The 20S proteasome activator PA28γ controls the compaction of chromatin

  1. Didier Fesquet
  2. David Llères
  3. Charlotte Grimaud
  4. Cristina Viganò
  5. Francisca Méchali
  6. Séverine Boulon
  7. Olivier Coux
  8. Catherine Bonne-Andrea
  9. Véronique Baldin

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Abstract

PA28γ (also known as PSME3), a nuclear activator of the 20S proteasome, is involved in the degradation of several proteins regulating cell growth and proliferation and in the dynamics of various nuclear bodies, but its precise cellular functions remain unclear. Here, using a quantitative FLIM-FRET based microscopy assay monitoring close proximity between nucleosomes in living human cells, we show that PA28γ controls chromatin compaction. We find that its depletion induces a decompaction of pericentromeric heterochromatin, which is similar to what is observed upon the knockdown of HP1β (also known as CBX1), a key factor of the heterochromatin structure. We show that PA28γ is present at HP1β-containing repetitive DNA sequences abundant in heterochromatin and, importantly, that HP1β on its own is unable to drive chromatin compaction without the presence of PA28γ. At the molecular level, we show that this novel function of PA28γ is independent of its stable interaction with the 20S proteasome, and most likely depends on its ability to maintain appropriate levels of H3K9me3 and H4K20me3, histone modifications that are involved in heterochromatin formation. Overall, our results implicate PA28γ as a key factor involved in the regulation of the higher order structure of chromatin.

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  1. Version published to 10.1242/jcs.257717
  2. eLife

    ##Author Response:

    All three reviewers agreed that establishing a link between a proteasome activator and heterochromatin stability was novel and intriguing. However, limited insight into the PA28-gamma mechanism of action (or possibly a new heterochromatin compaction mechanism) dampened reviewer enthusiasm. The reviewers offered many suggestions, including additional experiments, new controls, and structural changes to the Discussion, that we hope you find useful.

    We would like to thank the reviewers for their suggestions and comments, which we will take into account to improve our manuscript as much as possible. As stressed by reviewers, our manuscript highlights a new and unexpected function of a proteasome regulator, PA28γ, in the regulation of heterochromatin compaction. We also provide evidences that this unexpected function of PA28γ is independent of its proteasome regulatory function. Moreover, we can show that PA28γ is required at least for proper maintenance of heterochromatin regions dependent on HP1 proteins, thereby providing a clear insight into the PA28γ mechanism of action on chromatin.

    ###Reviewer #1:

    In the manuscript entitled, "The 20S proteasome activator PA28γ controls the compaction of chromatin," Fesquet et al. establish a functional link between PA28γ and chromatin compaction in human cells. Previous work established a role for PA28γ in DNA repair and in chromosome stability through mitotic checkpoint regulation; however, a role, if any, for PA28γ in heterochromatin establishment/maintenance was not known. The authors use an elegant LacO-GFP system combined with PA28γ knockdown to support the possibility that this nuclear activator contributes to DNA packaging of repetitive DNA. A nucleosome proximity assay offers additional support that the most compacted chromatin is most sensitive to loss of PA28γ. Using a truncated version of PA28γ, the authors show that this chromatin function appears to be independent of its interaction with the 20S proteasome. ChIP-qPCR suggests that PA28γ binds repetitive DNA and ChIP-qPCR of PA28γ knockdown cells lose H3K9me and H4K20me, two silent heterochromatin marks. In addition to these data, the authors also attempt to establish that PA28γ and HP1β may work together to support heterochromatin formation/maintenance. The manuscript reports several intriguing pieces of data that have the potential to open new areas of inquiry into proteasome components and accessory factors in chromatin organization and remodeling. The potency of these key experiments, however, were diluted by unconvincing co-localization assays, poorly controlled PLA and ChIP-qPCR assays, and a highly speculative Discussion. Moreover, key controls were missing for several experiments (detailed below) that would have otherwise established the heterochromatin-specificity of PA28γ. Finally, important potential functional consequences of heterochromatin disruption, including chromosome segregation defects, transposable element proliferation, and accumulation of DNA damage, were not addressed while there was a focus instead on cell cycle without clear interpretations.

    Major Comments:

    1. Figure 2: The co-localization experiments were unconvincing - HP1β and PA28γ foci decorate most of the nucleus, making inferences about significant overlap difficult to grasp.

    We fully agree with this criticism for Fig. 2A, in which classical indirect immunofluorescence (IF) and widefield microscopy were used. This is why in Fig. 2B, we set up a pre-extraction protocol of cells with a Triton X100 treatment, to remove almost all soluble proteins before cells fixation and IF. Then images were acquired as Z-stacks with an Airyscan confocal microscope, followed by a 3D reconstruction and analysis of the co-localization between PA28γ and PA28γ using Imaris co-localization software. This experiment highlighted that only a fraction of both endogenous proteins co-localize in the nucleus. Furthermore, we noted that the number of co-localization sites evidenced in Fig 2B (~32) was in the same range than the number of dots (~37) detected by another approach (is-PLA), suggesting that we can be confident with these results.

    I also found the significance of the PLA assays difficult to discern. When both factors are so abundant in the nucleus, it seems inevitable to observe loss of proximity when one 'partner' is depleted. How do these data demonstrate the specificity of this potential proximity? A clearer explanation would be helpful.

    PLA technique imposes numerous constraints to obtain a signal (i.e. distance less than 30-40 nm between the two epitopes, formation of a closed circular DNA template). As a control, we verified that the is-PLA approach gives specific signals between PA28γ and the 20S proteasome. Like PA28γ, the 20S proteasome is very abundant in the nucleus but only a small fraction of PA28γ interacts with the 20S proteasome by Co-IP (Jonik-Nowak, 2018). Consistent with this, less than 60 distinct PLA spots were detected in the nucleus between PA28γ and the 20S proteasome rather than a global nuclear PLA labeling suggesting that it is probably not the abundance of each protein tested that is responsible for PLA signal but rather, as suggested by this kind of techniques, their ability to interact.

    Note that the PIP30 data were a distraction from the main thread - I recommend removing or explaining more clearly.

    PIP30 is currently the only known regulator of PA28γ, for which we have previously shown a critical role in PA28γ interaction with different partners and localization (Jonik-Nowak, 2018). This is why we examined the potential requirement of PIP30 in this new chromatin regulatory function of PA28γ.

    1. The ChIP-qPCR data were certainly exciting but the absence of a negative control locus made me wonder how specific this result was to DNA repeats.

    As a control, we already used cyclin E2 promoter in our ChIP-qPCR for PA28γ. This led us to show that the detection of PPA28γ on heterochromatic repetitive DNA sequences is enriched by a factor of 2-3 as compared to this euchromatic loci bound by PA28γ. In a modified version of this manuscript, we will add other control loci located in euchromatin. We will also test these new loci as negative controls in ChIP-qPCR for H3K9me3 and H4K20me3.

    1. The LacO-GFP data are really cool. Why didn't the authors not attempt to rescue compaction with a PA28γ transgene as was done for the FLIM-FRET?

    Since, we could not obtain stable U20S-LacO-KO-PA28γ and -KO/KI-PA28γ cell lines, we decided to analyze the impact of PA28γ absence, using siRNA approaches. As it was shown that overexpression of PA28γ is sufficient to cause a disruption of Cajal Bodies (Cioce et al., 2006) and a decrease in the number of PML bodies (Zannini et al., 2009), and we also noticed in FRET-FLIM experiments that PA28γ expression level is critical for chromatin compaction, it is difficult to consider to overexpress a RNAi-resistant PA28γ protein in order to rescue the effect of the depleted endogenous protein.

    1. Cell cycle data would be much more interesting if the authors set up a priori predictions based on Figures 1-5.

    We agree with the comment, and we will correct it in the modified version of the manuscript.

    1. The absence of any report of PA28γ KD/KO on genome instability was surprising.

    As indicated in the manuscript, the potential effect of PA28γ depletion on genome stability has already been reported in the literature showing an increase in chromosomal instability (Zannini, 2008).

    Loss of heterochromatin integrity is expected to compromise chromosome transmission/transposable element expression or insertions. Do the repeats to which PA28γ localizes upregulate upon PA28γ KD or KO? Does DNA damage signaling increase at the loci? These functional consequences would be rather more explicable that the S-phase result reported.

    We did not detect any upregulation at these specific loci by RT-qPCR experiment using KO-PA28γ U2OS cells. Concerning the potential accumulation of DNA damage signaling at these loci in the absence of PA28γ, we have not studied this aspect because PA28γ depletion was reported to induce only a marked delay in DSB repair and not a DNA damage accumulation (Levy-Barda, 2011).

    1. The histone mark ChIP-qPCR, like the PA28γ ChIP-qPCR, lacks a negative control locus/loci, again undermining the inference of specificity of PA28γ on heterochromatin.

    We agree and these different control loci would be added in the modified version.

    1. The LLPS paragraph in the discussion was weak - consider removing.

    Yes, potentially. To be defined in the context of the modified version.

    1. The speculation of 20S into foci does not add and, to my mind, detracts from the focus of the Discussion.

    In the modified version of the manuscript, we will focus our study on endogenous proteins and therefore this aspect of the discussion concerning the 20S proteasome, and related to the overexpression of alpha4, will no longer be discussed.

    ###Reviewer #2:

    In this manuscript, Fesquet and colleagues describe an important role of the proteasome activator PA28-gamma in the compaction of chromatin. The authors first demonstrate that PA28-gamma colocalizes HP1-beta at nuclear foci induced by the ectopic expression of alpha-4 subunit of the 20S proteasome. They further show that a fraction of PA28-gamma colocalizes also with HP1-beta in cells without ectopic expression of the alpha-4. The authors then show that PA28-gamma is associated with heterochromatic regions and is required for the compaction of lacO array integrated at a pericentromeric region. They also performed the quantitative FLIM-FRET and demonstrate that PA28-gamma controls chromatin compaction in living cells, independently of its interaction with 20S proteasome. Finally, the authors show that PA29-gamma depletion leads to a decrease of heterochromatin marks, H3K9me3 and H4K20me3, at representative heterochromatic regions. From these findings they conclude that PA28-gamma contributes to chromatin compaction and heterochromatin formation.

    Although PA28-gamma has been identified as an alternative component associated with 20S proteasome, its physiological roles remain obscure. The present study demonstrates that PA28-gamma is involved in chromatin compaction and heterochromatin formation. The results presented are in most cases of high quality and convincingly controlled. I have the following concerns that should be addressed by the authors.

    Major points:

    1. For the localization study (Fig. 1), the authors first show the colocalization of alpha-4, PA28-gamma, and HP1-beta in the nuclear foci induced by ectopic expression of alpha-4-GFP. While the authors point out the similarity of cell-cycle dependent patterns between the alpa-4 induced foci and HP1-beta foci (lines 135-138), this argument seems to be poorly reasoned.

    We omitted to mention that we also tested the potential co-localization of alpha-4-GFP with different proteins associated with nuclear foci (SC35, PML PCNA, γH2AX) or BrdU-labelled replication foci without success, before to find a correlation with the accumulation of newly synthesized GFP-HP1β in nuclear foci.

    The authors previously showed that ectopically expressed CFP-tagged alpha-7, another core component of 20S, accumulates into discrete nuclear foci, and the foci are colocalized with SC35, a well-characterized member of nuclear speckle (Baldin et al. MCB 2008). Considering that both alpha-4 and alpha-7 are core components of 20S proteasome, it is highly likely that the alpha-4-GFP- accumulating nuclear foci are corresponding to the nuclear speckles. If so, HP1-beta foci should be distinct from that of alpha-4-GFP foci. The authors should test the relationship between alpha-4-GFP foci and nuclear speckles, and if this would be the case, it might be better to omit the colocalization data using cells expressing alpha-4-GFP (Fig. 1) and start by potential colocalization of PA28-gamma and HP1-beta in cells without expressing alpha-4-GFP (Fig. 2).

    As mentioned above alpha4-GFP did not co-localize with SC35, a marker of the nuclear speckles. When different alpha subunits of the 20S proteasome are overexpressed, only alpha7 and alpha4 show an accumulation in specific nuclear foci. This remains unclear but a possible explanation could be an alternative composition of alpha subunits in the 20S as previously reported for alpha4 (Padmanabhan A. et al., Assemnbly of an evolutionarily conserved alternative proteasome isoform in human cells, , 2016, Cell Reports). As this part of our study appears to confuse readers and to dilute the essential message of the manuscript, we are considering to exclude these data in the modified version.

    1. Although the functional link between PA28-gamma and chromatin compaction seems quite interesting, it remains unclear how it contributes to the establishment of repressive histone marks such as H3K9me3 and H4K20me3. While the authors clearly show that 20S-binding-deficient PA28-gamma mutant (PA28-gamma ∆C) could restore the chromatin compaction defect caused by PA28-gamma KO, it is also possible that PA28-gamma controls the stability of factors involved in heterochromatin assembly. To exclude this possibility the authors should test whether PA28-gamma KD/KD does not affect the protein levels of core histone modifying enzymes and HP1 proteins by immunoblotting.

    During this study we performed numerous immunoblots using anti-HP1 antibodies and we did not observe any significant variation of these proteins in KO-PA28γ cells. Furthermore, in an atempt to identify proteins whose stability could be controlled directly or indirectly by PA28γ, we performed a SILAC-based quantitative proteomic analysis comparing nuclear extracts from U2OS or HeLa wild type cells to U2OS- or HeLa KO-PA28γ cells. Under the tested conditions, we could not identify variation of the amount of factors involved in chromatin assembly, suggesting that the impact of PA28γ on chromatin organization is not driven by changes in the level of the important histone-modifying enzymes, nor core components of chromatin such as HP1 proteins.

    ###Reviewer #3:

    This manuscript explores the localization and function of a previously studied proteasome activator, PA28gamma. This protein is a nuclear activator of the 20S proteasome and is widely conserved during evolution, although largely absent in fungi. The authors report that (1) subunits of the 20S proteasome (alpha4 and alpha6) and GFP-tagged or endogenous PA28gamma colocalize with each other and with HP1beta in the nucleus, with HP1beta required for the localization of PA28gamma to nuclear foci, (2) depletion of PA28gamma results in decompaction of pericentromeric heterochromatin, and (3) use a FLIM-FRET based microscopy assay to show a broad role for PA28gamma in chromatin compaction, a function that PA28gamma shares with HP1beta. They also show that the C terminus of PA28gamma, which is required for its interaction with the 20S proteasome, is not required for its subnuclear localization or compaction functions, and that PA28gamma KO cells have reduced levels of H3K9me3 and H4K20me3 heterochromatin-associated histone modifications.

    The identification of a role for PA28gamma in heterochromatin compaction and heterochromatin maintenance is interesting and raises intriguing possibilities about the role of this protein and the 20S proteasome in heterochromatic domains. The study is largely descriptive and does not provide new mechanistic insight into heterochromatin or PA28gamma. Although the experiments in the paper are of high quality and well-executed, they basically amount to identification of a new factor that affects heterochromatin stability. The fact that PA28gamma is a proteasome activator provides no mechanistic insight since the 20S proteasome does not seem to be required for the heterochromatin compaction function of PA28gamma.

    The following suggestions may be helpful to the authors in preparing their manuscript for publication (in order of appearance).

    1. The IP experiments in Figure 1D should be performed in the presence of nuclease (DNase/RNase A or benzonase) to test whether the interactions are bridged by RNA or DNA.

    We actually tried several times to perform this IP experiments using notably benzonase. However, despite several attempts under various conditions, we could not obtain a clear and consistent answer to this question.

    1. Figure 2. What percentage of PA28gamma and HP1beta foci overlap in the absence of alpha4 overexpression?

    As indicated in the text, on average of 32 spot of co-localization between the two proteins were detected in Figure 2B and on average of 37 spots in is-PLA experiment (Figure 2C).

    1. Figure 3. Does decompaction result in loss of silencing of heterochromatin targets such as HERV-K, LINE1, alpha satellite etc? Ideally, the authors should perform RNA-seq to provide a more complete picture of changes in gene expression as a result of PA28gamma depletion.

    RT-qPCRs were performed on heterochromatin loci used for ChIP (HERV-K, L1 Line, SatII and alpha-sat) and no significant variation was observed. In order to determine whether the absence of PA28γ could affect gene expression, we performed a trancriptomic analysis using Affymetrix® Human Gene 2.1 ST Array Strip comparing mRNA expression in U2OS -WT and KO-PA28γ cells. This experiment revealed only very little variation between the two samples tested: 11 genes were up in KO-PA28γ (MFAP5 (Microfibrillar-associated protein 5), GLIPR1 (Glioma pathogenesis-related protein 1) and 9 that are still unannotated), and only 2 genes were significantly down: PSME3 (PA28γ) and MAGE-C1(Melanoma-associated antigen C1). These experiences led us to consider that PA28γ probably does not directly affect the level of transcription.

    1. Based on experiments with PA28gamma-deltaC, which does not interact with the 20S proteasome, the authors conclude that the 20S proteasome is not required for the PA28gamma-mediated chromatin compaction. Although their IP data (Figure 4E) seem persuasive, a more convincing experiment would be to also perform the FRET assay for compaction with knockdown of subunits of the proteasome.

    Knockdown of 20S proteasome subunits was not performed since in that condition all the proteasome family will be affected, and we already know that depletion of these proteins has several and pleiotropic effects (i.e. cell cycle progression), which could indirectly affect chromatin compaction.

    1. Figure 6. It is critical that the effects on histone modifications are evaluated using siRNA KD (or other transient KD methods) of PA28g to complement the KO results. PA28gamma KOs have many defects including genome instability and aneuploidy that may affect K9me3 and K20me3 indirectly.

    This is indeed a hypothesis that cannot be ruled out. But considering that these modifications (H3K9me3, H4K20me1/3) are crucial for the establishment of chromatin compaction and that the elimination of PA28γ (siRNA treatment) induces chromatin decompaction within 48h, it is reasonable to consider that the variation of these marks does not result from genome instability.

    1. In general, the manuscript would benefit from the addition of genome-wide approaches such as ChIP-seq to gain broader insight into PA28gamma localization and general compaction functions.

    We agree that the mapping of PA28γ distribution on non-repeated DNA sequences will be useful for the subsequent studies of PA28γ functions in DNA–related processes such as gene regulation. However, because of the difficulty to map HP1 proteins and heterochromatin regions by ChIP-seq, we do not believe that this approach will necessarily reinforce the current message of this first manuscript on the role of PA28γ in the regulation of heterochromatin compaction.

  3. eLife

    ###Reviewer #3:

    This manuscript explores the localization and function of a previously studied proteasome activator, PA28gamma. This protein is a nuclear activator of the 20S proteasome and is widely conserved during evolution, although largely absent in fungi. The authors report that (1) subunits of the 20S proteasome (alpha4 and alpha6) and GFP-tagged or endogenous PA28gamma colocalize with each other and with HP1beta in the nucleus, with HP1beta required for the localization of PA28gamma to nuclear foci, (2) depletion of PA28gamma results in decompaction of pericentromeric heterochromatin, and (3) use a FLIM-FRET based microscopy assay to show a broad role for PA28gamma in chromatin compaction, a function that PA28gamma shares with HP1beta. They also show that the C terminus of PA28gamma, which is required for its interaction with the 20S proteasome, is not required for its subnuclear localization or compaction functions, and that PA28gamma KO cells have reduced levels of H3K9me3 and H4K20me3 heterochromatin-associated histone modifications.

    The identification of a role for PA28gamma in heterochromatin compaction and heterochromatin maintenance is interesting and raises intriguing possibilities about the role of this protein and the 20S proteasome in heterochromatic domains. The study is largely descriptive and does not provide new mechanistic insight into heterochromatin or PA28gamma. Although the experiments in the paper are of high quality and well-executed, they basically amount to identification of a new factor that affects heterochromatin stability. The fact that PA28gamma is a proteasome activator provides no mechanistic insight since the 20S proteasome does not seem to be required for the heterochromatin compaction function of PA28gamma.

    The following suggestions may be helpful to the authors in preparing their manuscript for publication (in order of appearance).

    1. The IP experiments in Figure 1D should be performed in the presence of nuclease (DNase/RNase A or benzonase) to test whether the interactions are bridged by RNA or DNA.

    2. Figure 2. What percentage of PA28gamma and HP1beta foci overlap in the absence of alpha4 overexpression?

    3. Figure 3. Does decompaction result in loss of silencing of heterochromatin targets such as HERV-K, LINE1, alpha satellite etc? Ideally, the authors should perform RNA-seq to provide a more complete picture of changes in gene expression as a result of PA28gamma depletion.

    4. Based on experiments with PA28gamma-deltaC, which does not interact with the 20S proteasome, the authors conclude that the 20S proteasome is not required for the PA28gamma-mediated chromatin compaction. Although their IP data (Figure 4E) seem persuasive, a more convincing experiment would be to also perform the FRET assay for compaction with knockdown of subunits of the proteasome.

    5. Figure 6. It is critical that the effects on histone modifications are evaluated using siRNA KD (or other transient KD methods) of PA28g to complement the KO results. PA28gamma KOs have many defects including genome instability and aneuploidy that may affect K9me3 and K20me3 indirectly.

    6. In general, the manuscript would benefit from the addition of genome-wide approaches such as ChIP-seq to gain broader insight into PA28gamma localization and general compaction functions.

  4. eLife

    ###Reviewer #2:

    In this manuscript, Fesquet and colleagues describe an important role of the proteasome activator PA28-gamma in the compaction of chromatin. The authors first demonstrate that PA28-gamma colocalizes HP1-beta at nuclear foci induced by the ectopic expression of alpha-4 subunit of the 20S proteasome. They further show that a fraction of PA28-gamma colocalizes also with HP1-beta in cells without ectopic expression of the alpha-4. The authors then show that PA28-gamma is associated with heterochromatic regions and is required for the compaction of lacO array integrated at a pericentromeric region. They also performed the quantitative FLIM-FRET and demonstrate that PA28-gamma controls chromatin compaction in living cells, independently of its interaction with 20S proteasome. Finally, the authors show that PA29-gamma depletion leads to a decrease of heterochromatin marks, H3K9me3 and H4K20me3, at representative heterochromatic regions. From these findings they conclude that PA28-gamma contributes to chromatin compaction and heterochromatin formation.

    Although PA28-gamma has been identified as an alternative component associated with 20S proteasome, its physiological roles remain obscure. The present study demonstrates that PA28-gamma is involved in chromatin compaction and heterochromatin formation. The results presented are in most cases of high quality and convincingly controlled. I have the following concerns that should be addressed by the authors.

    Major points:

    1. For the localization study (Fig. 1), the authors first show the colocalization of alpha-4, PA28-gamma, and HP1-beta in the nuclear foci induced by ectopic expression of alpha-4-GFP. While the authors point out the similarity of cell-cycle dependent patterns between the alpa-4 induced foci and HP1-beta foci (lines 135-138), this argument seems to be poorly reasoned. The authors previously showed that ectopically expressed CFP-tagged alpha-7, another core component of 20S, accumulates into discrete nuclear foci, and the foci are colocalized with SC35, a well-characterized member of nuclear speckle (Baldin et al. MCB 2008). Considering that both alpha-4 and alpha-7 are core components of 20S proteasome, it is highly likely that the alpha-4-GFP- accumulating nuclear foci are corresponding to the nuclear speckles. If so, HP1-beta foci should be distinct from that of alpha-4-GFP foci. The authors should test the relationship between alpha-4-GFP foci and nuclear speckles, and if this would be the case, it might be better to omit the colocalization data using cells expressing alpha-4-GFP (Fig. 1) and start by potential colocalization of PA28-gamma and HP1-beta in cells without expressing alpha-4-GFP (Fig. 2).

    2. Although the functional link between PA28-gamma and chromatin compaction seems quite interesting, it remains unclear how it contributes to the establishment of repressive histone marks such as H3K9me3 and H4K20me3. While the authors clearly show that 20S-binding-deficient PA28-gamma mutant (PA28-gamma ∆C) could restore the chromatin compaction defect caused by PA28-gamma KO, it is also possible that PA28-gamma controls the stability of factors involved in heterochromatin assembly. To exclude this possibility the authors should test whether PA28-gamma KD/KD does not affect the protein levels of core histone modifying enzymes and HP1 proteins by immunoblotting.

  5. eLife

    ###Reviewer #1:

    In the manuscript entitled, "The 20S proteasome activator PA28γ controls the compaction of chromatin," Fesquet et al. establish a functional link between PA28γ and chromatin compaction in human cells. Previous work established a role for PA28γ in DNA repair and in chromosome stability through mitotic checkpoint regulation; however, a role, if any, for PA28γ in heterochromatin establishment/maintenance was not known. The authors use an elegant LacO-GFP system combined with PA28γ knockdown to support the possibility that this nuclear activator contributes to DNA packaging of repetitive DNA. A nucleosome proximity assay offers additional support that the most compacted chromatin is most sensitive to loss of PA28γ. Using a truncated version of PA28γ, the authors show that this chromatin function appears to be independent of its interaction with the 20S proteasome. ChIP-qPCR suggests that PA28γ binds repetitive DNA and ChIP-qPCR of PA28γ knockdown cells lose H3K9me and H4K20me, two silent heterochromatin marks. In addition to these data, the authors also attempt to establish that PA28γ and HP1β may work together to support heterochromatin formation/maintenance. The manuscript reports several intriguing pieces of data that have the potential to open new areas of inquiry into proteasome components and accessory factors in chromatin organization and remodeling. The potency of these key experiments, however, were diluted by unconvincing co-localization assays, poorly controlled PLA and ChIP-qPCR assays, and a highly speculative Discussion. Moreover, key controls were missing for several experiments (detailed below) that would have otherwise established the heterochromatin-specificity of PA28γ. Finally, important potential functional consequences of heterochromatin disruption, including chromosome segregation defects, transposable element proliferation, and accumulation of DNA damage, were not addressed while there was a focus instead on cell cycle without clear interpretations.

    Major Comments:

    1. Figure 2: The co-localization experiments were unconvincing - HP1β and PA28γ foci decorate most of the nucleus, making inferences about significant overlap difficult to grasp. I also found the significance of the PLA assays difficult to discern. When both factors are so abundant in the nucleus, it seems inevitable to observe loss of proximity when one 'partner' is depleted. How do these data demonstrate the specificity of this potential proximity? A clearer explanation would be helpful. Note that the PIP30 data were a distraction from the main thread - I recommend removing or explaining more clearly.

    2. The ChIP-qPCR data were certainly exciting but the absence of a negative control locus made me wonder how specific this result was to DNA repeats.

    3. The LacO-GFP data are really cool. Why didn't the authors not attempt to rescue compaction with a PA28γ transgene as was done for the FLIM-FRET?

    4. Cell cycle data would be much more interesting if the authors set up a priori predictions based on Figures 1-5.

    5. The absence of any report of PA28γ KD/KO on genome instability was surprising. Loss of heterochromatin integrity is expected to compromise chromosome transmission/transposable element expression or insertions. Do the repeats to which PA28γ localizes upregulate upon PA28γ KD or KO? Does DNA damage signaling increase at the loci? These functional consequences would be rather more explicable that the S-phase result reported.

    6. The histone mark ChIP-qPCR, like the PA28γ ChIP-qPCR, lacks a negative control locus/loci, again undermining the inference of specificity of PA28γ on heterochromatin.

    7. The LLPS paragraph in the discussion was weak - consider removing.

    8. The speculation of 20S into foci does not add and, to my mind, detracts from the focus of the Discussion.

  6. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 3 of the manuscript.

    ###Summary:

    All three reviewers agreed that establishing a link between a proteasome activator and heterochromatin stability was novel and intriguing. However, limited insight into the PA28-gamma mechanism of action (or possibly a new heterochromatin compaction mechanism) dampened reviewer enthusiasm. The reviewers offered many suggestions, including additional experiments, new controls, and structural changes to the Discussion, that we hope you find useful.

  7. Version published to 10.1101/716332 on bioRxiv