The Role of Med15 Sequence Features in Transcription Factor Interactions

  1. David G. Cooper
  2. Shulin Liu
  3. Emma Grunkemeyer
  4. Jan S. Fassler

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

No abstract available

Article activity feed

  1. Version published to 10.1080/10985549.2024.2436672
  2. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors performed extensive genetic analyses in yeast on the functions of Med15 regions under multiple stress conditions, linking cell growth phenotypes, gene expression and protein-protein interaction. Med15 is an activator-contacting subunit of the Mediator complex and the functions of yeast Med15 have been extensively studied. In particular, the authors attempted to understand the roles of a poly-Q region (Q1), its length and composition in stress response phenotypes, Med15-mediated gene expression, and interaction with transcription factor Msn2. Results from this work are consistent with several previous studies and revealed some new insights. The authors concluded that robust Med15 activities required the Q1 tract and the length of Q1 tract modulates activity in a context-dependent manner. While the study is well executed and the conclusions are generally sound, several concerns listed below should be addressed and some clarifications should be made.

    Major comments:

    1. Abstract, "We also observed that distinct glutamine tracts and Med15 phosphorylation affected the activities of the KIX domain". Fig. 1 shows the effects of KIXQ2Q3 deletion and p7 phosphor-dead mutant under Acetic acid and Ketoconazole treatment, but does not demonstrate that these domains or phosphorylation affects KIX domain activities.
    2. Is it known that Med15 is dephosphorylated under stress conditions other than osmotic challenge? Another explanation for D7P/D30P mutant results (Fig. 1B) might be that Med15 phosphorylation in unstressed cells is important for certain types of stress response (acetic acid and Keto). In contrast, the observation that D7P has no effects on osmotic stress (Fig. 1B) might suggest that phosphor-Med15 is dispensable for function. Some explanations on how to ascertain the roles of Med15 phosphorylation would be needed.
    3. Fig. 4, what is the rationale of analyzing basal expression rather than activated expression of Gcn4 and Msn2 dependent genes? Gal4 and Hap5 dependent genes could be measured as well, in order to complete the gene expression-phenotype correlation that the authors strive to make in this paper.
    4. Fig. 4, Error bars should be provided on gene expression analysis. Gcn4 and Msn2 target genes should be highlighted separately to facilitate comparisons.
    5. Results from Fig. 4 and 5 indicate that Spacer-Q1 and 12PQ-Q1, being the strongest interactors to Msn2, actually reduced HSP12 expression (a known Msn2 target gene). Some explanations would be needed. Page 12 Discussion paragraph 2 "Q1 substitutions that interfered with coiled-coil propensity had no effect on TF activity" would need to be revised and to include some discussions on this result.
    6. Fig. 5C, additional explanation is needed on how interaction rank is determined and how error bars are obtained.
    7. The idea that Q1 provides a molecular hinge to facilitate intramolecular interactions is interesting, but sounds like a possible scenario without further evidence. Are there any published structural studies on Med15/Mediator complex that might support this idea?

    Minor comments:

    1. SC-HLUM and SC-HLMU is used interchangeably in the legend and text. Please keep consistent. Explanations for these acronyms are not found in the Methods.
    2. Fig. 2, 3. AcOH should be Acetic acid, to be consistent with Fig. 1.
    3. Fig. 3B, error bars should be provided for growth measurements.

    Significance

    This work provided a detailed analysis on the roles of a specific poly-glutamine region in yeast Med15 functions and regulation. One conceptual advance of this work is that the structural flexibility rather than the sequence itself of Q1 tract proves to be critical for Med15 function. The ability to correlate Med15-Msn2 interaction with gene expression analysis demonstrated some technical novelty, given the power of genetic manipulation in yeast.

    Med15 is a key Mediator subunit contacting several sequence-specific transcription activators. Its interaction with a number of transcription activators in yeast such as Gcn4 and Gal4, was previously studied as referenced in this manuscript. This manuscript first provided a quite comprehensive genetic mutational analysis and confirmed several findings in previous studies. The identification of critical Med15 regions for acetic acid response (and Hap5-dependent gene activation) and the analysis of Msn2-Med15 interactions appear to be novel. Researchers interested in eukaryotic transcriptional regulation would benefit from reading this study.

    Field of expertise of this reviewer: mechanisms of transcriptional regulation, genetics, nuclear organization and function

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The Mediator complex, a multicomponent complex, regulates the interaction between transcription factors and RNA polymerase II using protein interactions. In particular, Intrinsically Disordered Regions (IDRs) with in Med15. Copper and Frassler made extensive mutations to the three IDRs that are characterized with high glutamate content (poly-Q), the KIX domain which interacts with transcription factors and the MAD which interacts with the rest of the Mediator complex. The three poly-Q repeats are adjacent to Activator Binding Domains (ABD). The impact of mutant Med15 was measured with growth assays, co-IPs with a transcription factor, and transcriptional activation of a reporter during different stresses. Med15 is particular important in stress responsive transcription rather than basal transcription because in yeast it is nonessential. It can be repress and activation translation of genes. Using a series of internal deletions and substitutions impact of the mutations was tested on by measuring growth of strains, expression of Med15 regulated genes, and interaction with Msn2, a transcription factor that regulates to various stresses. This work adds to the body of research confirming that multiple weak/ transient interaction domains regulate Med15 function.

    Major comments:

    1. Without knowing the protein levels of the different mutants, it is difficult to contribute deletions of different regions with phenotypes measured. Various internal deletions decrease Med15 protein levels (Jedidi et al. 2010) while other affect the integrity of the Mediator complex. This study did not measure mRNA or protein levels of their mutants. However, (Jedidi et al. 2010) used Myc-tagged Med15 which affects regulation of Med15 via SNF1 (Gallagher et al. 2020). In another study, Med15 was N-terminally tagged and protein levels of some deletions increased (Herbig et al. 2010). It's unknown if other tags such as HA, TAP or FLAG affect Med15 regulation via SNF1. This study used untagged Med15 expressed from the native promoter which avoids these complications. It's also unknown if the differences in Med15 deletions are from reduced transcription, translation, or protein stability. There are commercial antibody that may work (https://www.genetex.com/Product/Detail/Gal-11-S-cerevisiae-antibody/GTX64110). There are several commercial antibodies to human Med15 but the cross reactivity has not been tested.
    2. Quantification with ImageJ on spot assays is difficult because once growth has maxed out on the dense spots there is no resolution. Using more dilute spots is challenging because colony size is affected by the nearby colonies. The error bars are the mutants are large. Can quantitative growth curves be carried out in flasks or an automatic plate reader for better quantification?

    Minor comments:

    1. Why were the stress conditions chosen in figure 1B? These are only a subset of conditions that the med15 deletion is sensitive to. Aside from acetic acid the phenotypic profile of each deletion is similar. The bigger the deletion, the more severe the growth defect. The keto plate appears under loaded by comparing the number of colonies in the third dilution on the keto plate and the fourth spot on the YPD plate in the BY4742 (MED15 wild-type strain). Does keto lyse cells? Or was there that much variation between mutants? Perhaps the dose of keto needs to be calibrated. In figure 2B it looks like wildtype growth in keto was 70% of untreated growth.
    2. Figures 1C and 1D are not discussed in the results. The authors should remind what the hGR assay measures when discussing the results. How is it different from the GAL4 transcriptional reporter?
    3. In the D to A mutants some appear to be required for acetic acid tolerance. What was the pH of the media?
    4. The labels between Figure 1 and 2 are inconsistent. 90 mM acetic versus 80mM AcOH, YPGal versus YPGalactose, SD+LKHU versus SD+KLUH. The mutants on 0.97M NaCl at 37oC from figure 2A grew more than 0.9M NaCl at 38oC. Also, in the text it says 37 oC.
    5. Is the MED15 strain BY4742? In Figure 1 was it also transformed the pRS315? How was the plasmid maintained on the plates, specifically YPD?
    6. Genes such as GAL and URA3 should in italics.
    7. In the split Ub assays, was wildtype Msn2 and Med15 also present?
    8. There is inconsistently naming of media in Media and Phenotype Testing section. The media is called synthetic complete media is labeled SC-URA or SC-LEU and at times in Results its called SD+K or SC-HULM. Is SC with all amino acids and SD without any amino acids?
    9. The plasmid names in the supplemental table don't match the ones labeled in the figures.
    10. Why was ALG9 used for normalization of qRT PCR?
    11. The background of the strains is confusing. There appears to be two different med15 knockouts OY320 and JF1368. Which ones were used in which experiments? Some of the trains have a trp1 auxotrophic which affects stress response on it's own (González et al. 2008; Schroeder and Ikui 2019).

    Significance

    • General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed? Extensive mutational analysis of Med15.
    • Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...). This work confirms numerous other studies on the contribution of various Med15 domains on function.
    • Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field? Incorporation of human domain substitutions could influence how people outside the field would interpret how Med15 interacts with transcription factors.
    • Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Expert in using yeast genetics and natural genetic variation to address underlying mechanisms of stress response to environmental toxins with a particular focus on transcription factors and TORC1.
  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Major Comments

    The authors show that the amino acid content and length of Q1 affects transcription activity in a media-dependent way in a construct that includes Q1-ABD1 and a tailing Q/N rich region (Q1R). Briefly, different media conditions used as proxies for specific target TF activities varied in their sensitivity to the Q1 sequence content. However, the reason for this variation between target TF activities is not addressed, so the observations seem more anecdotal than insightful. One test performed suggests some of the Q1 sequence dependence may be due to changes in AD-ABD interactions, but this interesting possibility is not investigated further.

    A split-ubiquitin two-hybrid assay, meant to detect interactions between Msn2 TAD and Med15-Q1R, showed clear Q1 sequence/composition-dependence when changed from polyQ tracts. In particular, replacement with leucine-rich tracts (12L and RvHs) significantly reduced interactions (as inferred from growth requirements in Fig 5B). Q1 consisting of just 10 spacer residues, 0 to 24 Q residues, or PQ repeats all had quite similar results suggesting retention of some Msn2 and Med15 interactions. Replacement with a helix-forming sequence from NAB3 gave intermediate results. Again, no explanation was offered for the observation but it seems probable that the NAB3 Q1 system is no longer reporting on Msn2 Med15 interactions.

    The manuscript presents extensive assays, but a lack of consistency in conditions and constructs tested makes comparing different assays difficult. In particular, it would be valuable to have NAB3-Q1, FrHs-Q1, and RvHS-Q1 tested under conditions of high salt as that is indicated to be the Msn2 target condition (e.g. an additional result that would be presented in Fig 3B); this would be valuable to compare to the two-hybrid results. The relationship between Q1 polyQ length and Msn2 TAD-Med15 ABD1 binding is not clear from this assay as all had similar growth on the plates. A possible explanation for the inferred reduction in TAD-ABD1 binding in the leucine rich Q1 constructs is that this highly hydrophobic linker itself binds to ABD1 and is therefore self-inhibitory. There is also the unexplored/not discussed possibility that NAB3, 12L, and RvHs have off-target interactions that disrupt the TAD-ABD1 interactions.

    The framing of the study and the title of the manuscript strongly suggest that there might be a relationship between coiled-coil formation and transcription activity. This is the basis for selection of many of the Q1 sequences tested, with the premise of either increasing or disrupting coiled-coil structure. These 'propensities' are quantified in Supp Fig 1; however, a significant limitation of this interpretation is that these propensities are bulk properties that presume formation of homo-dimers or homo-trimers, a situation that is not shown to be relevant for Med15 at a promoter. This means that Q1 is potentially only one of the multiple partners required for coiled-coil formation. So even if a tested sequence has high coiled-coil propensity, that may not be the case in the actual biological systems at play here. Another consideration to be entertained is how different solvent conditions (different media) may affect coiled-coil propensity. An unanswered question is whether Q1 may form coiled-coil structure either with other regions of Med15 and/or with other Mediator subunits or even other co-factors entirely. This is a question implied by the title of this paper, but the data presented address neither intra- nor inter-molecular interactions of the polyQ regions (the two-hybrid study is designed to probe the ABD-AD interactions).

    A final proposed hypothesis was that Q1 acts as a hinge in a way analogous to what was reported for the huntingtin protein (ref 7). This is an attractive model but remains untested in this work. In particular, the Med15-Q1R construct used does not have multiple ABDs that would potentially be brought in close contact, so the results here cannot be interpreted as analogous to the huntingtin hinge model. Minor Comments:

    Please explain the choice of the 10-residue spacer instead of a 12-residue spacer.

    Page 14: "We observed that Q1 substitutions with increased coiled-coil propensity (Supplementary Figure 1) diminished TF activity while Q1 substitutions that interfered with coiled-coil propensity had no effect on TF activity (Fig. 3, 4), suggesting that the flexibility of the sequence is an important feature." There was no demonstration that those sequences in this context form CCs. There's no evidence of what is actually being modulated whether it's length, flexibility, or ability to interact with other regions of Med15 or even with other co-factors.

    Page 15: "We confirmed that Msn2-dependent activities of Med15 are encoded by the region containing the Q1 tract and ABD1 (aa 116-277) and found that the KIX domain alone could also mediate an interaction with Msn2 (Fig. 5). This contrasts with the Gcn4- or Gal4-dependent growth or stress responses which are the result of additive interactions with Med15 that are characterized by weak, highly dispersed, multivalent interfaces. While it is not yet entirely clear if the interaction with Msn2 is similarly multivalent, we have shown that either the KIX domain alone or the Q1R region alone of Med15 was sufficient with no evidence of additivity." These statements are unsupported. While Gcn4 and Gal4 transcription activity has been shown to depend on multiple AD-ABD interactions, none of the data reported here shows that Msn2 does not (as is stated here, which undermines the "contrasts" argument. Further, based on the assays presented in Figure 1B, Msn2, Gal4, and Gcn4 behave similarly for the various Med15 constructs.

    Page 16: "In all instances TF activity was reduced in the absence of the Med15 Q1 tract." This seems false based on the data presented. Met10 activity appears to have increased in Figure 4A.

    Page 16: Reference to Figure 2C and Figure 2B are mislabeled. Should be Figure 2D and 2C, respectively.

    Page 17: "The fact that residues at Q1 were not functionally constrained to be glutamine residues suggests the Q1 tract is not an interaction motif participating directly in protein-protein interactions." This is completely unsupported. There are no data presented that address interactions between Q1 and anything else.

    Figure 2: Not clear which assays were at 30{degree sign}C vs 22{degree sign}C as they are not labeled in the figure. In Figure 2A, the label med15 should be med15Δ.

    Figure 4: Interpretation of these results seems limited by only reporting YPD media conditions. May be helpful to include the conditions reported in Figure 1.

    Figure 6: It is not clear what some elements of this figure are meant to represent. Is saw tooth always polyQ? or Is ABD1 always blue and ABD2 is always red. What then are the loops? The general premise of this figure does not seem to be supported by the actual experiments performed.

    Supp Figure 3: "K is the Med15 fragment encompassing the KIX domain, aa 1-277." This aa range is KQ in the main text. Either the residue range is wrong, or the label is wrong.

    Significance

    This manuscript addresses an interesting topic. There appears to be a disconnect between the stated motivation and what was actually done. The large array of assays and conditions are difficult to compare, leaving the reader with a feeling that the authors have catalogued a lot of possibilities but that no generalizable or unifying insights are at hand. The attempt to present a model (Figure 6) is difficult to parse and is not directly supported by the data presented. Addressing the issues raised here could result in a work that is useful to the specific field of Med15 structure and function but of limited use at the moment to a wider audience.

  6. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time

  7. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors performed extensive genetic analyses in yeast on the functions of Med15 regions under multiple stress conditions, linking cell growth phenotypes, gene expression and protein-protein interaction. Med15 is an activator-contacting subunit of the Mediator complex and the functions of yeast Med15 have been extensively studied. In particular, the authors attempted to understand the roles of a poly-Q region (Q1), its length and composition in stress response phenotypes, Med15-mediated gene expression, and interaction with transcription factor Msn2. Results from this work are consistent with several previous studies and revealed some new insights. The authors concluded that robust Med15 activities required the Q1 tract and the length of Q1 tract modulates activity in a context-dependent manner. While the study is well executed and the conclusions are generally sound, several concerns listed below should be addressed and some clarifications should be made.

    Major comments:

    1. Abstract, "We also observed that distinct glutamine tracts and Med15 phosphorylation affected the activities of the KIX domain". Fig. 1 shows the effects of KIXQ2Q3 deletion and p7 phosphor-dead mutant under Acetic acid and Ketoconazole treatment, but does not demonstrate that these domains or phosphorylation affects KIX domain activities.
    2. Is it known that Med15 is dephosphorylated under stress conditions other than osmotic challenge? Another explanation for D7P/D30P mutant results (Fig. 1B) might be that Med15 phosphorylation in unstressed cells is important for certain types of stress response (acetic acid and Keto). In contrast, the observation that D7P has no effects on osmotic stress (Fig. 1B) might suggest that phosphor-Med15 is dispensable for function. Some explanations on how to ascertain the roles of Med15 phosphorylation would be needed.
    3. Fig. 4, what is the rationale of analyzing basal expression rather than activated expression of Gcn4 and Msn2 dependent genes? Gal4 and Hap5 dependent genes could be measured as well, in order to complete the gene expression-phenotype correlation that the authors strive to make in this paper.
    4. Fig. 4, Error bars should be provided on gene expression analysis. Gcn4 and Msn2 target genes should be highlighted separately to facilitate comparisons.
    5. Results from Fig. 4 and 5 indicate that Spacer-Q1 and 12PQ-Q1, being the strongest interactors to Msn2, actually reduced HSP12 expression (a known Msn2 target gene). Some explanations would be needed. Page 12 Discussion paragraph 2 "Q1 substitutions that interfered with coiled-coil propensity had no effect on TF activity" would need to be revised and to include some discussions on this result.
    6. Fig. 5C, additional explanation is needed on how interaction rank is determined and how error bars are obtained.
    7. The idea that Q1 provides a molecular hinge to facilitate intramolecular interactions is interesting, but sounds like a possible scenario without further evidence. Are there any published structural studies on Med15/Mediator complex that might support this idea?

    Minor comments:

    1. SC-HLUM and SC-HLMU is used interchangeably in the legend and text. Please keep consistent. Explanations for these acronyms are not found in the Methods.
    2. Fig. 2, 3. AcOH should be Acetic acid, to be consistent with Fig. 1.
    3. Fig. 3B, error bars should be provided for growth measurements.

    Significance

    This work provided a detailed analysis on the roles of a specific poly-glutamine region in yeast Med15 functions and regulation. One conceptual advance of this work is that the structural flexibility rather than the sequence itself of Q1 tract proves to be critical for Med15 function. The ability to correlate Med15-Msn2 interaction with gene expression analysis demonstrated some technical novelty, given the power of genetic manipulation in yeast.

    Med15 is a key Mediator subunit contacting several sequence-specific transcription activators. Its interaction with a number of transcription activators in yeast such as Gcn4 and Gal4, was previously studied as referenced in this manuscript. This manuscript first provided a quite comprehensive genetic mutational analysis and confirmed several findings in previous studies. The identification of critical Med15 regions for acetic acid response (and Hap5-dependent gene activation) and the analysis of Msn2-Med15 interactions appear to be novel. Researchers interested in eukaryotic transcriptional regulation would benefit from reading this study.

    Field of expertise of this reviewer: mechanisms of transcriptional regulation, genetics, nuclear organization and function

  8. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The Mediator complex, a multicomponent complex, regulates the interaction between transcription factors and RNA polymerase II using protein interactions. In particular, Intrinsically Disordered Regions (IDRs) with in Med15. Copper and Frassler made extensive mutations to the three IDRs that are characterized with high glutamate content (poly-Q), the KIX domain which interacts with transcription factors and the MAD which interacts with the rest of the Mediator complex. The three poly-Q repeats are adjacent to Activator Binding Domains (ABD). The impact of mutant Med15 was measured with growth assays, co-IPs with a transcription factor, and transcriptional activation of a reporter during different stresses. Med15 is particular important in stress responsive transcription rather than basal transcription because in yeast it is nonessential. It can be repress and activation translation of genes. Using a series of internal deletions and substitutions impact of the mutations was tested on by measuring growth of strains, expression of Med15 regulated genes, and interaction with Msn2, a transcription factor that regulates to various stresses. This work adds to the body of research confirming that multiple weak/ transient interaction domains regulate Med15 function.

    Major comments:

    1. Without knowing the protein levels of the different mutants, it is difficult to contribute deletions of different regions with phenotypes measured. Various internal deletions decrease Med15 protein levels (Jedidi et al. 2010) while other affect the integrity of the Mediator complex. This study did not measure mRNA or protein levels of their mutants. However, (Jedidi et al. 2010) used Myc-tagged Med15 which affects regulation of Med15 via SNF1 (Gallagher et al. 2020). In another study, Med15 was N-terminally tagged and protein levels of some deletions increased (Herbig et al. 2010). It's unknown if other tags such as HA, TAP or FLAG affect Med15 regulation via SNF1. This study used untagged Med15 expressed from the native promoter which avoids these complications. It's also unknown if the differences in Med15 deletions are from reduced transcription, translation, or protein stability. There are commercial antibody that may work (https://www.genetex.com/Product/Detail/Gal-11-S-cerevisiae-antibody/GTX64110). There are several commercial antibodies to human Med15 but the cross reactivity has not been tested.
    2. Quantification with ImageJ on spot assays is difficult because once growth has maxed out on the dense spots there is no resolution. Using more dilute spots is challenging because colony size is affected by the nearby colonies. The error bars are the mutants are large. Can quantitative growth curves be carried out in flasks or an automatic plate reader for better quantification?

    Minor comments:

    1. Why were the stress conditions chosen in figure 1B? These are only a subset of conditions that the med15 deletion is sensitive to. Aside from acetic acid the phenotypic profile of each deletion is similar. The bigger the deletion, the more severe the growth defect. The keto plate appears under loaded by comparing the number of colonies in the third dilution on the keto plate and the fourth spot on the YPD plate in the BY4742 (MED15 wild-type strain). Does keto lyse cells? Or was there that much variation between mutants? Perhaps the dose of keto needs to be calibrated. In figure 2B it looks like wildtype growth in keto was 70% of untreated growth.
    2. Figures 1C and 1D are not discussed in the results. The authors should remind what the hGR assay measures when discussing the results. How is it different from the GAL4 transcriptional reporter?
    3. In the D to A mutants some appear to be required for acetic acid tolerance. What was the pH of the media?
    4. The labels between Figure 1 and 2 are inconsistent. 90 mM acetic versus 80mM AcOH, YPGal versus YPGalactose, SD+LKHU versus SD+KLUH. The mutants on 0.97M NaCl at 37oC from figure 2A grew more than 0.9M NaCl at 38oC. Also, in the text it says 37 oC.
    5. Is the MED15 strain BY4742? In Figure 1 was it also transformed the pRS315? How was the plasmid maintained on the plates, specifically YPD?
    6. Genes such as GAL and URA3 should in italics.
    7. In the split Ub assays, was wildtype Msn2 and Med15 also present?
    8. There is inconsistently naming of media in Media and Phenotype Testing section. The media is called synthetic complete media is labeled SC-URA or SC-LEU and at times in Results its called SD+K or SC-HULM. Is SC with all amino acids and SD without any amino acids?
    9. The plasmid names in the supplemental table don't match the ones labeled in the figures.
    10. Why was ALG9 used for normalization of qRT PCR?
    11. The background of the strains is confusing. There appears to be two different med15 knockouts OY320 and JF1368. Which ones were used in which experiments? Some of the trains have a trp1 auxotrophic which affects stress response on it's own (González et al. 2008; Schroeder and Ikui 2019).

    Significance

    • General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed? Extensive mutational analysis of Med15.
    • Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...). This work confirms numerous other studies on the contribution of various Med15 domains on function.
    • Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field? Incorporation of human domain substitutions could influence how people outside the field would interpret how Med15 interacts with transcription factors.
    • Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. Expert in using yeast genetics and natural genetic variation to address underlying mechanisms of stress response to environmental toxins with a particular focus on transcription factors and TORC1.
  9. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Major Comments

    The authors show that the amino acid content and length of Q1 affects transcription activity in a media-dependent way in a construct that includes Q1-ABD1 and a tailing Q/N rich region (Q1R). Briefly, different media conditions used as proxies for specific target TF activities varied in their sensitivity to the Q1 sequence content. However, the reason for this variation between target TF activities is not addressed, so the observations seem more anecdotal than insightful. One test performed suggests some of the Q1 sequence dependence may be due to changes in AD-ABD interactions, but this interesting possibility is not investigated further.

    A split-ubiquitin two-hybrid assay, meant to detect interactions between Msn2 TAD and Med15-Q1R, showed clear Q1 sequence/composition-dependence when changed from polyQ tracts. In particular, replacement with leucine-rich tracts (12L and RvHs) significantly reduced interactions (as inferred from growth requirements in Fig 5B). Q1 consisting of just 10 spacer residues, 0 to 24 Q residues, or PQ repeats all had quite similar results suggesting retention of some Msn2 and Med15 interactions. Replacement with a helix-forming sequence from NAB3 gave intermediate results. Again, no explanation was offered for the observation but it seems probable that the NAB3 Q1 system is no longer reporting on Msn2 Med15 interactions.

    The manuscript presents extensive assays, but a lack of consistency in conditions and constructs tested makes comparing different assays difficult. In particular, it would be valuable to have NAB3-Q1, FrHs-Q1, and RvHS-Q1 tested under conditions of high salt as that is indicated to be the Msn2 target condition (e.g. an additional result that would be presented in Fig 3B); this would be valuable to compare to the two-hybrid results. The relationship between Q1 polyQ length and Msn2 TAD-Med15 ABD1 binding is not clear from this assay as all had similar growth on the plates. A possible explanation for the inferred reduction in TAD-ABD1 binding in the leucine rich Q1 constructs is that this highly hydrophobic linker itself binds to ABD1 and is therefore self-inhibitory. There is also the unexplored/not discussed possibility that NAB3, 12L, and RvHs have off-target interactions that disrupt the TAD-ABD1 interactions.

    The framing of the study and the title of the manuscript strongly suggest that there might be a relationship between coiled-coil formation and transcription activity. This is the basis for selection of many of the Q1 sequences tested, with the premise of either increasing or disrupting coiled-coil structure. These 'propensities' are quantified in Supp Fig 1; however, a significant limitation of this interpretation is that these propensities are bulk properties that presume formation of homo-dimers or homo-trimers, a situation that is not shown to be relevant for Med15 at a promoter. This means that Q1 is potentially only one of the multiple partners required for coiled-coil formation. So even if a tested sequence has high coiled-coil propensity, that may not be the case in the actual biological systems at play here. Another consideration to be entertained is how different solvent conditions (different media) may affect coiled-coil propensity. An unanswered question is whether Q1 may form coiled-coil structure either with other regions of Med15 and/or with other Mediator subunits or even other co-factors entirely. This is a question implied by the title of this paper, but the data presented address neither intra- nor inter-molecular interactions of the polyQ regions (the two-hybrid study is designed to probe the ABD-AD interactions).

    A final proposed hypothesis was that Q1 acts as a hinge in a way analogous to what was reported for the huntingtin protein (ref 7). This is an attractive model but remains untested in this work. In particular, the Med15-Q1R construct used does not have multiple ABDs that would potentially be brought in close contact, so the results here cannot be interpreted as analogous to the huntingtin hinge model. Minor Comments:

    Please explain the choice of the 10-residue spacer instead of a 12-residue spacer.

    Page 14: "We observed that Q1 substitutions with increased coiled-coil propensity (Supplementary Figure 1) diminished TF activity while Q1 substitutions that interfered with coiled-coil propensity had no effect on TF activity (Fig. 3, 4), suggesting that the flexibility of the sequence is an important feature." There was no demonstration that those sequences in this context form CCs. There's no evidence of what is actually being modulated whether it's length, flexibility, or ability to interact with other regions of Med15 or even with other co-factors.

    Page 15: "We confirmed that Msn2-dependent activities of Med15 are encoded by the region containing the Q1 tract and ABD1 (aa 116-277) and found that the KIX domain alone could also mediate an interaction with Msn2 (Fig. 5). This contrasts with the Gcn4- or Gal4-dependent growth or stress responses which are the result of additive interactions with Med15 that are characterized by weak, highly dispersed, multivalent interfaces. While it is not yet entirely clear if the interaction with Msn2 is similarly multivalent, we have shown that either the KIX domain alone or the Q1R region alone of Med15 was sufficient with no evidence of additivity." These statements are unsupported. While Gcn4 and Gal4 transcription activity has been shown to depend on multiple AD-ABD interactions, none of the data reported here shows that Msn2 does not (as is stated here, which undermines the "contrasts" argument. Further, based on the assays presented in Figure 1B, Msn2, Gal4, and Gcn4 behave similarly for the various Med15 constructs.

    Page 16: "In all instances TF activity was reduced in the absence of the Med15 Q1 tract." This seems false based on the data presented. Met10 activity appears to have increased in Figure 4A.

    Page 16: Reference to Figure 2C and Figure 2B are mislabeled. Should be Figure 2D and 2C, respectively.

    Page 17: "The fact that residues at Q1 were not functionally constrained to be glutamine residues suggests the Q1 tract is not an interaction motif participating directly in protein-protein interactions." This is completely unsupported. There are no data presented that address interactions between Q1 and anything else.

    Figure 2: Not clear which assays were at 30{degree sign}C vs 22{degree sign}C as they are not labeled in the figure. In Figure 2A, the label med15 should be med15Δ.

    Figure 4: Interpretation of these results seems limited by only reporting YPD media conditions. May be helpful to include the conditions reported in Figure 1.

    Figure 6: It is not clear what some elements of this figure are meant to represent. Is saw tooth always polyQ? or Is ABD1 always blue and ABD2 is always red. What then are the loops? The general premise of this figure does not seem to be supported by the actual experiments performed.

    Supp Figure 3: "K is the Med15 fragment encompassing the KIX domain, aa 1-277." This aa range is KQ in the main text. Either the residue range is wrong, or the label is wrong.

    Significance

    This manuscript addresses an interesting topic. There appears to be a disconnect between the stated motivation and what was actually done. The large array of assays and conditions are difficult to compare, leaving the reader with a feeling that the authors have catalogued a lot of possibilities but that no generalizable or unifying insights are at hand. The attempt to present a model (Figure 6) is difficult to parse and is not directly supported by the data presented. Addressing the issues raised here could result in a work that is useful to the specific field of Med15 structure and function but of limited use at the moment to a wider audience.

  10. Version published to 10.1101/2024.05.04.592524v1 on bioRxiv