The molecular processes that drive gene transcription are inherently noisy. This noise often manifests in the form of transcriptional bursts, producing fluctuations in gene activity over time. During cell fate specification, this noise is often buffered to ensure reproducible developmental outcomes. However, sometimes noise is utilized as a “bet-hedging” mechanism to diversify functional roles across a population of cells. Studies of bacteria, yeast, and cultured cells have provided insights into the nature and roles of noise in transcription, yet we are only beginning to understand the mechanisms by which noise influences the development of multicellular organisms. Here we discuss the sources of transcriptional noise and the mechanisms that either buffer noise to drive reproducible fate choices or amplify noise to randomly specify fates. Caenorhabditis elegans. Nearly all of the cells of the worm derive from stereotypical division patterns and gene expression that have been very well-mapped  and the MS2/MCP system  . Althougin situ hybridization and the MS2 hairpin system , they do not explain how genes in the same nucleus exhibit different kinetics. Promoter architecture, chromatin state, enhancer-promoter interactions, transcription factor copy number, and binding kinetics control different bursting parameters such as amplitude, duration, and frequency of bursting from individual loci  Figure . These dtrans-acting factor concentrations and binding affinities  and Mediator complex suggested a ng model  . In contrast, other experiments suggest that promoters have naturally evolved to produce noise. For example, synthetic Escherichia coli promoters were generated in the lab through directed evolution. These artificially evolved promoters drove gene expression at similar levels to their endogenous promoters . If the expression of Tat reaches a certain threshold, its production will be stabilized through a positive feedback loop, thereby promoting active replication rather than proviral latency , 2008. AStochastic fate choices also occur in multicellular organisms, but it is largely unclear how noise controls these decisions. Some preliminary work has been performed in hematopoietic cells, whose fates are influenced by the expression levels of the cell surface receptor Sca-1 . Noise iin vitro (Noise in gene expression can be utilized or buffered to control cell fates in both unicellular and multicellular organisms. Recent technological advances are allowing more in-depth studies of gene expression noise in multicellular organisms. Single molecule FISH and the MS2 hairpin system have been applied in several organisms, and the tracking of single RNA polymerases has been established in vitro . Future Drosophila retina. Precise signaling cues produce a crystalline pattern of ommatidia (ss RNA levels during development in response to repression from Klu. For example, since transcription is inherently stochastic, and Klu binding to the ss locus will vary between cells, the level of repression in each cell will be noisy. If each cell expresses ss at a different level, this could imply that a particular threshold is necessary for producing the terminal decisions to be on or off by creating a bistable system. These observations raise the intriguing possibility that Ss is regulated similarly to ComK in Bacillus subtilis and Tat in HIV-1, suggesting a general paradigm for stochastic fate choice that is conserved through higher organisms.One potential system for future investigation is the mmatidia . Despitemmatidia , 2010. Tmmatidia . Ss exprmmatidia . The trammatidia . It is pStochastic fate decisions are essential for development, pattern formation, and survival. The biological roles for noise in stochastic fate specification has been well-studied in viruses and bacteria, but its functions in eukaryotes are still being elucidated. With advances in quantitative imaging techniques, work on the roles of transcriptional bursting and noise in multicellular organisms is now accessible. Disruptions of stochastic fate specification mechanisms have been linked to human diseases including vision disorders, autism, anosmia, immunodeficiencies and lymphoma . ElucidaEU wrote and edited the manuscript. RJ edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Saccharomyces cerevisiae triggers rapid activation of a highly specific stress pathway that coordinately upregulates Hsf1 target genes and downregulates RP genes. Activation of Hsf1 target genes requires neo-synthesis of RPs, which accumulate in an insoluble fraction and presumably titrate a negative regulator of Hsf1, the Hsp70 chaperone. RP aggregation is also coincident with that of the RP gene activator Ifh1, a transcription factor that is rapidly released from RP gene promoters. Our data support a model in which the levels of newly synthetized RPs, imported into the nucleus but not yet assembled into ribosomes, work to continuously balance Hsf1 and Ifh1 activity, thus guarding against proteotoxic stress during ribosome assembly.Ribosome biogenesis is a complex and energy-demanding process requiring tight coordination of ribosomal RNA (rRNA) and ribosomal protein (RP) production. Given the extremely high level of RP synthesis in rapidly growing cells, alteration of any step in the ribosome assembly process may impact growth by leading to proteotoxic stress. Although the transcription factor Hsf1 has emerged as a central regulator of proteostasis, how its activity is coordinated with ribosome biogenesis is unknown. Here, we show that arrest of ribosome biogenesis in the budding yeast  When yeast cells are growing at top speed, they can make 2,000 new ribosomes every minute. These enormous molecular assemblies are the protein-making machines of the cell. Building new ribosomes is one of the most energy-demanding parts of cell growth and, if the process goes wrong, the results can be catastrophic. The proteins that make up the ribosomes themselves are sticky. Left unattended, they start to form toxic clumps inside the compartment that houses most of the cell’s DNA, the nucleus.A protein called Heat shock factor 1, or Hsf1 for short, plays an important role in the cell's quality control systems. It helps to manage sticky proteins by switching on genes that break down protein clumps and prevent new clumps from forming. Hsf1 levels start to rise whenever cells are struggling to keep up with protein production. If it is half-finished ribosomes that are causing the problem, cells can stop making ribosome proteins. The protein in charge of this in yeast is Ifh1. It is a transcription factor that sits at the front of the genes for ribosome proteins, switching them on. When yeast cells get stressed, Ifh1 drops away from the genes within minutes, switching them off again. Yet how this happens, and how it links to Hsf1, is a mystery.To start to provide some answers, Albert et al. disrupted the production of ribosomes in yeast cells and examined the consequences. This revealed a new rescue response, that they named the “ribosome assembly stress response”. Both Hsf1 and Ifh1 are sensitive to the build-up of unfinished ribosomes in the nucleus. As expected, Hsf1 activated when ribosome proteins started to build up, and switched on the genes needed to manage the protein clumps. The effect on Isfh1, however, was unexpected. When the unassembled ribosome proteins started to build up, it was the clumps themselves that pulled the Ifh1 proteins off the genes. The unassembled ribosomes proteins seemed to be stopping their own production. Low levels of clumped ribosome proteins in the nuclei of unstressed cells also helped to keep Hsf1 active and pull Ifh1 off the ribosome genes. It is possible that this provides continual protection against a toxic protein build-up.These findings are not only important for understanding yeast cells; cancer cells also need to produce ribosomes at a very high rate to sustain their rapid growth. They too might be prone to stresses that interrupt their ribosome assembly. As such, understanding more about this process could one day lead to new therapies to target cancer cells. Saccharomyces cerevisiae is a promising model system in which to uncover ancestral processes that might monitor ribosome assembly to regulate growth and protein homeostasis in eukaryotes.Ribosome assembly is the most energy demanding process linked to cell growth and requires coordinated production of processed ribosomal RNAs (rRNAs), ribosomal proteins (RPs) and ribosome biogenesis (RiBi) factors. This massive biosynthetic program permits rapidly growing yeast cells to produce about 2000 ribosomes per minute , which iHeat shock factor 1 (Hsf1) is a central actor in Protein Quality Control (PQC) and protein homeostasis (proteostasis) in eukaryotes, in both stressed and unstressed cell, and in pathological situations . NotablyS. cerevisiae, RP gene transcription is known to be tightly regulated according to growth conditions through the stress-sensitive transcription factor (TF) Ifh1. Thus, Ifh1 is rapidly released from RP promoters only minutes following inhibition of the conserved eukaryotic growth regulator Target Of Rapamycin Complex 1 (TORC1) kinase (Given the tremendous investment of cellular resources involved in ribosome production  and the ) kinase . Althoug) kinase , how IfhIn this study, we uncover a novel regulatory pathway, hereafter referred to as the Ribosomal Assembly Stress Response (RASTR), that allows rapid and simultaneous up-regulation of protein homeostasis genes and downregulation of RP genes following disruption of various steps in ribosome biogenesis . We show that RASTR is highly specific to the RP and Hsf1 regulons, with little or no effect on a much larger group of genes implicated in the Environmental Stress Response (ESR). Importantly, RASTR requires neo-synthesis of RPs following stress and is linked to the accumulation of RP aggregates, which we propose lead to Hsf1 activation, through chaperone competition, and to the sequestration of Ifh1 in an insoluble nucleolar fraction. Notably, we show that protein synthesis inhabitation via cycloheximide treatment leads to a transcriptional response opposite to that of RASTR, supporting a model in which unstressed cells constantly monitor nuclear levels of unassembled RPs and use this information to balance expression of Hsf1 target genes with those encoding RPs. Finally, we demonstrate that RASTR is the initial transcriptional response to inactivation of TORC1 kinase, supporting a key role for this regulatory pathway in the activation of a protein homeostasis transcriptional program that allows cells to cope with the proteotoxic consequences of disruptions to ribosome biogenesis.In an effort to better understand the role of the two major eukaryotic DNA topoisomerases in protein-coding gene transcription, we generated yeast strains in which Top1, Top2, or both of these enzymes are rapidly degraded by the auxin-induced degron (AID) method  and confSince upregulation of proteostasis-related genes and downregulation of RP genes are characteristic of many different stress responses, we decided to quantify the effect of Top1 depletion on transcription all gene groups that have been classified as part of the general ‘Environmental Stress Response’ ESR; , which iTOR1-1 mutation and is thus resistant to the normal physiological consequences of rapamycin treatment, which inactivates the growth-promoting TORC1 kinase (To explore the target(s) of this hypothetical signaling pathway at RP genes, we monitored by qPCR ChIP the promoter association of three TFs  that operate at the majority (>90%) of these 138 genes . Interes1 kinase . This ex1 kinase . Therefo1 kinase , since M1 kinase .mak1) defective in large ribosomal subunit production  depletion by addition of auxin to the medium  and treatment of cells with diazaborine. Utp8p is a member of the t-UTP subcomplex of 90S pre-ribosomes and its depletion inhibits rDNA transcription, leading to a reduction of the primary 35S pre-rRNA transcript and subsequent processing intermediates . In conttae2-Δ cells. We found that activation of two Hsf1 target genes (SSA1 and HSP42) and downregulation of two RP genes (RPL30 and RPL39) was unaffected by deletion of TAE2  and the other on a long timescale (~20 min). Interestingly, ifh1-ΔC promoter release is stable following Top1 depletion, suggesting that an unknown mechanism regulates Ifh1 during RASTR  that weakens its interaction with RP gene promoters. Remarkably, tom1-Δ is synthetically lethal with ifh1-AA  that triggers a similar growth defect  completely abolished both RP gene repression and activation of Hsf1 target genes . Indeed,-Δ cells . Similar-Δ cells . Conside-Δ cells .Taken together with the strong reduction of Hsf1 target gene activation following Ifh1 cytoplasmic anchoring, both in the presence and absence of topoisomerase degradation, our observations on the effect of cycloheximide highlight the interwoven nature of RP and Hsf1 target gene regulons and support the notion that unassembled, aggregated RPs constitute the primary RASTR-induced signal capable of regulating both Ifh1 and Hsf1 activities, albeit in an opposite direction. More generally, these data indicate that newly synthetized RPs, in both stressed and unstressed cells, operate as a central hub in coordinating the expression of RP genes themselves with the Hsf1-dependent activation of chaperone and proteasome genes.We next turned our attention to the potential involvement of RASTR during more general stress responses that might also rapidly affect ribosome assembly. In an initial set of experiments, we inactivated the conserved growth-promoting TORC1 kinase by treatment of cells with rapamycin, which is known to mimic a major part of the environmental stress response, including osmotic and redox stress, as well as carbon, nitrogen, phosphate or amino acid starvation . As repoTo explore this hypothesis further, we took advantage of our observation that cycloheximide treatment prevents RASTR activation by either Top1 or Top1/2 degradation  and treaThe effects of rapamycin treatment described above are fully consistent with our previous report demonstrating that regulation of RP gene transcription following TORC1 inactivation by rapamycin operates through two distinct mechanisms at short and long timescales, with the latter dependent on RNAPI activity and the CURI complex . The shoTo explore the possible generality of rapid RASTR-mediated shut-down of RP gene transcription in response to stress, we measured the transcriptional response to heat shock, which is known to transiently downregulate both RP gene and rRNA transcription . As expeIn this study, we demonstrate the existence of a regulatory mechanism, which we refer to as the ribosome assembly stress response, or RASTR, that allows yeast cells to specifically coordinate the activity of two TFs, Hsf1 and Ifh1, with the functional state of ribosome assembly. Our data and several previous reports suggest that rapid ribosome biogenesis is a potentially proteotoxic process, in large part due to accumulation of unassembled RPs, whose production needs to be carefully coordinated at the transcriptional level, at least in yeast, together with that of chaperones and proteasome components . In thisOur data indicate that disequilibrium at any step-in ribosome biogenesis  will lead to RASTR activation until the pool of unassembled RPs decreases. Consequently, proteasome inhibition , dNTP deS. cerevisiae and human cells  supplemented with 1 mM PMSF and 1x protease inhibitor cocktail (Roche). Samples were aliquoted in Eppendorf tubes and frozen. After thawing, the cells were broken using Zirconia/Silica beads (BioSpec). The lysate was spun at 13,000 rpm for 30 min at 4°C and the pellet was resuspended in 300 μl ChIP lysis buffer + 1 mM PMSF and sonicated for 15 min (30 s ON - 60 s OFF) in a Bioruptor (Diagenode). The lysate was spun at 7000 rpm for 15 min at 4°C. Antibody  was added to the supernatant and incubated for 1 hr at 4°C. Magnetic beads were washed three times with PBS plus 0.5% BSA and added to the lysates (30 μL of beads/300 μL of lysate). The samples were incubated for 2 hr at 4°C. The beads were washed twice with , once with AT2 buffer , once with AT3 buffer  and twice with TE. The chromatin was eluted from the beads by resuspension in TE + 1% SDS and incubation at 65°C for 10 min. The eluate was transferred to an Eppendorf tube and incubated overnight at 65°C to reverse the crosslinks. The DNA was purified using High Pure PCR Cleanup Micro Kit (Roche). DNA libraries were prepared using TruSeq ChIP Sample Preparation Kit (Illumina) according to manufacturer’s instructions. The libraries were sequenced using an Illumina HiSeq 2500 and the reads were mapped to the sacCer3 genome assembly using HTSStation  to a final concentration of 1 μg/ml (Strains used in this study are listed in  1 μg/ml . Depleti600 reached 0.4. Cells were spread on slides coated with an SC medium patch containing 2% glucose. Stacked images were recorded (Intelligent Imaging Innovations) at a spinning disc confocal inverted microscope (Leica DMIRE2) using the 100x oil objective and an Evolve EMCCD Camera (Photometrics).Cells were grown overnight at 30°C in SC medium (0.67% nitrogen base without amino acids (BD), 2% dextrose supplemented with amino acids mixture , adenine, and uracil). Cells were diluted and were harvested when OD600 units (50 ml) of exponentially growing cells were harvested, and cell pellets were frozen in liquid N2. The cell pellets were resuspended in 1 ml lysis buffer  and incubated at 30° C for 30 min. Chilled samples were treated by tip sonication  and centrifuged for 20 min at 600 g at 4°C. Aggregated proteins were pelleted at 16,000 g for 20 min at 4°C. After removing supernatants, insoluble proteins were washed once with Wash I buffer , and centrifuged at 16,000 g for 20 min at 4°C. Insoluble proteins were washed with Wash II buffer , pelleted and sonicated (2x for 10 s) in 40 μl of Wash II buffer. For analysis by SDS-PAGE (4–12% acrylamide) and subsequent western blotting, proteins were first boiled in Laemmli buffer. 1x of the total cell lysate and 25x of the insoluble pellet fraction were separated and analyzed by Coomassie Blue staining or immunoblotting. Proteins were identified by shotgun mass spectrometry analysis at the Functional Genomics Center Zurich  following TCA precipitation (20%) and acetone washing, according to posted procedures. Database searches were performed by using the Mascot  search program, using very stringent settings in Scaffold .Isolation of protein aggregates from yeast cells was performed as described previously  with sli2] supplemented with 50 µg/mL cycloheximide and collected again by centrifugation. Dry pellets were resuspended with approximately one volume of ice-cold buffer K supplemented with 1 mM DTT, 1 × Complete EDTA-free protease inhibitor cocktail (Roche), 0.1 U/µL RNasin (Promega) and 50 µg/mL cycloheximide. About 250 µL of ice-cold glass beads (Sigma) were added to 500 µL aliquots of the resuspended cells and cells were broken by vigorous shaking, three times 2 min, separated by 2 min incubations on ice. Extracts were clarified through two successive centrifugations at 13,000 rpm and 4°C for 5 min and quantified by measuring absorbance at 260 nm. About 30 A260 units were loaded onto 10–50% sucrose gradients in buffer K, and then centrifuged for 150 min at 39,000 rpm and 4°C in an Optima L-100XP Ultracentrifuge (Beckman-Coulter) using a SW41Ti rotor without brake. Following centrifugation, 18 fractions of 500 µl each were collected from the top of the gradients with a Foxy Jr. apparatus (Teledyne ISCO). The absorbance at 254 nm was measured during collection with a UA-6 device (Teledyne ISCO).Yeast cells growing exponentially were treated or not with auxin for 20 min. 50 µg/mL cycloheximide (Sigma) was added directly to the culture medium. Cells were collected by centrifugation, rinsed with buffer K -adenine (NET06300 Perkin Elmer) at 0, 10, 20 and 30 min following the addition of auxin. Cell pellets were frozen in liquid nitrogen. RNA extractions and Northern hybridizations were performed as previously described  are given in  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 reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Naama Barkai as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: John Woolford (Reviewer #2); Marlene Oeffinger (Reviewer #3).Thank you for submitting your article "A ribosome assembly stress response regulates transcription to maintain proteome homeostasis" 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:This report shows that depletion of Top1, which leads to arrest of Pol I elongation, evokes a specific induction of the Hsf1 regulon, and not the general ESR, which can be blocked by anchor away of Hsf1; and also evokes the rapid downregulation of nearly all ribosome protein genes (RPGs), which can be attributed to loss of the transcription factor Ifh1 from RPG promoters. They term this response RASTR for Ribosome Assembly Stress Response. Evidence is presented that the signal for both responses, Hsf1 target induction and RPG repression, is a defect in 60S ribosome biogenesis associated with accumulation of nuclear aggregates of unassembled RPs, which sequester Ifh1 from RPG promoters. They show that blocking new synthesis of RPs by anchor away of Ifh1 blocks induction of Hsf1 targets on depletion of Top1. Moreover, they show that cycloheximide (CHX) treatment blocks both aspects of RASTR, presumably by preventing accumulation of newly synthesized RPs in nuclear aggregates capable of sequestering Ifh1, . CHX also appears to block the RASTR that is triggered immediately by Rapamycin presumably owing to an immediate inhibition of rRNA processing before the onset of RP repression, leading to accumulation of RP aggregates that sequester Ifh1. However, again, this interpretation was not supported by direct evidence of nuclear aggregates of RPs and Ifh1 at early, but not later, times of Rapamycin treatment, which are eliminated by CHX treatment. The authors conclude that unassembled RPs in nuclear aggregates serve to activate the Hsf1 regulon and down-regulate RPG transcription by sequestering Ifh1 as a means of tightly regulating RP expression and responding to the toxicity of RP aggregates, which functions both in normal cells and during conditions of unbalanced RP and rRNA production when ribosome biogenesis is impaired, when TORC1 is acutely inhibited by Rapamycin, and during heat-shock.Essential revisions:Additional experiments are required to establish that the accumulation of ribosomal proteins and Ifh1 in nuclear aggregates is diminished in cells treated with cycloheximide (CHX) to justify the interpretations of multiple experiments in Figures 5-6, in which CHX is found to block RASTR in response to Top1 depletion or at the early time points of rapamycin treatment. The authors should also confirm that Ifh1 promoter occupancy is reduced in tom1 cells to bolster their interpretation of the genetic data in Figure 3D. Revisions of text are also needed throughout the paper to better explain the rationale behind certain experiments or their design, and to expand the Discussion to consider the signals for triggering RASTR , the conditions that lead to cessation of the response, and situations in WT cells in which it would be triggered .Reviewer #1:This report shows that depletion of Top1, which leads to arrest of Pol I elongation evokes a specific induction of the Hsf1 regulon, and not the general ESR, which is blocked by anchor away of Hsf1; and also to rapid downregulation of nearly all ribosome protein genes (RPGs), which can be attributed to loss of the transcription factor Ifh1 from RPG promoters. They term this response RASTR for Ribosome Assembly Stress Response. Evidence is presented that the signal for both responses, Hsf1 target induction and RPG repression, is a defect in 60S ribosome biogenesis associated with accumulation of nuclear aggregates of unassembled RPs, which sequester Ifh1 from RPG promoters. Unlike in cells treated with Rapamycin, in which Ifh1 also is sequestered, the removal of Ifh1 from RPG promoters in RASTR does not required the Ifh1 C-terminal domain. They show that blocking new synthesis of RPs by anchor away of Ifh1 blocks induction of Hsf1 targets on depletion of Top1. Moreover, they show that cycloheximide (CHX) treatment blocks both aspects of RASTR, presumably by preventing accumulation of newly synthesized RPs in nuclear aggregates capable of sequestering Ifh1, although this effect of CHX on aggregation of RPs and Ifh1 is not directly demonstrated. CHX also appears to block the RASTR that is triggered immediately by Rapamycin presumably owing to an immediate inhibition of rRNA processing before the onset of RP repression, leading to accumulation of RP aggregates that sequester Ifh1. However, again, this interpretation was not supported by direct evidence of nuclear aggregates of RPs and Ifh1 at early, but not later, times of Rapamycin treatment, which are eliminated by CHX treatment. The authors conclude that unassembled RPs in nuclear aggregates serve to activate the Hsf1 regulon and down-regulate RPG transcription by sequestering Ifh1 as a means of tightly regulating RP expression and responding to the toxicity of RP aggregates, which functions both in normal cells and during conditions of unbalanced RP and rRNA production when ribosome biogenesis is impaired, TORC1 is acutely inhibited by Rapamycin, and in heat-shock.The findings in this paper are significant in showing specific induction of the Hsf1 regulon, and not the general ESR, when ribosome biogenesis is impaired either by depleting Top1, known already and confirmed here to arrest Pol I elongation, or by interfering with 60S biogenesis in different ways; and in describing a mechanism for the concurrent reduction in RP transcription via sequestration of Ifh1 in nuclear aggregates of unassembled ribosomal proteins.- It was felt that direct evidence is needed that the accumulation of RPs and Ifh1 in nuclear aggregates is diminished in cells treated with cycloheximide (CHX) to justify their interpretations of the multiple experiments in Figures 5-6 in which CHX is found to block the RASTR in response to Top1 depletion or at the early time points of rapamycin treatment.- The authors should also show that Ifh1 promoter occupancy is reduced in tom1 cells to bolster their interpretation of the genetic data in Figure 3D.Reviewer #2:This is an interesting and timely manuscript describing discovery of the so-called Ribosome Assembly Stress Response (RASTR) pathway: When ribosome assembly is blocked, transcription by Hsf1 to activate protein homeostasis genes  is upregulated, and ribosomal protein gene transcription is down-regulated. Further experiments explain how this occurs. When ribosome assembly is blocked by aborting rRNA transcription or preventing assembly factor function, excess unassembled ribosomal proteins aggregate. This leads to sequestration of Hsp70, an inhibitor of Hsf1, as well as Ifh, which is necessary for transcription of RP genes.The RASTR mediated pathway is rapid and apparently short-lived, activated within 5 minor aborting assembly, but returning to previous levels of expression of r protein proteostasis genes within an hour. It is not yet known whether r proteins, Ifh1, and Hsp70 are present in separate pools of aggregates, or whether or how they traffic into or out of aggregates.Because ribosomal proteins are synthesized at such high levels in rapidly growing cells, RASTR represents a significant mechanism to prevent proteostasis when assembly is imbalanced by any means.Some data are presented to distinguish RASTR from other stress response pathways, and to distinguish long term responses from the short term RASTR pathway. I found this portion of the Results and Discussion difficult to follow at times. The manuscript could perhaps be made more clear, especially for a general readership, by including a short paragraph early in the Discussion clearly outlining in one place the different pathways. To what exactly might each pathway respond and why? For example, what are general means by which imbalance in synthesis of ribosome components could be created in wild type cells (or by mutation?) and what then enables a return to normal synthesis once RASTR is inactivated? Is it diminished pools of aggregates? Then what about the primary triggers for the pathway? It also might be worth repeating here exactly where and how the CURI complex fits in to these effects.Reviewer #3:In the submitted manuscript by Albert et al., the authors identify the Ribosomal Assembly Stress Response (RASTR), a novel regulatory pathway that responds to excess numbers of RPs and their aggregation, and allows a simultaneous up-regulation of protein homeostasis genes and down-regulation of RP genes upon disruption of ribosome biogenesis at different stages, providing a mechanism for the cell to counteract RP proteotoxicity.Overall, the data presented here is very interesting and experimentally sound. The data is mostly well-presented and the manuscript generally concise; however, a few points should be addressed by the authors.- Throughout, the authors focus on the accumulation and fate of large subunit RPs and in Figure 2C and D, only large subunit RPs are shown by western blot. Have the authors looked at small subunit RPs? Given that the stresses shown here affect pre-rRNA transcription and assembly of RPs with pre-rRNA is co-transcriptional, the sRPs should also be affected and involved in the RASTR. The authors should address this point in their Discussion at the very least.- The authors selected Utp8 and Utp13 to mimic a ribosome assembly stress response as observed with Top1 and Top1/2 deletion; why were these two proteins chosen specifically? This should be explained for an audience less familiar with ribosome biogenesis.- Along these lines, the authors say that disruption of ribosome biogenesis at several levels – pre-rRNA transcription/processing, assembly – will activate RASTR. What about later stages in assembly/processing than the one tested in the 90S complex? Is the response related to early RiBi pathways steps?- Figure 2—figure supplement 1D: tae2-Δ cells – the figure/experiment described in the text does not match the panel/legend – please make sure it is the correct data panel or labeled to be made clearer to the reader.- While the manuscript and the logic of experiments is in most cases obvious to a reader within the immediate field, it may be less so to a general/non-ribosome biogenesis readership. I would suggest the authors be more liberal and expansive with some of their explanations, one example being the choice of Utp8 and Utp13, but there are others throughout.- Figure 2A: labels for the northern probe(s) used should be added as well as labels for the precursors visualized.eLife. Your revised article has been favorably evaluated by Naama Barkai as the Senior Editor and a Reviewing Editor.Thank you for resubmitting your work entitled "A ribosome assembly stress response regulates transcription to maintain proteome homeostasis" for further consideration at The manuscript has been improved substantially but there are a few remaining issues that need to be addressed before acceptance, as outlined below:1) The new experiment presented in Figure 3D lacks the WT TOM1 control strain needed to establish that the Ifh1 foci occur only in the tom1 mutant cells.2) It appears that the data are mislabeled in the new Figure 5—figure supplement 1E-F.3) Please re-proof all new figures and revised text for correctness. Reviewer #1:[…] – It was felt that direct evidence is needed that the accumulation of RPs and Ifh1 in nuclear aggregates is diminished in cells treated with cycloheximide (CHX) to justify their interpretations of the multiple experiments in Figures 5-6 in which CHX is found to block the RASTR in response to Top1 depletion or at the early time points of rapamycin treatment.tom1-Δ strain . Remarkably, these nuclear structures rapidly diminish (20 min) after CHX treatment . Furthermore, we have now added data showing that the accumulation of Ifh1 in nuclear aggregates observed during RASTR  is abolished in the presence of cycloheximide (CHX) ,in agreement with the proposed model, now in Figure 8. Lastly, we observed during RASTR activation that Rpl25-eGFP rapidly forms a nuclear structure that is not observed in the presence of CHX . Taken together, we believe that our new results provide very clear evidence that CHX blocks (or strongly diminishes) the formation of both RP and Ifh1 aggregates following RASTR activation.We agree with the reviewer that these data are important for interpretation of the experiments shown in Figures 5 and 6. To address this issue, we first point out more clearly in the revised text that the Deshaies lab  have already shown by MS analysis that RP aggregation following proteasome inhibition is strongly diminished upon cycloheximide (CHX) addition. Since the aggregation assay that they used didn’t allow them to discriminate between cytosolic and nuclear aggregates we decided to use an alternative approach (live cell imaging) to observe accumulation of RPs and Ifh1 in protein aggregates. To this end, we expressed Super-Fold GFP-tagged versions of Ifh1 and Rpl25 and observed their aggregation (foci formation) in the presence or absence of CHX. Significantly, we found that Ifh1 forms small punctate structures in a - The authors should also show that Ifh1 promoter occupancy is reduced in tom1 cells to bolster their interpretation of the genetic data in Figure 3D.tom1-Δ cells, which showed that Ifh1 binding at RPL30 appears to be reduced compared to that in TOM1 cells . However, this reduction is small, and we are hesitant to place too much weight on quantitative comparisons of ChIP enrichment in different strains. Nevertheless, this small difference in Ifh1 binding is in agreement with the near wild-type growth rate of the tom1-Δ strain, suggesting that the level of Ifh1 promoter occupancy in tom1-Δ remains sufficient to promote near normal RP gene transcription. Importantly, though, cycloheximide treatment triggers a significant increase in Ifh1 promoter binding in tom1-Δ strains compared to wild type , consistent with the disappearance of Ifh1 nuclear aggregates upon CHX treatment in the tom1-Δ strain . These data support the idea that tom1-Δ does indeed increase the amount of Ifh1 trapped in an insoluble nuclear fraction that can be rapidly released upon CHX treatment to increase binding at RP promoters.As suggested by the reviewer, we performed Ifh1 ChIP in TOM1 and the hypo-morphic ifh1-AA allele  could be linked to the growth defect of this mutation, we examined another mutated allele of IFH1 (ifh1-6) that triggers a similar growth defect .Lastly, to exclude the possibility that the genetic interaction between tom1-Δ is synthetically lethal only with ifh1-AA , supporting the notion that this genetic interaction is directly linked to the ability of Ifh1 to bind RP gene promoters. Altogether, we believe that these data strongly support the idea that one part of the pool of Ifh1 is trapped in an insoluble nuclear fraction in absence of Tom1 and prevented from binding to RP gene promoters.Importantly, the ifh1-6 mutant protein remains bound at high levels to RP gene promoters even under stress conditions, whereas ifh1-AA interacts weakly with RP gene promoters, even under optimal growth conditions . Remarkably, Reviewer #2:This is an interesting and timely manuscript describing discovery of the so-called Ribosome Assembly Stress Response (RASTR) pathway: When ribosome assembly is blocked, transcription by Hsf1 to activate protein homeostasis genes  is upregulated, and ribosomal protein gene transcription is down-regulated. Further experiments explain how this occurs. When ribosome assembly is blocked by aborting rRNA transcription or preventing assembly factor function, excess unassembled ribosomal proteins aggregate. This leads to sequestration of Hsp70, an inhibitor of Hsf1, as well as Ifh, which is necessary for transcription of RP genes.The RASTR mediated pathway is rapid and apparently short-lived, activated within 5 minor aborting assembly, but returning to previous levels of expression of r protein proteostasis genes within an hour. It is not yet known whether r proteins, Ifh1, and Hsp70 are present in separate pools of aggregates, or whether or how they traffic into or out of aggregates.Because ribosomal proteins are synthesized at such high levels in rapidly growing cells, RASTR represents a significant mechanism to prevent proteostasis when assembly is imbalanced by any means.Some data are presented to distinguish RASTR from other stress response pathways, and to distinguish long term responses from the short term RASTR pathway. I found this portion of the Results and Discussion difficult to follow at times. The manuscript could perhaps be made more clear, especially for a general readership, by including a short paragraph early in the Discussion clearly outlining in one place the different pathways. To what exactly might each pathway respond and why? For example, what are general means by which imbalance in synthesis of ribosome components could be created in wild type cells (or by mutation?).These are excellent questions, and we agree that the original manuscript was overly concise, to the point where even well-informed general readers might have difficulty following the arguments. To improve clarity, we have expanded both the Discussion and Introduction, emphasizing more the identity and role of the CURI complex in balancing rRNA and RP production at extended periods following stress (TORC1 inhibition) and pointing out that RASTR is a mechanistically distinct and faster acting response. We also include speculation regarding the physiological and genetic perturbations that might activate these two different responses.What then enables a return to normal synthesis once RASTR is inactivated? Is it diminished pools of aggregates?As pointed out above, our new data strongly suggest that the return to normal RP and Hsf1 target gene expression is promoted by the diminution of pools of aggregates, of both Ifh1 and RPs. This is now clearly stated in the text.Then what about the primary triggers for the pathway? It also might be worth repeating here exactly where and how the CURI complex fits in to these effects.This is a good point. We add speculation in the Discussion to the effect that the long-term regulation through the CURI complex may be driven by the release of its two rRNA processing components, Utp22 and Rrp7 from nascent rDNA, freeing them for interaction with Ifh1.Reviewer #3:In the submitted manuscript by Albert et al., the authors identify the Ribosomal Assembly Stress Response (RASTR), a novel regulatory pathway that responds to excess numbers of RPs and their aggregation, and allows a simultaneous up-regulation of protein homeostasis genes and down-regulation of RP genes upon disruption of ribosome biogenesis at different stages, providing a mechanism for the cell to counteract RP proteotoxicity.Overall, the data presented here is very interesting and experimentally sound. The data is mostly well-presented and the manuscript generally concise; however, a few points should be addressed by the authors.- Throughout, the authors focus on the accumulation and fate of large subunit RPs and in Figure 2C and D, only large subunit RPs are shown by western blot. Have the authors looked at small subunit RPs? Given that the stresses shown here affect pre-rRNA transcription and assembly of RPs with pre-rRNA is co-transcriptional, the sRPs should also be affected and involved in the RASTR. The authors should address this point in their Discussion at the very least.This is a good point, and we agree that sRPs should be affected by and involved in RASTR. Consistent with this, we detect by mass spectrometry both large and small subunit RPs (43 Rpl and 30 Rps proteins) in the insoluble fraction following topoisomerases depletion  and we detect accumulation of a small subunit protein, Rps8, in trailing fractions of polysome gradients . We thank the reviewer for pointing this out.- The authors selected Utp8 and Utp13 to mimic a ribosome assembly stress response as observed with Top1 and Top1/2 deletion; why were these two proteins chosen specifically? This should be explained for an audience less familiar with ribosome biogenesis.We agree with the reviewer. We now explain in the revised text why these factors were selected. Please see our reply to comment #4 of the reviewer #2.- Along these lines, the authors say that disruption of ribosome biogenesis at several levels – pre-rRNA transcription/processing, assembly – will activate RASTR. What about later stages in assembly/processing than the one tested in the 90S complex? Is the response related to early RiBi pathways steps?NAR 46: 3140). The failure to recycle these proteins results in a rapid feedback at an early maturation step. We speculate that both early and late stages of assembly will rapidly activate RASTR but acknowledge that further work will be required to nail this down.We agree with the reviewer that this is an important point. In the manuscript we show that diazaborine, an inhibitor acting at the mid-late steps of 60S biogenesis, activates RASTR, suggesting that this pathway is not limited to early steps. Nevertheless, it’s important to note that blocking maturation of late cytoplasmic large subunit precursors results in a rapid depletion of early acting factors in the nucleolus due to trapping of these factors in the accumulating late populations  show that both sets of genes respond as in TAE2 wild-type cells, ruling out a role for RQC in RASTR .We thank the reviewer for catching this mistake. The text and figure now describe Rpb1 ChIP of two Hsf1 target genes and two RP genes following Top1/2 degradation in a - While the manuscript and the logic of experiments is in most cases obvious to a reader within the immediate field, it may be less so to a general/non-ribosome biogenesis readership. I would suggest the authors be more liberal and expansive with some of their explanations, one example being the choice of Utp8 and Utp13, but there are others throughout.We agree with the reviewer that the text is too concise and lacking in detail. We have extensively expanded the text to make both the experiments and their interpretation clearer.- Figure 2A: labels for the northern probe(s) used should be added as well as labels for the precursors visualized.We apologize for this omission. Please see reply to the comment #1 of the reviewer #2.Thank you for resubmitting your work entitled "A ribosome assembly stress response regulates transcription to maintain proteome homeostasis" for further consideration at eLife. Your revised article has been favorably evaluated by Naama Barkai as the Senior Editor and a Reviewing Editor.The manuscript has been improved substantially but there are a few remaining issues that need to be addressed before acceptance, as outlined below:1) The new experiment presented in Figure 3D lacks the WT TOM1 control strain needed to establish that the Ifh1 foci occur only in the tom1 mutant cells.TOM1 control in Figure 3D which establishes that Ifh1 does not form foci in wild-type cells. This was apparent from other images, but you’re right that it’s best to show the control directly in Figure 3D. Thanks for pointing this out.We have now included the 2) It appears that the data are mislabeled in the new Figure 5—figure supplement 1E-F.You’re absolutely right about the swapped labeling on Figure 5—figure supplement 1E-F, and we thank you for pointing out this mistake, which has now been corrected.3) Please re-proof all new figures and revised text for correctness.Checking everything again we realized that there were graphical mistakes in the cartoons of Figures 1, 4 and 5. These have been corrected and we have uploaded the revised figures. The rest of the manuscript and figures have been checked and appear to us to be correct.


Despite similar number of total interactions between cell types, intrachromosomal interaction profiles in heart but not liver demonstrate that genes forming promoter-to-transcription-end-site loops in the cardiac nucleus tend to be involved in cardiac-related pathways. The same analysis revealed an analogous organ-specific interaction profile for liver-specific loop genes. Investigation of A/B compartmentalization (marker of chromatin accessibility) revealed that in the heart, 66.7% of cardiac-specific genes are in compartment A, while 66.1% of liver-specific genes are found in compartment B, suggesting that there exists a cardiac chromatin topology that allows for expression of cardiac genes. Analyses of interchromosomal interactions revealed a relationship between interchromosomal interaction count and organ-specific gene localization (p = 2.2 × 10−16) and that, for both organs, regions of active or inactive chromatin tend to segregate in 3D space . 3D models of topologically associating domains (TADs) suggest that TADs tend to interact with regions of similar compartmentalization across chromosomes, revealing trans structural interactions contributing to genomic compartmentalization at distinct structural scales. These models reveal discordant nuclear compaction strategies, with heart packaging compartment A genes preferentially toward the center of the nucleus and liver exhibiting preferential arrangement toward the periphery. Taken together, our data suggest that intra- and interchromosomal chromatin architecture plays a role in orchestrating tissue-specific gene expression.Packaging of the genome in the nucleus is a non-random process that is thought to directly contribute to cell type-specific transcriptomes, although this hypothesis remains untested. Epigenome architecture, as assayed by chromatin conformation capture techniques, such as Hi-C, has recently been described in the mammalian cardiac myocyte and found to be remodeled in the setting of heart failure. In the present study, we sought to determine whether the structural features of the epigenome are conserved between different cell types by investigating Hi-C and RNA-seq data from heart and liver. Investigation of genes with enriched expression in heart or liver revealed nuanced interaction paradigms between organs: first, the log Before DNA was universally recognized as the genetic material, it was hypothesized that nuclear proteins may be responsible for how the same DNA does different things in the various cell types of a multicellular organism . Since aMore recently, the emergence of chromatin capture technology combined with next generation sequencing has enabled unprecedented analyses of endogenous chromatin structure with increasing levels of resolution –8. ChromThese observations raise the following question: if TADs are a conserved feature of epigenomes across cell types, where does the specificity in structure arise? Compounding this question is the fact that, until recently, chromatin conformation capture studies have been often carried out in either cell culture or whole tissue extracts, making it possible to evaluate neither terminally differentiated cells nor the cell type-specific nature of chromatin structure. While we understand transcriptome changes across multiple organs and disease states, a major gap in our basic understanding of organ function is how genome architecture varies between cells and how this relates to gene expression.To address these gaps in knowledge, we investigated chromatin structural differences between heart and liver, and how they relate to tissue-specific gene expression programs. Specifically, we studied the role of genomic interactions  in organ-specific gene architecture. The analysis reveals a concordance between interaction frequency and organ-specific gene expression between tissues. We also explored compartment differences between organs, demonstrating that gene expression paradigms in distinct tissues act concertedly with their organ-specific compartmentalization pattern to regulate function of the cell. Lastly, we show that more interchromosomal interactions exist at organ-specific genes, and that about half of such interactions bridge distinct compartments within both cardiac and liver nuclei. Together, these investigations reveal organ-specific chromatin conformations that may contribute to cell identity in heart and liver.GSE96693 (Control_HiC). Liver data (acquired from isolated hepatocyte nuclei) (GSE104129 (Hi-C reps1-5). This dataset comes from wild-type C57BL/6J mice whose hepatocyte nuclei were isolated via homogenization and then crosslinked in 1% formaldehyde in PBS and quenched in glycine  for Hi-C. Hi-C libraries for both datasets were generated using Hi-C protocols based on (Hi-C datasets from this study were downloaded from NCBI GEO: Isolated cardiac myocyte data  were dow nuclei)  were dowbased on , 15. Libentire chromosomes, at 5 kb resolution. Another benefit of Fit-Hi-C is that it reports a q-value for each interaction, and we can filter for significant (q < 0.01) ones. For significant interchromosomal interaction identification, we performed a similar Fit-Hi-C analysis, but with the following parameters to investigate interactions that are not on the same chromosome: –b 200 –r 5000 –x interOnly. Significant intra- and interchromosomal interactions (q < 0.01) at 5 kb resolution are quantified in Supplementary Table Hi-C datasets were run through the HiC-Pro analysis pipeline , versiont q < 0.0 ones. Fowindow.size = 3 as a parameter. We then used Population-based Genome Structure (PGS) software . For both heart and liver data, 100 kb resolution TADs were designated as being in compartment A or B based on the majority compartmentalization status of the 5 kb bins  that lie within each 100 kb resolution TAD.To generate 3D models of topologically associating domains, we first ran TopDom  version software  to genersoftware . This reGSE96693 (Control_RNAseq Replicates 1–3). Mouse (C57BL/6) whole liver RNA-seq data from ENCODE Portal were downloaded using ENCODE Data Coordination Center accession number ENCSR000BYS . Raw FASTQ files from two biological replicates of liver tissue were downloaded. For both cardiac and liver RNA-seq library prep, rRNA was depleted and polyA selection performed. For the bioinformatics analysis, raw paired-end FASTQ files were aligned to the mm10 reference genome (Ensembl release 81) using HISAT2 (RNA-seq data from this study were downloaded from NCBI GEO: Isolated cardiac myocyte data corresponding to our previous study  were dowg HISAT2  version g HISAT2  version g HISAT2  version g HISAT2  was thenq < 0.01) Fit-Hi-C interactions with the promoter  and the TES of a gene. Genes that have such interactions underwent KEGG analysis using KEGG.db  intrachromosomal Fit-Hi-C interactions . These findings suggest that structural organization in 3D underpins cell type specific transcriptomes through greater frequency of interactions , and the same is true for liver-specific genes in liver tissue  of cardiac-specific genes are found in compartment A (the accessible compartment), while the majority of liver-specific genes (61.9%) are found in compartment B (the less accessible compartment) Figure , left. Ca Figure  in the eCardiac- and liver-specific genes have increased accessibility and a larger number of intrachromosomal interactions at organ-specific genes. However, the distribution of intrachromosomal interactions across genomic features, as well as the compartment change of 5 kb bins in the genome, could contribute to this phenomenon in both organs. To investigate whether intrachromosomal interactions in heart and liver have different localization across genomic features , we performed an overlap of significant intrachromosomal Fit-Hi-C interaction anchors  with these regions. Notably, in both heart and liver, we observe an almost identical distribution of Fit-Hi-C anchors Figures . Intrachp = 5.2 × 10−9, Figure p = 2.7 × 10−23, Figure To understand how A/B compartmentalization differs between heart and liver, we determined which 5 kb bins of the genome have a difference in compartment status between heart and liver. Five percent of bins are in compartment B in the heart and A in the liver, while 7% of bins are in compartment A in the heart and B in the liver Figure , for a tq < 0.01) intrachromosomal Fit-Hi-C interactions that overlap both the promoter region  and the transcription end site (TES) of a gene . The same analysis on liver gene loops reveals enrichments for phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, allograft rejection, and tryptophan metabolism .We next sought to determine whether there are organ-specific gene loops that govern cardiac- or liver-specific organ function. Here we define gene loops as significant  interchromosomal interactions were identified in the cardiac and liver Hi-C datasets and are summarized in Supplementary Table p = 2.2 × 10−16, Fisher's exact test; Figure p = 1, Fisher's exact test; Figure Examination of interchromosomal interactions allows for exploration of regional apposition—and potentially regulation—between distinct chromosomes. Significant q < 0.0 interchrHiC data is informative to define regions of local interaction, but how these substructures of the epigenome arrange in 3D has remained an open question. We performed 3D reconstruction of cardiac and liver epigenomes based on HiC data, establishing models for how chromosomes fold and for how they associate with other chromosomes, using PGS . The appTo investigate the distribution of different chromatin features within the nuclear space, we divided the nuclear volume into 5 concentric shells in such a way that each shell contains 20% of the total number of TADs per structure. Based on their radial positions, all TADs in each of the 10,000 genome structures are then partitioned into the 5 shells. We then measured the probability for a TAD in a given subcompartment (A/B) to be localized in each of the concentric shells Figure . We obseTo gain a quantitative understanding of interchromosomal TAD-TAD colocalization, we studied the compartment composition at the interchromosomal boundaries. At each TAD position, we determined all TADs that are localized within a distance of 500 nm and are part of a different chromosome. We then determined the percentage of A/B compartment found in this group of inter-chromosomal TAD neighbors. The heart genome shows a high preference for TADs in the same chromatin compartment across chromosome boundaries, indicating a high level of compartmentalization across chromosome borders. In liver cells, we observe a different behavior. While TADs in subcompartment B also show a high preference to be in proximity to TADs in same state, TADs of state A do not show a preference for the same state, showcasing the different global organization of the genome in liver nuclei.We also calculated the average radial position of each TAD with respect to the nuclear center Figure . TADs coFinally, we examined the localization of chromatin from a gene centric view, determining the relative positioning of heart and liver specific genes in the different nuclei Figure . In agreHow chromatin structure underpins gene expression has ramifications across biology and medicine. In the cardiovascular realm, as in other areas of epigenomics research, this question has largely been answered from the perspective of histone modifications , 32, enhThe current study demonstrates that organ-specific genes preferentially localize in 3D in the nuclei of the organs in which they are transcribed. This conceptually straightforward hypothesis has never, to our knowledge, been tested experimentally and reveals a structural underpinning for cell type-specific transcriptomes. These observations also support the concept of transcriptional neighborhoods , or tranOur comparison of liver and cardiac chromatin structure reveals widespread differences in compartmentalization, some but not all of which coordinate with transcriptional behaviors that vary between the organs. This finding is intriguing, given the fact that altered compartmentalization following the development of pressure overload-induced cardiac hypertrophy and failure is very minor : localizThe results of the analysis of gene looping data were particularly revealing: heart and liver establish comparable numbers of promoter to TES gene loops, however this specific class of loops appears in different genes in the different organs. These findings support that at multiple scales, including the level of gene looping in addition to compartmentalization as mentioned above, structural organization of the epigenome is cell type specific.The majority of chromatin conformation capture studies that have emerged the past few years have focused exclusively on intrachromosomal interactions. The adult cardiac myocyte, which does not divide, is an interesting test case to explore the role of interchromosomal contact surfaces in genome function—principally, although not exclusively, via gene regulation. A liver HiC dataset of comparable sequencing depth afforded the opportunity to explore contrasting features of such interactions, should they exist, within the same genome housed in separate cells' nuclei. Both epigenomes exhibited similar levels of interchromosomal interactions and in both cases, they were enriched in genes associated with the function of that cell type. Combining these interactions with 3D renderings of genomes in heart and liver provided a unique opportunity to investigate differences in chromosome folding and nuclear organization. Several observations emerged: liver and heart cells not only package their genomes differently, but they appear to obey distinct general principles of organization, wherein heart genomes preferentially localize compartment A regions toward the center and compartment B regions toward the periphery, whereas liver cells do not exhibit this behavior. Future studies will investigate whether interchromosomal interaction surfaces participate in such behaviors as cell proliferation, whether they change with age or are dependent on developmental state, and what non-DNA molecules inhabit the surfaces of interchromosomal apposition, presumably orchestrating the reproducible formation of these structures.DC and TV conceived the study. DC performed bioinformatics and statistical analyses. DC and MR-G generated figures and diagrams. NH and FA generated and analyzed 3D genomic models. DC and TV wrote the paper. All authors approved the content of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously  across the viral genome. The results indicate that this E1A interaction with p300 is required for H3K18 and H3K27 acetylation at the E2early, E3, and E4 promoters and is required for TBP and Pol II association with the E2early promoter. In contrast, H3K18/27 acetylation was not required for TBP and Pol II association with the E3 and E4 promoters but was required for E4 transcription at a step subsequent to Pol II preinitiation complex assembly.How histone acetylation promotes transcription is not clearly understood. Here, we confirm an interaction between p300 and the adenovirus 2 large E1A activation domain (AD) and map the interacting regions in E1A by observing colocalization at an integrated IMPORTANCE Despite a wealth of data associating promoter and enhancer region histone N-terminal tail lysine acetylation with transcriptional activity, there are relatively few examples of studies that establish causation between these histone posttranslational modifications and transcription. While hypoacetylation of histone H3 lysines 18 and 27 is associated with repression, the step(s) in the overall process of transcription that is blocked at a hypoacetylated promoter is not clearly established in most instances. Studies presented here confirm that the adenovirus 2 large E1A protein activation domain interacts with p300, as reported previously , and that the resulting acetylation of H3K18/27 affects varied steps in transcription at different viral promoters. Proteins encoded in early region 1A (E1A) of human adenoviruses (Ad) have been studied extensively as model transcriptional regulators to uncover molecular mechanisms that control viral and cellular gene expression. Upon infection, E1A is the first viral region transcribed, and E1A pre-mRNA is alternatively spliced into mRNAs, designated 13S, 12S, and 9S mRNAs, as well as the less abundant 10S and 11S mRNAs  Fig. 1)Fig. 1).Large and small E1A both contain the N-terminal CR1 and CR2, which interact with the closely related nuclear lysine acetyltransferases CBP/p300 and RB family proteins  to reprogram host cell gene expression and induce S phase in quiescent host cells , 6 Fig.. The smadl312 (6) just C terminal to CR3  were later shown to be essential for activation of early viral promoters by native E1A  in transient-transfection assays  implicated in promoter binding via association with host cell sequence-specific transcription factors -derivative tagged proteins. We constructed Ad mutants with multialanine substitutions in E1A regions observed to be required for the interaction with p300 in this in vivo protein-protein interaction assay and analyzed transcription from early viral promoters after infection of human primary airway epithelial cells. Additionally, we used these same methods to further characterize the association of multisubunit mediator complexes with the E1A activation domain and propose a new explanation for the requirement of the CR3 C-terminal invariant region (aa 183 to 188) for activation. Using chromatin immunoprecipitation sequencing (ChIP-seq), we analyzed the association of modified histones, TBP, and Pol II with the early viral promoters after infection with wild-type (wt) and Ad5 mutants. We found that eliminating the interaction between the large E1A activation domain and p300 prevented H3K18/27ac at the adenovirus E2early, E3, and E4 promoters during the early phase of infection. The consequences of this failure to acetylate H3K18/27 in these promoter regions for PIC assembly and transcription were different at each of these viral promoters.To corroborate this conclusion by an independent method, we analyzed p300 interaction with E1A(aa 121–223) in vivo by confocal fluorescence microscopy of proteins fused to differently colored GFP derivatives. Expression vectors for YFP-p300 and LacI-mCherry alone or LacI-mCherry fused to an E1A fragment consisting of aa 121 to 223, E1A(aa 121–223), were transfected into CHO-A03.1 cells. These cells contain an amplified region of DNA with ∼10,000 lac operator sites (lacO) integrated into a single region of one chromosome , a region lacking the well-characterized large and small E1A p300/CBP binding site formed from the E1A N-terminal ∼12 aa plus E1A(aa 54–82) in CR1 , 25. To romosome . Expressy signal  and c. CcO array  to c. HocO array  to f, coin vivo colocalization assay.To identify the amino acids of large E1A required for this specific association with p300, we assayed colocalization of YFP-p300 with LacI-mCherry fused to deletions of E1A(aa 121–223) . Fusion In vivo colocalization, assayed as described above, was again used to observe the association of E1A deletion constructs with mediator subunit MED23, located in the tail of the mediator    associat to 178) . Interes to 178) . These r to 178)  and 3a a4-type zinc finger, is essential for activation , including a C0–178, intivation , 18. In tivation –18. To dpromoter  29). Th. Th4-typin vivo colocalization assay, E1A(aa 133–189) and E1A(aa 140–205), each lacking one of the two acidic peptides flanking CR3, displayed partial defects in activation, with ∼50% of the activity of E1A(aa 121–223)  did not affect p300 binding in our 121–223) . Deletio121–223)  resulted121–223) . The gre121–223)  to e. Thin vivo Cre-mediated site-specific recombination with the Ψ5 vector  of 20, we observed that HBTECs expressed considerably more wt than DM E1A protein (data not shown), possibly due to differences in protein stability caused by the introduced mutations. Therefore, in order to achieve equal levels of wt and DM E1A protein, cells were infected with the wt virus at an MOI of 5 and diluted with the E1A deletion mutant dl312 at an MOI of 95. Alternatively, cells were infected with the DM virus alone at an MOI of 100. Under these conditions, the levels of wt and mutant E1A proteins were similar at 12 and 18 h postinfection (p.i.) , derived from the natural Ad5 host tissue. We first analyzed the consequences of mutations in the DM, since deletion of both of the mutated regions in the DM was required to eliminate the interaction of E1A with p300 in the n (p.i.) . Furthern (p.i.) . An unandl312 viruses at 12 h p.i. and was only very modestly decreased at 18 h p.i. While E2, E3, and L3 RNA increased between 12 h and 18 h p.i. in response to wt E1A, E4 RNA nearly reached a plateau at 12 h  site, including all of the major E1A splice sites  site within the E1A 3′ exon  to 200]  to d. De to 200]  to d.lacO array in CHO-A03.1 cells , corresponding to a sequence found just C terminal to the Zn-finger region in large E1A proteins that is precisely conserved in all primate adenoviruses m et al. , contribBecause p300/CBP is primarily responsible for mediating the acetylation of histone H3 at lysines 18 (H3K18) and 27 (H3K27) , 8, 9, wy axis at the bottom in gray).To assay Pol II preinitiation complex (PIC) assembly, we also did ChIP-seq for TBP and Pol II. The TBP subunit of TFIID is the first protein to interact with a TATA box promoter during PIC assembly, while Pol II is one of the last . We cleaTo determine if the interaction of p300 with the E1A activation domain influences E2, E3, and E4 transcription via acetylation of histone H3 during infection, we also performed ChIP-seq for H3K18ac, H3K27ac, and H3K9ac in virus-infected HBTECs expressing equal concentrations of wt and DM E1A protein at 12 and 18 h p.i. We observed dramatic decreases in H3K18/27ac peaks at the E2early, E3, and E4 promoters in cells expressing DM E1A compared to wt E1A . ChIP-sePol II and TBP ChIP-seq experiments were subsequently performed to determine if these differences in H3 acetylation were associated with differences in PIC assembly on the early viral promoters. Striking decreases in TBP and Pol II association induced by DM E1A compared to wt E1A correlated well with the observed decreases in H3K18/27ac at the E2early promoter. Apparently, the failure of the E1A DM to induce H3K18/27 acetylation at the E2early promoter completely blocked RNA polymerase II PIC assembly on the E2early promoter. In the same cells infected with the E1A DM and as discussed above in relation to the E2early promoter, H3K18/27ac also was virtually eliminated at the E3 promoter, with a TSS only 418 bp to the right of the E2early TSS. However, in contrast to the nearby E2early promoter, at the E3 promoter TBP association was only slightly reduced compared to that of cells expressing the same concentration of wt E1A, and there was no apparent decrease in RNA polymerase II association. These ChIP-seq data correlated well with the much greater effect of the E1A DM mutations on expression of E2early RNA compared to that of E3 RNA .Surprisingly, and of considerable interest, TBP and Pol II association with the E4 promoter were only minimally decreased by the DM mutations , even thAlthough there is a well-established correlation between promoter and enhancer region histone N-terminal tail lysine acetylation and transcriptional activity , the mecin vivo, we confirm an interaction between the strong E1A activation domain included in E1A(aa 133–205) and the acetyltransferase p300, as reported by Pelka et al. 6, and an (AP)6 substitution was inactive  with p300 were mapped to two acidic peptides flanking CR3, aa 133 to 138 (DDEDEE) and aa 189 to 200 (EPEPEPEPEPEP). This EP repeat was previously shown to contribute to maximal E1A activation of the early adenovirus promoters in transient-transfection assays with native E1A as opposed to Gal4DBD-E1A fusions and was named auxiliary region 1 , 17. Thefunction  and e. Ifunction , 17, wasfunction . The gre with p30function , in coimfunction , 7, 25.4-Zn-finger region of large E1A(aa 140–178)  infected at low MOI with wt mouse adenovirus I (MAVI) failed to express the MAV early mRNAs, similar to the phenotype observed for E1A-minus MAVI in wt MEFs   is requi domains . The sam domains . These rn domain . Further wt MEFs .in vivo, we analyzed nuclear colocalization of LacI-CFP-E1A fragment fusions with YFP-MED6 at the lacO array in CHO-A03.1 cells  that are invariable in all primate adenoviruses  stimulates the assembly of a PIC on a template with Gal4-binding sites . Present3 stimuladl312 virus and cells infected with dl312 alone  H3K9ac in the absence of H3K18/27ac may be sufficient for PIC assembly at the E3 promoter.While hypoacetylated H3K18/27ac was observed at the E4 promoter in cells expressing DM E1A, high levels of H3K9ac as well as TBP and Pol II association were observed. This suggests that H3K18/27ac is not required for PIC assembly at the E4 promoter. However, H3K9ac at the E4 promoter, which continues to occur in cells expressing the E1A DM, may be sufficient for Pol II PIC assembly. Surprisingly, in cells infected with the E1A DM, we noted a clear discrepancy between the TBP and Pol II ChIP-seq signals at the E4 promoter and the qRT-PCR data for E4 mRNA. Pol II and TBP peaks were not reduced at the E4 promoter in DM-infected cells , yet H3KH3K9ac peaks were present at the E2early, E3, and E4 promoters in addition to H3K18/27ac and were decreased after infection with the DM at the E2early promoter . This inThese patterns of H3 acetylation at early viral promoters in response to multialanine substitutions of subdomains of the E1A activation domain indicate that H3K18/27ac by p300/CBP is required for maximal H3K9 acetylation and the assembly of Pol II PICs at the E2early promoter. In contrast, Pol II PIC assembly on the E3 and E4 promoters is associated with H3K9ac but not H3K18/27ac. In the case of E3, this is sufficient for PIC assembly and moderate transcription. At the E4 promoter, the E1A activation domain-p300/CBP interaction is not required for TBP and Pol II association but is required for H3K18/27ac and transcription. This may be because elongation of paused Pol II at the E4 promoter is dependent on H3K18/27ac. By eliminating the association of the E1A activation domain with p300, we have revealed striking diversity in transcriptional regulation of the viral genome early in infection, including nuanced roles for p300/CBP at each early viral promoter.in vivo colocalization assays and luciferase reporter transient-transfection assays were constructed from plasmid NYE127  were grown at 37°C in a BronchiaLife medium complete kit  in a 5% COTotal RNA extracted from HTBECs using a PureLink RNA minikit (Ambion) was reverse transcribed with random hexamer priming using Superscript III (Invitrogen). RNA was treated with DNase I with a DNA-free kit (Ambion). Quantitative reverse transcription-PCRs (qRT-PCRs) were carried out with the Applied Biosystems 7500 real-time PCR system with FastStart universal SYBR green master mix (Roche). All values were normalized to 18S RNA levels.Preparation of cross-linked HBTEC chromatin, sonication, and immunoprecipitation was as described in reference Antibodies included H3K9ac  and H3K18ac (814), prepared and validated as described previously , and Ku-GSE116772.DNA sequencing data related to