SMC5/6-Mediated Plasmid Silencing is Directed by SIMC1-SLF2 and Antagonized by LT

Reviewer #1 (Public review):

SMC5/6 is a highly conserved complex able to dynamically alter chromatin structure, playing in this way critical roles in genome stability and integrity that include homologous recombination and telomere maintenance. In the last years, a number of studies have revealed the importance of SMC5/6 in restricting viral expression, which is in part related to its ability to repress transcription from circular DNA. In this context, Oravcova and colleagues recently reported how SMC5/6 is recruited by two mutually exclusive complexes (orthologs of yeast Nse5/6) to SV40 LT-induced PML nuclear bodies (SIMC/SLF2) and DNA lesions (SLF1/2). In this current work, the authors extend this study, providing some new results. However, as a whole, the story lacks unity and does not delve into the molecular mechanisms responsible for the silencing process. One has the feeling that the story is somewhat incomplete, putting together not directly connected results.

(1) In the first part of the work, the authors confirm previous conclusions about the relevance of a conserved domain defined by the interaction of SIMC and SLF2 for their binding to SMC6, and extend the structural analysis to the modelling of the SIMC/SLF2/SMC complex by AlphaFold. Their data support a model where this conserved surface of SIMC/SLF2 interacts with SMC at the backside of SMC6's head domain, confirming the relevance of this interaction site with specific mutations. These results are interesting but confirmatory of a previous and more complete structural analysis in yeast (Li et al. NSMB 2024). In any case, they reveal the conservation of the interaction. My major concern is the lack of connection with the rest of the article. This structure does not help to understand the process of transcriptional silencing reported later beyond its relevance to recruit SMC5/6 to its targets, which was already demonstrated in the previous study.

(2) In the second part of the work, the authors focus on the functionality of the different complexes. The authors demonstrate that SMC5/6's role in transcription silencing is specific to its interaction with SIMC/SLF2, whereas SMC5/6's role in DNA repair depends on SLF1/2. These results are quite expected according to previous results. The authors already demonstrated that SLF1/2, but not SIMC/SLF2, are recruited to DNA lesions. Accordingly, they observe here that SMC5/6 recruitment to DNA lesions requires SLF1/2 but not SIMC/SLF2. Likewise, the authors already demonstrated that SIMC/SLF2, but not SLF1/2, targets SMC5/6 to PML NBs. Taking into account the evidence that connects SMC5/6's viral resistance at PML NBs with transcription repression, the observed requirement of SIMC/SLF2 but not SLF1/2 in plasmid silencing is somehow expected. This does not mean the expectation has not to be experimentally confirmed. However, the study falls short in advancing the mechanistic process, despite some interesting results as the dispensability of the PML NBs or the antagonistic role of the SV40 large T antigen. It had been interesting to explore how LT overcomes SMC5/6-mediated repression: Does LT prevent SIMC/SLF2 from interacting with SMC5/6? Or does it prevent SMC5/6 from binding the plasmid? Is the transcription-dependent plasmid topology altered in cells lacking SIMC/SLF2? And in cells expressing LT? In its current form, the study is confirmatory and preliminary. In agreement with this, the cartoons modelling results here and in the previous work look basically the same.

(3) There are some points about the presented data that need to be clarified.

Reviewer #2 (Public review):

Oracová et al. present data supporting a role for SIMC1/SLF2 in silencing plasmid DNA via the SMC5/6 complex. Their findings are of interest, and they provide further mechanistic detail of how the SMC5/6 complex is recruited to disparate DNA elements. In essence, the present report builds on the author's previous paper in eLife in 2022 (PMID: 36373674, "The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers") by showing the role of SIMC1/SLF2 in localisation of the SMC5/6 complex to plasmid DNA, and the distinct requirements as compared to recruitment to DNA damage foci. Although the findings of the manuscript are of interest, we are not yet convinced that the new data presented here represents a compelling new body of work and would better fit the format of a "research advance" article. In their previous paper, Oracová et al. show that the recruitment of SMC5/6 to SV40 replication centres is dependent on SIMC1, and specifically, that it is dependent on SIMC1 residues adjacent to neighbouring SLF2.

Other comments

(1) The mutations chosen in Figure 1 are quite extensive - 5 amino acids per mutant. In addition, they are in many cases 'opposite' changes, e.g., positive charge to negative charge. Is the effect lost if single mutations to an alanine are made?

(2) In Figure 2c, it isn't clear from the data shown that the 'SLF2-only' mutations in SMC6 result in a substantial reduction in SIMC1/SLF2 binding.

(3) In the GFP reporter assays (e.g. Figure 3), median fluorescence is reported - was there any observed difference in the percentage of cells that are GFP positive?

(4) The potential role of the large T antigen as an SMC5/6 evasion factor is intriguing. However, given the role of the large T antigen as a transcriptional activator, caution is required when interpreting enhanced GFP fluorescence. Antagonism of the SMC5/6 complex in this context might be further supported by ChIP experiments in the presence or absence of large T. Can large T functionally substitute for HBx or HIV-Vpr?

(5) In Figure 5c, the apparent molecular weight of large T and SMC6 appears to change following transfection of GFP-SMC5 - is there a reason for this?

Reviewer #3 (Public review):

Summary:

This study by the Boddy and Otomo laboratories further characterizes the roles of SMC5/6 loader proteins and related factors in SMC5/6-mediated repression of extrachromosomal circular DNA. The work shows that mutations engineered at an AlphaFold-predicted protein-protein interface formed between the loader SLF2/SIMC1 and SMC6 (similar to the interface in the yeast counterparts observed by cryo-EM) prevent co-IP of the respective proteins. The mutations in SLF2 also hinder plasmid DNA silencing when expressed in SLF2-/- cell lines, suggesting that this interface is needed for silencing. SIMC1 is dispensable for recruitment of SMC5/6 to sites of DNA damage, while SLF1 is required, thus separating the functions of the two loader complexes. Preventing SUMOylation (with a chemical inhibitor) increases transcription from plasmids but does not in SLF2-deleted cell lines, indicating the SMC5/6 silences plasmids in a SUMOylation dependent manner. Expression of LT is sufficient for increased expression, and again, not additive or synergistic with SIMC1 or SLF2 deletion, indicating that LT prevents silencing by directly inhibiting 5/6. In contrast, PML bodies appear dispensable for plasmid silencing.

Strengths:

The manuscript defines the requirements for plasmid silencing by SMC5/6 (an interaction of Smc6 with the loader complex SLF2/SIMC1, SUMOylation activity) and shows that SLF1 and PML bodies are dispensable for silencing. Furthermore, the authors show that LT can overcome silencing, likely by directly binding to (but not degrading) SMC5/6.

Weaknesses:

(1) Many of the findings were expected based on recent publications.

(2) While the data are consistent with SIMC1 playing the main function in plasmid silencing, it is possible that SLF1 contributes to silencing, especially in the absence of SIMC1. This would potentially explain the discrepancy with the data reported in ref. 50. SLF2 deletion has a stronger effect on expression than SIMC1 deletion in many but not all experiments reported in this manuscript. A double mutant/deletion experiments would be useful to explore this possibility.

(3) SLF2 is part of both types of loaders, while SLF1 and SIMC1 are specific to their respective loaders. Did the authors observe differences in phenotypes (growth, sensitivities to DNA damage) when comparing the mutant cell lines or their construction? This should be stated in the manuscript.

(4) It would be desirable to have control reporter constructs located on the chromosome for several experiments, including the SUMOylation inhibition (Figures 5A and 5-S2) and LT expression (Figure 5D) to exclude more general effects on gene expression.

(5) Figure 5A: There appears to be an increase in GFP in the SLF2-/- cells with SUMOi? Is this a significant increase?

(6) The expression level of SFL2 mut1 should be tested (Figure 3B).

Author response:

This study builds on, extends, and experimentally validates results/models from our previous study. Our and others’ data implicated SMC5/6, PML nuclear bodies (PML NBs), and SUMOylation in the transcriptional repression of extrachromosomal circular DNA (ecDNA). Moreover, multiple viruses were found to express early genes that combat SMC5/6-based repression through targeted proteasomal degradation (e.g. Hepatitis B virus HBx and HIV-1 Vpr). Thus, our analysis of the roles of the foregoing in plasmid repression yields a coherent set of results for the field to build on.

In our previous study we presented a model, but no supportive ecDNA silencing data, suggesting that distinct SMC5/6 subcomplexes, SIMC1-SLF2 and SLF1/2, separately control its transcriptional repression and DNA repair activities. In this study we experimentally validate that prediction using an ecDNA silencing assay and SMC5/6 localization analysis following DNA damage.

Our study further reveals the unexpected dispensability of PML NBs in the silencing of simple plasmid DNA, a departure from current dogma. This raises important questions for the field to address in terms of the silencing mechanisms for different ecDNAs across different cell types. Despite the dispensability of SUMO-rich PML NBs, SUMOylation is required for ecDNA repression. Lastly, the SV40 LT antigen early gene product counteracts ecDNA silencing. These results used genetic epistasis arguments to implicate SUMO and LT in SMC5/6-based transcriptional silencing. We provide provisional responses for some of the reviewer’s general comments below.

Public Reviews:

Reviewer #1 (Public review):

SMC5/6 is a highly conserved complex able to dynamically alter chromatin structure, playing in this way critical roles in genome stability and integrity that include homologous recombination and telomere maintenance. In the last years, a number of studies have revealed the importance of SMC5/6 in restricting viral expression, which is in part related to its ability to repress transcription from circular DNA. In this context, Oravcova and colleagues recently reported how SMC5/6 is recruited by two mutually exclusive complexes (orthologs of yeast Nse5/6) to SV40 LT-induced PML nuclear bodies (SIMC/SLF2) and DNA lesions (SLF1/2). In this current work, the authors extend this study, providing some new results. However, as a whole, the story lacks unity and does not delve into the molecular mechanisms responsible for the silencing process. One has the feeling that the story is somewhat incomplete, putting together not directly connected results.

Please see the introductory overview above.

(1) In the first part of the work, the authors confirm previous conclusions about the relevance of a conserved domain defined by the interaction of SIMC and SLF2 for their binding to SMC6, and extend the structural analysis to the modelling of the SIMC/SLF2/SMC complex by AlphaFold. Their data support a model where this conserved surface of SIMC/SLF2 interacts with SMC at the backside of SMC6's head domain, confirming the relevance of this interaction site with specific mutations. These results are interesting but confirmatory of a previous and more complete structural analysis in yeast (Li et al. NSMB 2024). In any case, they reveal the conservation of the interaction. My major concern is the lack of connection with the rest of the article. This structure does not help to understand the process of transcriptional silencing reported later beyond its relevance to recruit SMC5/6 to its targets, which was already demonstrated in the previous study.

Demonstrating the existence of a conserved interface between the Nse5/6-like complexes and SMC6 in both yeast and human is foundationally important and was not revealed in our previous study. It remains unclear how this interface regulates SMC5/6 function, but yeast studies suggest a potential role in inhibiting the SMC5/6 ATPase cycle. Nevertheless, the precise function of Nse5/6 and its human orthologs in SMC5/6 regulation remain undefined, largely due to technical limitations in available in vivo analyses. The SIMC1/SLF2/SMC6 complex structure likely extends to the SLF1/2/SMC6 complex, suggesting a unifying function of the Nse5/6-like complexes in SMC5/6 regulation, albeit in the distinct processes of ecDNA silencing and DNA repair. There have been no studies to date (including this one) showing that SIMC1-SLF2 is required for SMC5/6 recruitment to ecDNA. Our previous study showed that SIMC1 was needed for SMC5/6 to colocalize with SV40 LT antigen at PML NBs. Here we show that SIMC1 is required for ecDNA repression, in the absence of PML NBs, which was not anticipated.

(2) In the second part of the work, the authors focus on the functionality of the different complexes. The authors demonstrate that SMC5/6's role in transcription silencing is specific to its interaction with SIMC/SLF2, whereas SMC5/6's role in DNA repair depends on SLF1/2. These results are quite expected according to previous results. The authors already demonstrated that SLF1/2, but not SIMC/SLF2, are recruited to DNA lesions. Accordingly, they observe here that SMC5/6 recruitment to DNA lesions requires SLF1/2 but not SIMC/SLF2.

Our previous study only examined the localization of SLF1 and SIMC1 at DNA lesions. The localization of these subcomplexes alone should not be used to define their roles in SMC5/6 localization. Indeed, the field is split in terms of whether Nse5/6-like complexes are required for ecDNA binding/loading, or regulation of SMC5/6 once bound.

Likewise, the authors already demonstrated that SIMC/SLF2, but not SLF1/2, targets SMC5/6 to PML NBs. Taking into account the evidence that connects SMC5/6's viral resistance at PML NBs with transcription repression, the observed requirement of SIMC/SLF2 but not SLF1/2 in plasmid silencing is somehow expected. This does not mean the expectation has not to be experimentally confirmed. However, the study falls short in advancing the mechanistic process, despite some interesting results as the dispensability of the PML NBs or the antagonistic role of the SV40 large T antigen. It had been interesting to explore how LT overcomes SMC5/6-mediated repression: Does LT prevent SIMC/SLF2 from interacting with SMC5/6? Or does it prevent SMC5/6 from binding the plasmid? Is the transcription-dependent plasmid topology altered in cells lacking SIMC/SLF2? And in cells expressing LT? In its current form, the study is confirmatory and preliminary. In agreement with this, the cartoons modelling results here and in the previous work look basically the same.

We agree, determining the potential mechanism of action of LT in overcoming SMC5/6-based repression is an important next step. It will require the identification of any direct interactions with SMC5/6 subunits, and better methods for assessing SMC5/6 loading and activity on ecDNAs. Unlike HBx, Vpr, and BNRF1 it does not appear to induce degradation of SMC5/6, making it a more complex and interesting challenge. Also, the dispensability of PML NBs in plasmid silencing versus viral silencing raises multiple important questions about SMC5/6’s repression mechanism.

(3) There are some points about the presented data that need to be clarified.

Reviewer #2 (Public review):

Oracová et al. present data supporting a role for SIMC1/SLF2 in silencing plasmid DNA via the SMC5/6 complex. Their findings are of interest, and they provide further mechanistic detail of how the SMC5/6 complex is recruited to disparate DNA elements. In essence, the present report builds on the author's previous paper in eLife in 2022 (PMID: 36373674, "The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers") by showing the role of SIMC1/SLF2 in localisation of the SMC5/6 complex to plasmid DNA, and the distinct requirements as compared to recruitment to DNA damage foci. Although the findings of the manuscript are of interest, we are not yet convinced that the new data presented here represents a compelling new body of work and would better fit the format of a "research advance" article. In their previous paper, Oracová et al. show that the recruitment of SMC5/6 to SV40 replication centres is dependent on SIMC1, and specifically, that it is dependent on SIMC1 residues adjacent to neighbouring SLF2.

We agree, this manuscript fits the Research Advance model, which is the format that this manuscript was submitted in.

Reviewer #3 (Public review):

Summary:

This study by the Boddy and Otomo laboratories further characterizes the roles of SMC5/6 loader proteins and related factors in SMC5/6-mediated repression of extrachromosomal circular DNA. The work shows that mutations engineered at an AlphaFold-predicted protein-protein interface formed between the loader SLF2/SIMC1 and SMC6 (similar to the interface in the yeast counterparts observed by cryo-EM) prevent co-IP of the respective proteins. The mutations in SLF2 also hinder plasmid DNA silencing when expressed in SLF2-/- cell lines, suggesting that this interface is needed for silencing. SIMC1 is dispensable for recruitment of SMC5/6 to sites of DNA damage, while SLF1 is required, thus separating the functions of the two loader complexes. Preventing SUMOylation (with a chemical inhibitor) increases transcription from plasmids but does not in SLF2-deleted cell lines, indicating the SMC5/6 silences plasmids in a SUMOylation dependent manner. Expression of LT is sufficient for increased expression, and again, not additive or synergistic with SIMC1 or SLF2 deletion, indicating that LT prevents silencing by directly inhibiting 5/6. In contrast, PML bodies appear dispensable for plasmid silencing.

Strengths:

The manuscript defines the requirements for plasmid silencing by SMC5/6 (an interaction of Smc6 with the loader complex SLF2/SIMC1, SUMOylation activity) and shows that SLF1 and PML bodies are dispensable for silencing. Furthermore, the authors show that LT can overcome silencing, likely by directly binding to (but not degrading) SMC5/6.

Weaknesses:

(1) Many of the findings were expected based on recent publications.

Please see introductory paragraphs above.