Previous studies have shown that repeated influenza vaccination can enhance susceptibility to subsequent infection with a drifted influenza virus strain. This paper seeks to further understanding of the interactions between influenza viruses and specific immune cells that accompany this phenomenon. The paper argues that repeated vaccination increases susceptibility to infection only in the context of a residual immunity induced by prior vaccination or infection. The results of computational analysis indicate that this is a dynamic consequence of interactions between vaccines, influenza viruses and specific immune cells. In particular, mathematical modelling was used to show that in the presence of residual immunity conferred by a vaccine administered in Canada in the 2013–2014 influenza season, the 2014–2015 season vaccine enhanced susceptibility to infection. Such infection enhancement occurs when the 2014–2015 vaccine boosts suppressive T-regulatory cells induced by the 2013–2014 vaccine, decreasing the strength of antibody responses to the infecting strain. Overall, the study suggests probable characteristics of infecting viruses and vaccines that make repeated influenza infections and vaccinations detrimental. Previous exposures of the immune system to antigens facilitate prompt responses to future infections with the same antigens. The body accomplishes this by using immunological memory cells derived from the previous exposures to the antigens. Besides direct natural infections from disease causing organisms resulting in what is termed as natural immunity, vaccinations intended to stimulate specific immune responses are also means to expose the body to antigens to confer resistance to future infections. The latter is an efficient medical intervention that has been successfully applied to eradicate mortality from some communicable diseases over the years . HoweverParticularly, this has been well reported in the studies of influenza since 1979 when Hoskins and colleagues revealed that repeated influenza vaccinations only provided a suboptimal protection in the long term . In thatUsing a novel mathematical model, this study investigates the immunological dynamics underlying these particularly troubling observations in influenza. More specifically, separate systems of models designed to capture the immune responses under various conditions of repeated vaccinations and infections are conveyed in a system of differential equations. In this way, it will be possible to ascertain the consequences of synergies of influenza viruses, specific immune cells and vaccines to describe the immune response in both repeated infections and vaccinations. With an elucidated immune response under these conditions, this paper further provides possible implications of findings on the effectiveness of influenza vaccines.. 2. 2.1The model reproduces the consequences of the dynamic interactions between the immune agents and vaccination during influenza epidemic. It comprises of a subset of the components of the immune system, the regulation of immune response to influenza virus, T-regulatory cells (tregs) and the target cells of the virus.The influenza virus infects epithelial cells of the lungs which results in the activation of lymphocytes through mechanisms involving antigen presentation by antigen presenting cells ,13. WhenV1, if the adaptive immune system has never encountered any antigen which cross-reacts with V1, specific naive lymphocytes will be activated [V2, which cross-reacts with V1, is encountered by the immune system in future infection, then a subset of V1-specific lymphocytes, including tregs will be re-activated in addition to the activation of naive lymphocytes by V2. The reactivated tregs will suppress the presentation of V2-specific antigens by dendritic cells and decrease antigen dose loading of V2 accessible for activating V2-specific B cells and specific T-helper cells [3V that cross-reacts with V1 and V2 is encountered, then the strength of humoral response to 3V will depend on activated V1- and V2-specific B cells and naive lymphocytes elicited by V3 [V1- and V2-specific lymphocytes compared with naive lymphocytes [Particularly, in line with previous studies –20, thisctivated ,22. Whener cells . If a sted by V3 . Note thphocytes . These fphocytes –20. In ti, infects target cells (Ei) at a rate EViβ leading to the activation of B cells (Bi) and tregs (Ri). The activated virus-specific B cells also produce IgG Abs (with concentration Ai pg ml−1) that react to the virus. As has been indicated, previous knowledge [D). However, antigen presentation is suppressed by activated tregs [R) and antigen dose loaded (D). These motivated equations (2.3) and (2.4) in the model. Equations (2.1) and (2.2) mark the dynamics of the number of healthy target cells and the number of infected target cells respectively over time. The presentation of virus antigens by dendritic cells results in the activation of B cells, which produce virus-specific antibodies. This dynamic relationship informed the formulations of equations (2.5) and (2.6) of the model. In addition, the infecting virus titre  at any time will be expected to be a function of the number of infected cells and the concentration of the neutralizing Abs as entailed in equation (2.7). The mathematical formulations based on these facts founded the following model:i or j identifies the specific virus strain.With the background of these aspects of the adaptive immunity which has also been the main focus of previous studies –20, the nowledge  permits ed tregs  which isvc) and this is given by:An addition to this model is the extension to simulate influenza vaccination. Particularly, in vaccinations, there are no rates of non-specific virus clearance and infections of vaccine strains. These are particularly significant since immunizations are given wholly to individuals and the vaccine strains do not replicate. Therefore, a new equation accounting for these variations in equation (2.7) excludes rate of non-specific virus clearance  the exposition of the immune system to specific influenza virus strains and then elicits its response to a cross-reactive virus in an epidemic. In the repeated infections, the case of three cross-reactive virus strains, namely he model . Consequepidemic .50 ml−1, respectively [In the absence of any infections, there will be no infected cell, virus-specific B cells, tregs, antigen dose to be loaded by dendritic cells, and virus-specific antibodies at the beginning of the experiment. This is indicated by the initial conditions of the variables in the system. However, the initial number of uninfected target cells and the available concentration of infecting virus at the start of the experiments were set to 200 000 and 1500 EIDectively ,27. The ectively .. 3. 3.1V1 for 28 days. This was followed by another vaccination with vaccines derived from the strain V2 for another 28 days. The second group (single vaccination) was vaccinated with only one vaccine derived from strain V1 for 28 days. Each group and a naive group without any prior vaccination are then infected with equal amounts of virus titre of the challenge strain, e (epidemic strain), that cross-reacts with each vaccine strain. The peak viral load in each group is derived for different rates of cross-reactivity between vaccine strains (vaccine–vaccine cross-reactivity) and between vaccine strains and epidemic strain (vaccine–epidemic strain cross-reactivity). Although 10% and 20% rates of vaccine–vaccine cross-reactivity are considered, the vaccine–epidemic strain cross-reactivity was varied between 10% and 90%. On the other hand, to determine the effects of prior infections on immune response in an epidemic, simulations of sequential infections were performed in another two different groups with virus strains in a mathematical model.To examine the effects of repeated vaccinations on the immune cells, experiments in two groups were simulated using the proposed model. In the first group (repeated vaccination), the first vaccination was performed with the vaccine strain derived from the virus V1 for 28 days followed by infection with strain V2 for another 28 days. In the second group (single infection), the model simulated the infection with V1 alone for 28 days. The simulation in this second group was allowed for another 28 days before the challenge infections were applied. Each group is then challenged with e (epidemic strain) infection for 28 days. Here, while cross-reactive rates of 10% and 20% between infecting strains prior to the epidemic infections (prior strains cross-reactivity) were used, the cross-reactivity between epidemic and previous infecting strains (prior–epidemic strain cross-reactivity) was varied from 10% to 90%. In each experiment of this study, the immune response in terms of the activated effective tregs, effective B cells and Abs as well as the peak viral loads are derived during the challenge infection. The peak virus load in each experiment was calculated as the peak amount of the challenge infection minus the challenge dose [In the first group (repeated infection), the sequential infection was simulated with strain nge dose .. 3.2a). In the experiment with vaccine–vaccine cross-reactivity of 20%, higher viral loads were found over the period of increasing the vaccine–epidemic cross-reactivity in as much as it was falling in repeated vaccination (figure 3b). This was to be expected for different vaccine–vaccine cross-reactivity. The viral loads increased in the repeated vaccination as a result of lower effective lymphocytes activated to react to the epidemic permitting virus to replicate. Higher viral loads in repeated vaccination are indicative that they are more susceptible to the influenza epidemic infections compared with single vaccination. These observations were not different in repeated sequential infections as they had the highest viral loads compared with single infection although they were characterized by lower viral loads compared with repeated vaccination (figure 3c). However, although no viral load was found in each infection group beyond prior–epidemic strain cross-reactivity of 35%, it was found that the single infection had much lower values with no viral load from 20% in all experiments (figure 3c). When prior infections cross-reacted at 10%, no viral load was observed at prior–epidemic strain cross-reactivity of 30% or more. Nonetheless, the absence of viral loads could only be found after a prior–epidemic strain cross-reactivity of 35% in the case when prior infections cross-react at 20% (figure 3c). For the experiment in which prior infections cross-react at 20%, the viral loads at the early epidemic infection require an increased amount of activated lymphocytes which is achieved when the prior–epidemic strain cross-reactivity increases beyond 30%.For a vaccine–vaccine cross-reactivity of 10%, viral loads in repeated vaccination were reducing for vaccine–epidemic strain cross-reactivity up to 70% as did single vaccination . Howeverfigure 3d). This consistent decrease was anticipated since the viral loads in single and repeated infections decreased together (c). Increasing the prior–epidemic strains cross-reactivity enhances the activation of effective lymphocytes to react to the epidemic strain. This reduces the viral loads in each infection. However, as the prior–epidemic cross-reactivity exceeds 16%, the single infection has relatively very small and constant viral loads compared with the decrements observed in the repeated infection. This increases the ratios above 20% until the prior–epidemic strain cross-reactivity is 22% (d). The higher viral loads in repeated infections in the experiment involving prior strains that cross-react at 20% accounted for higher ratios as the cross-reactive rate between the epidemic strain and the prior infections increases to 22%. These results suggest that the viral loads in single infection only exceed half of viral loads in repeated infection while the prior–epidemic strain cross-reactivity is not more than 6%. However, it is largely reduced when the cross-reactive rate between the epidemic strain and the prior infections exceeds 6%. Thus, repeated infection group becomes more susceptible to the epidemic infections because they have higher viral loads compared with single infections.To further explore the viral loads, the study investigates their ratios within the range of cross-reactivity where significant viral loads were found in the repeated infection. The ratios represent the fraction of viral loads of single infection contained in repeated infection. Overall, the ratios decreased to less than 10% as the prior–epidemic strain cross-reactivity increased to 16% d. This ctogether c. Increay is 22% d. The hi. 3.3During vaccinations, there are lower amounts of activated lymphocytes compared with prior infections . These dc). The higher numbers of activated effective tregs in the repeated infections are accumulated from reactivation of the tregs derived from the two prior infections that cross-react with the current epidemic strain. Nevertheless, the rising numbers of tregs in all experiments as prior–epidemic strain cross-reactivity increased resulted from increasing reactivated tregs for higher cross-reactive rates. Higher vaccine– or prior–epidemic cross-reactivity increases the reactivated tregs derived in reaction to the epidemic. As a result, the effective tregs increase during the epidemic as more tregs are activated in respect of epidemic strain. Unlike repeated vaccination, repeated infections in which the prior strains (V2 and V1) cross-react at 20% had higher tregs compared with single prior infection . However, as B cells produce effective Abs for clearing virus, it was expected to decrease after the peak amount of B cells since virus clearance reduces antigens loaded for B cell activation [figure 5c). During the early days of epidemic strain infection, lower antigen dose was loaded by dendritic cells to activate specific lymphocytes leading to lower numbers of B cells in both vaccination and sequential infection experiments at that time  produced during the epidemic infection are lower compared with the 10% vaccine–vaccine cross-reactivity figures  and 6. Ffigure 5tivation . The peativation . The moda). This, particularly, higher effective Abs at the early days in repeated infection results from sufficient antigen dose loading by dendritic cells for higher production of Abs in the repeated vaccination where vaccines cross-react at 10%. Nevertheless, the Abs in both repeated and single vaccinations increase to a maximum from day 3 to day 15 after which their levels decline as a result of virus clearance . In this case, antigen presentation by dendritic cells is greatly suppressed by the higher numbers of effective tregs for production of Abs in repeated infection compared with single infection. The observation is well noted in the role of tregs to dynamically control the primary immune response [c). Overall, lower levels of effective Abs were found in repeated infections compared with single infection as a result of relatively lower amounts of B cells. These suggest that Abs produced in response to the epidemic result in a better protection in single infection compared with repeated infection. In essence, the dynamics of Abs production make repeated infection more susceptible during the epidemic compared with single infection because the suboptimal reaction to the virus leads to the higher viral loads.Similarly, results of effective Abs produced in repeated infections are lower compared with single infection c. In thiresponse –38. As i. 3.4V1 for 28 days followed by V2 for another 28 days. The host was then injected with the challenge virus, e (epidemic strain). The experiments are repeated for 100 pairs of cross-reactive rates. Each pair of values of cross-reactivity represents the chances that lymphocytes derived by V1 will recognize e (1-eV cross-reactivity) and the chances that lymphocytes derived by V2 infection will recognize e (2-eV cross-reactivity), respectively. The peak virus load for each experiment and the number of effective tregs are then derived.The sensitivity of viral loads to cross-reactive rates was examined by studying the effects of sequential infections of several cross-reactive strains on viral loads using the mathematical model. This was simulated in a sequential infection with 1-eV cross-reactivity and 2-eV cross-reactivity increased (a). Changes in viral loads as the 1-eV cross-reactivity increases indicate that viral loads are very sensitive to the 1-eV cross-reactivity. Increasing the V1-e cross-reactivity increased the effective Abs that reacted to the virus leading to the decrease in viral loads. On the other hand, while the V2-e cross-reactivity was increased, viral loads decreased significantly for cross-reactivity exceeding 30% (a). The decrease in the viral loads was a result of increase in effective Abs that react to the virus as the 2-eV cross-reactivity increases.The viral loads decreased as the ncreased a. Changeding 30% a. The de2-eV cross-reactivity increased (b). The increase in the effective tregs as V2-e cross-reactivity increases is achieved as a result of reactivating V2-specific tregs that complement the effective tregs derived during the epidemic. Nevertheless, while varying the V1-e cross-reactivity, only small increments in tregs in some instances of V2-e cross-reactivity were observed (b). The V1-derived tregs directly add to the effective tregs activated during the epidemic infection. Therefore higher V2-e cross-reactive rate increases the effective tregs derived at the epidemic infection by reactivating more V2-specific tregs. However, V2-derived tregs occur in relatively larger amounts so that higher 1-eV cross-reactivity will be required to observe its accompanying changes during the epidemic. The consequences are that the changes in tregs that occurred due to increasing 1-eV cross-reactivity required higher V2-e cross-reactivity. These results further confirm earlier observation that the immune response is sensitive to changes in V1-e cross-reactivity as it does to V2-e cross-reactivity.In investigating the roles of tregs in relation to antigens and regulation of the immune response, tregs were expected to be sensitive to the cross-reactive rates between infecting strains ,40. Thisncreased b. The inobserved b. The V1. 3.5Epidemiological findings following influenza vaccinations linked individuals who had been vaccinated with variable risks of influenza infection in an epidemic –7. In thIn order to allow a direct comparison of the study with the work of Skowronski and colleagues , additio. 4Sometimes, rather than boosting immunity, repeated vaccination make vaccine recipients more susceptible to epidemic infections compared with single vaccination. These have been reported in the case of seasonal influenza infestation over the past years –7. TheseThe study identifies higher viral loads in repeated vaccinations compared with single vaccination as a factor contributing to the susceptibility of the vaccine recipients during influenza epidemics. In vaccination, the activated effective lymphocytes that react to the epidemic strain are reduced resulting in higher viral loads compared with sequential infections. The fraction of sites on the surfaces of dendritic cells that load antigens of the epidemic strains are reduced in the vaccinations. This lower antigen dose to be loaded by dendritic cells is to activate effective B cells and Abs. Insufficient effective Abs allow virus to replicate leading to high viral loads. Although repeated vaccinations could enhance the production of B cells and Abs, the cross-reactive rates between vaccine strains were very crucial during the epidemic infections. In particular, vaccine strains that cross-react at 20% culminate in decreasing antigens loaded by dendritic cells for activating the effective B cells and to produce Abs in the epidemic. Therefore, there are lower effective B cells and Abs during the epidemic compared with experiments in which vaccine strains cross-react at 10% figures  and 6.The study further investigated the immune response to repeated infections. Individuals who have experienced repeated prior infections are highly susceptible to infections during the epidemic compared with those with single prior infection, which have much lower viral loads. In repeated infections, a cross-reactive epidemic strain reactivates tregs derived from previous infections. This increases the effective tregs activated in response to the epidemic in repeated infections. Also, increasing the prior–epidemic strain cross-reactivity increases the number of reactivated tregs which cumulates the effective tregs derived during the epidemic infection. Therefore, the effective number of tregs during the epidemic increases as the cross-reactivity between the prior infections and the epidemic strain increases . A seconConsequently, during the epidemic infection, numbers of effective tregs are produced in excess in the repeated infections involving prior infecting strains that cross-react at 20% compared with 10% cross-reactive rate. The higher effective tregs decrease the antigen dose of epidemic strain loaded by dendritic cells to activate lymphocytes. This leads to lowering the activated number of effective B cells and the production of Abs during the epidemic infections. As the epidemic progresses, higher antigen dose are loaded by dendritic cells in both groups (single and repeated infections) resulting in the activation of higher B cells and Abs production until reduction in viral loads as Abs react to the virus figures  and 6. Bd). However, this is decreased as the cross-reactivity between the prior infections and the epidemic strain increases since these increments allow more Abs to be produced to react to the virus. The presence of higher viral loads in the repeated infection for a very low prior–epidemic strain cross-reactivity make them more susceptible to infection compared with single infection. On the detrimental effect of excess suppression of antigen dose loading by tregs, it has been suggested that increasing antigen dose can diminish the effect of tregs in order to allow for activation of sufficient lymphocytes in response to antigens [The presence of higher Abs in single infections provides better reaction to the epidemic strain resulting in lowering viral loads. Specifically, 20% cross-reactivity between the prior infections and the epidemic strain is sufficient to increase the effective B cells to produce Abs capable of completely clearing the epidemic strains in the single infection. The effective Abs are increased by increasing the cross-reactivity between the prior infections and the epidemic strain to 30% or more for a complete virus clearance in repeated infections. In addition, when prior infections cross-react with the epidemic strain at a rate less than 6%, effective lymphocytes activated in the early days of repeated infections are in very low amounts. This allowed the repeated infections to have more than twice the viral loads in single infections as virus replicates d. Howeveantigens . In thisThe findings of this study suggest that repeated vaccination could enhance immune response provided that the vaccine strains cross-react at rates which do not exceed 10%. Nonetheless, as antigen dose of dendritic cells increases as the epidemic progresses with virus replication, more Abs are produced (in single vaccination) to react to the virus (compared with the repeated vaccination). The presence of higher effective B cells and effective Abs produced as the epidemic progresses beyond day 3 provide better protection for individuals with single vaccination compared with those who receive repeated vaccinations. These results support the previous studies that found that repeated vaccination was not effective in the long term ,49. In p. 5Overall, the study suggests probable characteristics of vaccine or infection strains (viruses) that make repeated vaccination/infection detrimental. In particular, the results of the study suggest that individuals with single vaccination are less susceptible to infections during the epidemic compared with those who receive repeated vaccination with residual immunity gained from first vaccination. It has been shown that the higher effective Abs produced to react to the epidemic infection make the immune response more effective in single prior vaccination and infection. The integration of literature and simulations with the models are indicative that the immune response may be compromised in repeated vaccination during the influenza epidemic compared with those with lesser vaccination. In repeated vaccinations, although immunity may be enhanced at the early days of the epidemic with vaccine strains that cross-react at 10% or less, those with single vaccination will always have better protection in the long term. The findings of this study call for further investigation and considerations in vaccine designs and administration. Particularly, since antigen dose loading by dendritic cells are reduced in repeated vaccination, this can be heightened by using adjuvanted vaccines so as to improve the activated effective lymphocytes during the epidemic. This could have been applied during the 2014–2015 influenza season where most of the vaccines were not adjuvanted. This study investigated the dynamics of the immune response to influenza infection and use can be made of the methods in studying other variable pathogens.Click here for additional data file.


Arabidopsis thaliana TPL to enable repression of a synthetic auxin response circuit in Saccharomyces cerevisiae (yeast). Here, we leveraged the yeast system to interrogate the relationship between TPL structure and function, specifically scanning for repression domains. We identified a potent repression domain in Helix 8 located within the CRA domain, which directly interacted with the Mediator middle module subunits Med21 and Med10. Interactions between TPL and Mediator were required to fully repress transcription in both yeast and plants. In contrast, we found that multimer formation, a conserved feature of many corepressors, had minimal influence on the repression strength of TPL.The plant corepressor TOPLESS (TPL) is recruited to a large number of loci that are selectively induced in response to developmental or environmental cues, yet the mechanisms by which it inhibits expression in the absence of these stimuli are poorly understood. Previously, we had used the N-terminus of  Drosophila Groucho (Gro) and mammalian transducing-like enhancer (TLE)  .In plants, the role of Gro/TLE-type corepressors is played by TOPLESS (TPL), TOPLESS-RELATED (TPR1-4), LEUNIG (LUG) and its homolog (LUH), and High Expression of Osmotically responsive genes 15 (HOS15) . Plant cSaccharomyces cerevisiae (yeast) by porting individual components of the Arabidopsis auxin nuclear response  behave differently , and the second, more potent domain to Helix 8, which falls within the CRA domain. Full repression by Helix 8 required the Mediator complex, specifically direct interaction with Med21 and Med10. The Med21 residues that interact with TPL are the same ones required for transcriptional repression by the yeast corepressor Tup1. In addition, we found that multimerization of TPL was not required for repression in yeast or in plants. Our yeast results were validated with plant assays and extended to include evidence that interaction with the middle domain of Mediator was required for TPL repression of the auxin-regulated development program giving rise to lateral roots. Our findings point to a conserved functional connection between Tup1/TPL corepressors and the Mediator complex that together create a repressed state poised for rapid activation.Here, we leveraged the power of yeast genetics to interrogate the mechanism of TPL repression. Using AtARCSc   . We choscytoSUS) . PutativcytoSUS) , HistonecytoSUS) , and thecytoSUS)  and MED2cytoSUS) . We did cytoSUS) . HDAC incytoSUS) ; howevercytoSUS) . The absArabidopsis  completely abrogated AtMED21 binding (AAAA mutations into the Helix 3 through Helix 8 context (H3-H8-QuadAAAA) in the AtARCSc largely phenocopied a deletion of Helix 8  enabling transcriptional activation . The conAtARCS  using the Versatile Genetic Assembly System  and higher enrichment of MED21-FRB at the active PMA1 promoter (approximately eightfold). Next, we introduced the fully repressed SPARC plasmid containing TPLN188 (SPARCN188) into a library of Anchor Away yeast strains that allow specific depletion of Mediator components  partially activates genes that are normally repressed by Tup1 , so we fPL H1-H5 . No mutaPL H1-H5 , we remoPL H1-H5 .N188 in Δ3ScMed21 or Δ5ScMed21 mutants showed elevated reporter transcription when compared to strains carrying wild-type ScMED21  into the Anchor Away SPARC strains . The comexamined  while inexamined . When wiexamined , whereasexamined . iΔ3Med2examined . In the examined .tpl-1 altered a single amino acid in the ninth helix of the TPL-N terminus (N176H) that induces aggregation of TPL and its homologs (TPR1-4), reducing total activity  that abrogated the ability of the CRA domain (Helix 6 and Helix 7) to form inter-TPL interactions . As thisTo make a fully monomeric form of TPL, we introduced mutations into the dimerization interface of the LisH domain in the context of LDimer. We first mutated one of a pair of interacting residues (F15) to a series of amino acids  in the context of either LDimer , or H1-2To further push the LDimer towards a monomeric form, we introduced two additional mutations  and Arabidopsis. Bimolecular fluorescence complementation (BiFC) confirmed the interaction between TPL and MED21 (H8QuadA). Similarly, the Δ5AtMED21 N-terminal truncation eliminated interaction with full-length TPL (AAA) and in the degron (P306S)  mutant, which carries an auxin-resistant form of IAA14 (mED (with no TPL fusion) or TPLN188 (with no IAA14 fusion) had no effect on lateral root production , to repression by TPL . We did activity . Loss ofAtARCSc could facilitate a resolution to this apparent contradiction by targeting repression to a single synthetic locus. We focused our initial efforts on the analysis of the N-terminal portion of TPL, which has multiple known protein-protein interaction surfaces (AtARCSc identified two independent repression domains (A review of the current literature on corepressors gives the conflicting impressions that (a) corepressor function is broadly conserved and (b) that every organism (and perhaps even every corepressor) has a distinct mode for transcriptional repression . We hopesurfaces . Experim domains , and we  domains . Four am domains . Indeed, domains . Contrar domains , leaving domains .Corepressors coordinate multiple mechanisms of repression through discrete protein interactions, leading to robust control over eukaryotic transcription by combining repression modalities. Corepressor function has variously been linked to (a) altering chromatin confirmation, often through interaction with histone-modifying proteins or histone proteins themselves, (b) direct interference with transcription factor binding or function, and (c) physical spreading of long-range oligomeric corepressor complexes across regions of regulatory DNA . DissectThe synthetic system used here allowed us to build on this model and further refine our understanding of TPL’s repressive activity. In our experiments, we see a similar set of conditions, with TPL recruited to the DNA-bound transcriptional activator (ARF), and several possible mechanisms of repression. Unlike Tup1, we have subdivided the TPL protein to identify interactions between TPL and individual protein interactors with no effect on yeast function. In these experiments, we can eliminate the possibility that TPL blocks ARF activation by directly blocking the transcription factor activation domains because we see a loss of repression only when TPL-MED21 binding is eliminated through specific point mutations . Our estThe Mediator complex is a multi‐subunit complex that connects DNA‐bound transcription factors and the RNA Pol II complex to coordinate gene expression . The yeaArabidopsis .All codes are available through Github: AtMED21 promoter  using PCR and Golden Gate cloning strategy described in Construction of TPL-IAA3 and TPL-IAA14 fusion proteins was performed by Golden Gate cloning as described in Fluorescence measurements were taken using a Becton Dickinson (BD) special order cytometer with a 514 nm laser exciting fluorescence that is cut off at 525 nm prior to photomultiplier tube collection . Events were annotated, subset to singlet yeast using the FlowTime R package . A totalAtARCSc to eliminate any repetitive DNA sequences  into the acceptor plasmid by Golden Gate reaction using designed BsmBI sites engineered into the acceptor plasmid and the primers used to amplify the CEN/ARS (see https://www.addgene.org/Christopher_Grefen/). Interaction between bait and prey proteins was evaluated using a modified version of the split ubiquitin technique (Standard yeast drop-out and yeast extract–peptone–dextrose plus adenine (YPAD) media were used, with care taken to use the same batch of synthetic complete (SC) media for related experiments. A standard lithium acetate protocol  was usednces see , using a/ARS see . For theechnique . After 2600 = 0.6 and incubated until cultures reached OD600 ∼1. Cells were harvested by centrifugation. Cells were lysed by vortexing for 5 min in the presence of 200 µl of 0.5 mm diameter acid washed glass beads and 200 µl SUMEB buffer  per 1 OD unit of original culture. Lysates were then incubated at 65° for 10 min and cleared by centrifugation prior to electrophoresis and blotting. Antibodies: anti-HA-HRP , anti-FLAG , anti-FRB  at a final concentration of 400 μM at 18°C overnight. Bacteria cultures were centrifuged and the pellets were resuspended in the buffer A , where cells were lysed by sonication. His-tagged AtTPL188 (wt and mutants) bacteria pellets were resuspended in buffer A with EDTA-free antiprotease (Roche). The soluble fractions recovered after sonication were passed through a Ni-sepharose  column previously washed with buffer A, and the bound proteins were eluted with buffer A with 250 mM imidazole. A second purification step was carried out on Gel filtration Superdex 200 10/300 GL  equilibrated with buffer A.All multimer-deficient TPL proteins were expressed in 600 0.5 (~1E7 cells/ml) using selective media, harvested, and resuspended in 200 μl extraction buffer  with protease inhibitors. Cells were lysed by vortexing 3 × 1 min full speed with 100 μl of 0.5 mm Acid Washed Glass Beads, clarified by centrifugation , and supernatant was mixed with 1 ml IP buffer  with protease inhibitors and incubated with 100 μl of IgG sepharose at 25°C for 2 hr with rotation. The beads were washed 1× with IP buffer and 2× with IP-wash buffer  with protease inhibitors. Protein was eluted with 50 μl of SUME  buffer +0.005% bromophenol blue by incubation at 65°C for 10 min and run on handmade 12% acrylamide SDS-PAGE gels, and western blotted accordingly. Co-IPs from tobacco were performed on leaves 2 days after injection as described in Co-IP from yeast was performed using the cytoSUS strains. Cultures were grown to ODN. benthamiana plants grown at 22°C under long days (16 hr light/8 hr dark) on soil (Sunshine #4 mix) as per H8QuadA – proteins .BiFC experiments were performed on 3‐week‐old proteins . In all proteins . InjectiYeast were inoculated in liquid YPD and grown at 30°C with shaking at 225 rpm. After 18 hr of growth, cultures were diluted 1:50 in fresh YPD to a final volume of 200 ml. 4 hr post-dilution, cells were treated with 20 ml of fix solution  for 20 min at room temperature with shaking. Cultures were further treated with 36 ml of 2.5 M glycine for 5 min to quench the cross-linking. Cells were then pelleted at 4°C, washed twice with ice-cold TBS, and flash-frozen in liquid nitrogen. Cells were lysed in breaking buffer  on a bead beater before sonication. Samples were processed in a Bioruptor Plus sonication device at 50% for 30 cycles. Following centrifugation (to pellet cellular debris), the supernatant was used for the IP reaction. Biotin-conjugated Anti-HA  coupled to Streptavidin-coated Dynabeads Streptavidin conjugated magnetic beads  was used to probe for HA-tagged TPLN188. Anti-FRB coupled to Protein A Dynabeads  was used for ChIP on FRB-tagged yeast proteins. Following elution from the beads, samples were incubated overnight at 65°C to reverse cross-links. DNA was purified using a Monarch PCR purification kit (NEB). qPCR for three independent replicates was performed using iQ SYBR Green Supermix (Biorad) in a C100 thermocycler fitted with a CFX96 Real-Time Detection System (Biorad). To calculate fold enrichment, the delta CT (dCT) between input and IP was calculated for each sample for both the control locus  for 3 hr at room temperature with rotation. Strain density was normalized to an OD600 of 1 for each strain in the final mixture of strains before injection into tobacco leaves. Leaves were removed, and eight different regions were excised using a hole punch, placed into a 96-well microtiter plate with 100 µl of water. Each leaf punch was scanned in a 4 × 4 grid for yellow and red fluorescence using a plate scanner . Fluorescence data was quantified and plotted in R (ggplots). For Arabidopsis thaliana experiments using the GAL4-UAS system  seeds were obtained from the Arabidopsis Biological Resource Center . CRISPR/CAS9-based mutations in AtMED2 were generated as described in AtMED21 that introduces a single base-pair insertion of G at nucleotide 214 after the A of the start codon (i214G). This mutation alters the amino acid sequence starting at residue 25 and creates an early stop codon after 11 random amino acids  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Acceptance summary:In this study, Leydon et al., use an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein the authors determine two short α helical regions that act as independent repression domains. They provide evidence that the target of one of these domains is the N-terminal region of the Med21 subunit of the mediator complex. Chromatin immunoprecipitation experiments, anchor-away loss of function and co-immunoprecipitation assays indicate that Tpl mediated repression involves formation of a promoter complex comprising the mediator complex along with several general transcription factors, but lacking RNA polymerase II. The authors also show that Tpl-Med21 interactions are involved in Tpl mediated repression in plants.Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]eLife. Your article has been reviewed by 3 peer reviewers, including Irwin Davidson as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Lucia Strader (Reviewer #3).Thank you for submitting your work entitled "Structure-function analysis of Arabidopsis TOPLESS reveals conservation of repression mechanisms across eukaryotes" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in This is an interesting study that uses an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein the authors determine two short α helical regions that act as independent repression domains. They provide evidence that the target of one of these domains is the N-terminal region of the Med21 subunit of the mediator complex. They then use the phylogenetic relationships amongst the Tpl proteins to identify a short repression domain in the human TBL1 protein.eLife.Despite defining Tpl repression domains at amino acid resolution and at least in case of Helix 8 one of its targets, the referees were not convinced that the study provided novel insights into the mechanisms of repression. Almost all of the experiments are based on the same AtARC(Sc) system and the cytoSUS interaction assay, but were not supported by more direct biochemical interaction assays or ChIP experiments to address key events in preinitiation complex formation and RNA polymerase II recruitment that may underlie the repressive mechanism. The referees feel that such insight is required for the study to be of general interest to readers of Reviewer #1:This is an interesting study that uses an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein the authors determine two short α helical regions that act as independent repression domains. They provide evidence that the target of one of these domains is the N-terminal region of the Med21 subunit of the mediator complex. They then use the phylogenetic relationships amongst the Tpl proteins to identify a short repression domain in the human TBL1 protein.Several issues should be addressed.One of the major conclusions of this study is that the repression domain in helix 8 functions through interaction with the N-terminal domain of Med21. While all of the genetic setup provides strong evidence for this hypothesis, it would have been important to demonstrate this interaction using an independent method, co-immunoprecipitation, recombinant proteins etc. The authors could also address whether Tpl is capable of capturing the entire Med complex or only a sub-complex comprising Med21.A second major issue is that while the study defines repression domains at amino acid resolution and at least in case of Helix 8 one of its targets, the reader is still left with questions concerning the mechanism of repression. Could the authors perform ChIP studies under the appropriate experimental conditions to assess how the Helix1 and Helix 8 repression domains affect pre-initiation complex formation when they are targeted to a promoter. Do they affect PIC formation Pol II recruitment, promoter release or some other process? It seems the authors have developed a complex and robust genetic reporter system to analyse domains involved in repression. This system can be further exploited to address more mechanistic questions and perhaps even their kinetics. This is particularly important when considering helix 8 and interaction with the Med21. It is not clear to the reader, how this interaction results in repression. This aspect should be further investigated.Reviewer #2:A. thaliana TPL corepressor using a synthetic auxin response circuit (ARC) in the yeast S. cerevisiae reported in their previous paper (Pierre-Jerome 2014 PNAS). This artificial system combines plant components including TPL domain fused to IAA domain with auxin-responsive plant promoter and relies on the use of the yeast degradation and transcriptional machinery allowing to monitor the repression and response to auxin of the reporter expression. Based on published structure, the authors identified two domains of TPL corepressor that independently contribute to repression in this system. One of these domains interacts with Med21 Mediator subunit in cytoSUS interaction assay. The authors provide a lot of work with many constructions to study different domains or point amino acid substitutions in TPL-mediated auxin-responsive repression. The question of molecular mechanisms of transcription repression is certainly very important biological question that still remains poorly answered. However, the work did not provide any mechanistic insights and the artificial AtARC(Sc) system and the cytoSUS interaction assay used did not allow to address the repression mechanism in physiological context. Moreover, the role of Mediator complex through its CDK8 kinase module in TPL-mediated repression with a change in Mediator composition previously reported by Furutani laboratory (Ito 2016 PNAS) has not been addressed and integrated with regards to TPL-Med21 interaction.In this manuscript, the authors studied the specific domains of the plant On my opinion, the work is more suitable for a specialized journal. Many experiments and more direct evidences are needed to support the author conclusions. Without additional evidence, their title that contains "conservation of repression mechanisms" is not supported by experiments. An extensive experimental work should be done to provide at least some mechanistic insights on repression of auxin-responsive gene promoters that could not be achieved within 2 months.S. cerevisiae proteins of degradation and transcriptional machinery as an argument for conservation of the mechanisms. At the same time, the interaction assay with plant proteins in the yeast is presented as a direct interaction, but one could not exclude the involvement of endogenous proteins in this assay.1. The synthetic auxin response circuit (ARC) system is heterological making difficult the interpretation of the results. This artificial system combines plant components including TPL domain fused to IAA domain with auxin-responsive plant promoter and relies on the use of the endogenous, yeast degradation and transcriptional machinery. It was used previously to analyze the implication of different components of auxin response regulation from different plants and their functional annotation or for synthetic biology purposes. However, in this work the use of this system, especially to propose a molecular mechanism of transcription repression and to investigate the implication of Mediator complex is not sufficient and should be completed by additional experiments in plants in more physiological conditions (see below). The authors presented the fact that this system relies on endogenous A. thaliana.2. A direct analysis of gene expression and direct genetic experiments in plants are missing. It should be feasible since many genetic, genomic and biochemical tools are available in 3. The interaction cytoSUS assay is not sufficient to prove the direct interaction between TPL and Med21 Mediator subunit and should be confirmed by additional biochemical experiments with purified proteins or plant crude extracts . In general, TPL interaction with Mediator complex should be analyzed.4. A direct analysis of Mediator composition, especially with regards to the publication by Ito and colleagues  demonstrating the role of Mediator CDK8 kinase module in auxin-responsive repression with TPL-CDK8 module interaction and dissociation of this module for active transcription, is necessary. More generally, the role of this Mediator module in repression mechanisms should be taken into account.5. The recruitment of transcriptional machinery including Mediator to promoter and regulatory regions of auxin-responsive genes should be analyzed by ChIP. RNA polymerase II recruitment and state should be directly tested in native system.6. Physiological consequences of the changes in TPL domains and TPL-Med21 interaction should be studied.7. The results are difficult to follow. First of all, the AtARC(Sc) system should be described at the beginning of the results indicating exactly which construction was used from the previous paper of the authors (Pierre-Jerome 2014 PNAS) and emphasizing the possibility to measure reporter fluorescence to evaluate reporter repression and also the response of the system to auxin (induction in the presence of auxin). The results should be profoundly rewritten for clarity and the figure organization should be readjusted.For example, why the first figure cited in the results is Figure 1C?Why some but not all constructions are summarized on Figure 1B with corresponding repression index and auxin induction level? The main figure summarizing all truncations tested would help.In general, Figure 1 is a complex mix between different panels and constructions.8. The authors did not provide any direct evidence to claim that "these results indicate that the CRA domain (H3-H8) requires contact with MED21 to drive repression" . No direct evidence was provided that TPL interaction with Med21 is involved in repression. Med21 is not the only interacting partner of TPL. Moreover, amino acid substitutions in TPL-N188 used in cytoSUS assay that reduced TPL-Med21 interaction also reduced TPL-IAA3 interaction.9. The protein level of Med21 in wt is lower compared to Med21∆3 and ∆5  that should be explained/commented to indicate how this could influence the interpretation of the results.10. The part of the manuscript on Med21 Anchor Away constructions in AtARC(Sc) system seems very methodological. The combined system is extremely artificial and heterological with three synthetic constructions . There are too many modifications and treatments . This is the problem especially since Med21 is a part of the Mediator complex. What happens with Mediator in these conditions? The results are difficult to interpret. The complicated experimental procedure is not clear. The result description is unclear with mistakes . Why no differences occur between mock and +Rapa for ∆5-MED21-FRB in blue on Figure 4 D? In general, the potential conclusions from this part are not clear.Alternative strategies, for example point Med21 mutations directly tested in plants should be considered.A. thaliana and native gene expression? Finally, they decided to use transgenic Arabidopsis lines and obtained a negative result suggesting that multimer formation is not required for repression.11. The part of the results on TPL multimerization and potential conclusions are unclear. It is not well integrated into the manuscript. The authors indicate a caveat in the use of heterological systems that further illustrates the limitations of their use and difficult interpretation of the results. The authors also indicate that "there are important differences between the synthetic and native systems" . This is the problem for result interpretation. It is not clear why the authors then used another conditional expression system in another plant (tobacco)? (p. 19) Why they use this additional model instead of 12. The last part on minimal repression domains in synthetic circuits is not within the general scope of the manuscript. It is not clear why this part was included. One of the possibilities would be to focus the manuscript on synthetic biology purposes and to choose an appropriate journal.13. Many sentences in discussion are not supported by the results. For example, the authors should not say that their result "suggests a fundamental conservation in at least one corepressor mechanism across species" . No mechanistic insights were provided and conservation from yeast to plants was not directly addressed. No recruitment experiments were done for TLP, Mediator and RNA polymerase II. No direct experiments were done to demonstrate that TPL-Med21 interaction is directly involved in repression and to propose a molecular mechanism or to integrate their data into previously proposed model for Cdk8 module-containing Mediator. Mediator complex composition and its implication was not directly addressed.More appropriate references should be cited for the main roles of Mediator complex.No evidence for stabilization of Mediator complex by TPL were provided.It is completely incorrect to say that in plants and yeast there is "more primitive form of pausing".Reviewer #3:In this manuscript, authors seek to resolve conflicting models for corepressor function using the elegant synthetic auxin response system. Auxin signaling is governed by a de-repression paradigm and is ideally suited to interrogate co-repressor function – in this case, the TOPLESS (TPL) co-repressor. Several contradicting models have been put forward for the mechanism of TPL-mediated gene repression, ranging from a requirement for protein oligomerization for activity, interaction with distinct partners, and even which regions of the protein are required for repressive activity. Leydon et al., use the yeast-based synthetic auxin response system to interrogate these models using a single reporter locus, allowing for straight-forward examination of TPL function.Strengths of this study include the use of a simplified model to study a complex problem and the broad implications for study results . There were a few pieces of data that I found confusing, which I have outlined below. Clarifying these points would strengthen the manuscript.1. In Figure 1E, it appears that IAA3 alone can repress the reporter to some degree, or at least the maximal reporter activity cannot be achieved with IAA3 present. However, a lack of increased reporter expression with auxin treatment suggests that IAA3 degradation is insufficient to allow for maximal reporter expression . I find this to be a curious result; can the authors expand on this?2. In many cases, experiments to differentiate between variant effects on TPLN activity versus TPLN protein stability are lacking. This data would be helpful for interpreting some of the results. Note: this is included for some assays/variants, but not others.3. It seems that the images presented for the split ubiquitin assays are stitched together from multiple images. This should be made clear in both the figure legend. In addition, it should be clear whether these were performed as a single experiment or images put together from distinct experiments. If from distinct experiments, I suggest that these be repeated as a single experiment.4. I don't understand why N188 repression is not relieved by auxin treatment, when both the H1 and H3-H8 truncations are relieved. Maybe this is explained in the text, but if so, I missed this.5. I also don't understand why H3-H8-QuadAAA is more auxin responsive than H3-H8.6. I am confused by the data presented in Figure 4D. The text on lines 354-354 suggest that auxin sensitivity of the system is being examined, but it is not apparent that auxin treatment is involved in either the figure or the legend.eLife. Your article has been reviewed by 3 peer reviewers, including Irwin Davidson as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and James Manley as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Lucia Strader (Reviewer #3).Thank you for submitting your article "Repression by the Arabidopsis TOPLESS corepressor requires association with the core Mediator complex" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.The referees agreed that overall this new version of the manuscript is much improved over the original and many important issues have been addressed. However, a number of serious issues remain. If the authors do decide to submit a revision, it is essential that these concerns are fully addressed.Essential Revisions:1. As previously requested, the authors included ChIP experiments to directly show the presence of pre-initiation complex components at the repressed promoter. Nevertheless, it is not clear how the ChIP experiments were quantified, what is enrichment over background, this is not explained in the text. In addition, it is essential to include ChIP controls at an actively transcribed promoter, this is an especially important positive control for RA polymerase II that was not detected at the repressed promoters. Furthermore, Med21 Mediator subunit and TPLN188 were analyzed on chromosome-integrated AtARC locus  while other ChIP experiments for Mediator subunits, some GTFs and Pol II were done with SPARC plasmid. Chromosomal ACT1 gene body is completely inappropriate as a background for Pol II ChIP, since this region is enriched for Pol II. Appropriate control regions, regulatory, core promoter and transcribed regions, as well as experiments with untagged control strains should be added. Percentage of IP over input values should be presented for untagged control strains and for several regions . Sequences and positions of qPCR primers should be indicated. Mediator is mainly enriched on regulatory (UAS) regions, GTFs are bound on core promoters and Pol II signal in yeast is mostly on transcribed regions. Well-identified UAS enriched by Mediator should be added as positive controls and for comparison. The growth conditions should be clearly indicated to specify that they correspond to repressed state. The ChIP occupancy was analyzed only in transcriptionally repressed state and no results were provided for transition to the active state. This should be analyzed in detail.2. The co-immunoprecipitation experiments in plant extracts lack a negative control to conclude on the specificity of CoIP signal. For Figure 6B, a control IP without HA tag or antibody should be added to evaluate non-specific binding of YFP-tagged TPL to beads. A condition Med21-YFP-HA "-" YFP-TPL "-" could not serve as a negative control, since no detection with anti-GFP is possible. Using actin depletion as a specificity control is not sufficient and not an accepted control. For CoIP in Figure 6 —figure supplement 1A, a negative control is not appropriate. A control IP is needed to evaluate non-specific binding of YFP-tagged proteins to beads. Furthermore, it would be important to perform co-immunoprecipitation of the Tpl quad AAAA mutant with Med21 to confirm loss of interaction and of Tpl with the N-terminal deletions of Med21.3. It would be appropriate to remove the dimerization/multimerization experiments either completely or at least relegating them to the supplementary data and move the estradiol inducible experiments  to main figures. This data appears more pertinent to the message of the paper than the ability of Tpl to form tetramers. Also, the colours in Figure 4 Sup. 3 are difficult to distinguish, can this be improved? [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:This is an interesting study that uses an elegant multi-component genetic system to address the mechanisms of repression by the Arabadopsis TOPLESS (Tpl) protein. Taking advantage of the genetic tools and knowledge of the structure of the Tpl protein the authors determine two short α helical regions that act as independent repression domains. They provide evidence that the target of one of these domains is the N-terminal region of the Med21 subunit of the mediator complex. They then use the phylogenetic relationships amongst the Tpl proteins to identify a short repression domain in the human TBL1 protein.We thank the reviewer for their positive comments.Several issues should be addressed.One of the major conclusions of this study is that the repression domain in helix 8 functions through interaction with the N-terminal domain of Med21. While all of the genetic setup provides strong evidence for this hypothesis, it would have been important to demonstrate this interaction using an independent method, co-immunoprecipitation, recombinant proteins etc. The authors could also address whether Tpl is capable of capturing the entire Med complex or only a sub-complex comprising Med21.We thank the reviewer for this recommendation, and we have extensively addressed the protein-protein interaction question through the inclusion of several independent methods in both yeast , and plants .A second major issue is that while the study defines repression domains at amino acid resolution and at least in case of Helix 8 one of its targets, the reader is still left with questions concerning the mechanism of repression. Could the authors perform ChIP studies under the appropriate experimental conditions to assess how the Helix1 and Helix 8 repression domains affect pre-initiation complex formation when they are targeted to a promoter. Do they affect PIC formation Pol II recruitment, promoter release or some other process? It seems the authors have developed a complex and robust genetic reporter system to analyse domains involved in repression. This system can be further exploited to address more mechanistic questions and perhaps even their kinetics. This is particularly important when considering helix 8 and interaction with the Med21. It is not clear to the reader, how this interaction results in repression. This aspect should be further investigated.We fully agree that mechanism of repression is of great interest, and we appreciate the suggestion to investigate the nature of the repressed state. To this end, we performed two types of experiments. First, we performed ChIP-qPCR using Anchor.Away strains for multiple Mediator subunits, basal transcription factors and RNA Polymerase II--importantly all fused to the same epitope tag. These experiments indicated that: (1) MED21 is indeed associated with the synthetic auxin circuit promoter, and (2) there is significant enrichment of the core Mediator complex and PIC components (minus PolII) at both our synthetic TPL-regulated locus and at a Tup1bound loci (ScSUC1). Second, we used an even more expanded set of strains for Anchor Away analysis in the presence of the Auxin circuit. These experiments connected components from all of the core Mediator subcomplexes to maintenance of transcriptional repression. These two assays complement each other, and greatly strengthen the model that TPL (and likely Tup1) facilitate pre-assembly of the PIC in the absence of PolII, and that this assembly is required for repression.Reviewer #2:S. cerevisiae reported in their previous paper (Pierre-Jerome 2014 PNAS). This artificial system combines plant components including TPL domain fused to IAA domain with auxin-responsive plant promoter and relies on the use of the yeast degradation and transcriptional machinery allowing to monitor the repression and response to auxin of the reporter expression. Based on published structure, the authors identified two domains of TPL corepressor that independently contribute to repression in this system. One of these domains interacts with Med21 Mediator subunit in cytoSUS interaction assay. The authors provide a lot of work with many constructions to study different domains or point amino acid substitutions in TPL-mediated auxin-responsive repression. The question of molecular mechanisms of transcription repression is certainly very important biological question that still remains poorly answered. However, the work did not provide any mechanistic insights and the artificial AtARC(Sc) system and the cytoSUS interaction assay used did not allow to address the repression mechanism in physiological context. Moreover, the role of Mediator complex through its CDK8 kinase module in TPL-mediated repression with a change in Mediator composition previously reported by Furutani laboratory (Ito 2016 PNAS) has not been addressed and integrated with regards to TPL-Med21 interaction.In this manuscript, the authors studied the specific domains of the plant A. thaliana TPL corepressor using a synthetic auxin response circuit (ARC) in the yeast On my opinion, the work is more suitable for a specialized journal. Many experiments and more direct evidences are needed to support the author conclusions. Without additional evidence, their title that contains "conservation of repression mechanisms" is not supported by experiments. An extensive experimental work should be done to provide at least some mechanistic insights on repression of auxin-responsive gene promoters that could not be achieved within 2 months.As summarized at the top of this document, we took the critiques from Reviewer 2 very seriously, and have added a number of new experiments to address their concerns. Below, we will detail the specific experiments. Please note that we have also changed the title of the manuscript to address the concern that our claims were too bold for our findings.S. cerevisiae proteins of degradation and transcriptional machinery as an argument for conservation of the mechanisms. At the same time, the interaction assay with plant proteins in the yeast is presented as a direct interaction, but one could not exclude the involvement of endogenous proteins in this assay.1. The synthetic auxin response circuit (ARC) system is heterological making difficult the interpretation of the results. This artificial system combines plant components including TPL domain fused to IAA domain with auxin-responsive plant promoter and relies on the use of the endogenous, yeast degradation and transcriptional machinery. It was used previously to analyze the implication of different components of auxin response regulation from different plants and their functional annotation or for synthetic biology purposes. However, in this work the use of this system, especially to propose a molecular mechanism of transcription repression and to investigate the implication of Mediator complex is not sufficient and should be completed by additional experiments in plants in more physiological conditions (see below). The authors presented the fact that this system relies on endogenous We thank the reviewer for their recommendations. We have extensively addressed the protein-protein interaction question through the inclusion of several independent methods in both yeast , and plants . We have also added additional experiments in plants that support the importance of the TPL-MED21 interaction for control of auxin-regulated development.A. thaliana.2. A direct analysis of gene expression and direct genetic experiments in plants are missing. It should be feasible since many genetic, genomic and biochemical tools are available in We appreciate the reviewer’s desire for more evidence of the relevance of the yeast work for understanding plant biology, and have added a number of experiments with this concern in mind. We have also made many changes to the text to make sure that our claims are stated clearly and conservatively. As both TPL and MED21 play critical roles in a large and diverse set of gene regulatory networks, it is actually quite challenging to address their direct impacts (or the impact of their interaction) on the expression of any one gene or developmental program. To address this significant challenge, we have employed a variety of complementary methods in the synthetic and in the native contexts to accumulate evidence that supports (but could have just as easily have refuted) our model. We have employed more genetic and biochemical assays in the revised text, including cell-type-specific repression and expression lines in plants, as well as BIFC and Co-IP tests for interactions. We did not perform gene expression analysis on these lines, as it would be unlikely to work given that the driver is only expressed in ~5% of root cells .3. The interaction cytoSUS assay is not sufficient to prove the direct interaction between TPL and Med21 Mediator subunit and should be confirmed by additional biochemical experiments with purified proteins or plant crude extracts . In general, TPL interaction with Mediator complex should be analyzed.As described above, we have extensively addressed the protein-protein interaction question through the inclusion of several independent methods in both yeast , and plants . These methods all support the conclusion that TPL and MED21 interact in yeast and in plants. As also described above, we added ChIP and Anchor Away experiments to probe the role of other components of the Mediator complex.4. A direct analysis of Mediator composition, especially with regards to the publication by Ito and colleagues  demonstrating the role of Mediator CDK8 kinase module in auxin-responsive repression with TPL-CDK8 module interaction and dissociation of this module for active transcription, is necessary. More generally, the role of this Mediator module in repression mechanisms should be taken into account.We agree with the reviewer that the role of the MED13 component of the CDK8 kinase module in TPL-based repression is of great interest. We took two complimentary approaches to probe this relationship. First, we used an Anchor Away experiment in yeast to deplete CDK8 from the nucleus in real time. We found that, in contrast to the marked relief of repression we observed with depletion of core Mediator components, the removal of CDK8 had only a minimal effect. Second, we crossed the dominant effect TPLN188-IAA14 fusion plant lines into the gct-5 background (a MED13 loss of function), and carefully compared the ability of med13 loss of function to influence repression of lateral root development. The choice of med13 allele for these experiments is critical, as other mutations that are in the latter portion of the gene body have a dominant effect in reverting the loss of lateral root phenotype in the solitary root mutant see . Because5. The recruitment of transcriptional machinery including Mediator to promoter and regulatory regions of auxin-responsive genes should be analyzed by ChIP. RNA polymerase II recruitment and state should be directly tested in native system.We have now included ChIP-qPCR on the yeast circuit to answer this question.6. Physiological consequences of the changes in TPL domains and TPL-Med21 interaction should be studied.We agree with the reviewer that understanding the physiological role of the TPL-MED21 interaction is critical . We have included several new experiments that address this concern . The first experiment was to introduce the Helix 8 quadruple point mutant (that abrogates MED21 binding) into the same driver line used for the lateral root suppression assay. The transgenic lines carrying the UAS-TPLN188-Helix8Quad construct produce lateral roots, demonstrating that the Helix 8 residues (and thus TPLMED21 interaction) are required for full repression of target genes. To complement this experiment, we introduced UAS-MED21 with and without N-terminal deletions  into the same driver line. We hypothesized that N-terminal deletions when expressed over the wild-type allele might have a dominant effect on lateral root development. Indeed, we observed an increase in lateral root density compared to wild type. These two reciprocal experiments help to pinpoint the importance of the TPLMED21 interaction in a specific auxin-regulated plant process: lateral root development.7. The results are difficult to follow. First of all, the AtARC(Sc) system should be described at the beginning of the results indicating exactly which construction was used from the previous paper of the authors (Pierre-Jerome 2014 PNAS) and emphasizing the possibility to measure reporter fluorescence to evaluate reporter repression and also the response of the system to auxin (induction in the presence of auxin). The results should be profoundly rewritten for clarity and the figure organization should be readjusted.For example, why the first figure cited in the results is Figure 1C?Why some but not all constructions are summarized on Figure 1B with corresponding repression index and auxin induction level? The main figure summarizing all truncations tested would help.In general, Figure 1 is a complex mix between different panels and constructions.We thank the reviewer for this observation, and we have revised the manuscript to focus only on Helix 8 and its interaction with MED21 to allow us to simplify the story. We have also worked to simplify the language and readability of the manuscript overall. Figure 1 is now streamlined accordingly. We have also included more schematics throughout to help guide the reader.8. The authors did not provide any direct evidence to claim that "these results indicate that the CRA domain (H3-H8) requires contact with MED21 to drive repression" . No direct evidence was provided that TPL interaction with Med21 is involved in repression. Med21 is not the only interacting partner of TPL. Moreover, amino acid substitutions in TPL-N188 used in cytoSUS assay that reduced TPL-Med21 interaction also reduced TPL-IAA3 interaction.We have included two experiments that focus on this interaction in planta. First, we performed UAS-TPL-IAA14 experiments with the TPL Helix 8 quad mutations which break TPL’s interaction with MED21. This was sufficient to break the repression of IAA14 controlled genes, and resulted in more lateral root production. Second, we created UAS-MED21 lines with either the wild-type N-terminus, or Nterminal deletions that break TPL-MED21 interaction. These lines show increases in lateral root production, and reciprocally demonstrate that the TPL-MED21 interaction is required for the repression of lateral root development.9. The protein level of Med21 in wt is lower compared to Med21∆3 and ∆5  that should be explained/commented to indicate how this could influence the interpretation of the results.We understand the concern over the protein level in these assays; however, if anything it is more striking that the protein level of the Med21∆3 and ∆5 are slightly higher and yet still do not interact with TPLN188. This demonstrates that the proteins in this assay are: (a) expressed and stable, and (b) that protein stability is not responsible for the loss of protein interaction.10. The part of the manuscript on Med21 Anchor Away constructions in AtARC(Sc) system seems very methodological. The combined system is extremely artificial and heterological with three synthetic constructions . There are too many modifications and treatments . This is the problem especially since Med21 is a part of the Mediator complex. What happens with Mediator in these conditions? The results are difficult to interpret. The complicated experimental procedure is not clear. The result description is unclear with mistakes . Why no differences occur between mock and +Rapa for ∆5-MED21-FRB in blue on Figure 4 D? In general, the potential conclusions from this part are not clear.Alternative strategies, for example point Med21 mutations directly tested in plants should be considered.It was clear that our original submission was challenging to follow and included a number of methods that might not be familiar to all of our readers. We have worked hard to improve readability and accessibility throughout, including adding explanations for why we believed certain approaches were the best choice for answering particular questions. There are a few areas of specific concern here that need clarification. First, the Anchor away system has been extensively utilized in yeast to characterize and understand the mechanism of Mediator’s function at promoters . In our case, the only novel component is the Auxin Response Circuit. Therefore, we do not agree that because the circuit is complicated it is not relevant to transcription. We would argue that the dynamic nature of transcription greatly complicates the interpretation of phenotypes of stable point mutations in Mediator components. We strongly believe that our approaches and findings in yeast add unique and highly relevant insights into this complicated process, as well as providing guidance for follow-up work in plants.A. thaliana and native gene expression? Finally, they decided to use transgenic Arabidopsis lines and obtained a negative result suggesting that multimer formation is not required for repression.11. The part of the results on TPL multimerization and potential conclusions are unclear. It is not well integrated into the manuscript. The authors indicate a caveat in the use of heterological systems that further illustrates the limitations of their use and difficult interpretation of the results. The authors also indicate that "there are important differences between the synthetic and native systems" . This is the problem for result interpretation. It is not clear why the authors then used another conditional expression system in another plant (tobacco)? (p. 19) Why they use this additional model instead of We have extensively edited the relevant sections of the manuscript to increase clarity of our logic and conclusions.12. The last part on minimal repression domains in synthetic circuits is not within the general scope of the manuscript. It is not clear why this part was included. One of the possibilities would be to focus the manuscript on synthetic biology purposes and to choose an appropriate journal.We thank the reviewer for this constructive suggestion, and have removed the discussion of synthetic circuitry in plants from the manuscript13. Many sentences in discussion are not supported by the results. For example, the authors should not say that their result "suggests a fundamental conservation in at least one corepressor mechanism across species" . No mechanistic insights were provided and conservation from yeast to plants was not directly addressed. No recruitment experiments were done for TLP, Mediator and RNA polymerase II.We have now addressed this concern via both ChIP and Anchor away experiments in yeast, as described above.No direct experiments were done to demonstrate that TPL-Med21 interaction is directly involved in repression and to propose a molecular mechanism or to integrate their data into previously proposed model for Cdk8 module-containing Mediator.We have now directly tested this in experiments in plants, see previous comment aboveMediator complex composition and its implication was not directly addressed.We have now included this via both ChIP and Anchor away experiments in yeast, as described above.More appropriate references should be cited for the main roles of Mediator complex.We have carefully reviewed the references cited, and would welcome any specific suggestions for further improvement.No evidence for stabilization of Mediator complex by TPL were provided.We have addressed this by ChIP which makes it is clear that the Mediator core complex is co-bound with TPLN188 at promoters in yeast.It is completely incorrect to say that in plants and yeast there is "more primitive form of pausing".This sentence has been removed from the newest version of the manuscript.Reviewer #3:In this manuscript, authors seek to resolve conflicting models for corepressor function using the elegant synthetic auxin response system. Auxin signaling is governed by a de-repression paradigm and is ideally suited to interrogate co-repressor function – in this case, the TOPLESS (TPL) co-repressor. Several contradicting models have been put forward for the mechanism of TPL-mediated gene repression, ranging from a requirement for protein oligomerization for activity, interaction with distinct partners, and even which regions of the protein are required for repressive activity. Leydon et al., use the yeast-based synthetic auxin response system to interrogate these models using a single reporter locus, allowing for straight-forward examination of TPL function.Strengths of this study include the use of a simplified model to study a complex problem and the broad implications for study results .We thank the reviewer for their positive comments.There were a few pieces of data that I found confusing, which I have outlined below. Clarifying these points would strengthen the manuscript.1. In Figure 1E, it appears that IAA3 alone can repress the reporter to some degree, or at least the maximal reporter activity cannot be achieved with IAA3 present. However, a lack of increased reporter expression with auxin treatment suggests that IAA3 degradation is insufficient to allow for maximal reporter expression . I find this to be a curious result; can the authors expand on this?We agree that this result gets at a very interesting observation, namely that in circuits where the reporter gene is repressed, it is often activated to a higher level than in control lines where there is no repression (constitutive activation). In our experiments in yeast, IAA3 does not provide any repressive function unless it is fused to TPL, and the increase in activation above this level is due to the switch from repressed to activated state.2. In many cases, experiments to differentiate between variant effects on TPLN activity versus TPLN protein stability are lacking. This data would be helpful for interpreting some of the results. Note: this is included for some assays/variants, but not others.This is something that concerned us as well, and we have tried to address it wherever possible. Our hands are tied with respect to the addition of epitope tags in the case of certain experiments. While an epitope tag can be added to the N188 full length protein at the N terminus, the number and length of tags does diminish repression activity. When we use further truncations, the presence of an epitope tag can drastically affect repression. We have tried to include protein levels whenever the inclusion of a tag does not itself change repression strength.3. It seems that the images presented for the split ubiquitin assays are stitched together from multiple images. This should be made clear in both the figure legend. In addition, it should be clear whether these were performed as a single experiment or images put together from distinct experiments. If from distinct experiments, I suggest that these be repeated as a single experiment.All experiments within a figure panel are from the same experiment, but were cropped together to conserve space. We have added white lines to indicate this on the panels, and have included text within the figure legend to explain this.4. I don't understand why N188 repression is not relieved by auxin treatment, when both the H1 and H3-H8 truncations are relieved. Maybe this is explained in the text, but if so, I missed this.We have included a description of our previous experiments from prior publications that discuss the relevant results that lead to our understanding of why the N188 repressed circuit is not relievable by auxin addition. This is on page 4, lines 78-85.5. I also don't understand why H3-H8-QuadAAA is more auxin responsive than H3-H8.We agree that this is an interesting and somewhat surprising result. One way to explain this pattern is that the loss of interaction with Med21 weakens the repression state, as evidenced by higher reporter expression in the absence of auxin. Adding auxin sensitizes the system by decreasing the level of the TPL-fusion, thereby revealing the decrease in stability of this complex and allowing activation at a lower level of auxin. In the context of the entire TPLN188 with the QuadA mutations, we would argue that the second repression domain (H1) and the intact interaction with Med10 are sufficient to maintain a stable repression state, even when the level of TPL is decreased with auxin addition.6. I am confused by the data presented in Figure 4D. The text on lines 354-354 suggest that auxin sensitivity of the system is being examined, but it is not apparent that auxin treatment is involved in either the figure or the legend.We thank the reviewer for this correction, which is remedied in the current version of the manuscript.[Editors’ note: what follows is the authors’ response to the second round of review.]Essential Revisions:1. As previously requested, the authors included ChIP experiments to directly show the presence of pre-initiation complex components at the repressed promoter.a) Nevertheless, it is not clear how the ChIP experiments were quantified, what is enrichment over background, this is not explained in the text.eLife paper PMID: 30681409). It is described here: https://www.thermofisher.com/us/en/home/life-science/epigenetics-noncoding-rnaresearch/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html Briefly, the δ CT (dCT) between input and IP is calculated for each sample for both the control locus  and the target locus . The δ δ CT (ddCT) is then identified for the (Ct IP) – (Ct control locus) to create the non-specific adjustment. Then the fold enrichment is calculated (2DDCt).We quantified our ChIP experiments using a method applied extensively for such assays in yeast ” or “Fold enrichment over background (Chr.V gene free region)”. We have also included a supplementary ChIP-qPCR figure to assist in guiding the reader .b) In addition, it is essential to include ChIP controls at an actively transcribed promoter, this is an especially important positive control for RA polymerase II that was not detected at the repressed promoters.PMA1 (PMID: 3005867). ChIP assays in yeast grown under similar conditions as we are using for our analysis have previously detected enrichment of transcriptional machinery at the PMA1 locus, and it is frequently used as a housekeeping gene (PMID: 16461706). We tested our ChIP samples at the PMA1 promoter using validated qPCR primer sets, and detected a significant enrichment of Mediator and GTFs, consistent with previous reports (PMID: 28699889). Thus, we are confident that our ChIP conditions and anti-FRB antibodies are able to capture transcriptionally active loci. The data is shown in Figure 4D. Please see Point D below for experiments examining Pol-II.We appreciate this suggestion, and have now tested the enrichment of our proteins of interest at the promoter of the essential plasma membrane ATPase gene c) Furthermore, Med21 Mediator subunit and TPLN188 were analyzed on chromosome-integrated AtARC locus  while other ChIP experiments for Mediator subunits, some GTFs and Pol II were done with SPARC plasmid.It is absolutely true that genes on plasmids and those integrated into the genome may behave differently. It is for this exact reason that we carefully validated our results in multiple independent assays  in multiple species. We created the SPARC because shared prototrophy genes make it impossible to combine the Anchor Away strains with the integrated ARC as originally constructed. To be scrupulously careful in drawing our main conclusion, we created the integrated strain with TPLN188-HA and MED21-FRB in a non-Anchor Away genetic background to confirm that we saw similar enrichment patterns whether we performed ChIP on MED21-FRB at an integrated locus or on the SPARC plasmid. Because we see that (i) the SPARC behaves similarly to the ARC with respect to repression, and (ii) that we could ChIP MED21-FRB at both the integrated ARC and the plasmid SPARC, we feel comfortable using the SPARC for ChIP assays on other components of the transcriptional machinery.Given that even this level of caution still left the reviewer with some concerns, we performed additional experimental validations of both the integrated locus and the SPARC plasmid. These results now appear in Figure 4 —figure supplement 2. We find a general similarity in ChIP results between the two reporter locations (SPARC or ARC), and that the ChIP trends track the expression level of the reporter (YFP). Please find the specific description of the experiments in the sections below.d) Chromosomal ACT1 gene body is completely inappropriate as a background for Pol II ChIP, since this region is enriched for Pol II. Appropriate control regions, regulatory, core promoter and transcribed regions, as well as experiments with untagged control strains should be added.SUC1 promoter as compared to the clear enrichment at the promoter of PMA1. This indicates that the anti-FRB ChIP was successful, and that the Pol-II is not enriched at the repressed promoters. These data now appear as part of Figure 4D.We have repeated this analysis for the RNA Polymerase II ChIP samples using an alternative primer set. The Chr.V non-genic control primer is the well-characterized gene-free region on Sc chromosome V, utilized in PMID: 22156212. We saw a similar profile for Pol-II at the repressed gene loci at the SPARC reporter promoter and the endogenously Tup1-repressed e) Percentage of IP over input values should be presented for untagged control strains and for several regions .We appreciate the reviewer’s suggestion, and have several responses. First, we added an explanation of how ‘Fold enrichment over the background is calculated’ . We have also shown this graphically by comparing the percent input calculation to the Fold enrichment over background.This normalization to control allows us to plot ChIPs from different antibodies/IPs onto the same graph. We show this in Figure 4C as it will allow better comparisons to be made with the new Figure 4D, and Figure 4L and the new supplemental ChIP figure .We have also performed ChIPs with the control strains that do not have any FRB tag present. These data demonstrate that there is no enrichment of the SPARC promoter or relevant control loci .The second part of this question is addressed in our response to point G below, as it is thematically linked.f) Sequences and positions of qPCR primers should be indicated.All sequences are included in the provided oligo sequences supplementary file and are labelled with their use as “ChIP-qPCR”. We have updated this list to include all new primer sets. We have also created a map of the ARC promoter, the sequence of which is identical in both the integrated ARC and the SPARC plasmid. This is included in Figure 4 —figure supplement 2.g) Mediator is mainly enriched on regulatory (UAS) regions, GTFs are bound on core promoters and Pol II signal in yeast is mostly on transcribed regions. Well-identified UAS enriched by Mediator should be added as positive controls and for comparison.ACT1 gene body, >700 base pairs downstream of the start codon) is appropriate to test this level of resolution for most cases except for Pol-II, as discussed above. As a note, this figure again highlights why it is critical to normalize the data to a control region. It is only with this step that allows us to plot the data in a single graph, which we think makes for optimal readability and straightforward comparisons.Two pieces of information are important to consider in addressing this issue. The first is that our sonication conditions generate DNA libraries that are centered at ~500 base pairs in size. The second is that the promoter we are analyzing here is very short , and indeed this is a similar size to many other genes in the yeast genome. Therefore, the UAS and core promoter cannot be easily untangled without a finer resolution technique such as ChIP-seq, using an alternative fragmentation technique. We tested the resolution of our assays using a series of primers along the gene body, and confirmed that our ‘peaks’ are quite broad . A primer set ~500 base pairs downstream of the ‘UAS’-site still shows a residual enrichment of both TPL and MED21. We will refer to the region we are assaying as the Promoter as opposed to an UAS due to the resolution of our chromatin preparation protocol. Our control gene (the h) The growth conditions should be clearly indicated to specify that they correspond to repressed state.We have put headers on the figures to make sure that the growth conditions and repression state are clear. We have also added language to the figure legends to further guide the reader.i) The ChIP occupancy was analyzed only in transcriptionally repressed state and no results were provided for transition to the active state. This should be analyzed in detail.We understand the reviewer’s concerns about what the normal enrichment of Mediator / GTFs / Pol-II is at an ARC promoter in the absence of TPL repressor, or in other words what happens at the active ARC promoter. If we understand the reviewer’s comments, we think that one of our statements  led to this concern. While we intended to indicate that the concentration of Mediator complex that is actively recruiting Pol-II at the promoter is increasing, we were not clear. We have changed the text to clarify this (Pg.28 lines 656-660). The sentence now reads: “As suggested by Ito and colleagues  and supported by our synthetic system, auxin-induced removal of TPL is sufficient to induce changes in the activity of the Mediator complex”.PMA1 promoter into this figure panel as well as the control, highlighting the lines where there is no repression (No TPL – dark grey). We detect a high abundance of Med21 at the active SPARC promoter, as compared to the repressed promoter. The well-documented PMA1 promoter has an enrichment of MED21, and is unaffected by the presence of the SPARC. The Δ5Med21 does not fully rescue the association of Med21 to the promoter, however this is roughly similar to the change in the transcriptional output of the SPARC when helix 8 is absent ~1.6x . The Δ5Med21 protein is integrating into Mediator complexes (White bars), however it appears slightly less enriched at the PMA1 promoter compared to wild-type Med21, supporting the phenotypic analysis that the Δ5Med21 mutant has slower transcription promoting activity .We agree that it is critical to evaluate the change in Mediator abundance at the ARC promoter when it is active. We would first indicate that the original Figure 4L did indeed test this question specifically. We observed a greater enrichment of MED21 at unrepressed promoters. However, to provide additional information, we have added the analysis of the PMA1 shows a similar enrichment profile. This indicates that the abundance, and not the composition of the Mediator complex, is altered at the SPARC promoter, with the most striking difference being the recruitment of Pol-II. We have edited our language to make this distinction explicit.To more directly answer the concern over changes in Mediator composition between repressed and active SPARC promoters, we performed ChIP-qPCR in SPARC lines that have no TPL (active). To do this we had to purchase more anti-FRB, which was from a separate lot number from the prior experiments leading to less effective pull-downs. While we observed a lower total enrichment of FRB tagged proteins at all loci compared to the previous experiments , the trends are entirely consistent. The enrichment of Mediator components MED18 and MED14 at the SPARC promoter is increased under active conditions, whereas CDK8 is absent. Both TBP1 and Pol-II show high levels of enrichment at the active SPARC promoter. The positive control promoter of 2. The co-immunoprecipitation experiments in plant extracts lack a negative control to conclude on the specificity of CoIP signal. For Figure 6B, a control IP without HA tag or antibody should be added to evaluate non-specific binding of YFP-tagged TPL to beads. A condition Med21-YFP-HA "-" YFP-TPL "-" could not serve as a negative control, since no detection with anti-GFP is possible. Using actin depletion as a specificity control is not sufficient and not an accepted control. For CoIP in Figure 6 —figure supplement 1A, a negative control is not appropriate. A control IP is needed to evaluate non-specific binding of YFP-tagged proteins to beads. Furthermore, it would be important to perform co-immunoprecipitation of the Tpl quad AAAA mutant with Med21 to confirm loss of interaction and of Tpl with the N-terminal deletions of Med21.We thank the reviewers for their comments on the specificity of the Co-IP signal. We repeated the Co-IPs using the same protocols with the indicated controls. In these experiments, we see no non-specific binding between the TPL-YFP fusion protein and the beads. The Figure 6 panel B has been replaced .3. It would be appropriate to remove the dimerization/multimerization experiments either completely or at least relegating them to the supplementary data and move the estradiol inducible experiments  to main figures. This data appears more pertinent to the message of the paper than the ability of Tpl to form tetramers. Also, the colours in Figure 4 Sup. 3 are difficult to distinguish, can this be improved?We thank the reviewers for this suggestion and have given the question of the ‘message of the paper’ a great deal of thought. While the significance of a result is in the eye of the beholder, we see this study as an investigation into the mechanism and functional unit of repression by TPL. While the data on multimerization is not directly relevant to the first of these objectives, it is of central concern for the second. The prevailing thought has been that multimerization is required for repression, and certainly there is data that higher-order multimers-of-multimers exist and have functional roles (PMID: 28630893). To our knowledge, this is the first time that a study has been able to engineer a variant of the TPL N-terminus capable of testing the ability of a monomer of TPL to repress. Therefore, the ability of a monomer of TPL to repress is a striking and novel finding. Other researchers outside of our lab have expressed their interest in this finding as well, indicating to us that these results are integral to the main message of the manuscript. While we appreciate the reviewer’s enthusiasm for the estradiol experiments. We see these results as being supplementary proof that Med21 and TPL interaction has a functional role in repression that is not caused by an off-target effect due to the method of analysis in yeast. Therefore, while we respect the reviewer’s opinion, and have discussed this extensively among the authors, we have left the distribution of main and supplementary figures unchanged.