Proteasome gene expression is controlled by coordinated functions of multiple transcription factors

  1. Jennifer E. Gilda
  2. Asrafun Nahar
  3. Dharanibalan Kasiviswanathan
  4. Nadav Tropp
  5. Tamar Gilinski
  6. Tamar Lahav
  7. Dina Alexandrovich
  8. Yael Mandel-Gutfreund
  9. Soyeon Park
  10. Shenhav Shemer

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Abstract

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7–10 days after denervation), it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g., type-2 diabetes, cancer).

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  1. Version published to 10.1083/jcb.202402046
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    Reply to the reviewers

    We appreciate the thoughtful comments of the reviewers. We have revised the manuscript according to these comments as detailed below.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Efficient proteostasis in cells demands efficient clearing of damaged or misfolded proteins, and an important pathway involved in such clearance is the ubiquitin-proteasome pathway. In this system, proteins are tagged with ubiquitin to target them for degradation by the 26S proteasome complex. The conventional 26S proteasome complex consists of a core particle (CP or 20S proteasome) and one or two regulatory particles (RP, or 19S proteasome) to form the singly or doubly-capped proteasome, respectively. Proteasome assembly is a well-orchestrated process that requires proper stoichiometry of proteasome subunits and dedicated proteasome assembly chaperones. This is maintained by fine-tuning their transcriptional and translational regulation.

    This manuscript elucidates an important aspect of how the different proteasome components are transcriptionally regulated upon denervation in mouse muscles for timely and efficiently assembling 26S proteasome. The authors present data that point out towards the model whereby a two-phase transcriptional program (early: day 3-7 and late: day 10-14) activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. This involves the coordinated functions of two transcription factors, PAX4 and alpha-PAL(Nrf1) which were important for both early and late phase of the transcriptional program. Their roles were not redundant as loss of one transcription factor was sufficient to prevent induction of various proteasome genes in muscle after denervation.

    In summary, the authors report a novel bi-phasic mechanism elevating proteasome production in vivo, which involves the coordinated functions of two transcription factors, PAX4 and alpha-PAL(Nrf1).

    Major points:

    1. It is not clear why PAX4 and alpha-PAL(Nrf1) are both fully required for the transcriptional induction of some proteasome genes upon denervation (with good overlap), while only PAX4 is important for increased proteasome assembly. The authors speculate that this could be due to a stoichiometry problem but an alternative scenario where translation is increased upon alpha-PAL(Nrf1) inhibition would also be possible. This would explain why, for example, the induction of PSMC1 gene expression upon denervation is abolished upon alpha-PAL(Nrf1) inhibition (Fig. 5C) while the protein level is still increased (Fig. 6H). Is that also true for PSMD5 and Rpn9? Could it also be that the loss of function of alpha-PAL(Nrf1) is too detrimental for the muscle so that they induce an alternative stress response pathway increasing proteasome subunit translation?

    We thank the reviewer for this comment. To better clarify this important point, we conducted further experiments to examine the differential effects between PAX4 vs. α-PALNRF1 on proteasome assembly chaperons (Fig. S4b). Our new data show that PAX4 promotes the induction of the assembly chaperone, PSMD5 (S5b) at 3 days after denervation (Fig. S4B). This induction is critical for the increase in PSMD5 protein levels because PAX4 knockout results in decreased PSMD5 protein levels at both 3 and 10 days after denervation (Fig. 4K). α-PALNRF1, however, does not affect the mRNA levels of this chaperone (Fig. S4A). This new result strengthens our conclusion that induced expression of assembly chaperones by PAX4 is key to raising proteasome levels after denervation.

    We cannot rule out an indirect effect of α-PALNRF1 knock-down on protein synthesis, and therefore this potential alternative mechanism is now discussed in the text. It appears unlikely, however, that α-PALNRF1 knock-down is too detrimental to muscle as we do not find any evidence phenotypically for any type of stress or abnormalities.

    1. Pax4 controls Rpt1-2 transcription and these two Rpt proteins form a pair. As Rpt4 is also regulated by Pax4, is Rpt5 also controlled by Pax4?

    We believe the reviewer meant to request the data for Rpt4, because the data for Rpt5 was already included in original Fig. 4G-H. Therefore, we repeated the RT-PCR analysis of PAX4 KO mouse muscles for Rpt4 and now show that its induction requires PAX4 at 10 d after denervation, just when proteasome content is increased (Fig. 4G). At 3 d after denervation, Rpt4 induction is probably regulated by other transcription factors because its mRNA levels at this early phase were similar in muscles from WT and PAX4 KO mice (Fig. 4H). These data, strengthen our conclusions that coordinated functions of multiple transcription factors control proteasome gene expression in vivo. In future studies, we will investigate the specific mode of cooperation and mechanisms by which various transcription factors and co-factors collaborate to enhance the expression of proteasome genes in the early and delayed stages of gene expression within a living organism.

    What about the assembly chaperone for these two pairs: PSMD5 and p27? It would be very interesting to know if there is a transcriptional coregulation based on proteasome assembly intermediates.

    The referee raises an important point, which we also discuss in the text. We now present data showing that PAX4 promotes the induction of the assembly chaperon PSMD5 at 3 d after denervation (Fig. S4B), correlating nicely with the observed changes in protein levels of this chaperon (Fig. 4K). The expression of PSMD9 (p27) however, does not require neither PAX4 nor α-PALNRF1 (Fig. S4). Consequently, we conclude that PAX4 promotes proteasome biogenesis by promoting PSMD5 induction, and in the absence of α-PALNRF1 proteasome subunits can still efficiently assemble into the proteasomes (even though their expression is reduced), due to the induced expression and increased action of the assembly chaperone PSMD5. Our data highlight the intricacy in controlling proteasome levels, through transcriptional regulation of proteasome genes and assembly chaperones during muscle atrophy. We now further document and discuss the regulation of proteasome biogenesis by these two transcription factors in the text and Discussion (p.28).

    1. Fig. 4J: PSMD5 and PSMD13 are not tested in Fig. 4A, G and H. This needs to be done if the authors want to draw the parallel mRNA-protein levels, as in their conclusion. Moreover, the protein levels seem to be much more induced than the mRNA levels, could that be due to increased translation? This could be discussed.

    We accepted this thoughtful suggestion and now present the mRNA levels for PSMD5 and PSMD13 in Figs. 4A, G and H and Fig. S4. The new data does not change our conclusion that protein abundance largely correlate with the transcript levels (Figs. 2 and 4K).

    The reviewer raises an important question that we hope to resolve in the future. As we point out in the revised Discussion section, “the substantial rise in protein levels compared to mRNA levels after denervation suggests potential increased protein translation due to PAX4 loss. Whether PAX4 regulates protein synthesis and thus can affect protein levels beyond gene expression are intriguing questions for future research”.

    1. The conclusion is not correct in this sentence: "Moreover, analysis of innervated and 10 d denervated muscle homogenates from WT, alpha-PAL(Nrf1) KD or PAX4/alpha-PAL(Nrf1) KD mice by native gels and immunoblotting or LLVY-cleavage indicated that loss of both transcription factors is necessary to effectively block accumulation of active assembled proteasomes on denervation (Fig. 6H)". This is not correct, as the loss of PAX4 is sufficient to block accumulation of active assembled proteasomes on denervation (Fig. 4K). So, it could just be that alpha-PAL(Nrf1) KD has no effect on the induction of proteasome assembly after denervation and that all the effect of the double mutant is due to PAX4 loss. This needs to be corrected.

    We thank the reviewer for this thoughtful comment. The text has been revised accordingly.

    Minor points:

    1. I would rephrase the sentence "baseline at 14 d after denervation and showed a sustained low mRNA levels until 28 d (Fig. 2A-F).", as the mRNA levels are still significantly higher that the basal levels for most proteasome genes. Same for the sentence: "RNA sequencing (RNA-Seq) analysis of TA muscles at 14 d after denervation indicated that expression of most proteasome genes is low at 14 d (Fig. S1)". Expression is low compared to what and not being induced doesn't mean they are low. This needs to be rephrased.

    We revised the text accordingly and thank the reviewer for these suggestions.

    1. Microscopy images need more explanation: define the green and red channel and what they are used for in the legend.

    The legends have been updated as requested.

    1. Columns have moved from the Table 2.

    The tables have now been submitted as separate files.

    1. Fig. S3: RT-PCR on NRF-1(NFE2L1) need to be performed to see the extent of inhibition by shRNA.

    We thank the reviewer for this important comment. The data, which was added as new Fig. S3A, shows an efficient knockdown of NRF-1NFE2L1 with shNFE2L1.

    1. In the sentence: "PAX4 maintaining subunit stoichiometry for increased proteasome assembly.", could it be due to the much higher levels of PSMB8, 9 and 10 immunoproteasome subunits upon alpha-PAL(Nrf1) KD (Fig. 6F)?

    We addressed this aspect in Major Point #1, regarding the difference between PAX4 and α-PALNRF1; please see our response. As for the Reviewer’s comment concerning Fig. 6F, we think that the increased expression of PSMB 8, 9, and 10 in α-PALNRF1-KD compared to the double KD or PAX4 KO further suggests a distinct cooperative interaction between these transcription factors in promoting proteasome expression, assembly, and function, which we plan to thoroughly investigate in future separate studies. However, the increased expression of PSMB 8, 9, and 10 can affect the composition of the CP (by replacing their normal ounterpart), but not the RP assembly. CP and RP are known to assemble separately with their own dedicated chaperones; RP and CP then associate to complete the assembly of proteasome holoenzyme (RP-CP complex). Thus, it is unlikely that increased CP assembly alone would increase overall RP-CP assembly.

    **Referees cross-commenting**

    All other comments are relevant.

    Reviewer #1 (Significance (Required)):

    Overall, the work is impactful and timely, reporting the participation of a novel transcription factor, alpha-PAL(Nrf1), along with PAX4, in regulating the transcription of proteasome genes and the subsequent assembly of conventional proteasomes in mouse muscle upon denervation. One limitation is that alpha-PAL(Nrf1) kockdown is only inhibiting proteasome genes expression but proteasome assembly, the reason being still unknown. Most of the conclusions drawn in the manuscript are supported by the experimental data. Better understanding how proteasome homeostasis is regulated upon stressful conditions is an important fundamental aspect of proteasome biology. I would support publication of this manuscript providing the more specific concerns listed are addressed.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The main limitation of this study is that is based on a single model of muscle atrophy: that induced by cut of the sciatic nerve. Another one will nicely complement the findings as fasting atrophy or cancer cachexia model, to see if the two phase is recapitulated with regard to proteasome modulation.

    The referee raises an interesting point, but as we explained throughout the manuscript, we did not use denervation in this study as a model for atrophy but rather as an in vivo model system to investigate mechanisms of protein degradation and proteasome homeostasis in a whole organism in vivo. The reason we selected denervation as an in vivo model for accelerated proteolysis is due to the gradual nature of muscle loss, which allows us to dissect the various phases of proteasome homeostasis effectively. Fasting, as an alternative model, is too rapid for addressing the specific questions that we asked in this study. In addition, in the rapid atrophy induced by fasting the primary physiological mechanism to increase protein degradation in vivo is believed to be through post-synthetic modification of proteasomes, rather than the production of new proteasomes (VerPlank et al., 2019). In future separate studies, we will thoroughly investigate whether the mechanisms discovered here are applicable to other types of atrophy (e.g. diabetes, aging, cancer). The obtained results will be published and fully discussed separately, in part because covering all types of atrophy within a single paper is impractical and goes beyond the scope of the current manuscript.

    Another major concern is that the author do not measure over time during denervation atrophy the mRNA and protein content expression of the two transcription factors that they found crucial in the proteasome induction and assembly.

    We agree with the reviewer that time course would strengthen our conclusions that the two transcription factors are important for proteasome gene induction and assembly. We have added these data showing that PAX4 (Fig. 4I) and α-PALNRF-1 (Fig. 6E) both accumulate in the nucleus at 7 d after denervation, just when proteasome content is maximal (Fig. 3A) and protein breakdown is accelerated (Cohen 2009; Volodin 2017; Aweida 2021). The mRNA levels of PAX4 were presented as original Fig. 4F and indicate that PAX4 is induced already at 3 d after denervation. We have added new RT-PCR data for α-PALNRF-1 showing that α-PALNRF-1 is induced at 7 d and 10 d after denervation (Fig. 6D).

    Major and minor concerns are as follows:

    Typos now and then are present all over the text, as holoemzyme shall be replaced with holoenzyme on page 9, on page 12 proteasome is misspelled on mid page, as well as cellls. By cotrast shall be corrected on page 19. References on page 22 shall be formatted.

    We have corrected the typographical errors.

    • reference 29 on page 7 seems out of context together with the sentences it is coupled with.

    The reference is appropriately located within the text in terms of context, and precisely aligns with the sentence to which it is associated. Reference 29 (Boos 2019) describes a cellular state in which all proteasome genes rise simultaneously.

    • muscle electroporation of plasmid shall be replaced by AAV9 injection that causes less inflammation and more expressing fibers

    We do not understand and see no basis for the referee’s assertion that the “muscle electroporation of plasmid shall be replaced by AAV9 injection”. On the contrary, the electroporation methodology is widely used by many labs because of its many advantages. This in vivo gene transfection approach is extremely useful to study transient gene (or shRNA) effects in adult muscles, while avoiding the developmental effects of genes (or shRNA) that are often seen in transgenic or knockout animals (e.g., the inducible knockout of α-PALNRF-1 caused lethality, see Fig. 6B-C).

    In addition, the electroporation technique offers great advantages from its speed and major cost savings. We have been using it routinely in our lab for in vivo studies, and articles using it from many laboratories worldwide have appeared in all major journals, e.g. see our papers in Nature Communications, J Cell Biol, PNAS, EMBO rep, and papers from late Alfred Goldberg (Harvard), Marco Sandri (Padova, Italy), Jeff Brault (Indiana Univ.) and others. In all studies included in this manuscript that involve electroporation, contrary to the reviewer’s impression, there was no damage or inflammation to the muscles, and we routinely examined histological sections. Finally, for our studies, we always use muscles that are at least ~70% transfected, which has proven adequate for observing gene effects in mouse muscle. In each experiment, transfected muscles are always compared and analyzed in parallel to control muscles (transfected with scrambled shLacz control). In fact, the validity of the in vivo electroporation technique is further confirmed herein by our investigations of transgenic inducible knock-down mice, showing similar effects on proteasome gene expression.

    • the shGankyrin data shall be complemented with overexpression of the same chaperone to see the effects of proteasome expression and assembly.

    We understand the reviewer’s concern but do not believe that such an experiment is necessary since it is well known and there is already extensive evidence in the literature showing that the chaperon Gankyrin is essential for proteasome assembly (Kaneko et al. Cell 137, 914–925, May 29, 2009 (DOI 10.1016/j.cell.2009.05.008). Thus, various Gankyrin mutants have often been used as an inactive control for proteasome assembly in vitro and in vivo (Kaneko et al. Cell 137, 914–925, May 29, 2009 (DOI 10.1016/j.cell.2009.05.008). In fact, Gankyrin’s known function in ensuring not only the proper subunit composition, but also proper conformation of the proteasome holoenzyme (Lu et al., Mol Cell. 2017 Jul 20;67(2):322-333.e6).

    • another important transcription factor driving MuRF1 expression is Twist and it is totally ignored in the discussion, please add it.

    We regret this oversight. We did not mean to slight any authors, although our major new discoveries and focus is on proteasome genes and not MuRF1. However, to satisfy the reviewer, we now discuss in the text Twist and other transcription factors (including SMAD2/3, glucocorticoid receptors and NFkB) capable of inducing the major atrophy-related genes (among them MuRF1).

    • WB in Fig 2 shall be complemented by one in the Supp with more replicates per timepoint

    We accepted this thoughtful suggestion and now present blots from additional normal and atrophying denervated mouse muscle samples as new Fig. S1B. This approach, however, does not change any of our conclusions.

    • please justify why only PSMD10 (gankyrin) has been silenced and not any of the others (POMP, PSMD5, PSMD9)

    We silenced PSMD10 (Gankyrin) as a representative RP assembly chaperone, since it is better characterized than the other RP assembly chaperones (PSMD5 and PSMD9). We kept POMP (a CP assembly chaperone) intact. Since the formation of one proteasome holoenzyme (RP2-CP) requires two RPs and one CP, increasing proteasome assembly is expected to be more demanding for RP assembly than CP. This led us to predict that disrupting RP assembly should be sufficient to block the induced proteasome assembly. This prediction is supported by our data (Fig. 3), and this justification was also added to the revised text to enhance clarity.

    The originality is limited by the fact that Pax4 was already shown to have a role in muscle atrophy and drives the expression of p97 by the same authors. I would be curious to see if treatments in vitro know to induce the proteasome as starvation etc acts through the biphase mechanism showed in this paper, to understand how extendable to other kinds of atrophy is.

    We respectfully disagree that the originally of the present findings is limited, because previously we validated a single proteasome subunit (Rpt1) as a target gene for PAX4 (Volodin 2017), and here we discover novel global coordination of proteasome gene expression by multiple transcription factors.

    As we mention above, muscle denervation was used here as an in vivo model system of catabolic conditions. Unlike prior reports that were limited to cultured cells, our studies focus on the physiological setting in vivo to reveal mechanisms of proteasome homeostasis. In any case, regulation of proteasome gene expression by multiple transcription factors in other types of atrophy has not been investigated but is possible because common transcriptional adaptations activate protein breakdown in different types of muscle atrophy, including a coordinated induction of numerous components of the ubiquitin proteasome system (Jagoe 2002; Lecker 2004; Gomes 2001). In future independent research, we intend to investigate if the two-phase mechanism reported here can in fact be generalized to other atrophy (or stress) conditions.

    Reviewer #2 (Significance (Required)):

    The authors Gilda and co-workers made a great attempt to dissect the induction of proteasome activity during denervation muscle atrophy and discovered a two-phase process which involves two transcription factors Pax4 and NRF1. The manuscript is clearly written and the experiments fully delineated.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Using denervated mouse muscle as a model, Gilda et al. demonstrated that a two-step transcriptional program operates in the process of muscle atrophy after denervation and that proteasome expression-induced enhancement of protein degradation is important. Gilda et al. clarified that the transcription factors PAX4 and PAL/NRF-1 act on this proteasome expression induction and that the induction of these transcription factors and the expression induction of the proteasome gene cluster after denervation are necessary for muscle atrophy using an in vivo mouse model. The experiments were logically designed, and the results presented are considered clear and reliable. However, some of the descriptions in the text lack accuracy and courtesy, and some experiments require additional data to support and strengthen the author's claims. In particular, it is unclear whether PAX4, FOXO3, and NRF-1 work together or whether they have distinct functions. Although the authors claim that there are two stages of proteasome expression induction after denervation, this remains unclear. The authors should clarify the differences in target sequence or target genes and the substitutability of each transcription factor.

    Major comments: 1: In Figure 3A, the results of the immunoblot of SDS-PAGE against 20S proteasome subunits should also be shown to confirm the increase in proteasome activity and amount.

    We would like to clarify this aspect. We show the increased levels of proteasome holoenzyme complex (RP2-CP) by immunoblotting of the native gel, rather than SDS-PAGE gel. This is because the blots of the native gel can assess the levels of the actual proteasome complex, not simply subunit levels in their denatured state as in SDS-PAGE; SDS-PAGE cannot distinguish between free subunits and ones that are incorporated into the proteasome.

    If proteasome activity was increased due to some other mechanisms, proteasome levels would remain relatively constant, while proteasome activity would have increased. However, this is not the case here since our data demonstrates that both RP2-CP activities and levels peak at day 7. Furthermore, the in-gel peptidase assay (Fig. 3A panel b) directly tests the 20S CP activity within the proteasome holoenzyme (RP2-CP complex) using the fluorogenic model substrate, LLVY-AMC. The 20S CP is activated for substrate degradation, only upon its association with RP (RP2-CP complex), since RP opens the substrate entry gate of the 20S. Free 20S itself is inactive, as its gate for substrate entry is closed; for this reason, free 20S can be detected, only after its substrate entry gate is artificially opened by SDS (see free 20S in panel b, but not in panel a).

    2: In Figure 3, the reviewer assumed the conflict between the results of peptidase activity and SDS-PAGE in 14d. Therefore, quantification and statistical analysis should be performed on the results of proteasome peptidase activity and immunoblots to clarify the relationships between proteasome activity and amounts. Immunoblotting against ubiquitin is also needed to confirm the requirement and efficiency of proteasome induction.

    As the reviewer pointed out, it might seem discrepant that peptidase activity at 14 d denervation is lower than its peak at 7d (Fig. 3A, panel a), but SDS-PAGE signal for proteasome subunits seems still high (Fig. 3A, panel d, Rpn2). SDS-PAGE detects total cellular content of proteasome subunits (free subunits as well as ones assembled within proteasomes). However, at any given moment, these subunits are not only in the proteasome holoenzyme complex, but also in different assembly intermediates. When proteasome subunits are transcriptionally induced as in this study, proteasome assembly process is also increased. However, proteasome assembly is a multi-step process, and the fold-induction for each specific subunit is different (Fig. 2A-B). This means that the rate of a certain assembly step would be differently affected for a given subunit, depending on their fold-induction. For this reason, some subunits seem to exist at a high level at 14d (e.g. Fig. 3A, panel d, Rpn2), but they are not yet incorporated into the proteasome complex, because they might be still undergoing assembly process.

    As for the ubiquitin blot, it can be a good indicator for proteasome activity, when proteasome activity is decreased than normal. In such situations, ubiquitinated proteins accumulate (i.e. their signals increase as compared to control), due to their deficient degradation. However, our present study pertains to the opposite situation, where proteasome activity is increased in degrading ubiquitinated proteins. In normal cells, ubiquitinated proteins are hardly detectable due to their rapid degradation. Thus, when proteasome activity is greater than normal, ubiquitinated protein levels will be further decreased than normal. Data become unreliable when the signals are below the detection threshold. For this reason, we provided functional readouts involving the number of muscle fibers (for example, Fig. 3D).

    3: In Figure 3C, the sample labels of shGankyrin and shLacZ are repeated. Would it be mislabeled?　In addition, NATIVE PAGE immunoblot analysis against Gankyrin and proteasome subunits are needed to prove the knockdown efficiency and to reveal the assembly defect of proteasome by Gankyrin knockdown.

    To present our findings more clearly, we show one of each sample in the revised figure, rather than the duplicates as in the previous figure (Fig. 3C). We also included the immunoblot data to show that Gankyrin knockdown disrupts proteasome assembly, as seen by the reduced proteasome complex activity and level (Fig. 3C, panels a, b, c, lane 3, see RP2-CP). In Gankyrin knockdown samples, proteasome holoenzyme complex exhibited smeary appearance (Fig. 3C, panel c, see bracketed region in lane 3), as opposed to a discrete band in the controls (lanes 1, 2). This smeary appearance reflects more heterogeneous proteasome populations, due to defects in their composition and/or conformation. This is in line with Gankyrin’s known function in ensuring not only the proper subunit composition, but also proper conformation of the proteasome holoenzyme (Lu et al., Mol Cell. 2017 Jul 20;67(2):322-333.e6).

    4: In Figures 4A, 4G, 4H, 4J, and 4K, the results of shPAX4 against innervated muscle should be shown to estimate the contribution of PAX4 in steady-state conditions. To clarify the innervated muscle-specific function of PAX4, histological analysis and quantification of proteasome gene expression in multiple organs in PAX4 KO mice are needed.

    The reviewer raises an interesting point, but as we explained above, we concentrate here on the major new discovery that multiple transcription factors increase proteasome content in a catabolic condition *in vivo, *correlating directly with the accelerated protein loss. Regulation of the basal levels of proteasome in normal conditions in various types of cells and tissues is certainly an important issue meriting in depth study and will be the subject for future studies, but it is beyond the scope of this lengthy paper. This point is now discussed in the revised text.

    The tissue distribution of PAX4 and the detailed description of the phenotype of KO mice are also needed to understand and evaluate the role of PAX4 in muscle.

    We added the requested data about PAX4 distribution as Fig. 4I. These data shows that PAX4 accumulates in the nucleus already at 3 d after denervation. Furthermore, we are happy to add further information about the knock-out mouse model. The requested information and a detailed description of how PAX4 KO mice were generated were added to the text. The PAX4 KO mice showed no abnormalities and did not appear in any way different from the wild type littermates.

    5: In Figure 4C, immunoblot analysis against PAX4 is essential to confirm the PAX4 protein knockout.

    We agree and representative blots were added to Fig. 4C.

    6: In Figure 5, peptidase activity and immunoblotting in NATIVE PAGE are needed to reveal the contribution of FOXO3 and NRF-1 in denervated muscle as shown in Figure 4.

    The requested data for FOXO3 using FOXO3 dominant negative (as in Fig. 5A-B) were added as new Fig. 5C-D, showing no effect on proteasome content by FOXO3 inhibition. These new data are consistent with our findings that the expression of only two proteasome subunit genes was affected by FOXO3 inhibition at 10 d after denervation (Fig. 5B). The data for α-PALNRF-1 and the effects of its knockdown on proteasome content and activity were shown as original Fig. 6H (now Fig. 6J).

    The expression of FOXO3 and NRF-1 should also be shown by RT-PCR and immunoblotting as shown in Figure 4.

    We thank the reviewer for this thoughtful suggestion, and as requested, we now show representative blots of transfected muscles to support the graphical data (Figs. 5C-F). These data confirm the efficient expression of HA-FOXO3ΔC or FLAG-α-PALNRF-1 dominant negative inhibitors in transfected muscles. It is important to note that these inhibitors are mutant forms designed to interfere with the normal function of the wild-type endogenous FOXO3 or α-PALNRF-1 proteins, without affecting their transcript levels. Given this mechanism, we believe that Western blotting is a more appropriate technique for assessing their impact, as it provides direct insights into protein expression. In the revised main text and methods, we have now clarified this point.

    Similar to previous comments, the expression of the dominant negative form of Foxo3 and NRF-1 should be performed in innervated muscles to reveal the significance and specificity of Foxo3 and NRF-1 function in denervated muscles.

    As mentioned above, regulation of the normal basal levels of proteasomes is certainly an important issue meriting in depth study and will be the subject for future studies, but it is beyond the scope of this lengthy paper, which focuses on the mechanisms increasing protein content in catabolic conditions in vivo. With respect to FOXOs, there is a large literature on its regulation and roles in normal muscle (please see papers by late Alfred L Goldberg, Marco Sandri and others). Under normal conditions FOXO3 is largely inactive via phosphorylation by insulin-PI3K-AKT signaling (Stitt 2004; Latres 2005; Zhao 2007).

    7: In Figure 6D, the list of genes should be served especially about 27 genes and 69 genes that show common features between NRF-1 KD and PAX4 KO.

    The requested data is now presented as new Table 4.

    8: In Figure 6F, the list of genes that change expression in PAX4 and NRF-1 KD mice is needed.

    We agree and the requested data has now been added to table 5.

    9: In Figure 6H, immunoblotting against ubiquitin is needed to evaluate the contribution of proteasome induction to protein degradation.

    We clarified this aspect in the Major Point #2. Please see our response.

    10: This study lacks the detailed mechanisms by which PAX4, Foxo3, and NRF-1 regulate the expression of proteasome genes. The contribution of these transcription factors is revealed by experiments, but the specific sequence that these transcription factors bind and how transcription factors are induced in denervated muscles is not clarified. As shown in the figures, the ChIP assay provides convincing results, but the detailed sequence or map of the promoter region of proteasome genes must be shown in the figures to clarify the target sequences of NFE2L1 and PAX4, FOXO3, and NRF-1. In addition, the luciferase assay would support the results of the ChIP assay.

    Again, the reviewer raises an important question that we plan to resolve in the future. As mentioned, our findings strongly suggest a novel coordinated mechanism involving multiple transcription factors that control proteasome content in catabolic states in vivo. The enclosed revised manuscript primarily focuses on elucidating the contributions of individual transcription factors (α-PALNRF-1, PAX4, NRF-1NFE2L1 and FOXO3) to the induction of proteasome genes, revealing a significant overlap in genes regulated by multiple transcription factors. The specific mode of cooperation among these and other transcription factors and cofactors is certainly an important question for future studies, but it is beyond the scope of this lengthy paper. In the revised text we have now clarified this point (page 27). In addition, we agree that clarifying how the transcription factors are induced in denervated muscles merits some considerations and a paragraph was added to the Discussion (page 26) concerning possible mechanisms. For example, it is possible that the transcription factor STAT3 is involved in PAX4 induction because, based on previous microarray and ChIP data in cultured NIH3T3 cells, PAX4 was identified as a target gene of STAT3 (Snyder et al., 2008), and STAT3 becomes activated after denervation (Madaro et al., 2018).

    We are delighted that the reviewer found the results obtained through the ChIP assay convincing. Given the extensive scope of our investigation and rigorous analyses of dozens of genes, it is not feasible to generate luciferase-encoding plasmids for all of them. However, in response to the reviewer's request, we have carried out predictions of the binding sites of the 4 transcription factors within the minimal promoter regions (300 up- and 1000 down-stream to TSS) of the 64 proteasome sequences. The predicted binding sites are now listed in Table 2A-D. These new data further support our key findings that multiple transcription factors control proteasome gene expression in a catabolic physiological state in vivo.

    11: The results of the loss of transcription factors are well done, but the authors should also try to estimate the effect of overexpression of transcription factors in muscle. If the overexpressed transcription factors cause proteasome induction and muscle fiber mass reduction, these results strongly support the importance of transcription factor-mediated proteasome enhancement.

    We understand the reviewer’s comment but do not believe that such an experiment is necessary to support our key findings about proteasome gene induction by multiple transcription factors in vivo. In fact, we have specifically refrained from pursuing overexpression studies in this context due to the apparent coordination and some potential interdependence between the functions of PAX4 and α-PALNRF-1 transcription factors in inducing proteasome genes. Manipulating one specific gene through overexpression could potentially disrupt this delicate coordination and yield misleading results.

    In addition, there are several limitations of gene overexpression in mouse muscle, as it may not be as efficient and does not represent physiological conditions. Therefore, to validate gene functions in a physiological setting in vivo, we generated transgenic animals with the gene of interest specifically knocked-out or knocked-down. Utilizing transgenic mice lacking the gene of interest, though time-consuming, is a widely accepted and common approach that proves to be the most suitable method for specifically demonstrating the involvement of a particular gene in a physiological process, enabling a targeted and controlled investigation of its role and providing valuable insights into its contribution to the observed effects.

    Minor comments:

    12: The authors should describe the inducible KO mice more carefully and correctly. In the Results section on P12, the description of "whole body Cre+ mice" confuses the readers in understanding the mechanism of inducible Cre-mediated KO.

    We agree and have added the information requested about the KO mice to the main text and a detailed description in the methods section.

    13: In Figures 6B and 6C, the number of mice and the meaning of the asterisk should be described correctly. Is it statistically significant?

    We agree. By accident the number of mice and sign for statistical significance were omitted during processing. The correct sign was added to Fig. 6B-C, and the number of mice used, and the meaning of asterisks were added to the corresponding legend. N=10 mice per condition. **, P

    14: There is no description of Figure 6E in the manuscript. The authors should include it.

    In the original version of this paper, we refer to Fig. 6E in the text on pages 21 and 25. Also, the presented illustration is fully described in the corresponding legend.

    Reviewer #3 (Significance (Required)):

    This paper clarified a novel mechanism of proteasome induction by transcription factors in denervated muscles other than Nrf1 (NFE2L1), which has been shown to contribute to the induction of proteasome gene expression in cultured cells. This is an important paper for expanding the understanding of the field. It is also important because it has demonstrated the potential for new therapeutic targets in diseases such as type 2 diabetes and cancer.

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    Referee #3

    Evidence, reproducibility and clarity

    Using denervated mouse muscle as a model, Gilda et al. demonstrated that a two-step transcriptional program operates in the process of muscle atrophy after denervation and that proteasome expression-induced enhancement of protein degradation is important. Gilda et al. clarified that the transcription factors PAX4 and PAL/NRF-1 act on this proteasome expression induction and that the induction of these transcription factors and the expression induction of the proteasome gene cluster after denervation are necessary for muscle atrophy using an in vivo mouse model. The experiments were logically designed, and the results presented are considered clear and reliable. However, some of the descriptions in the text lack accuracy and courtesy, and some experiments require additional data to support and strengthen the author's claims. In particular, it is unclear whether PAX4, FOXO3, and NRF-1 work together or whether they have distinct functions. Although the authors claim that there are two stages of proteasome expression induction after denervation, this remains unclear. The authors should clarify the differences in target sequence or target genes and the substitutability of each transcription factor.

    Major comments:

    1. In Figure 3A, the results of the immunoblot of SDS-PAGE against 20S proteasome subunits should also be shown to confirm the increase in proteasome activity and amount.
    2. In Figure 3, the reviewer assumed the conflict between the results of peptidase activity and SDS-PAGE in 14d. Therefore, quantification and statistical analysis should be performed on the results of proteasome peptidase activity and immunoblots to clarify the relationships between proteasome activity and amounts. Immunoblotting against ubiquitin is also needed to confirm the requirement and efficiency of proteasome induction.
    3. In Figure 3C, the sample labels of shGankyrin and shLacZ are repeated. Would it be mislabeled?　In addition, NATIVE PAGE immunoblot analysis against Gankyrin and proteasome subunits are needed to prove the knockdown efficiency and to reveal the assembly defect of proteasome by Gankyrin knockdown.
    4. In Figures 4A, 4G, 4H, 4J, and 4K, the results of shPAX4 against innervated muscle should be shown to estimate the contribution of PAX4 in steady-state conditions. To clarify the innervated muscle-specific function of PAX4, histological analysis and quantification of proteasome gene expression in multiple organs in PAX4 KO mice are needed. The tissue distribution of PAX4 and the detailed description of the phenotype of KO mice are also needed to understand and evaluate the role of PAX4 in muscle.
    5. In Figure 4C, immunoblot analysis against PAX4 is essential to confirm the PAX4 protein knockout.
    6. In Figure 5, peptidase activity and immunoblotting in NATIVE PAGE are needed to reveal the contribution of FOXO3 and NRF-1 in denervated muscle as shown in Figure 4. The expression of FOXO3 and NRF-1 should also be shown by RT-PCR and immunoblotting as shown in Figure 4. Similar to previous comments, the expression of the dominant negative form of Foxo3 and NRF-1 should be performed in innervated muscles to reveal the significance and specificity of Foxo3 and NRF-1 function in denervated muscles.
    7. In Figure 6D, the list of genes should be served especially about 27 genes and 69 genes that show common features between NRF-1 KD and PAX4 KO.
    8. In Figure 6F, the list of genes that change expression in PAX4 and NRF-1 KD mice is needed.
    9. In Figure 6H, immunoblotting against ubiquitin is needed to evaluate the contribution of proteasome induction to protein degradation.
    10. This study lacks the detailed mechanisms by which PAX4, Foxo3, and NRF-1 regulate the expression of proteasome genes. The contribution of these transcription factors is revealed by experiments, but the specific sequence that these transcription factors bind and how transcription factors are induced in denervated muscles is not clarified. As shown in the figures, the ChIP assay provides convincing results, but the detailed sequence or map of the promoter region of proteasome genes must be shown in the figures to clarify the target sequences of NFE2L1 and PAX4, FOXO3, and NRF-1. In addition, the luciferase assay would support the results of the ChIP assay.
    11. The results of the loss of transcription factors are well done, but the authors should also try to estimate the effect of overexpression of transcription factors in muscle. If the overexpressed transcription factors cause proteasome induction and muscle fiber mass reduction, these results strongly support the importance of transcription factor-mediated proteasome enhancement.

    Minor comments:

    1. The authors should describe the inducible KO mice more carefully and correctly. In the Results section on P12, the description of "whole body Cre+ mice" confuses the readers in understanding the mechanism of inducible Cre-mediated KO.
    2. In Figures 6B and 6C, the number of mice and the meaning of the asterisk should be described correctly. Is it statistically significant?
    3. There is no description of Figure 6E in the manuscript. The authors should include it.

    Significance

    This paper clarified a novel mechanism of proteasome induction by transcription factors in denervated muscles other than Nrf1 (NFE2L1), which has been shown to contribute to the induction of proteasome gene expression in cultured cells. This is an important paper for expanding the understanding of the field. It is also important because it has demonstrated the potential for new therapeutic targets in diseases such as type 2 diabetes and cancer.

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    Referee #2

    Evidence, reproducibility and clarity

    The main limitation of this study is that is based on a single model of muscle atrophy: that induced by cut of the sciatic nerve. Another one will nicely complement the findings as fasting atrophy or cancer cachexia model, to see if the two phase is recapitulated with regard to proteasome modulation.

    Another major concern is that the author do not measure over time during denervation atrophy the mRNA and protein content expression of the two transcription factors that they found crucial in the proteasome induction and assembly.

    Major and minor concerns are as follows:

    Typos now and then are present all over the text, as holoemzyme shall be replaced with holoenzyme on page 9, on page 12 proteasome is misspelled on mid page, as well as cellls. By cotrast shall be corrected on page 19. References on page 22 shall be formatted.

    • reference 29 on page 7 seems out of context together with the sentences it is coupled with.
    • muscle electroporation of plasmid shall be replaced by AAV9 injection that causes less inflammation and more expressing fibers
    • the shGankyrin data shall be complemented with overexpression of the same chaperone to see the effects of proteasome expression and assembly
    • another important transcription factor driving MuRF1 expression is Twist and it is totally ignored in the discussion, please add it.
    • WB in Fig 2 shall be complemented by one in the Supp with more replicates per timepoint
    • please justify why only PSMD10 (gankyrin) has been silenced and not any of the others (POMP, PSMD5, PSMD9)

    The originality is limited by the fact that Pax4 was already shown to have a role in muscle atrophy and drives the expression of p97 by the same authors. I would be curious to see if treatments in vitro know to induce the proteasome as starvation etc acts through the biphase mechanism showed in this paper, to understand how extendable to other kinds of atrophy is.

    Significance

    The authors Gilda and co-workers made a great attempt to dissect the induction of proteasome activity during denervation muscle atrophy and discovered a two-phase process which involves two transcription factors Pax4 and NRF1. The manuscript is clearly written and the experiments fully delineated.

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    Referee #1

    Evidence, reproducibility and clarity

    Efficient proteostasis in cells demands efficient clearing of damaged or misfolded proteins, and an important pathway involved in such clearance is the ubiquitin-proteasome pathway. In this system, proteins are tagged with ubiquitin to target them for degradation by the 26S proteasome complex. The conventional 26S proteasome complex consists of a core particle (CP or 20S proteasome) and one or two regulatory particles (RP, or 19S proteasome) to form the singly or doubly-capped proteasome, respectively. Proteasome assembly is a well-orchestrated process that requires proper stoichiometry of proteasome subunits and dedicated proteasome assembly chaperones. This is maintained by fine-tuning their transcriptional and translational regulation.

    This manuscript elucidates an important aspect of how the different proteasome components are transcriptionally regulated upon denervation in mouse muscles for timely and efficiently assembling 26S proteasome. The authors present data that point out towards the model whereby a two-phase transcriptional program (early: day 3-7 and late: day 10-14) activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. This involves the coordinated functions of two transcription factors, PAX4 and alpha-PAL(Nrf1) which were important for both early and late phase of the transcriptional program. Their roles were not redundant as loss of one transcription factor was sufficient to prevent induction of various proteasome genes in muscle after denervation.

    In summary, the authors report a novel bi-phasic mechanism elevating proteasome production in vivo, which involves the coordinated functions of two transcription factors, PAX4 and alpha-PAL(Nrf1).

    Major points:

    1. It is not clear why PAX4 and alpha-PAL(Nrf1) are both fully required for the transcriptional induction of some proteasome genes upon denervation (with good overlap), while only PAX4 is important for increased proteasome assembly. The authors speculate that this could be due to a stoichiometry problem but an alternative scenario where translation is increased upon alpha-PAL(Nrf1) inhibition would also be possible. This would explain why, for example, the induction of PSMC1 gene expression upon denervation is abolished upon alpha-PAL(Nrf1) inhibition (Fig. 5C) while the protein level is still increased (Fig. 6H). Is that also true for PSMD5 and Rpn9? Could it also be that the loss of function of alpha-PAL(Nrf1) is too detrimental for the muscle so that they induce an alternative stress response pathway increasing proteasome subunit translation?
    2. Pax4 controls Rpt1-2 transcription and these two Rpt proteins form a pair. As Rpt4 is also regulated by Pax4, is Rpt5 also controlled by Pax4? What about the assembly chaperone for these two pairs: PSMD5 and p27? It would be very interesting to know if there is a transcriptional coregulation based on proteasome assembly intermediates.
    3. Fig. 4J: PSMD5 and PSMD13 are not tested in Fig. 4A, G and H. This needs to be done if the authors want to draw the parallel mRNA-protein levels, as in their conclusion. Moreover, the protein levels seem to be much more induced than the mRNA levels, could that be due to increased translation? This could be discussed.
    4. The conclusion is not correct in this sentence: "Moreover, analysis of innervated and 10 d denervated muscle homogenates from WT, alpha-PAL(Nrf1) KD or PAX4/alpha-PAL(Nrf1) KD mice by native gels and immunoblotting or LLVY-cleavage indicated that loss of both transcription factors is necessary to effectively block accumulation of active assembled proteasomes on denervation (Fig. 6H)". This is not correct, as the loss of PAX4 is sufficient to block accumulation of active assembled proteasomes on denervation (Fig. 4K). So, it could just be that alpha-PAL(Nrf1) KD has no effect on the induction of proteasome assembly after denervation and that all the effect of the double mutant is due to PAX4 loss. This needs to be corrected.

    Minor points:

    1. I would rephrase the sentence "baseline at 14 d after denervation and showed a sustained low mRNA levels until 28 d (Fig. 2A-F).", as the mRNA levels are still significantly higher that the basal levels for most proteasome genes. Same for the sentence: "RNA sequencing (RNA-Seq) analysis of TA muscles at 14 d after denervation indicated that expression of most proteasome genes is low at 14 d (Fig. S1)". Expression is low compared to what and not being induced doesn't mean they are low. This needs to be rephrased.
    2. Microscopy images need more explanation: define the green and red channel and what they are used for in the legend.
    3. Columns have moved from the Table 2.
    4. Fig. S3: RT-PCR on NRF-1(NFE2L1) need to be performed to see the extent of inhibition by shRNA.
    5. In the sentence: "PAX4 maintaining subunit stoichiometry for increased proteasome assembly.", could it be due to the much higher levels of PSMB8, 9 and 10 immunoproteasome subunits upon alpha-PAL(Nrf1) KD (Fig. 6F)?

    Referees cross-commenting

    All other comments are relevant.

    Significance

    Overall, the work is impactful and timely, reporting the participation of a novel transcription factor, alpha-PAL(Nrf1), along with PAX4, in regulating the transcription of proteasome genes and the subsequent assembly of conventional proteasomes in mouse muscle upon denervation. One limitation is that alpha-PAL(Nrf1) kockdown is only inhibiting proteasome genes expression but proteasome assembly, the reason being still unknown. Most of the conclusions drawn in the manuscript are supported by the experimental data. Better understanding how proteasome homeostasis is regulated upon stressful conditions is an important fundamental aspect of proteasome biology. I would support publication of this manuscript providing the more specific concerns listed are addressed.

  6. Version published to 10.1101/2023.04.12.536627v2 on bioRxiv
  7. Version published to 10.1101/2023.04.12.536627v1 on bioRxiv