimer. They could also use the PICT assay to monitor Pol I:Rrrn3 complex levels and show that these are down-regulated in starved cells, although not to the same extent as promoter occupancy of these proteins measured by ChIP. They examine the kinetics of formation/dissociation of the Pol I dimer and Pol I:Rrn3 complexes by PICT in the transition from growth to starvation and back to growth, which are complex and multiphasic and show that at least to some extent the two processes are not tightly coupled in time. They then switch gears and present two cryo-EM structures, a Pol I::Rrn3 complex at 7.7 Å resolution, which appears to be similar in all important respects to a higher resolution structure (4.8 Å) of the same complex recently published by another group; and a Pol I monomer at 4.9 Å, of higher resolution than one also published this year. Comparison of their Pol I monomer to the crystallized dimer gives some insights into the transition from the presumably inactive dimer to active monomer, but these transitions were either not dramatic or were not well explained in the text/figures. The Pol I:Rrn3 complex is highly relevant to Pol I activation and they go on to perform a limited structure-function analysis of this interaction, showing that it is impaired if the A14 subunit (which interacts extensively with Rrn3) is simply deleted from Pol I. the Pol I:Rrn3 complex assembly and recruitment are either diminished or enhanced if the unstructured C-terminal tail of A43 is removed or the internal unstructured "TA-loop" of A14 is removed, respectively. The effect of deleting A14 entirely could be indirect, as it also interacts extensively with A43, and the opposing effects of deleting A43-CTT or A14 TA-loop, while interesting, are difficult to rationalize because they are unstructured segments that don't make known contacts with Rrn3. Unfortunately, they did not make any "surgical" alterations to A14 or A43 at points of direct contact with Rrn3 in an attempt to validate the physiological relevance of the cryoEM structure for understanding assembly of the Pol I:Rrn3 complex.General critique:Previous work on Pol I in yeast has shown that Rrn3 binding to the enzyme is required for Pol I recruitment to the promoter, and that Pol I can form homodimers in solution and was crystallized as a homodimer with structural features indicating an inactive conformation. This paper uses the PICT assay to provide convincing evidence that Pol I homodimers can form in cells, but only in stress conditions including starvation for carbon and nitrogen, arrest of translation elongation by cycloheximide, or treatment with a drug shown to impair ribosome biogenesis. The evidence that homodimerization occurs in cells is firm, and the fact that it was observed only in starved/stressed cells supports its role as a regulatory mechanism for inactivation of Pol I under conditions where new ribosome synthesis is unwanted. Unfortunately, however, there is no direct evidence establishing its importance in down-regulating Pol I function. In fact, the kinetics of homodimerization versus PolI:Rrn3 interaction shown in The PICT assay also gives evidence of reduced Pol I:Rrn3 association in starved cells by ~50%, and there is an even greater reduction in Pol I/Rrn3 occupancy of the Pol I promoter measured by ChIP. However, the Western in The cryo-EM analysis of the Pol I monomer at 4.9 Å resolution and the Pol I::Rrn3 complex at 7.7 Å resolution are valuable, although another group recently published a Pol I-Rrn3 complex at higher resolution (4.9 Å) that seems to contain all of the same key features and allows the same insights into this important intermediate in Pol I activation as those described here-thus diminishing the significance of the current structure of the complex. The higher resolution of their monomeric Pol I compared to that recently published by another group, should allow a superior comparison between the monomeric and dimer Pol I , from which they conclude that the transition from dimer to monomer "involves partial cleft closure and increased flexibility of critical motifs". However, the relevant figure  does notThe experiments showing that deleting the A14 subunit weakens Pol I:Rrn3 interaction by PICT assay and reduces Pol I/Rrrn3 occupancy at the promoter by ChIP assay are valuable, but they have not ruled out the possibility that removing this subunit perturbs other Pol I subunits, e.g. A43 with which it seems to interact with extensively, and thus impairs Pol I interaction with Rrn3 indirectly by disrupting A43:Rrn3 contacts rather than eliminating the A14:Rrn3 interactions evident in the cryoEM structure. It is even possible that the steady state level of Pol I is reduced by the A14 deletion and contributes to the reduced yield of Pol I:Rrn3 interactions and Pol I recruitment. Thus, the effects of the A14 subunit deletion on Pol I integrity and abundance should have been examined. But even more importantly, the authors should have used the molecular details of the cryoEM structure to more surgically disrupt the A14:Rrn3 interaction by truncating or substituting helix 2 of A14 in a way designed not to disrupt A14 interactions with other Pol I subunits. This seems like a missed opportunity to exploit their structure.eLife, even if the authors can address all of the shortcomings in the experiments and interpretations mentioned above or below.In summary, the paper represents a collection of three different lines of work joined by the theme of regulating Pol I:Rrn3 interaction. The evidence for homodimer formation in cells is interesting and valuable but it is unclear that it constitutes an important means of impeding Pol I:Rrn3 association and promoter recruitment rather than being a byproduct of the loss of this complex by other means that serves only to protect Pol I from degradation. The cryoEM structures are valuable, but a Pol I:Rrn3 complex of higher resolution was already published. The structure-function analysis of A43 and A14 did not exploit the cryoEM structure, as it involved deletions of large segments that are not present at their interfaces with Rrn3. The result that deleting the TA loop in A14 leads to greater Pol I:Rrn3 association and promoter occupancy is the most interesting finding, but the molecular mechanism is not obvious from the structure. Taken together, the paper is not a strong candidate for Other major criticisms:The PICT approach requires Rapamycin, which is known to impair ribosome biogenesis. It's unclear from the genotypes provided whether both alleles of TOR1 in the diploid strains they used are the tor1-1 allele conferring resistance to Rap, which would seem to be required. Even if this is so, it seems important to show directly, if it's not in the literature already, that treatment of their PICT strain with rapamycin has no effect on Pol I recruitment or 35S pre-rRNA synthesis.It should be stipulated that the model in Reviewer #2:The manuscript has two distinct parts, the first to study the in vivo formation of the inactive PolI dimer and its role in nutrient regulation of transcription in yeast, the second to present 4.9A Cryo-EM structures of yeast PolI and the PolI-Rrn3 complex. This reviewer does not feel competent to judge the validity or significance of these structures and so I will limit my review to the data on PolI dimer formation as a regulatory mechanism, The authors use an adaption technique called PICT (Protein interactions from Imaging of Complexes after Translocation) that uses the ability of Rapamycin to mediate an interaction between FKBP and FRB. In this adaption of PICT, FKBP is fused to Tub4 and RFP, and FRB to the large subunit of PolI. Addition of rapamycin then causes FRB-PolI to be recruited to the RFP tagged spindle poles. The authors then study the co-localization of a GFP-PolI fusion with the RFP tag as a measure of PolI dimerization in rapid growth in rich medium and after nutrient withdrawal. Similarly, and Rrn3-GFP fusion is used to study the formation of the active PolI-Rrn3 complex. A rapamycin resistant yeast strain is used to avoid the known effects of this drug on the Tor pathway.The technical approach is interesting and still novel. The question of whether or not an inactive PolI dimer forms in vivo and plays a part in regulation of the ribosomal RNA genes is an important one. This said, I found the data inadequate to support the key claims that nutrient deprivation or drug induced inhibition of protein synthesis or ribosome assembly leads to a significant accumulation of inactive PolI dimers and that could be reversed by refeeding or drug withdrawal.Already from The same problems occur in For these reasons, it is my opinion that the data do not convincingly demonstrate PolI dimerization in vivo or that it is modulated on nutrient deprivation, transcription inactivation or inhibition of protein synthesis.The evidence for an in vivo interaction between PolI and Rrn3 in The time course data in In short, the questions are interesting, the technique fascinating, but the data do not support the claims and interpretations.Reviewer #3:eLife.It's been known for quite a while that the Rrn3 transcription factor is needed for RNA Polymerase I activity. Several very recent papers, including this one, have shown that the structure of Rrn3 bound to Pol I appears mutually exclusive with dimerization of Pol I. This report goes beyond to the recent Nature Communications papers from Cramer and Schultz in providing in vivo evidence for this model of regulation. Using an improved fluorescent co-localization protocol (PICT), Pol I homodimers and Rrn3-Pol I heterodimers can be assayed under various conditions. Several treatments known to repress ribosomal transcription or to inhibit translation result in a shift from heterodimers to Pol I homodimers. Overall, I think this is an interesting and important paper that would be appropriate for Specific comments:For the PICT results, it's sometimes not clear how the individual cell panels illustrate the numbers shown in the bar graphs. For example, in In the last paragraph of the subsection “Dynamics in the assembly of Pol I complexes in response to nutrient availability”, the paper states that in the second stage after addition of nutrients, the pol I homodimers disappear, but with no increase in the Rrn3/pol I complex. Where do those polymerase molecules go, if not into the Rrn3 complex?I like the idea in final paragraph of the Discussion, which draws parallels between the Rrn3-pol I and Mediator – pol II interactions. However, use of the word "holoenzyme" in the pol II system has been very messy and I would avoid it. To my mind, a holoenzyme should have all the activities needed for promoter recognition and transcription initiation. There were some early papers proposing a pre-assembled holoenzyme of pol II with all the general transcription factors, but those models haven't held up. Later papers started misusing holoenzyme to mean the Mediator – pol II complex. For similar reasons, I don't think Rrn3-pol I would qualify as a holoenzyme. I would avoid the word altogether so as not to distract from the concept that factor interactions with the stalk and surrounding regions might help bring RNA polymerases to the promoter.eLife. Your revised article has been favorably evaluated by Kevin Struhl as the Senior Editor, Alan Hinnebusch as the Reviewing Editor and two reviewers.Thank you for resubmitting your work entitled "The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription" for further consideration at The majority of the remaining issues have been raised by the Reviewing editor, as described below:The authors have substantially improved the manuscript in several respects to address all of the major criticisms. They obtained a new mutation in the A43 CTT that specifically impairs homodimerization without affecting interaction with Rrn3, and showed that this mutation dampened the loss of Pol I:Rrn3 complexes from promoter DNA in starved cells, thereby providing evidence that dimerization is an important component of the down-regulation of Pol I recruitment and transcription. They better explained the partial cleft closure observed in their cryoEM structure of the Pol I monomer. They provided evidence that the A14 deletion does not simply destabilize Pol I as the means of impairing complex formation with Rrn3 and promoter binding of the complex. Importantly, they also added additional mutants that implicate helix 2 of the A14 subunit in Rrn3 association with Pol I, exploiting novel predictions of their Pol I:Rrn3 structure. They have also better explained the quantification of results from the PICT assay. As such, there are no remaining major criticisms that would require any additional experimentation to address.However, there are two major issues with their description of the results.First, their description of the new coIP experiment shown in Second, in the Discussion: the statement that "initial transcriptional inactivation is mainly driven by Pol I:Rrn3 disassembly." does not seem justified. The experiments in Reviewer #2:My major criticisms of the original manuscript concerned the use of the PICT technique and more precisely the analysis of images. The authors have tried to address these concerns, but it is clear that their quantification of Pol1 dimer and Pol1-Rrn3 complex formation is subject to considerable noise. This said, they now provide a detailed quantification protocol and have attempted to better explain the steps used to eliminate problems caused by the very high and variable GFP background signal. They have also improved their ChIP data, clearly validating their ChIP signal for Rrn3.Given the novelty of the approach and the importance of better understanding Pol1 dimerization and whether or not it has a regulatory role, I feel that on balance the data should now be published.Reviewer #3:eLife.The revised version of this manuscript does a much better job explaining the PICT assay, and now that I understand how to look at the data I find that the authors have made a persuasive case. These experiments are very important in showing that yeast RNA pol I uses controlled dimerization, in competition with Rrn3 binding, to regulate transcription in response to nutrients and translational status. This work is nicely complemented by some structures that confirm and extend recent work from other groups. I am in favor of publication in  Essential revisions:The consensus of the reviewers is that the structural information in the second half of the paper is valuable, even if mostly confirmatory of other recently published structures, as are the structure-function studies, even if somewhat limited in scope. However, the structural insights into the transition from presumably inactive dimer to active monomer gleaned from the structures were either not dramatic or not well explained in the text/figures and should be highlighted better. In addition, the possible effects of the A14 subunit deletion on Pol I integrity and abundance would have to be examined.We thank the reviewers for the positive comments regarding the scientific value of the structural results. Following the Editor recommendation on the Pol I dimer to monomer structural description, we have included three major changes in our revised version: (i) text revision to more clearly highlight the structural transition, (ii) two additional panels in Regarding the results obtained using the PICT assay in the first part of the paper, there are important concerns about whether the results are sufficient to conclude, as the title of the paper implies, that formation/disassembly of the homodimer versus Pol I-Rrn3 complexes are important events regulating rDNA transcription in response to nutrient availability. All three reviewers had difficulty understanding how the PICT results have been quantified; Rev. #2 had the most serious concerns, and has explained the difficulties thoroughly in his/her review. The large halo of background GFP signal compared to the small amount associated with the tethered RFP makes quantification of the co-localizing PICT signal highly questionable; and the images in We would like to thank all reviewers for their comments on the PICT assay. We apologize for the lack of information on how the images from PICT assays were analyzed, and for missing figures illustrating this. We have now detailed the procedure in the ‘Materials and methods’ section and also included a clear definition of the recruitment score used to quantify PICT (see comments to reviewers 1 and 2 for a deeper explanation). In addition, we appended a new figure supplement  with a sRegarding the influence of complex formation and disruption on rDNA transcriptional regulation, we have performed new experiments that shed light on our interpretation. To support our claim that homodimerization influences Pol I transcription, we designed a new mutant  that specifically abolishes Pol I ability to homodimerize  and 3. USpecific recommendations:1) It would be very helpful if they had an independent assay(s) for dimerization. Two choices. First, they could formaldehyde crosslink under the various in vivo situation and do a co-IP with essentially the same constructs they already have. Second, they could replace the targeting tags with Gal4, which would allow Gal4-Pol I fusions to targeted to Gal4 binding sites (assayed by ChIP). If the model is correct, one should see the non-Gal4 version of Pol I associating with Gal4 binding sites under starvation. Both of these approaches might be better for kinetics and quantification, but the real virtue of them is that they are independent assays for dimerization.We thank the Editor for this useful recommendation. We have performed co-IP experiments after crosslinking on a diploid strain harboring TAP and MYC-tags at the C-termini of each A190 allele. After clarification of whole cell extracts to separate the soluble and chromatin insoluble fractions, we used both fractions to pull down A190-TAP with IgG resin. Thereafter, pulled down proteins were de-crosslinked at 65 ºC for several hours and we analyzed co-IPed A190-MYC by western blot. As shown in “In addition, we performed co-immunoprecipitation experiments after crosslinking, using a diploid strain where one A190 allele was tagged with TAP (A190-TAP) and the second with MYC (A190-MYC). Homodimerization could be observed in the soluble fraction of starved cells only . Overall2) The authors haven't demonstrated "regulation", so their title needs to be softened. What they have "shown" is that the dimerization and change in complexes is associated with regulation.Following the Editor recommendation and in the view of the new experiments, we have now changed the title to “The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription”, which more accurately reflects the significance of our results.Reviewer #1:[…] General critique:Previous work on Pol I in yeast has shown that Rrn3 binding to the enzyme is required for Pol I recruitment to the promoter, and that Pol I can form homodimers in solution and was crystallized as a homodimer with structural features indicating an inactive conformation. This paper uses the PICT assay to provide convincing evidence that Pol I homodimers can form in cells, but only in stress conditions including starvation for carbon and nitrogen, arrest of translation elongation by cycloheximide, or treatment with a drug shown to impair ribosome biogenesis. The evidence that homodimerization occurs in cells is firm, and the fact that it was observed only in starved/stressed cells supports its role as a regulatory mechanism for inactivation of Pol I under conditions where new ribosome synthesis is unwanted. Unfortunately, however, there is no direct evidence establishing its importance in down-regulating Pol I function. In fact, the kinetics of homodimerization versus PolI:Rrn3 interaction shown in We thank the reviewer for this useful suggestion. In spite of the challenge, we succeeded in finding a mutant that abolishes homodimerization while maintaining Pol I–Rrn3 levels . When this strain is cultured in starving medium, ChIP experiments show that the promoter levels of Pol I and Rrn3 are higher than in the WT strain. This proves that homodimerization is required for full down-regulation of Pol I recruitment/transcription and improves the significance of our results. To improve simplicity for the reader, we eliminated the former A43ΔCt mutant (Δ277-326) and used the same nomenclature for our more surgical mutant, so that in the revised manuscript A43ΔCt corresponds to A43Δ307-326. The results of this mutant appear at two sections in the revised manuscript:“Therefore, we studied Pol I dimerization upon partial deletion of this structural element . In this mutant, Pol I homodimerization is impaired , confirm“To further investigate the effect of starvation on Pol I transcription, we used our A43 C-terminal truncation abolishing Pol I dimerization. […] Furthermore, in these conditions, association of Pol I along the rDNA gene and of Rrn3 at the promoter increase about 6-fold with respect to the wild-type.”In addition, we studied the behavior of a chimeric strain where A43 is covalently fused to Rrn3 , so that all Pol I is complexed to Rrn3 and no free monomeric or dimeric Pol I can form. This strain presents higher association levels of A190 along the entire rDNA gene and recovers faster from starvation than the wild-type. This indicates that, when Pol I dimers cannot form and Pol I–Rrn3 levels are increased, rDNA transcription is upregulated in starving cells. The corresponding manuscript section now reads:“We also used a strain expressing a Pol I–Rrn3 chimera that cannot form Pol I homodimers . […] Moreover, recovery of CARA from nutrient-depleted medium is faster than for wild-type cells .”The PICT assay also gives evidence of reduced Pol I:Rrn3 association in starved cells by ~50%, and there is an even greater reduction in Pol I/Rrn3 occupancy of the Pol I promoter measured by ChIP. However, the Western in We agree that the PICT technique does not provide evidences about the affinity of the interaction between Pol I and Rrn3, but only about relative levels of the complex. As the reviewer pointed out, the levels of Rrn3 drop upon starvation. This suggests that degradation of Rrn3 might be a mechanism that the cell employs to regulate Pol I–Rrn3 levels during the first stages of starvation. We performed western-blots of Rrn3 and A190 at different time points . Althoug“We show that nutrient depletion induces a rapid reduction in the levels of Pol I–Rrn3, which correlates in time with a marked decrease in promoter association of both Pol I and Rrn3. […] This is in agreement with previous observations of rDNA transcription inactivation by inhibition of TOR signalling .”The cryo-EM analysis of the Pol I monomer at 4.9 Å resolution and the Pol I::Rrn3 complex at 7.7 Å resolution are valuable, although another group recently published a Pol I-Rrn3 complex at higher resolution (4.9 Å) that seems to contain all of the same key features and allows the same insights into this important intermediate in Pol I activation as those described here-thus diminishing the significance of the current structure of the complex. The higher resolution of their monomeric Pol I compared to that recently published by another group, should allow a superior comparison between the monomeric and dimer Pol I , from which they conclude that the transition from dimer to monomer "involves partial cleft closure and increased flexibility of critical motifs". However, the relevant figure  does notPartial cleft closure in the dimer to monomer transition is half the closure required to achieve the transcription-competent state and, importantly, no further closure is observed upon Rrn3 binding. Both observations highlight the relevance of this conformational change. In order to improve clarity in our text, we have included a more detailed explanation of this structural transition that involves two events: (i) increased flexibility of 3 domains, the stalk, the DNA-mimicking loop and the A12.2 C-terminal Zn-ribbon; and (ii) partial cleft closure by approach of the clamp and protrusion domains. The revised manuscript incorporates these changes in the subsection “Cryo-EM structures of monomeric Pol I alone and in complex with Rrn3”. As a second major change, we have included new panels in Regarding crystal contacts, domains that become flexible upon monomerization  are not directly involved in crystal maintenance. Moreover, the cryo-EM structure of Pol I dimers in solution at 7.5 Å resolution is virtually identical to that of the crystals . Therefore, it is not possible that these critical motifs are fixed in the dimer structure as a result of the crystal contacts. A sentence stating this has been included in our revised manuscript:“When compared with the crystal structure of dimeric Pol I , which is essentially identical to dimeric Pol I in solution , our monomeric Pol I structure presents a rearranged cleft entrance where the clamp coiled-coil and the protrusion approach by about 4 Å .”The experiments showing that deleting the A14 subunit weakens Pol I:Rrn3 interaction by PICT assay and reduces Pol I/Rrrn3 occupancy at the promoter by ChIP assay are valuable, but they have not ruled out the possibility that removing this subunit perturbs other Pol I subunits, e.g. A43 with which it seems to interact with extensively, and thus impairs Pol I interaction with Rrn3 indirectly by disrupting A43:Rrn3 contacts rather than eliminating the A14:Rrn3 interactions evident in the cryoEM structure. It is even possible that the steady state level of Pol I is reduced by the A14 deletion and contributes to the reduced yield of Pol I:Rrn3 interactions and Pol I recruitment. Thus, the effects of the A14 subunit deletion on Pol I integrity and abundance should have been examined. But even more importantly, the authors should have used the molecular details of the cryoEM structure to more surgically disrupt the A14:Rrn3 interaction by truncating or substituting helix 2 of A14 in a way designed not to disrupt A14 interactions with other Pol I subunits. This seems like a missed opportunity to exploit their structure.To rule out the possibility that mutations of A14 perturb other Pol I subunits, we performed western-blot analysis against several subunits in the complex . For theIn summary, the paper represents a collection of three different lines of work joined by the theme of regulating Pol I:Rrn3 interaction. The evidence for homodimer formation in cells is interesting and valuable but it is unclear that it constitutes an important means of impeding Pol I:Rrn3 association and promoter recruitment rather than being a byproduct of the loss of this complex by other means that serves only to protect Pol I from degradation. The cryoEM structures are valuable, but a Pol I:Rrn3 complex of higher resolution was already published. The structure-function analysis of A43 and A14 did not exploit the cryoEM structure, as it involved deletions of large segments that are not present at their interfaces with Rrn3. The result that deleting the TA loop in A14 leads to greater Pol I:Rrn3 association and promoter occupancy is the most interesting finding, but the molecular mechanism is not obvious from the structure. Taken together, the paper is not a strong candidate for eLife, even if the authors can address all of the shortcomings in the experiments and interpretations mentioned above or below.We believe that the three lines of work and the diversity of methods that we employed to study the influence of Pol I complexes on transcriptional activation represent a major strength of our manuscript. Overall, the manuscript has been improved in different ways:1) Pol I homodimerization is now confirmed by Co-IP after crosslinking experiments . Moreove2) New mutants allow specific targeting of Pol I complexes to discern the contribution of each assembly in Pol I regulation. As mentioned, we have further exploited our Pol I–Rrn3 structure to design mutants that show the biological relevance of subunit A14. Helix-α2 mutants specifically disrupt Pol I–Rrn3 assembly and show that Pol I–Rrn3 complexes are required for proper Pol I activation in growing cells. Moreover, cells expressing a mutated form of A14 lacking the TA-loop, show higher levels of Pol I–Rrn3, both in growing and starved cells . Since R3) We used ChIP to study the dynamics of Pol I activation upon perturbation in nutrient availability (further described below). Down regulation of Pol I upon nutrient deprivation follows a similar pattern than Pol I–Rrn3 disassembly. This is in agreement with the mutagenic analysis and suggests that the levels of Pol I–Rrn3 dictate activation of Pol I transcription in growing cells and the first minutes upon nutrient deprivation. Upon re-feeding, rDNA association follows a linear behavior, different to the dynamics of Pol I–Rrn3 assembly. We hypothesize that this reflects the combination of Pol I release from homodimers and the increment in the levels of Pol I–Rrn3.In conclusion, we believe that our manuscript represents a significant advance in the field.Other major criticisms:The PICT approach requires Rapamycin, which is known to impair ribosome biogenesis. It's unclear from the genotypes provided whether both alleles of TOR1 in the diploid strains they used are the tor1-1 allele conferring resistance to Rap, which would seem to be required. Even if this is so, it seems important to show directly, if it's not in the literature already, that treatment of their PICT strain with rapamycin has no effect on Pol I recruitment or 35S pre-rRNA synthesis.tor1-1 alleles as well as the two fpr1 alleles deleted. Following the reviewer’s suggestion, we performed ChIP analysis in our tor1-1 mutant strain and concluded that Rapamycin treatment has no effect on Pol I association to the rDNA promoter. The corresponding ChIP experiment has been included as a new panel A in We have clarified the genotype in “Since Pol I transcription is down-regulated by rapamycin, all subsequent experiments were performed in rapamycin-insensitive strains carrying the tor1-1 mutation , so that addition of this compound has no effect on Pol I association to rDNA promoters .”We conducted ChIP experiments at similar time intervals as PICT after nutrients were severely altered. Because performing ChIP experiments at many time points is very challenging, we chose time points that cover the different stages observed in complex assembly by PICT. Our results show that rDNA promoter association of Pol I and Rrn3 extinguishes and recovers with kinetics in the same time scale as changes observed in the levels of Pol I homodimers and Pol I–Rrn3 complexes detected by PICT. Upon nutrient deprivation, promoter association drops exponentially and correlates with changes in Pol I–Rrn3 levels. The rate curve resembles that of Pol I–Rrn3 clearance, but the latter only reaches 40% of the initial value, in accordance with the overall levels of Rrn3 as shown by western-blot. This is in agreement with the experiments of A14 mutants and the Rrn3-A43 fusion strain (see above). Altogether, the data show that Pol I–Rrn3 levels influence Pol I activation in growing cells and during the first minutes upon starvation, but they are not sufficient to regulate Pol I inactivation in long-term starved cells. Upon nutrient re-supplementation, promoter association increases steadily while Pol I–Rrn3 formation follows different dynamics, where Pol I–Rrn3 levels grow at an early stage, remain constant during a second stage and then increase again up to growing levels. This suggests that Pol I homodimers represent a rapid enzyme supply to generate Pol I–Rrn3 complexes. This is supported by the observation that cells where Pol I homodimerization is impaired (new A43ΔCt mutant) present higher levels of Pol I and Rrn3 at the promoter and of the former within the gene. This mutant also suggests that in cells starved for 2 hours, Pol I homodimerization accounts for about 60% of Pol I inactivation, according to Pol I promoter. The description of the new ChIP experiments has been included in the revised manuscript, which now reads:“ChIP experiments performed at an equivalent regime show that promoter association of both A190 and Rrn3 drops by two thirds within the first 10 minutes from starvation but requires about 2 hours to reach completion , left.  UpoReviewer #2:The manuscript has two distinct parts, the first to study the in vivo formation of the inactive PolI dimer and its role in nutrient regulation of transcription in yeast, the second to present 4.9A Cryo-EM structures of yeast PolI and the PolI-Rrn3 complex. This reviewer does not feel competent to judge the validity or significance of these structures and so I will limit my review to the data on PolI dimer formation as a regulatory mechanism, The authors use an adaption technique called PICT (Protein interactions from Imaging of Complexes after Translocation) that uses the ability of Rapamycin to mediate an interaction between FKBP and FRB. In this adaption of PICT, FKBP is fused to Tub4 and RFP, and FRB to the large subunit of PolI. Addition of rapamycin then causes FRB-PolI to be recruited to the RFP tagged spindle poles. The authors then study the co-localization of a GFP-PolI fusion with the RFP tag as a measure of PolI dimerization in rapid growth in rich medium and after nutrient withdrawal. Similarly, and Rrn3-GFP fusion is used to study the formation of the active PolI-Rrn3 complex. A rapamycin resistant yeast strain is used to avoid the known effects of this drug on the Tor pathway.The technical approach is interesting and still novel. The question of whether or not an inactive PolI dimer forms in vivo and plays a part in regulation of the ribosomal RNA genes is an important one. This said, I found the data inadequate to support the key claims that nutrient deprivation or drug induced inhibition of protein synthesis or ribosome assembly leads to a significant accumulation of inactive PolI dimers and that could be reversed by refeeding or drug withdrawal.Already from This reviewer mentions a list of points that are key to correctly interpret the PICT assay. We agree that the Methods section and some of the figures lack clarity and might have been misleading. For this reason we appreciate his/her comments and have modified the manuscript to address them as follows:tor1-1 mutant and deleted fpr1) and that express A190-FRB-GFP (OGY0346). These cells showed that some A190-FRB-GFP signal is also observed through the entire nucleus, which the reviewer called GFP “halo”. Since the anchoring platform (Tub4-RFP-FKBP) resides in the nuclear envelope, depending on the orientation from where each cell is imaged, the RFP spot corresponding to the anchor might be surrounded by more or less of this GFP halo. Since in the in focus field of view the nucleus of each cell is oriented randomly, we expect a high variability. As shown in The vast majority of A190 accumulates in a sub-compartment of the nucleus. We imaged the perimeter of the nucleus using the Nic96-RFP marker (a component of the nuclear pore complex) in cells with the genetic background used for PICT . As expected, there was no significant difference between the recruitment score determined in growing and starved cells.Our image analysis workflow, which is now fully detailed in As mentioned, the mean GFP halo intensity is comparable in growing and starved cells. Since the quantification of the recruitment score is done on multitude of cells (see comment to reviewer 1), the cell-to-cell variability is averaged out. For this reason, it is not necessary to take into account this GFP signal in order to establish relative differences between our samples.The optical system of our microscope ensures that bleed-through from the GFP to the RFP channel is negligible since we have used specific combination of optimized beam splitters, excitation and emission filters (cubes) for each fluorophore type and since the images were recorded sequentially. As mentioned earlier, the vast majority of the A190-GFP signal is in the nucleolus. If there was bleed-through from the GFP to the RFP channel, we should see this reflected in an apparent RFP signal localizing in this sub-compartment of the nucleus. Among other controls, the blank experiments  show thaIn summary, detection of Pol I homodimers by PICT cannot result from artifacts caused by the GFP halo, the incubation in different medium or bleed-through between the GFP and the RFP channels.The same problems occur in For these reasons, it is my opinion that the data do not convincingly demonstrate PolI dimerization in vivo or that it is modulated on nutrient deprivation, transcription inactivation or inhibition of protein synthesis.We have engineered anchoring platforms at the spindle pole body (SPB). During the cell cycle, SPB duplicates and separates to the daughter and mother cell. Therefore, cells hold 1 or 2 anchoring platforms depending on the cell cycle stage they are at during imaging. Starved cells also hold 1 or 2 anchoring platforms. When holding 2 anchoring platforms, the observed anchor-to-anchor distance spread is large and depends on the cell cycle stage at the moment of nutrient deprivation. These differences, however, are averaged out in our quantifications because the overall PICT recruitment score is computed over a minimum of 67 cells for each biological replicate . We apologize because some of the images we had chosen presented highly varying anchor to anchor distances, suggesting that samples had been analyzed at different cell cycle stages. We have now chosen cell images to correct this misunderstanding. The new Following the reviewer’s suggestion, we have performed a quantitative PICT assay to estimate the recruitment score of Pol I homodimers upon diazaborine and cycloheximide treatment in growing cells . As mentThe evidence for an in vivo interaction between PolI and Rrn3 in We agree with this reviewer that previous figures did not illustrate well the spread of recruitment typically observed in PICT experiments. As mentioned, we have modified the figures to include a zoomed inset around the anchors of a representative cell. In this inset we have color-overlaid segmented spots in the GFP channel, the RFP channel and the intersection of both channels . As we have shown for cells expressing A190-GFP, the GFP signal around the anchor in cells expressing Rrn3-GFP is similar in both growing cells and starved cells . Rrn3-GFRegarding disparity between Pol I–Rrn3 levels and Pol I and Rrn3 promoter association in starved cells, we agree that reduction of Pol I–Rrn3 levels are not sufficient to fully account for inactivation of rDNA transcription. However, we now present a more surgical mutation of the A43 C-terminal tail (new A43ΔCt mutant) where Pol I dimerization is impaired  but the To fulfill the reviewer’s requirement, we have performed ChIP experiments at two different regions within the rDNA gene, i.e. 18S and 25S . Our resThe time course data in We agree with the reviewer that PICT does not tell about absolute levels of protein complexes but it does reflect relative changes between different time points. As mentioned above, we have now included time-course ChIP experiments showing that, upon refeeding, Pol I is rapidly associated to promoters to activate transcription. Thus, in spite of an increment in the assembly of Pol I–Rrn3 complexes, these will also rapidly disassemble as a result of rRNA synthesis. Therefore, Pol I–Rrn3 levels as seen by PICT are not expected to rise until Pol I–Rrn3 assembly is faster than Pol I incorporation to rDNA, which we observe only at the later stages of recovery from starvation. The absolute quantification of Pol I dimer copies goes beyond the scope of this study. Nonetheless, our novel A43ΔCt mutant shows that Pol I homodimerization is responsible for about 60% of Pol I inhibition in cells starved for 2 hours, highlighting the relevance of this regulatory mechanism.In short, the questions are interesting, the technique fascinating, but the data do not support the claims and interpretations.Reviewer #3:[…] Specific comments:For the PICT results, it's sometimes not clear how the individual cell panels illustrate the numbers shown in the bar graphs. For example, in We have now presented more clear images of cells so that the quantification can be more easily examined. Please refer to responses to reviewer 2 for a thorough explanation on how PICT images have been quantified. The methodology followed to analyze and quantify these assays had not been explained accurately. We have extended the description in the Materials and methods section. We have also modified all the figures to clarify the PICT experiments. Since we had article space limitation and since the spots of the recruited prey-GFP at the anchoring platforms are small, we could not show many cells for each treatment. Instead we selected cells that were representative of each sample. We have adjusted the contrast to facilitate the identification of the recruited prey-GFP. Finally, we have updated the image to include a zoomed inset of the area around the anchoring platforms and also used a color overlay that labels pixels according to segmented spots in both channels.In the last paragraph of the subsection “Dynamics in the assembly of Pol I complexes in response to nutrient availability”, the paper states that in the second stage after addition of nutrients, the pol I homodimers disappear, but with no increase in the Rrn3/pol I complex. Where do those polymerase molecules go, if not into the Rrn3 complex?As explained for reviewer 2, we interpret that these molecules are engaged in active transcription, so they should be loaded along the rDNA gene. This would explain that there is no apparent change in the overall levels of Pol I–Rrn3 in the middle stage of recovery from starvation.I like the idea in final paragraph of the Discussion, which draws parallels between the Rrn3-pol I and Mediator – pol II interactions. However, use of the word "holoenzyme" in the pol II system has been very messy and I would avoid it. To my mind, a holoenzyme should have all the activities needed for promoter recognition and transcription initiation. There were some early papers proposing a pre-assembled holoenzyme of pol II with all the general transcription factors, but those models haven't held up. Later papers started misusing holoenzyme to mean the Mediator – pol II complex. For similar reasons, I don't think Rrn3-pol I would qualify as a holoenzyme. I would avoid the word altogether so as not to distract from the concept that factor interactions with the stalk and surrounding regions might help bring RNA polymerases to the promoter.We have now re-written this section to remove the Pol I holoemzyme concept, while keeping the comparison with the Pol II and bacterial systems. The revised manuscript includes a revised paragraph at the end of the Discussion.[…] However, there are two major issues with their description of the results.First, their description of the new coIP experiment shown in We thank the Reviewing editor for this useful recommendation. In addition to a more detailed description of the coIP experiments, labelling of lanes 8 and 9 in “In addition, we performed co-immunoprecipitation experiments after crosslinking, using a diploid strain where one A190 allele was tagged with TAP (A190-TAP) and the second with MYC (A190-MYC). […] The absence of histone H3 in the soluble fraction indicates that there is no contamination from the chromatin insoluble fraction .”Second, in the Discussion: the statement that "initial transcriptional inactivation is mainly driven by Pol I:Rrn3 disassembly." does not seem justified. The experiments in We agree with the view of the Reviewing editor and have now incorporated this concept in the Discussion section, as well as in the new “When nutrients are depleted, Pol I–Rrn3 levels and promoter association drop exponentially whereas Pol I only homodimerizes subsequently. […] At a later stage, Pol I homodimerization remains a major factor limiting transcription.”“


Aims and method The mean delay for bipolar disorder diagnosis is 10 years. Identification of patients with previous hypomania is challenging, sometimes resulting in misdiagnosis. The aims of this study were: (a) to estimate the proportion of primary care patients with depression currently taking antidepressants who have undiagnosed bipolar disorder and (b) to compare a brief 3-item manic features questionnaire with the Hypomania Checklist (HCL-13). The sample comprised patients with a recorded diagnosis of depression, either on long-term antidepressant therapy or with previous multiple courses of antidepressants.Results Of 149 participants assessed, 24 (16.1%) satisfied criteria for bipolar disorder. Areas under the curve (AUC) for the 3-item questionnaire and the HCL-13 were similar  but positive predictive values (PPV) were low.Clinical implications Bipolar disorder may be underdiagnosed in primary care. A 3-item questionnaire could be used by general practitioners to screen for bipolar disorder in their patients with depression. Our focus on these brief assessment instruments was stimulated by the need for questionnaires that could be used easily in primary care consultations, which are usually no longer than 10–15 min.In this context, we aimed to estimate unrecognised bipolar disorder within a sample of primary care patients with depression who were taking antidepressant medications, as well as to evaluate the potential clinical utility of two short screening questionnaires for bipolar disorder: the 13-item Hypomania Checklist (HCL-13)Practice managers of 9 primary care practices in the west of Scotland identified from their databases 2633 patients who (a) had a recorded diagnosis of depression and (b) were either currently taking long-term antidepressants (more than 12 months) or had had 3 or more courses of antidepressants in the past 5 years. Then, 1860 potentially eligible patients were reviewed by their general practitioner (GP) for eligibility and 1833 written invitations were sent by post, with participant information sheets, on behalf of the research team. There were 204 people who responded to this invitation and 151 were interviewed, giving a response rate of 8.23%. The study was approved by the West of Scotland Research Ethics Committee .13 in person in order to obtain a gold-standard diagnosis. An assessment of practice notes was also conducted to corroborate findings from the SCID-1 assessment and to clarify medication regimens. Participants completed the HCL-13 and a brief 3-item bipolar screening questionnaire. This 3-item questionnaire (with a maximum score of 6) was based on the three core diagnostic features for bipolar disorder taken from DSM-5: elevated mood, severe irritability and overactivity.14 The questions and scoring were as follows:Have you ever had a period of time when you were feeling so good, ‘high’, excited or ‘hyper’ that other people thought you were not your normal self or you were so ‘hyper’ that you got into trouble? (Definitely no (score 0), perhaps yes (score 1), definitely yes (score 2).)What about a period of time when you were so irritable that you found yourself shouting at people or starting fights or arguments? (Definitely no (score 0), perhaps yes (score 1), definitely yes (score 2).)What about a period of time when you were physically much more active than usual, for example, when you had lots of different projects on the go at the same time? (Definitely no (score 0), perhaps yes (score 1), definitely yes (score 2).)A research nurse completed the Structured Clinical Interview for DMS-IV (SCID-1)t-test, chi-squared test and chi-squared test for association, and were conducted using SPSS version 21 for Windows. To assess the likely clinical usefulness of the HCL-13 and 3-item questionnaires, in terms of differentiating between bipolar disorder and major depressive disorder (MDD), we calculated sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) using MedCalc and verified these manually. Positive and negative clinical utility measures were calculated using an online calculator constructed by the developer of the MedCalc test (www.psycho-oncology.info/cui.html).15Analyses included the Student's n = 149 had a DMS-IV diagnosis of bipolar disorder  and the remainder had a diagnosis of MDD .Of those who had a full diagnostic assessment, two participants were excluded because their SCID results suggested a primary diagnosis of alcohol-related mood problems. In total, 24 participants from our final sample of v. 6.61, P= 0.001) and on the 3-item questionnaire .There were no significant differences between the bipolar disorder group and the MDD group in terms of age, gender distribution and socioeconomic status  . Howevern = 125) and bipolar disorder (n = 24). For the HCL-13, an area under the curve (AUC) of 0.72 (95% CI 0.61–0.84) demonstrates a ‘fair’ overall ability of the questionnaire to discriminate effectively between the two groups. Similarly, an AUC of 0.79 (95% CI 0.69–0.89) for the 3-item questionnaire also demonstrates a ‘fair’ overall ability. An AUC of over 0.80 is considered to demonstrate a ‘good’ overall ability to discriminate.16The receiver operating characteristics (ROC) curves in Similarly, a threshold score of 4 on the 3-item questionnaire had a sensitivity of 83.33%, specificity of 64.23% and PPV of only 31.25%. At a higher threshold of 5 points, the sensitivity was 66.67%, specificity was 78.05%, PPV was 37.21% and NPV was 92.31%. At a lower threshold of 3 points, sensitivity was 91.67%, specificity was 43.09%, PPV was 23.91% and NPV was 93.36%. Therefore, a threshold of 4 points was chosen to give the best balance between these different parameters.The positive clinical utility – the ability of the test to confirm cases of bipolar disorder – was poor for both tests. The negative clinical utility a measure of screening and excluding bipolar disorder, was slightly better for the 3-item questionnaire than the HCL-13 at our threshold values: 0.611 (95% CI 0.541–0.682) compared with 0.582 (0.509–0.655). These thresholds were chosen to give the best balance between sensitivity, specificity, PPV, NPV and positive and negative clinical utilityet al10 found a prevalence of 7.3% whereas Smith et al found a prevalence of 9.6%. Both studies assessed patients with depressive disorder who had been prescribed antidepressant medication. It is possible that the addition in our study of participants who had previously been prescribed multiple courses of antidepressants led to a higher prevalence estimate for bipolar disorder, because unrecognised bipolar disorder is more common in patients with more severe and enduring depression.17One of the goals of this study was to estimate how common DMS-IV bipolar disorders might be in a sample of primary care patients taking antidepressant medication, specifically those patients who were either taking antidepressant therapy for more than 12 months or who had had multiple courses of antidepressants over the preceding 5 years. We found that 16.1% of our sample had bipolar disorder. This rate is higher than in previous literature from the UK. In samples of primary care patients, Hughes 18 In a review of brief screening instruments for depressive disorder in a low-income country, Hanlon et al19 concluded that the low PPV at acceptable sensitivity levels may preclude their use in clinical settings.We also aimed to compare the HCL-13 and a brief 3-item questionnaire in terms of their ability to differentiate between patients with MDD and bipolar disorder. We found that the AUC for HCL-13 was 0.72, while for the 3-item questionnaire it was slightly higher, at 0.79. For both tests the ability to discriminate between MDD and bipolar disorder in terms of sensitivity and specificity was reasonable, but PPVs were low. This is a function of the low prevalence of bipolar disorder in primary care setting, but represents a potential limitation in terms of the usefulness of these instruments to GPs in everyday clinical practice.8Nevertheless, we would argue that there may be some use in primary care for these brief screening instruments alongside additional assessments, for example whether patients have a strong family history of mood disorder. The 3-item questionnaire in particular may be useful to GPs in terms of fulfilling the NICE requirement to assess all patients with depression for a history of manic features. The high NPV of 95% means that clinicians may find this useful for excluding a diagnosis of bipolar disorder in their patients with depression. The NICE guidance states that the ideal instrument should be brief, easy to administer and to score, and should be able to be interpreted without extensive and specialist training This was a reasonably large study that took a systematic approach to screening patients in primary care settings. We used definitions of bipolar disorder and MDD based on formal diagnostic classifications by using SCID assessment. The study included a range of people from different social backgrounds, with the majority living in some of the most deprived areas of Scotland. However, it may have been helpful to have more baseline demographic information on patients, such as ethnicity, family history of bipolar disorder and age at onset of depression, and the study may be subject to recall bias because it relied on the patient's recall of prior episodes of manic symptoms rather than a corroborative history. There may also be an issue of selection bias, because GPs were able to exclude certain participants if they felt that they were not suitable for this study As a result of this, and the fact that only one method of recruitment was used in this study, there was a relatively small final sample given the number of invitations sent, which may have led to ascertainment bias. Moreover, the SCID interviewer was not masked to HCL-13 and 3-item scores, which may also have been a source of bias.Another potential limitation is that the 3-item questionnaire had no requirement for a minimum duration of symptoms. It is possible that individuals with brief periods of affective instability, such as those with borderline personality disorder, would be inclined to respond positively to these questions. Similarly, we did not take a history of alcohol or drug use, and while we did exclude alcohol or substance-induced mood disorders, the use of psychoactive substances could have led to false positives with the 3-item questionnaire.A brief 3-item questionnaire may be clinically useful for GPs who wish to screen for manic features in patients with MDD. This could prompt more detailed assessment, such as an appointment with a relative or friend to obtain a collateral history before assessing the need for a referral to secondary care. Further studies are required in larger samples to assess the clinical usefulness of this test in screening, ideally without the issues of recruitment faced in this study It may also be helpful to develop the 3-item questionnaire further, perhaps with the addition of other items such as the duration of symptoms. While the addition of items would lead to a more statistically sound test, it would also take longer to administer such a test, which may make it less clinically useful.A proportion of primary care patients with MDD, perhaps as many as 1 in 5, may have undiagnosed bipolar disorder. For busy clinicians working in a time-restricted environment, we suggest that a brief 3-item questionnaire may be a useful screening tool for bipolar disorder and a first step towards a more comprehensive assessment.


Post-hoc planned comparisons revealed that MEPs at N20+2 and N20+4 were inhibited compared to unconditioned MEPs. However, the level of inhibition (SAI) was similar in the PAIN and NEUTRAL conditions. This suggests that the interplay between pain and sensorimotor integration is not mediated through direct and rapid pathways as assessed by SAI, but rather might involve higher-order integrative areas.Sensorimotor integration is altered in people with chronic pain. While there is substantial evidence that pain interferes with neural activity in primary sensory and motor cortices, much less is known about its impact on integrative sensorimotor processes. Here, the short latency afferent inhibition (SAI) paradigm was used to assess sensorimotor integration in the presence and absence of experimental cutaneous heat pain applied to the hand. Ulnar nerve stimulation was combined with transcranial magnetic stimulation to condition motor evoked potentials (MEPs) in the first dorsal interosseous muscle. Four interstimulus intervals (ISI) were tested, based on the latency of the N20 component of the afferent sensory volley . In the PAIN condition, MEPs were smaller compared to the NEUTRAL condition ( Moving our limbs and interacting within the world involves the generation of a motor intention and motor commands, monitored by feedback arising from proprioceptive, tactile and visual information. This constant integration of sensorimotor information is essential to maintain a coherent representation of our body as well as to adapt to new conditions. When movement and sensation are discordant, however, sensorimotor integration can break down. For example, after amputation, phantom hand movements lead to activity in muscles that do not normally control the hand ,2,3, whiSupport for Harris’ hypothesis comes from research with chronic pain patients showing that artificially creating sensorimotor disturbances can increase pain ,6 and thSensory inputs influence motor cortex activity through several pathways of varying complexity, including direct monosynaptic connections from the sensory to motor cortex ,16. Relan = 8) had less SAI than control subjects in the M1 representation of the infraspinatus muscle, an important dynamic stabiliser of the shoulder  × 100. Therefore positive values reflect facilitation and negative values reflects inhibition related to the presence of the conditioning stimulus. Means are presented with standard deviations in parentheses and Pearson correlations were used to explore relationships between variables. All statistical analyses were computed using SPSS 13.0  for Windows.The peak-to-peak amplitude of all MEPs was measured using a custom-made program (Spike2). For each condition, data from the two blocks were pooled and analysed using a two-way repeated measures ANOVA with factors Condition  and Stimulation Interval (MEPThe average stimulation intensity to evoke an M-wave amplitude of between 5% and 10% of the M-Max was 12.5 (2.6) mA. The mean latency of the N20 evoked by electrical stimulation of the ulnar nerve was 20.5 (1.0) ms. The average TMS intensity that evoked a MEP of approximately 1 mV in the FDI was 42.7% (8.3) of the maximal stimulator output. This corresponded to 122 (11) % of the resting motor threshold. The thermal stimulation was reported as painful by all subjects, and the mean pain intensity across the two PAIN blocks was 3.7/10 (2.0) on the numerical rating scale.test) and at each of the four ISIs (MEPcond) between the peripheral stimulus and the TMS pulse. Visual inspection of this figure shows that MEPs were smaller in the PAIN than in the NEUTRAL condition, and that for both conditions MEP amplitudes were inhibited (relative to MEPtest) at the N20+2 and N20+4 ISIs. MEP amplitudes averaged across all blocks and all subjects  %) and that they varied across Stimulation Intervals . Post-hoc analyses showed that MEPs were significantly reduced compared to MEPtest at two ISIs: N20+2 ms (p < 0.001) and N20+4 ms, (p = 0.002). There was no interaction between Condition and Stimulation Interval (p = 0.189), indicating that PAIN did not affect SAI.subjects  showed acond amplitude relative to MEPtest separately for each ISI and each Condition. The substantial overlap of the two curves illustrates not only the absence of an interaction, but also that the percentage change in the MEP amplitude was similar in both conditions. To investigate whether this effect was present only in the averaged data, or whether the amount of inhibition exhibited by each subject was indeed very similar in both conditions, we plotted the percentage of inhibition averaged across N20+2 and N20+4 (the two ISIs that were significantly different from MEPtest) in the NEUTRAL condition (average SAI = −41.6% (28.4)) against the same average measured during the PAIN condition (average SAI = −41.4% (27.8)) (right panel of p = 0.002) between the amount of SAI in each condition, with SAI in the NEUTRAL condition explaining 71.6% of the variance in SAI in the PAIN condition. We interpret this as evidence that the absence of a difference in SAI between the NEUTRAL and PAIN conditions cannot simply be explained by high intra-subject variability in SAI. Rather, the amount of inhibition for a given subject was very similar in both conditions .Finally, we explored whether the percentage of inhibition of MEPparadigm , no signIn this study we investigated whether heat pain applied to the hand interferes with sensorimotor integration of a hand muscle as measured with SAI. The results showed (1) that muscle responses were inhibited in the presence of pain; (2) that significant inhibition was observed when the TMS pulse occurred 2 or 4 ms after the arrival of the afferent volley in S1; and (3) that the percentage of SAI was similar in both the presence and absence of heat pain.test amplitudes were expected in the PAIN condition, we could have increased the stimulation intensity to ensure comparable MEPtest amplitudes in the NEUTRAL and PAIN conditions. Instead we chose to keep the stimulation intensity constant across conditions because previous studies showed that the amount of SAI in FDI is robust for MEPtest amplitudes between 0.2 and 1 mV [test is above 130% of the motor threshold [test amplitude during pain did not result in a floor effect  which could have limited our ability to observed pain-related modulation in SAI, as MEPtest amplitudes of 1 mV and 0.5 mV (amplitudes close to what we observed in our NEUTRAL and PAIN conditions) result in similar amounts of SAI [The observation that heat pain inhibited FDI motor responses by approximately 40% is consistent with several other studies showing that experimental cutaneous and muscle hand pain reduce the corticospinal excitability of hand muscles ,27,28,29and 1 mV ,35. Althhreshold . It is is of SAI . This ids of SAI ).Despite the fact that acute pain has been shown to alter sensory processing and reduce corticospinal output ,29,30,31One limitation of the TMS technique used in the present study is that it cannot discriminate whether the reduction in MEP amplitude observed in the presence of pain was due to cortical or spinal mechanisms. However, the results of previous studies using complementary techniques such as transcranial electrical stimulation or H-reflex testing suggest that at least part of these effects can be attributed to a modulation of motor cortex excitability ,25,26,27The lack of evidence that either experimental cutaneous (present study) or muscle pain  affects In conclusion, the present study showed that while cutaneous heat pain of short duration applied to the hand can cause a marked decrease in the corticospinal excitability of a hand muscle, it does not affect SAI. It remains possible that longer pain durations might affect SAI, but at this point the evidence for altered SAI under conditions of acute or chronic pain remains limited. More studies are needed to assess whether other parietal-motor pathways are involved in the interactions between sensorimotor integration and pain that are observed in behavioural and clinical studies.


The varnishes will be applied on all permanent teeth, once a week for 4 weeks and they will be reapplied only once 6 and 12 months after the study begins. Two calibrated examiners will carry out the clinical examination  at baseline, before the first application, after the 1st, 6th, 12th, and 18th month of the study begins. Furthermore, quantitative fluorescence changes will be measured using Quantitative Light-Induced Fluorescence (QLF). The degree of patient satisfaction with the treatment will also be computed. The data will undergo statistical analysis (P<.05).This randomized, controlled, parallel and single-blind clinical trial involves 63 children, 6-7 years old, living in Bauru, São Paulo, Brazil. Children were selected according to their caries activity  and randomly divided into the following treatment categories: 4% TiF4 on the prevention and treatment of carious lesions by comparing it to NaF varnish. The subjects are under 1 month evaluation and the dropout was about 8%. No differences between the treatments have been detected at the first month so far (P>.05).This ongoing study is funded by funding agencies from Brazil . We expect to confirm the efficacy of TiF4 varnish can be marketed and applied at the individual level and used in community programs to control dental caries.If our hypothesis is confirmed, TiFBrazilian Clinical Trials Registry: RBR-5VWJ4Y; http://www.ensaiosclinicos.gov.br/rg/?q=RBR-5VWJ4Y (Archived by WebCite at http://www.webcitation.org/6wUurEnm7) Fluoride varnishes are a feasible approach for preventing and treating carious lesions at the individual level and in public health programs, due to its good cost-benefit compared to initial carious lesions restorations, when they eventually progress to cavitation and have a significant negative impact in quality of life ,2.2] and titanium tetrafluoride [TiF4]) [Due to the polarization of caries  and ineq [TiF4]) ,7.4 is more effective than NaF at reducing demineralization and improving remineralization , Colgate-Palmolive). The oral hygiene kit will be replaced every 3 months during the study.All children received instruction on cariogenic diets and oral hygiene, and were given supervised tooth brushing. The researchers provided new toothbrushes , dental floss , and fluoride toothpastes  or when a healthy surface is transformed into an active lesion (cavitated or not). The regression is defined as when the initial active white spot lesion is transformed into an inactive carious lesion or healthy surface. The data (degrees of freedom [df] = baseline Nyvad’s score – 1 month Nyvad’s score) was analyzed using a Kruskal-Wallis test.QLF is applied to measure the changes in the enamel fluorescence of white spot lesions and to quantify its reversal or progression. A xenon arc lamp is used as a light source, and an optical filter system, producing blue light with a maximum wavelength of 370 nm, is connected to the microscope by a liquid light guide . The fluorescence emitted by the tooth is collected with a charged coupled device (CCD)-video microcamera  equipped with high pass yellow filter (γ>520 nm) to exclude any excitation or ambient light that may reach the detector and a special dental mirror to reflect the image of the lesion connected to the camera .2) that is the sum of all points within the lesion with fluorescence loss > 5%; and 2) the mean fluorescence loss  [After drying the tooth surface (for 5 s), images of clinically detected white spot lesions are obtained by QLF, in a completely dark environment. A computer program  is utilized to display, store, browse, and analyze the images. The QLF parameters are: 1) the area of the lesion  .The QLF analysis was performed at baseline and after 1 month. The differences between the 1 month and baseline values were calculated as follows: ΔWS area = WS area baseline – WS area after 1 month (the same for ΔΔF), where ΔWS is the variation of the white spot lesion area and ΔΔF is the variation of the mean fluorescence loss. The data were analyzed using the Kruskal-Wallis test. This analysis will be repeated after the 6th, 12th, and 18th months of the treatment by BMS.The data will be subjected to statistical analysis using the GraphPad Instat and Prism (version 5.0 software) for Windows . Firstly, the data will be checked for normality and homogeneity. A parametric or a similar nonparametric test will be applied to compare the treatments with respect to: 1) the prevention of new carious lesions (ICDAS and Nyvad indexes); 2) regression or progression of previous active white spot lesions using the Nyvad index; 3) regression or progression  of previously active white spot lesions using QLF .This protocol refers to an ongoing clinical study funded by the São Paulo Research Foundation (FAPESP-2015/14149-1) and the National Council for Scientific and Technological Development (CNPq-401313/2016-6).Treatment, Clinical Examination and Quantitative Light-Induced Fluorescence4 varnish compared to one based on NaF for the prevention and treatment of carious lesions at the end of the present study (18 months) as we have previously found under in vitro and in situ protocols.We expect to confirm the efficacy of the TiF4 varnish.Previous systematic reviews have shown no significant differences between the anticaries performances of fluoride (mainly NaF) included in different products such as gel, varnish and toothpaste ,2. Howev4 into a varnish allows for a longer contact time with enamel, thereby improving the reaction of titanium with the tooth apatite and facilitating the formation of a glaze-like layer on the tooth surface that is rich in titanium dioxide, hydrated titanium phosphate and CaF2[4 varnish is able to enhance the enamel fluoride uptake compared to a NaF varnish [4 contact with soft tissues compared to a rinse solution, reducing the possibility of cytotoxicity due to its low pH [4 varnish presents similar levels of toxicity to murine fibroblast lineage (NIH/3T3) cells as a NaF varnish [4 varnish, patient satisfaction was evaluated using a simple, but effective tool for self-rated child pain [Incorporating TiF and CaF2,10. Due s low pH . A recen varnish . To checild pain .Previous studies have shown that the application of fluoride varnish once a week for 4 consecutive weeks  has been effective in accelerating remineralization of white spot lesions . On the The most common method for detecting caries is the visual-tactile technique by using the ICDAS, Nyvad and DMFT indexes. However, other noninvasive techniques for the detection of early carious lesions have been developed, such as QLF and DIAGNOdent, which are especially used for research purposes . The traThe QLF is a sensible quantitative clinical method with good repeatability and reproducibility, requiring a smaller number of participants  compared to the visual analysis . The QLF4 varnish, results obtained at the first month of this study demonstrate that the differences between TiF4 and NaF did not reach significance, due to the slight changes in the visual analysis and in the fluorescence loss. If the TiF4 varnish proves to be better at controlling dental caries compared to the NaF version by the end of our study, it shall be marketed and distributed at the individual level and to community programs, in order to prevent and treat dental carious lesions in children in the future.Despite our hypothesis on the effectiveness of the TiF