Interplay between acetylation and ubiquitination of imitation switch chromatin remodeler Isw1 confers multidrug resistance in Cryptococcus neoformans

  1. Yang Meng
  2. Zhuoran Li
  3. Tianhang Jiang
  4. Tianshu Sun
  5. Yanjian Li
  6. Xindi Gao
  7. Hailong Li
  8. Chenhao Suo
  9. Chao Li
  10. Sheng Yang
  11. Tian Lan
  12. Guojian Liao
  13. Tong-Bao Liu
  14. Ping Wang
  15. Chen Ding

  • Curated by eLife

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    eLife assessment

    This study links chromatin remodeling with antifungal drug resistance in Cryptococcus neoformans. The work is important because it reveals a new facet of how drug resistance can emerge and associates. The work presented is well done but the story is incomplete since there are questions about methods and association that need to be addressed. Establishing a link between chromatin remodeling and antifungal resistance is a finding that would be of interest to infectious disease researchers, cell biologists, and drug developers.

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Abstract

Cryptococcus neoformans poses a threat to human health, but anticryptococcal therapy is hampered by the emergence of drug resistance, whose underlying mechanisms remain poorly understood. Herein, we discovered that Isw1, an imitation switch chromatin remodeling ATPase, functions as a master modulator of genes responsible for multidrug resistance in C. neoformans . Cells with the disrupted ISW1 gene exhibited profound resistance to multiple antifungal drugs. Mass spectrometry analysis revealed that Isw1 is both acetylated and ubiquitinated, suggesting that an interplay between these two modification events exists to govern Isw1 function. Mutagenesis studies of acetylation and ubiquitination sites revealed that the acetylation status of Isw1 K97 coordinates with its ubiquitination processes at Isw1 K113 and Isw1 K441 through modulating the interaction between Isw1 and Cdc4, an E3 ligase. Additionally, clinical isolates of C. neoformans overexpressing the degradation-resistant ISW1 K97Q allele showed impaired drug-resistant phenotypes. Collectively, our studies revealed a sophisticated acetylation-Isw1-ubiquitination regulation axis that controls multidrug resistance in C. neoformans .

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  1. eLife

    Author Response

    Reviewer #2 (Public Review):

    Weaknesses:

    1)The authors demonstrate that Isw1 has a role in responding to antifungals in Cryptococcus. However, it is not clear if changes in Isw1 stability represent a general response to stress. This study would have benefited from experiments to test: (1) if levels of Isw1 change in response to other stressors (e.g., heat, osmotic, or oxidative stress) and (2) if loss of Isw1 impacts resistance to other stressors.

    A series of experiments were conducted to illustrate and measure phenotypic traits associated with virulence. These traits encompassed capsule formation, melanin synthesis, cell proliferation under stressful conditions, and Isw1 expression levels in response to diverse environmental stimuli. Please see Figure 3a, 3b, 3c, Figure 3-figure supplement 1 and line 237-241.

    1. The authors demonstrate a critical role in the acetylation of K97 and ubiquitination of K441 in regulating Isw1 stability. Additionally, this study shows that K113 is also likely involved in this process. However, it appears that K113 can be either acetylated or ubiquitinated, and it is, thus, less clear if one of the two modifications or both modifications is critical at this residue. Additional experiments may be required to answer this question. This study would have benefited from an additional discussion on the results related to the modification of K113.

    We express our genuine gratitude for this insightful critique pertaining to the K113 site. In our study, we observed the presence of acetylation and ubiquitination changes at the K113 site in our mass spectrometry data. This finding suggests that a proportion of Isw1 is acetylated, while another proportion of Isw1 is ubiquitinated. In order to analyze the K113 function, a series of experiments were conducted, involving the production of triple, double, and single mutations at positions K89, K97, and K113. In addition, the utilization of K-to-R (mimicking deacetylation) and K-to-Q (mimicking acetylation) methodologies was implemented. To elucidate the significance of the acetylation modification of K113, a series of mutants were created. The K-to-R mutation was employed to indicate the deacetylation and deubiquitylation status, while the K-to-Q mutation was utilized to represent the acetylation and deubiquitylation status. In our dataset, it was shown that neither the single mutation of K113 K-to-R nor K-to-Q exhibited any discernible drug resistance phenotype. This finding suggests that, within the physiological context of the Isw1 protein, both post-translational modifications (PTMs) of K113 had minimal or no impact on the regulation of drug resistance. The reason for this phenomenon is because the acetylation modification of K97 imitates the process of ubiquitination of Isw1, hence reducing the interaction between Isw1 and Cdc4, which is an E3 ligase. Hence, the ubiquitination of K113 does not play a crucial role in the regulation of Isw1 protein stability under conditions where K97 is completely acetylated. Nevertheless, upon deacetylation of K97, we observed a notable increase in the abundance of Isw1 protein when K113 is substituted with R. This finding strongly supports the notion that ubiquitination of K113 plays a crucial role in maintaining the stability of the Isw1 protein. Hence, in the case of K97 acetylation, the PTM modifications of K113 are not required for maintaining Isw1 protein levels. However, in the event of K97 deacetylation, the ubiquitination of K113 becomes crucial in regulating protein stability. Considering the intricate post-translational modification (PTM) regulation observed at the K113 site, it would be advantageous to generate antibodies specific to K113ac and K113ub in order to comprehensively investigate the functional role of K113 in the regulatory processes. Nevertheless, the presence of antibodies targeting site-specific ubiquitination is infrequent in scientific literature. We regret any confusion that may have arisen from the previous remark and have made revisions to the manuscript to address this issue. Please refer to line 485-500.

    3)The authors demonstrate that overexpression of ISW1 in select clinical isolates of Cryptococcus increases sensitivity to antifungals. However, these experiments would have benefited from additional controls, such as including overexpression of ISW1 in the wild-type strain (H99) and antifungal-sensitive isolate (CDLC120).

    In response to your concern, we successfully generated the strains as required. In the revised manuscript, we demonstrated that the overexpression of the stable variant of Isw1 in H99 and CDLC120 strains induces heightened susceptibility to antifungal drugs. Please see Figure 8e, 8i and line 404-413.

    Reviewer #3 (Public Review):

    1. ISWI chromatin remodellers are well-characterised in many organisms. How many ISWI proteins does Cryptococcus contain? Why did the authors focus on ISWI?

    We express our gratitude for this criticism. The identification of Isw1 was conducted as a further investigation building upon the findings presented in our previously published data (Li Y, 2019). In prior research, the acetylome in C. neoformans was comprehensively analyzed, and a series of knockout strains were created to investigate the relationship between fungal pathogenicity and acetylation. The Isw1 mutant has been discovered as a modifier of drug resistance. The identification of fungal paralogs of ISW genes was initially observed in Saccharomyces cerevisiae, a species of yeast that has experienced genome duplication. This process involves two paralogs, Isw1 and Isw2, which emerged as a result of the whole genome duplication event (Kellis M, 2004; Tsukiyama T, 1999; Wolfe KH, 1997). Because C. neoformans has not gone through the complete genome duplication event, its genome only encodes one copy of ISW gene. Please see line 129-134..

    1. What is the ISWI protein complex(es)? The Mass-Spec analysis should reveal this.

    Prior research conducted on Saccharomyces cerevisiae has provided evidence that the ISWI complex is comprised of several subunits, namely Isw1, Ioc genes, Itc1, Chd1, and Sua7 (Mellor J, 2004; Smolle M, 2012; Sugiyama and Nikawa, 2001; Vary JC Jr, 2003; Yadon AN, 2013). Upon a thorough examination of the C. neoformans genome, we have not been able to identifying a similar the IOC gene family. This absence likely suggests an evolutionary loss of the IOC gene family in C. neoformans, as suggested on the FungiDB website. However, C. neoformans has Itc1, Chd1, and Sua7. While we concur with the aforementioned statement on the capability of Mass-Spec data to elucidate potential protein-protein interactions and aid in the identification of subunits within the ISWI complex, it is important to acknowledge that the PTM Mass-Spec methodology is solely employed for the purpose of identifying potential sites of protein modification. In order to comprehensively investigate the cryptoccocal ISWI complex, we conducted a standardized Isw1-Flag protein immunoprecipitation procedure, followed by Mass-Spec analysis. In the present study, a total of 22 proteins that interact with Isw1 were found in our experimental data. Among these proteins, 11 have been previously reported to be associated with the regulatory networks including Isw1. In the mass spectrometry results, the protein Itc1 was found to be co-immunoprecipitated with the protein Isw1. Although the Mass-Spec analysis did not reveal the presence of Chd1 and Sua7, our study demonstrated that Chd1 can be coimmunoprecipitated with Isw1 through the utilization of co-IP and immunoblotting techniques. However, no interaction between Isw1 and Sua7 was shown utilizing any of these methods. In brief, cryptococcal ISWI regulatory machinery is distantly related to that from S. cerevisiae. Please see Figure 2 and line 206-219.

    1. Is Cryptococcus ISWI a transcriptional activator or repressor?

    We regret the erroneous representation of Isw1 in the prior iteration of the manuscript. The misclassification of Isw1 as a transcriptional regulator has been identified, since it has been determined to function as a chromatin remodeler instead. The text has been suitably revised in accordance with academic standards. In the revised publication, we have presented a comprehensive transcriptome analysis of the isw1 Δ strain under both FLC treatment and no treatment conditions. This analysis offers valuable insights into the gene regulatory patterns associated with Isw1. In our dataset, we observed that Isw1 exerts a negative regulatory effect on the expression of genes that encode drug pumps, while simultaneously exerting a positive regulatory effect on the expression of genes that are essential for 5-FC resistance. Moreover, the ChIP-PCR study demonstrated the binding of Isw1 to the promoter regions of genes of interest. Hence, the chromatin remodeler Isw1 has a dual role, wherein it both facilitates the activation of certain genes and suppresses the expression of others, in response to varying forms of drug resistance. Please see line 142-153.

    1. Is ISWI function in drug resistance linked to its chromatin remodelling activity?

    In order to investigate the potential role of Isw1 on chromatin activity in the modulation of multidrug resistance, we have conducted protein truncation experiments. Specifically, we deleted the DNA binding domain, the helicase domain, and the SNF2 domain, which have been previously shown to regulate Isw1 chromatin activity in the model organism S. cerevisiae (Grune T, 2003; Mellor J, 2004; Pinskaya M, 2009; Rowbotham SP, 2011). The new data demonstrated that all truncation variants of Isw1 mutants had a growth phenotype consistent with that of the deletional strain isw1Δ. In addition, the levels of gene expression observed in these strains were also similar to those observed in the deletion strain isw1Δ. This finding provides evidence that the regulation of the drug resistance mechanism is influenced by these critical domains involved in modifying chromatin activities. Moreover, the Isw1-Flag strain was utilized to conduct chromatin immunoprecipitation and PCR experiments, which revealed that Isw1 exhibits the ability to directly bind to the promoter regions of target genes. The new findings added evidence substantially supporting the hypothesis that the Isw1 chromatin activity plays a crucial role in modulating its protein function, and acting as a central regulator of drug resistance in C. neoformans. Please see revised Figure 1g, 1h, 1i and line 186-199 in the revised manuscript text.

    1. Does ISWI interact with chromatin? If so, which are ISWI-target genes? Does drug treatment modulate chromatin binding?

    To effectively tackle this concern, we have pursued two distinct approaches to demonstrate the chromatin regulatory effects of Isw1. In this study, the DNA binding domain was deliberately removed through genetic manipulation. The data presented indicates that the Isw1 mutants with shorter variations exhibited a growth phenotype that was characterized by multidrug resistance. This growth phenotype correlates with the growth phenotype obtained in the isw1Δ deletion strain. Additionally, it was observed that the levels of gene expression in the strain were comparable to those detected in the deletion strain isw1Δ. This discovery offers empirical support for the notion that the control of the drug resistance mechanism is indeed impacted by the DNA binding capability of Isw1. Furthermore, the Isw1-Flag strain was employed to perform chromatin immunoprecipitation and PCR assays, demonstrating the direct binding capacity of Isw1 to the promoter regions of target genes. The results obtained from this comprehensive analysis of the revised data offer significant evidence for the proposition that Isw1 interacts with chromatin and that its chromatin activity plays a pivotal role in modulating its protein function. This interaction serves as a central regulatory mechanism for drug resistance in C. neoformans. Furthermore, a transcriptome analysis was performed on both wildtype and isw1 deletion strains in the absence of FLC therapy. Upon comparing the results obtained from two unique experimental settings, specifically those with and without FLC administration, a notable disparity in the control of gene expression between these two situations was identified. In the context of the isw1 deletion strain exposed to FLC treatment, a set of 21 genes, including those belonging to the ABC/MFS family and efflux pumps, displayed significant changes in their gene expression patterns. In particular, a total of 9 genes exhibited downregulation, whilst 12 genes displayed upregulation. In contrast, in the absence of FLC supplementation, a total of 9 genes exhibited alterations in gene expression, with 3 genes showing downregulation and 6 genes showing upregulation. Therefore, the Isw1 protein plays a crucial role in the activation of certain genes, while simultaneously having a suppressive effect on other genes. Hence, the Isw1 undergoes a reconfiguration of its regulatory apparatus in response to drugs. Despite that the performance of ChIP-seq analysis was necessary in this study, it was observed that the treatment of fungal cells resulted in a notable decrease in the abundance of the Isw1 protein. This decrease can be attributed to the activation of Isw1 protein degradation. Consequently, there was an insufficient amount of Isw1 protein available for successful enrichment and subsequent ChIP-seq analysis (please see Figure 4a and 4c). However, the data collected collectively have demonstrated the idea that Isw1 serves as a crucial master regulator of drug resistance in C. neoformans. The text has undergone revisions in order to present our findings in a precise and thorough manner. Please see Figure 1c, 1g, Supplementary File 2, and line 145-153, 186-188.

  2. eLife

    eLife assessment

    This study links chromatin remodeling with antifungal drug resistance in Cryptococcus neoformans. The work is important because it reveals a new facet of how drug resistance can emerge and associates. The work presented is well done but the story is incomplete since there are questions about methods and association that need to be addressed. Establishing a link between chromatin remodeling and antifungal resistance is a finding that would be of interest to infectious disease researchers, cell biologists, and drug developers.

  3. eLife

    Reviewer #1 (Public Review):

    In this study, the authors found that the chromatin remodeling complex mutant isw1Δ of the fungal pathogen Cryptococcus neoformans is resistant to multiple different antifungal drugs. The mutant, however, is fully virulent in a mouse model. By comparing transcript changes of the wild type and the mutant when treated with antifungal fluconazole, they found that many transporter genes are differentially expressed in the isw1Δ mutant. Consistently, they showed reduced expression of genes involved metabolism of another antifungal 5-FC and a lower level of cellular accumulation of 5-FC in the isw1Δ mutant, which likely contributes to its 5-FC resistance. They found that the Isw1 protein is degraded mostly through ubiquitination and identified K97 deacetylation as being critical for drug resistance/protein degradation. Then they mutated nine E3 ubiquitination ligase genes and identified Cdc4 to be responsible for Isw1 degradation. Lastly, they showed that Isw1 is low in some clinical isolates that are modestly resistant to antifungals. The evidence of the interplay between acetylation status and ubiquitination of Isw1 is strong. The finding that reduced Isw1 increases drug resistance also fits the growing interest in studying epigenetic regulation of drug resistance in fungal pathogens. One area that needs to be strengthened is the potential clinical relevance of Isw1 reduction in drug resistance.

  4. eLife

    Reviewer #2 (Public Review):

    Cryptococcus neoformans is an important human pathogen, particularly in immunocompromised individuals. Like many fungal pathogens, resistance to antifungal drugs can emerge quickly in Cryptococcus. Understanding the mechanisms by which fungi develop resistance to antifungals will support new treatment strategies and, potentially, identify new drug targets. In this manuscript, Meng et al. describe a novel role for the conserved ATP-dependent chromatin remodeling factor, Imitation Switch (Isw1) in responding to antifungals in Cryptococcus. The authors first find that loss of Isw1 increases resistance to multiple antifungals and changes expression levels of genes potentially involved in antifungal resistance using functional genetics and cell growth assays. Next, the authors use mass spectrometry data (data generated in this study and public data) to identify ubiquitinated and acetylated sites of Isw1. The authors use this information to carry out an extensive series of western blot experiments using point mutations and chemical perturbations to dissect the contribution of specific modified sites of Isw1. Here, they identify important roles for the acetylation of K97 and ubiquitination of K113 and K441 in Isw1 stability. Lastly, the authors present evidence that clinical isolates of Cryptococcus that have increased antifungal resistance may have defects in Isw1 stability and that overexpressing ISW1 reduces antifungal resistance.

    Strengths:

    The authors present novel data that Isw1 is involved in responding to antifungals and that changes in Isw1 stability may lead to antifungal resistance. These results are of particular interest to the fungal pathogen research community and add to the general understanding of antifungal resistance.

    The authors present exciting data on post-translation modification (i.e., acetylation and ubiquitination) of Isw1, how those modifications contribute to Isw1 stability, and the regulatory interplay between modifications. Considering that Isw1 is broadly conserved across eukaryotes, these results are, potentially, of broad interest and raise questions outside of pathogen biology to be addressed in future research. For example, are the residues characterized in this study conserved in other Isw1 homologs, are they similarly modified, and is regulating the stability of Isw1 (or other chromatin remodeling factors) a general strategy for responding to external signals?

    Weaknesses:

    The authors demonstrate that Isw1 has a role in responding to antifungals in Cryptococcus. However, it is not clear if changes in Isw1 stability represent a general response to stress. This study would have benefited from experiments to test: (1) if levels of Isw1 change in response to other stressors (e.g., heat, osmotic, or oxidative stress) and (2) if loss of Isw1 impacts resistance to other stressors.

    The authors demonstrate a critical role in the acetylation of K97 and ubiquitination of K441 in regulating Isw1 stability. Additionally, this study shows that K113 is also likely involved in this process. However, it appears that K113 can be either acetylated or ubiquitinated, and it is, thus, less clear if one of the two modifications or both modifications is critical at this residue. Additional experiments may be required to answer this question. This study would have benefited from an additional discussion on the results related to the modification of K113.

    The authors demonstrate that overexpression of ISW1 in select clinical isolates of Cryptococcus increases sensitivity to antifungals. However, these experiments would have benefited from additional controls, such as including overexpression of ISW1 in the wild-type strain (H99) and antifungal-sensitive isolate (CDLC120).

  5. eLife

    Reviewer #3 (Public Review):

    This study focuses on the role of the chromatin remodeller ISWI in Cryptococcus. The authors show that a) ISWI modulates Cryptococcus' ability to grow in the presence of antifungal drugs and b) ISWI post-translational modifications (Acetylation and Ubiquitination) regulate ISWI protein stability. The observation that post-translational modifications regulate ISWI activity and stability is exciting and it could unveil novel mechanisms to rapidly and reversibly regulate the response to antifungal drug treatments. However, the study lacks a fundamental characterisation of ISWI. This information is essential to understand the mechanistic regulations of ISWI in Cryptococcus and how it mediates drug response. The following are questions that should be addressed:

    1. ISWI chromatin remodellers are well-characterised in many organisms. How many ISWI proteins does Cryptococcus contain? Why did the authors focus on ISWI?
    2. What is the ISWI protein complex(es)? The Mass-Spec analysis should reveal this.
    3. Is Cryptococcus ISWI a transcriptional activator or repressor?
    4. Is ISWI function in drug resistance linked to its chromatin remodelling activity?
    5. Does ISWI interact with chromatin? If so, which are ISWI-target genes? Does drug treatment modulate chromatin binding?

  6. Version published to 10.1101/2022.12.30.522307v1 on bioRxiv