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.”“


VL30-1 gene is transcribed as a long noncoding RNA (lncRNA) with an essential host function in an epigenetic transcription switch (ETS) that regulates transcription of multiple genes, including proto-oncogenes that control cell proliferation and oncogenesis. The ETS involves the tumor suppressor protein PSF that has a DNA-binding domain (DBD) and two RNA-binding domains (RBDs). The DBD binds to promoters that have a DBD-binding sequence and switches off transcription, and the RBDs bind lncRNAs that have a RBD-binding sequence, releasing PSF and switching on transcription. VL30-1 lncRNA has two RBD-binding sequences, apparently acquired by mutations during retrotranspositions of the founder retrovirus, which drive proto-oncogene transcription and oncogenesis via the ETS. VL30-1 lncRNA is a seminal example of the key role of endogenous retroviruses (ERVs) and their retroelements in the evolution of transcription regulatory systems.Here I review the properties of the mouse retroelement VL30-1, which apparently derived from retrotranspostions of a founder VL30 retrovirus that infected the mouse germline after the mouse–human speciation. The  Escherichia coli,1 focused attention on protein-coding genes as the fundamental functional component of all genomes, as affirmed in Monod’s statement that ‘anything found to be true for E. coli must also be true for the elephant.’ Although it was known that mammalian genomes also contained DNA that did not encode any proteins, such DNA was usually called useless or selfish.3 The revelation from whole-genome sequencing that protein-coding genes comprise only a minuscule part of a mammalian genome, ~2% of the human and mouse genomes,4–6 was a wake-up call to understand how most of the genomic DNA survived evolutionary selection for the fittest organisms. An associated revelation was that ~8–10% of the human and mouse genomes consist of ERVs presumably derived from retroviral infections of the mammalian germlines.7–9 An ERV initially functions as a selfish DNA that integrates at multiple genomic sites via successive cycles of duplicative retrotansposition (DRT), involving integration, transcription, reverse transcription and integration at another genomic site, which must eventually be suppressed in order for the host to survive, while the ERV must acquire a beneficial host function to survive as a component of the host genome.The operon model of gene regulation, a founding concept of molecular biology proposed by Jacob and Monod in 1961 based on their studies with 10 a member of the VL30 ERV family11 that probably originated from an infection of the mouse germline by a founder retrovirus after mouse–human speciation, as there are no VL30-related sequences in the human genome. The mouse genome currently is estimated to contain 150–200 VL30-related sequences, ranging from a full-length 5–6-kbp VL30 gene that has the features of an ERV, notably 5′ and 3′ long terminal repeats (LTRs), to a single ‘solo’ LTR.12 In the full-length VL30 genes sequenced so far, including VL30-1, the internal DNA flanked by the LTRs contains multiple mutations, including stop codons in all three reading frames, which block translation of the retroviral proteins required for further DRT cycles. Although the DRT cycles are suppressed, at least some of the full-length VL30 genes, including VL30-1, are transcribed as a lncRNA with a poly-A tail and are exported to the cytoplasm.Here I discuss the remarkable properties of a mouse ERV called VL30-1,10 Further studies showed that the increase in metastatic potential was caused by a novel mechanism of gene regulation involving the protein PSF13 and a PSF-binding RNA.14–17 PSF contains a DBD and two homologous RBDs . Consequently, another PSF-binding lncRNA RNA, probably MALAT-1,21 was needed before VL30-1 lncRNA was available, to prevent binding of PSF to proto-oncogenes during early development. As VL30-1 lncRNA binds more effectively to PSF than MALAT-1 lncRNA (unpublished data), it could have co-opted the role of MALAT-1 lncRNA as the major PSF-binding RNA in the mouse ETS, providing an explanation for the surprising finding that MALAT-1 lncRNA is dispensable for mouse development and survival.23 I propose that the beneficial function of VL30-1 lncRNA, which was needed for its evolutionary survival in the mouse genome, was achieved in this way, providing a seminal example of the importance of retroviruses and their retroelement descendants in shaping the evolution of epigenetic systems for regulating gene transcription.The


In organogenesis, cell types are specified from determined precursors as morphogenetic patterning takes place. These events are largely controlled by tissue-specific transcription factors. These transcription factors must function within the context of chromatin to activate or repress target genes. Recent evidence suggests that chromatin-remodeling and -modifying factors may have tissue-specific function. Here we review the potential roles for chromatin-remodeling and -modifying proteins in the development of the mammalian heart.


The Epstein–Barr nuclear antigen 3 (EBNA3) family of proteins, comprising EBNA3A, EBNA3B, and EBNA3C, play pivotal roles in the asymptomatic persistence and life-long latency of Epstein–Barr virus (EBV) in the worldwide human population. EBNA3-mediated transcriptional reprogramming of numerous host cell genes promotes in vitro B cell transformation and EBV persistence in vivo. Despite structural and sequence similarities, and evidence of substantial cooperative activity between the EBNA3 proteins, they perform quite different, often opposing functions. Both EBNA3A and EBNA3C are involved in the repression of important tumour suppressive pathways and are considered oncogenic. In contrast, EBNA3B exhibits tumour suppressive functions. This review focuses on how the EBNA3 proteins achieve the delicate balance required to support EBV persistence and latency, with emphasis on the contribution of the Allday laboratory to the field of EBNA3 biology. The lymphotropic gamma-herpesvirus Epstein–Barr virus (EBV) asymptomatically and persistently infects the majority of the worldwide human population. Despite being a ubiquitous virus, it is one of the most transforming viruses identified, and is aetiologically associated with a variety of B cell malignancies, including Burkitt’s lymphoma (BL), Hodgkin’s lymphoma (HL), and diffuse large B cell lymphoma (DLBCL), alongside post-transplant lymphoproliferative disease, nasopharyngeal carcinomas, and some gastric carcinomas . Upon inThe latency-associated EBNA3 family of proteins comprises 3 large proteins called EBNA3A, EBNA3B, and EBNA3C, each composed of over 900 amino acids. These non-redundant proteins are thought to have arisen from tandem duplication events during the evolution of old world lymphocryptoviruses. Their genes are structurally similar, with each consisting of a short 5′ coding exon and long 3′ coding exon, and share limited but significant sequence homology. Each of the EBNA3 proteins features a proline rich region, as well as a “homology domain” of approximately 220–230 amino acids near the N-terminus, in which there is 20–30% identity between the EBNA3s  technology, allowing detailed study of the EBNA3 proteins in the context of infection, B cell transformation and maintenance of viral latency. EBNA3 knockouts (KO) have been generated using mutations and deletions ,9, alongEBNA3 proteins are well characterised regulators of transcription. Exon-microarray analysis using the EBV-negative Burkitt’s lymphoma (BL) cell line BL31 infected with mutant EBNA3 viruses revealed that the EBNA3 proteins transcriptionally regulate over 1000 host cell genes. A substantial number of these genes show cooperative regulation between multiple EBNA3 proteins. Most of this co-regulation involves EBNA3C working in cooperation with either EBNA3A or EBNA3B or both, with only modest cooperation between EBNA3A and EBNA3B in the absence of EBNA3C. Moreover, deletion of EBNA3C had the most significant impact on host gene regulation, with gene repression dominating differential host gene expression . ChIP anBoth EBNA3A and EBNA3C are considered oncogenic. In an early demonstration of this, EBNA3C was shown to cooperate with oncogenic (Ha-)Ras during transformation of primary rat embryo fibroblasts . The oncAlthough the EBNA3 proteins regulate many target genes and robustly associate with chromatin, they do not bind DNA directly. Instead, the EBNA3 proteins bind many cell transcription factors to facilitate viral regulation of host genes. Of these factors, the Notch signalling pathway DNA-binding factor RBP-Jκ  was the first to be identified as a binding partner for all EBNA3s ,29, withSTK39 [RBP-Jκ is, however, absent from many known EBNA3 binding sites as revealed by ChIP-seq experiments exploring EBNA3 genome-wide localization ,18,38,39STK39 . It is cCXCL10/CXCL9 [ADAM28/ADAMDEC1 [ADAM28-ADAMDEC1 locus by promoting a long-range interaction between an intergenic enhancer and their respective promoters [WEE1, leading to gene repression [BCL2L11 (BIM) and CTBP2 repression are thought to result from EBNA3-mediated disruption of enhancer–promoter interactions [All EBNA3s localise to chromatin with histone marks characteristic of enhancer elements ,18,38,3910/CXCL9 , ADAM28/ADAMDEC1 ). Specifromoters ,38. Addiractions . Furtherractions . TogetheThe ability of the EBNA3s to mediate both host gene activation and repression was clear from a series of microarray experiments that interrogated the transcriptional profile of cells with or without expression of each EBNA3 ,26,43. TADAM28/ADAMDEC1 with repression [ADAM28/ADAMDEC1 with facilitation, and at BCL2L11 with disruption [How the different outcomes of repression or activation are determined for EBNA3A and EBNA3C is unclear. One could envisage that the EBNA3 proteins act always in the same way, and the chromatin context of each gene determines the outcome, but this has not yet been shown. It seems that the outcome of looping  is not the only determinant. There can be facilitation of looping and two different outcomes, for example, miRNA221/222 with activation  and ADAMpression ; in bothsruption , under EINK4a in B cells, but the mechanistic basis of this requirement remains uncertain [Regulatory mechanisms of EBNA3C are much better characterised than those of EBNA3A, and less is known about EBNA3B, with the current lack of an EBNA3B-ERT2 conditional virus limiting investigation. Part of their action is explained by their antagonism with the viral transactivator EBNA2 for binding to RBP-Jκ. This, however, is not the whole story, because of EBNA3 binding elsewhere and EBNA3A and EBNA3C also acting as activators themselves. Several transcription cofactors that could facilitate regulation have been found to interact with EBNA3A and EBNA3C. These include the histone deacetylases (HDAC1 and HDAC2) and the co-repressor CtBP for EBNA3A, and HDAC1/2, CtBP, SIN3 transcription regulator family member A (SIN3A), nuclear receptor co-repressor (NCoR) and histone acetyltransferase p300 for EBNA3C ,46,47,48ncertain . The p300 histone acetyltransferase is a co-activator, and its association with EBNA3C might explain EBNA3C’s ability to activate. Enrichment of p300 at EBNA3C-activated gene AICDA was found in response to EBNA3C presence , but morHDACs, NCoR, and SIN3A are generally thought of as repressors, and are all associated with histone deacetylation—a primary epigenetic mark associated with repression. In all cases of EBNA3-mediated repression investigated on a gene-by-gene basis, histone deacetylation has been observed. Importantly, when the process of histone deacetylation was tracked together with establishment of repression, they were found to be concurrent ,41.EBNA3-mediated gene silencing through association with transcription factors often involves localising polycomb repressive complexes 1 and 2  to target genes. Interestingly, PRC1 component BMI1 was found to be recruited to genes during establishment of repression, with kinetics that matched those of histone deacetylation, suggesting that it might be important at the initial stage . BMI1 knIn contrast to PRC1, the involvement of PRC2 in repression of EBNA3 target genes is well established, and can often result in deposition of the repressive histone mark H3K27me3, catalysed by the methyltransferase subunit (EZH2) of PRC2 ,41,51,52The apparent complexity of EBNA3 mechanisms of action is probably the result of the many transcription factors they seem to interact with. Regardless, general principles have emerged. Their association with chromatin is usually mediated by more than one DNA-binding transcription factor, probably in a locus-specific manner. They are mostly recruited to enhancer elements, distal to the promoters they regulate, which means that changes in chromatin architecture are necessary for their action. This can be either by enhancing or disrupting chromatin loop formation. There is evidence for direct activation only by EBNA3A and EBNA3C, whereas EBNA3B seems to act only as a repressor. The regulation they confer is epigenetic, primarily through changes in histone acetylation, which in some cases lead to changes in histone methylation with additive effects on repression.A complex system of phosphorylation and feedback loops regulates cell division to prevent aberrant proliferation and cancer progression. EBNA3A and EBNA3C have been shown to circumvent these pathways by interfering with intrinsic cell cycle associated factors that prevent anti-proliferation and suicidal response pathways. The best characterised is EBNA3A- and EBNA3C-mediated repression of various cyclin-dependent kinase inhibitors (CDKIs) . DeregulINK4a was first shown using a recombinant virus containing EBNA3C fused to oestrogen receptor. This showed that functional EBNA3C is essential to restrain expression of p16INK4a [INK4a mRNA and protein, with correspondingly reduced levels of hyperphosphorylated forms of the retinoblastoma protein (Rb), a cessation of proliferation, and more cells in the G1 phase of the cell cycle [INK4a levels were ablated by transfection of a plasmid expressing EBNA3C and by stabilisation of the conditional EBNA3C [CDKN2A gene encodes p16INK4a, and also  encodes p14ARF, which is also repressed by EBNA3C [INK4a protein interacts with the cyclin dependent kinases CDK4 and CDK6, preventing phosphorylation of the retinoblastoma (Rb) protein leading to cell cycle arrest, whereas p14ARF interacts with MDM2, resulting in p53 activation and subsequent cell cycle arrest or apoptosis [INK4a and p14ARF [INK4a when both EBNA3A and EBNA3C are inactivated, with elevated levels of p16INK4a mRNA compared to when EBNA3A or EBNA3C are inactivated independently [INK4a repression during B cell transformation by EBV was highlighted by evidence that EBNA3C becomes unnecessary for transformation and sustained proliferation when p16INK4a is functionally inactive. In an elegant experiment, Skalska et al. showed that EBNA3C-null LCLs can be readily produced from B cells derived from an individual with a homozygous deletion in CDKN2A that inhibits all known p16INK4a functions. This demonstrated that EBNA3C-mediated repression of p16INK4a plays a crucial role in the transformation of B cells by EBV [EBV-mediated repression of the CDKI and tumour suppressor p16p16INK4a . Inactivll cycle ,11,43. Iy EBNA3C . Howeverpoptosis . Analysid p14ARF ,26,54. Tendently . The impINK4a through epigenetic chromatin modification, via deposition of the polycomb-associated repressive mark H3K27me3, and reduction of the activation mark H3K4me3 at the CDKN2A locus [CDKN2A by EBNA3s is maintained in the absence of functional p16INK4a, further indicating CDKN2A is a direct target of EBV [CDKN2A by EBNA3A and EBNA3C was shown to require interaction with the transcriptional co-repressor CtBP [CDKN2A locus, alongside increased H3K4me3. The resulting increases in p16INK4a mRNA and protein are thought to contribute to the impaired outgrowth of primary B cells infected with EBV containing mutated CtBP binding sites in EBNA3A and EBNA3C, and the reduced proliferative capacity of resulting LCLs [ChIP analysis indicates EBNA3A and EBNA3C directly repress p162A locus ,54. Moret of EBV . The chrsor CtBP . CtBP issor CtBP ). EBNA3Asor CtBP ,47. Mutasor CtBP ,44,47. Iing LCLs .CDKN2A, EBNA3 proteins have also been shown to repress the adjacent gene CDKN2B. This gene encodes the related INK4 family protein p15INK4b, which performs similar functions to p16INK4a [INK4a repression [INK4b mRNA transcription has been reported following EBNA3C inactivation in conditional systems, demonstrating EBNA3C involvement in p15INK4b repression [CDKN2A, ChIP analyses have indicated EBNA3A and EBNA3C bind near the transcription start sites of the CDKN2B locus, suggesting EBNA3A and EBNA3C play a direct repressive role [INK4b and p16INK4a, EBNA3A and EBNA3C have also been shown to repress the INK4 family member p18INK4c through PRC2-mediated deposition of H3K27me3 . EBNA3A and EBNA3C were recently shown to mediate transactivation of the oncogenic miR cluster miR221 and miR222 (miR221/222), leading to repression of the CDKI p57 p27KIP1 . KIP2 and p27KIP1; transcriptional activation of the miRNAs by EBNA3A and EBNA3C leads to repression of p57KIP2, and to a lesser extent, p27KIP1. This provides a novel indirect method of cell cycle regulation mediated by EBNA3A and EBNA3C gene activation [Screening LCLs, where EBNA3A and EBNA3C were either conditionally expressed or knocked out, identified modified expression of various miRNAs by the EBNA3 proteins. Expression of the oncogenic miR221/222 cluster was shown to be reduced when either EBNA3A or EBNA3C was non-functional, with a significant increase in miR221/222 mRNA when EBNA3A and EBNA3C were conditionally activated. ChIP analysis confirmed EBNA3A and EBNA3C binding sites within an enhancer element of the pri-miR221/222 locus, from which miR221 and miR222 are processed. ChIP analysis further revealed an altered epigenetic profile in which the transcriptional activation markers H3K4me3, H3K9ac, H3K27ac, and phospho-Ser5 Pol II were elevated at the transcription start site only when EBNA3A and EBNA3C were functionally expressed. Furthermore, chromosome confirmation capture established that long range chromatin interactions between EBNA3A and EBNA3C binding sites within the predicted enhancer site of pri-miR221/222 and its promoter only occur when EBNA3A and EBNA3C are functionally active. The miRNA221/222 cluster targets the CDKIs p57tivation . KIP1 and p57KIP2, EBNA3A and EBNA3C have also been associated with repression of the CDKI and CIP/KIP family member p21WAF1/CIP1 [KIP2 and p27KIP1, EBNA3C works directly though interactions with the oncogenic kinase Pim-1 to result in proteasomal degradation of p21WAF1/CIP1 [INK4a is the dominant factor in EBNA3A- and EBNA3C-mediated cell cycle control during B cell transformation by EBV [Alongside indirect repression of p27AF1/CIP1 ,60. In cAF1/CIP1 . There in by EBV . HoweverBCL2L11 [MYC, which is expressed by B cells following EBV infection and induces hyperproliferation. A hallmark of BL is translocation of MYC and subsequently constitutively elevated expression [ARF–MDM2–p53 pathway) and it has been shown that even loss of a single BIM allele accelerates B lymphomagenesis [Concurrent with manipulation of CDKI and cell cycle control, EBNA3A and EBNA3C further facilitate virally-induced proliferation and oncogenesis by repressing apoptotic responses to viral infection and transformation. Using a panel of EBNA3KO viruses in BL31 BL-derived cells, EBNA3A and EBNA3C were found to cooperatively repress the tumour suppressor and pro-apoptotic BCL-2-family member BIM (Bcl-2-interacting mediator of cell death) encoded by BCL2L11 . EBNA3-mBCL2L11 ,43. BIM BCL2L11 ,62,63. Fpression . BIM is agenesis . The epiagenesis ,66,67.BCL2L11 locus, facilitating transcriptional repression [BCL2L11 transcriptional start site was confirmed by ChIP analysis, with later studies showing concurrent EBNA3A binding sites [Analogous with the epigenetic repression of INK4 family proteins, detailed ChIP analysis of EBV-positive B cells indicated that EBNA3-mediated repression of BIM involves modification of chromatin marks. This includes a reduction of histone acetylation and deposition of the H3K27me3 repressive mark within the pression . Direct ng sites ,42. Furtng sites . A requing sites ,42. EBNAINK4a and BIM, alongside others mentioned in this article, EBNA3C has been shown to attenuate the DNA damage response (DDR), which functions to limit aberrant proliferation by inducing senescence and apoptosis [INK4a expression seen following primary infection [INK4a and BIM activation, is subsequently prevented by the expression of functional EBNA3C and EBNA3A, which help to prevent apoptosis and cell cycle arrest pathways and allow outgrowth of LCLs [SKP2 and c-Myc [In addition to EBNA3A- and EBNA3C-mediated repression of the important tumour suppressors p16poptosis . The DDRpoptosis . Inductinfection . This isnfection ,53. The  of LCLs ,53,68. Fnd c-Myc ,74,75,76Despite structural and partial sequence homology, EBNA3B has distinctly different functions from those of the oncogenic EBNA3A and EBNA3C. It is completely dispensable in vitro, and B cells infected with EBV in the absence of EBNA3B exhibit elevated proliferative capacity compared to WT ,27,77. TnullIL2Rγ mice engrafted with human immune system components, which are susceptible to EBV infection, those infected with EBNA3B-KO exhibited increased splenomegaly and a higher frequency of large tumour masses than WT and EBNA3B-revertant infections after four weeks. These tumours destroyed splenic architecture, and by immunohistochemistry were monomorphic and highly proliferative, resembling the activated B cell (ABC) subtype of DLBCL. Furthermore, tumours derived from EBNA3B-deficient infections lacked infiltrating T cells. This was despite these infections priming a stronger systemic immune response compared to WT and revertant virus infections, with a higher percentage of splenic T cells, including CD8+ve T cells capable of killing EBV-positive cells in vitro [In contrast to the oncogenic functions of EBNA3A and EBNA3C, EBNA3B appears to have tumour suppressive activity in vivo . In NOD-in vitro . This isin vitro . Togethein vitro . Despite the seemingly opposite basic functions of EBNA3B compared to EBNA3A and EBNA3C, EBNA3B cooperatively regulates some genes with EBNA3C, sometimes with the additional contribution of EBNA3A . This isINK4c, encoded by CDKN2C [PRDM1 [Aside from manipulating regulation of cell cycle factors, microarray analysis has also indicated that EBNA3 proteins may alter the regulation of various factors associated with B cell differentiation . Recently CDKN2C ,78,79,80C [PRDM1 ,81,82,83INK4c and BLIMP-1 mRNA and protein [INK4b and p16INK4a, functional expression of either EBNA3A or EBNA3C individually is largely sufficient to repress p18INK4c and BLIMP-1 transcription [INK4c and BLIMP-1 in EBNA3A/EBNA3C-null cells were physiologically comparable to those of plasma cell models, and transcription of the BLIMP-1 target genes XBP-1, LMO2, and SPIB responded as expected to the elevated BLIMP-1 levels. Concurrent with the roles of p18INK4c and BLIMP-1 in promoting plasma cell differentiation, flow cytometric analysis of EBNA3A/EBNA3C-null cells showed the development of a plasma cell-like phenotype, with elevated surface markers characteristic of plasma cells, including CD138 and CD38, alongside elevated IgG and IgM expression. This indicates that EBNA3A and EBNA3C have evolved, not only as oncogenic factors, but also as repressors of plasma cell differentiation [Using a recombinant virus, in which EBNA3A and EBNA3C are both expressed conditionally, simultaneous inactivation of EBNA3A and EBNA3C during primary B cell infection was shown to lead to elevated levels of p18 protein . In contcription ,43,54,58BCL2L11, CDKN2A, and CDKN2B loci, ChIP analysis showed EBNA3A and EBNA3C bind around the transcriptional start sites of the CDKN2C (p18INK4c) and PRDM1 (BLIMP-1) genes, where they mediate deposition of H3K27me3 catalysed by the EZH2 component of the PRC2 complex. This leads to stable epigenetic reprogramming of these plasma cell differentiation associated genes [INK4b and p16INK4a, which can be reversed by inactivation of either EBNA3A or EBNA3C, regulation of p18INK4c and BLIMP-1 was shown to be irreversible by eight days post infection, with inactivation of EBNA3A and EBNA3C failing to lead to reactivation of either gene, even after thirty days [INK4c and BLIMP-1 by EBNA3 proteins are presently unknown, but may involve additional chromatin modifications. The differential regulation of these target genes highlights the complexity of EBNA3A and EBNA3C appropriation of the polycomb system.As with the ed genes . In contrty days ,15,43,54INK4c and BLIMP-1 appears to be independent of EBNA3B [EBNA3A- and EBNA3C-mediated repression of the plasma cell differentiation factors p18f EBNA3B . HoweverThe complex cooperative and antagonistic functions of the EBNA3 proteins, encompassing the critical epigenetic reprogramming of target genes, are central to achieving EBV persistence and latency. EBV infection stimulates proliferation of infected B cells to facilitate differentiation. This aberrant cell proliferation is permitted due to EBNA3A- and EBNA3C-mediated repression of cell cycle arrest pathways and suppression of pro-apoptotic signals. As B cell differentiation may lead to plasma cells, EBV must also promote transition through the germinal centre to achieve latency in memory B cells. EBNA3A and EBNA3C thus also suppress plasmacytoid differentiation, supporting the creation of a long-lived reservoir of EBV-infected memory B cells. It is possible that this process is further supported by EBNA3B, through T cell interactions and additional regulation within the germinal centre. In addition, EBNA3B plays a role in suppressing the detrimental effects of EBNA3A- and EBNA3C-mediated oncogenic functions. Because the EBNA3s largely epigenetically reprogram genes, the regulation of those genes is sustained following the sequential silencing of viral gene expression during germinal centre transition. This ensures the memory B cell pathway is favoured during latency progression, but it is possible that long-term effects may contribute to the prevalence of EBV-associated cancers. The complex essential roles of EBNA3A, EBNA3B and EBNA3C within EBV infection thus appear to be delicately balanced, allowing this potent oncogenic virus to achieve ubiquitous and life-long latent infection in the worldwide population.


In the past years, there has been a growing attention towards crafting personalized cancer therapies based on epigenetic targets. In particular, the field made a major leap forward triggered by the possibility to pharmacologically target the activity of BRD4, a chromatin reader of the BET family of bromodomain proteins. This remarkable interest stemmed from the early promises held by inhibitors like JQ1 and I-BET151, which showed strong therapeutic efficacy in selectively inhibiting the expression of cellular oncogenes  x Kelongation, where K is the elongation rate. If K decreases and stalled-Pol2 increases then elongation is buffered). Conversely, highly expressed genes, which in order to support robust transcription have maximized RNApol2 recruitment, are kinetically limited by elongation and thus are unable to mount such compensatory responses.This because low/moderately expressed genes will re-adjust to the drop in transcription by increasing the recruitment of RNApol2 on their promoters, thus reaching a new steady state; here increased promoter-paused RNApol2 compensates for decreased elongation and allows steady mRNA synthesis (this is based on the following: the amount of elongating RNApol2 = [Pol2This implies an inherent balancing mechanism that on the one hand provides transcriptional buffering capabilities to moderately expressed genes but on the other poses an intrinsic liability on highly transcribed genes. As a corollary, this may explain the selective sensitivity of cancer cells to general transcriptional inhibitors, since these cells, by being addicted to high rates of mRNA synthesis, would be impaired in mounting compensatory responses. This mechanism of transcriptional buffering provides a rationale for the evaluation of inhibitors of transcription as selective therapeutic agents and highlights the need for further deepening our understanding on transcription as an Achille’s heel of tumor cells.


Enhancer regions producing eRNAs decreased in HD mouse striatum were associated with genes involved in striatal neuron identity. Consistently, they were enriched in striatal super-enhancers. Moreover, decreased eRNA expression in HD mouse striatum correlated with down-regulation of associated genes. Additionally, a significant number of RNA Polymerase II (RNAPII) binding sites were lost within enhancers associated with decreased eRNAs in HD vs control mouse striatum. Together, this indicates that loss of RNAPII at HD mouse enhancers contributes to reduced transcription of eRNAs, resulting in down-regulation of target genes. Thus, our data support the view that eRNA dysregulation in HD striatum is a key mechanism leading to altered transcription of striatal neuron identity genes, through reduced recruitment of RNAPII at super-enhancers.Epigenetic and transcriptional alterations are both implicated in Huntington’s disease (HD), a progressive neurodegenerative disease resulting in degeneration of striatal neurons in the brain. However, how impaired epigenetic regulation leads to transcriptional dysregulation in HD is unclear. Here, we investigated enhancer RNAs (eRNAs), a class of long non-coding RNAs transcribed from active enhancers. We found that eRNAs are expressed from many enhancers of mouse striatum and showed that a subset of those eRNAs are deregulated in HD  Huntington’s disease (HD), a progressive neurodegenerative disease affecting primarily medium spiny neurons of the striatum, leads to cognitive, motor and mood impairments. As for several neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases, neuronal dysfunction in HD correlates with epigenetic changes, particularly changes in histone modifications123456i.e. genes essential to the function and identity of striatal neurons21012e.g. DRD1, DRD2, KCNJ4, RGS9, DARPP32) required for proper regulation of striatal neuron activity. It is considered that transcriptional down-regulation underlies dysfunction of striatal neurons, preceding neuronal death, and may thus be a key mechanism of HD striatal pathogenesis18Transcriptional dysregulation in HD is tissue-dependent and most extensive in the striatum89124Using genome-wide scale approaches, we previously showed that the enhancer mark H3K27 acetylation (H3K27ac) was selectively decreased at super-enhancers, a category of broad enhancers regulating cell type-specific identity genes, and this event correlated with decreased expression of super-enhancer target genesi.e. transcription of long non-coding RNAs, called enhancer RNAs (eRNAs)15151617181820Regulation of enhancer activity involves enhancer transcription, Here we show that dysregulation of enhancer transcription is extensive in HD mouse striatum. Our data further indicate that eRNA dysregulation in HD mouse striatum results from altered recruitment of RNAPII at super-enhancers and underlies down-regulation of striatal marker genes. Thus, we provide new insights into the epigenetic mechanism underlying repression of striatum-specific identity genes.i.e. non-coding RNAs synthesized from enhancers, we first excluded signals within genic regions, defined as the interval starting 3 kb upstream of the transcription start site and ending 10 kb downstream of the transcription termination site, since they might result from polymerase read-through of genic transcripts  data previously generated in the striatum of control and HD R6/1 transgenic micepts ref. . Analysipts ref. . Noticeapts ref. . Decreasq-RT-PCR  and S1A. control  and S1A.ps1 mRNA , suggestted eRNA , suggestWe investigated whether down-regulated eRNAs in R6/1 striatum were enriched in genes implicated in specific functions. Gene ontology analysis (GO) using GREATKnca4, Kcnab1 and Kcnj4  contained striatal markers , including striatal voltage-gated potassium channels  enhancer activity had no broad impact on gene up-regulation in HD striatum, since up-regulated eRNAs in R6/1 striatum poorly overlapped with super-enhancers and their target genes were not enriched in up-regulated genes . Howeveret al. reported extensive transcription within super-enhancersUsing a cellular model of inflammation, Hah We analyzed eRNA transcription in basal conditions – as opposed to stimulus-induced conditions – and found that many eRNAs (6068) were transcribed from mouse striatal enhancers. This result is consistent with previous study, which identified 8990 and 7779 eRNAs in mouse cortex and cerebellum, respectivelyet al. previously identified neuronal stimulus-responsive enhancers using a genome-wide approach16Using primary cortical cultures, Kim Fos is low in neurons. Remarkably, enhancers that associated with reduced eRNA transcription in HD mouse striatum were enriched in a DNA motif recognized by another transcription factor, SRF Srf transcription is reduced in R6/1 striatum vs WT comparisons) or from single striatum of Q140 and WT littermate mice (for Q140 vs WT comparisons). Biological replicates (4 to 6) were performed for each group. Tissues were finely cut with a razor blade and total RNA was extracted using TRIzol reagent (Invitrogen). An additional DNAse treatment (Euromedex) was added before RNA purification by phenol/chloroform extraction and ethanol precipitation. cDNA synthesis was performed either on 0.5 μg of total RNA  or on 2 μg of total RNA for strand-specific reverse transcription . Gene-specific primers are available upon request. qRT-PCR analysis was performed on a Bio-Rad iCycler System (CFX) using SsoAdvanced SYBR Green Supermix (Bio-Rad). qRT-PCR conditions were 30 s at 95 °C, followed by 45 cycles of 5 s at 95 °C and 20 s at 63 °C. RT controls were performed by the omission of RNA template or RT enzyme. A specific standard curve was performed in parallel for each gene, and each sample was quantified in duplicate. Data were analyzed by gene regression using iCycler software and normalized to Gapdh or 36B4 levels.Each sample was prepared from single striatum of R6/1 and WT littermate mice  genes annotated by Ensembl (release 78) as: IG_C_gene, IGD_gene, IG_J_gene, IG_LV_gene, IG_V_pseudogene, Mt_rRNA, Mt_tRNA, polymorphic_pseudogene, protein_coding, pseudogene, rRNA, TR_V_gene and TR_V_pseudogene. We extended the region corresponding to those genes to 3 kb upstream of the transcription start site and 10 kb downstream of the transcript end site, in order to minimize signal from polymerase read-through from genic transcripts  was made using FIMOThe Z-score was computed this way:where:−x is the observed value (number of motif occurrence)−μ is the mean of the number of occurrences (computed on randomly selected data)−σ is the standard deviation of the number of occurrences of motifs (computed on randomly selected data)From the Z-scores, P values for each motif were computed. The P values were corrected for multi-testing using a method previously describedP values < 0.05 were considered).Functional enrichments analyses were performed using the tools DAVID and/or GREAT22GalaxEast  was used to integrate data relative to eRNA, mRNA, H3K27ac (including super-enhancers) and RNAPII signals.RNA-seq and ChIP-seq data used for eRNA analysis are accessible through GEO (accession number GSE59572).How to cite this article: Le Gras, S. et al. Altered enhancer transcription underlies Huntington’s disease striatal transcriptional signature. Sci. Rep.7, 42875; doi: 10.1038/srep42875 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.