PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes

  1. Abdoulie O Touray
  2. Rishi Rajesh
  3. Tony Isebe
  4. Tamara Sternlieb
  5. Mira Loock
  6. Oksana Kutova
  7. Igor Cestari

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Abstract

African trypanosomes evade host immune clearance by antigenic variation, causing persistent infections in humans and animals. These parasites express a homogeneous surface coat of variant surface glycoproteins (VSGs). They transcribe one out of hundreds of VSG genes at a time from telomeric expression sites (ESs) and periodically change the VSG expressed by transcriptional switching or recombination. The mechanisms underlying the control of VSG switching and its developmental silencing remain elusive. We report that telomeric ES activation and silencing entail an on/off genetic switch controlled by a nuclear phosphoinositide signaling system. This system includes a nuclear phosphatidylinositol 5-phosphatase (PIP5Pase), its substrate PI(3,4,5)P3, and the repressor-activator protein 1 (RAP1). RAP1 binds to ES sequences flanking VSG genes via its DNA binding domains and represses VSG transcription. In contrast, PI(3,4,5)P3 binds to the N-terminus of RAP1 and controls its DNA binding activity. Transient inactivation of PIP5Pase results in the accumulation of nuclear PI(3,4,5)P3, which binds RAP1 and displaces it from ESs, activating transcription of silent ESs and VSG switching. The system is also required for the developmental silencing of VSG genes. The data provides a mechanism controlling reversible telomere silencing essential for the periodic switching in VSG expression and its developmental regulation.

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  4. Version published to 10.7554/elife.89331.4 on eLife
  5. Version published to 10.7554/elife.89331 on eLife
  6. Version published to 10.7554/elife.89331.3 on eLife
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    Author Response

    The following is the authors’ response to the previous reviews

    Reviewer #1 (Public Review):

    Comments on the original submission:

    Trypanosoma brucei undergoes antigenic variation to evade the mammalian host's immune response. To achieve this, T. brucei regularly expresses different VSGs as its major surface antigen. VSG expression sites are exclusively subtelomeric, and VSG transcription by RNA polymerase I is strictly monoallelic. It has been shown that T. brucei RAP1, a telomeric protein, and the phosphoinositol pathway are essential for VSG monoallelic expression. In previous studies, Cestari et al. (ref. 24) has shown that PIP5pase interacts with RAP1 and that RAP1 binds PI(3,4,5)P3. RNAseq and ChIPseq analyses have been performed previously in PIP5pase conditional knockout cells, too (ref. 24). In the current study, Touray et al. did similar analyses except that catalytic dead PIP5pase mutant was used and the DNA and PI(3,4,5)P3 binding activities of RAP1 fragments were examined. Specifically, the authors examined the transcriptome profile and did RAP1 ChIPseq in PIP5pase catalytic dead mutant. The authors also expressed several C-terminal His6-tagged RAP1 recombinant proteins (full-length, aa1300, aa301-560, and aa 561-855). These fragments' DNA binding activities were examined by EMSA analysis and their phosphoinositides binding activities were examined by affinity pulldown of biotin-conjugated phosphoinositides. As a result, the authors confirmed that VSG silencing (both BES-linked and MES-linked VSGs) depends on PIP5pase catalytic activity, but the overall knowledge improvement is incremental. The most convincing data come from the phosphoinositide binding assay as it clearly shows that N-terminus of RAP1 binds PI(3,4,5)P3 but not PI(4,5)P2, although this is only assayed in vitro, while the in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al (ref. 24) already. Considering that many phosphoinositides exert their regulatory role by modulate the subcellular localization of their bound proteins, it is reasonable to hypothesize that binding to PI(3,4,5)P3 can remove RAP1 from the chromatin. However, no convincing data have been shown to support the author's hypothesis that this regulation is through an "allosteric switch".

    Comments on revised manuscript:

    In this revised manuscript, Touray et al. have responded to reviewers' comments with some revisions satisfactorily. However, the authors still haven't addressed some key scientific rigor issues, which are listed below:

    1. It is critical to clearly state whether the observations are made for the endogenous WT protein or the tagged protein. It is good that the authors currently clearly indicate the results observed in vivo are for the RAP1-HA protein. However, this is not as clearly stated for in vitro EMSA analyses. In addition, in discussion, the authors simply assumed that the c-terminally tagged RAP1 behaves the same as WT RAP1 and all conclusions were made about WT RAP1.

    There are two choices here. The authors can validate that RAP1-HA still retains RAP1's essential function as a sole allele in T. brucei cells (as was recommended previously). Indeed, HA-tagged RAP1 has been studied before, but it is the N-terminally HA-tagged RAP1 that has been shown to retain its essential functions. Adding the HA tag to the C-terminus of RAP1 may well cause certain defects to RAP1. For example, N-terminally HA-tagged TERT does not complement the telomere shortening phenotype in TERT null T. brucei cells, while C-terminally GFP-tagged TERT does, indicating that HA-TERT is not fully functional while TERT-GFP likely has its essential functions (Dreesen, RU thesis). Although RAP1-HA behaves similar to WT RAP1 in many ways, it is still not fully validated that this protein retains essential functions of RAP1. By the way, it has been published that cells lacking one allele of RAP1 behave as WT cells for cell growth and VSG silencing (Yang et al. 2009, Cell; Afrin et al. 2020, mSphere). In addition, although RAP1 may interact with TRF weakly, the interaction is direct, as shown in yeast 2-hybrid analysis in (Yang et al. 2009, Cell).

    Alternatively, if the authors do not wish to validate the functionality of RAP1-HA, they need to add one paragraph at the beginning of the discussion to clearly state that RAP1-HA may not behave exactly as WT RAP1. This is important for readers to better interpret the results. In addition, the authors need to tune down the current conclusions dramatically, as all described observations are made on RAP1-HA but not the WT RAP1.

    The results with RAP1-HA are consistent with previous knowledge of RAP1 interactions with telomeric proteins and DNA. Hence, the C-terminal HA-tagged RAP1 seems, by all measures, functional. Nevertheless, to make it clear for the reader, we added a note in the discussion, lines 244-246: “Although we showed that C-terminal HA-tagged RAP1 protein has telomeric localization (Cestari et al. 2015, PNAS) and interactions with other telomeric proteins (Cestari et al. 2019 Mol Cell Biol); we cannot rule out potential differences between HA-tagged and non tagged RAP1.”

    For a similar reason, the current EMSA results truly reflect how C-terminally His6-tagged RAP1 and RAP1 fragments behave. If the authors choose not to remove the His6 tag, it is essential that they use "RAP1-His6" to refer to these recombinant proteins. It is also essential for the authors to clearly state in the discussion that the tagged RAP1 fragments bind DNA, but the current data do not reveal whether WT RAP1 binds DNA. In addition, the authors incorrectly stated that "disruption of the MybL domain sequence did not eliminate RAP1-telomere binding in vivo" (lines 165-166). In ref 29, deletion of Myb domain did not abolish RAP1-telomere association. However, point mutations in MybL domain that abolish RAP1's DNA binding activities clearly disrupted RAP1's association with the telomere chromatin. Therefore, the current observation is not completely consistent with that published in ref 29.

    We stated in line 149-150 “…we expressed and purified from E. coli recombinant 6xHistagged T. brucei RAP1 (rRAP1)”. To clarify to the authors, we replaced rRAP1 with rRAP1-His throughout the manuscript and figures. As for the statement that “disruption of the MybL domain sequence did not eliminate RAP1-telomere binding in vivo" (lines 165-166).”. We removed the statement from the manuscript.

    1. There is no evidence, in vitro or in vivo, that binding PI(3,4,5)P3 to RAP1 causes conformational change in RAP1. The BRCT domain of RAP1 is known for its ability to homodimerize (Afrin et al. 2020, mSphere). It is therefore possible that binding of PI(3,4,5)P3 to RAP1 simply disrupts its homodimerization function. The authors clearly have extrapolated their conclusions based on available data. It is therefore important to revise the discussion and make appropriate statements.

    We did not state that PI(3,4,5)P3 causes RAP1 conformational changes. We discussed the possibility. We stated in lines 199-201: “PI(3,4,5)P3 inhibition of RAP1-DNA binding might be due to its association with RAP1 N-terminus causing conformational changes that affect Myb and MybL domains association with DNA.” This is a reasonable discussion, given the data presented in the manuscript.

    Reviewer #2 (Public Review):

    In this manuscript, Touray et al investigate the mechanisms by which PIP5Pase and RAP1 control VSG expression in T. brucei and demonstrate an important role for this enzyme in a signalling pathway that likely plays a role in antigenic variation in T. brucei. While these data do not definitively show a role for this pathway in antigenic variation, the data are critical for establishing this pathway as a potential way the parasite could control antigenic variation and thus represent a fundamental discovery.

    The methods used in the study are generally well-controlled. The authors provide evidence that RAP1 binds to PI(3,4,5)P3 through its N-terminus and that this binding regulates RAP1 binding to VSG expression sites, which in turn regulates VSG silencing. Overall their results support the conclusions made in the manuscript. Readers should take into consideration that the epitope tags on RAP1 could alter its function, however.

    There are a few small caveats that are worth noting. First, the analysis of VSG derepression and switching in Figure 1 relies on a genome which does not contain minichromosomal (MC) VSG sequences. This means that MC VSGs could theoretically be mis-assigned as coming from another genomic location in the absence of an MC reference. As the origin of the VSGs in these clones isn't a major point in the paper, I do not think this is a major concern, but I would not over-interpret the particular details of switching outcomes in these experiments.

    We agree with the reviewer and thus made no speculations on minichromosomes. The data analysis must rely on the available genome, and the reference genome used is well-assembled with PacBio sequences and Hi-C data (Muller et al. 2018, Nature).

    Another aspect of this work that is perhaps important, but not discussed much by the authors, is the fact that signalling is extremely poorly understood in T. brucei. In Figure 1B, the RNA-seq data show many genes upregulated after expression of the Mut PIP5Pase (not just VSGs). The authors rightly avoid claiming that this pathway is exclusive to VSGs, but I wonder if these data could provide insight into the other biological processes that might be controlled by this signaling pathway in T. brucei.

    We published that the inositol phosphate pathway also plays a role in T. brucei development (Cestari et al. 2018, Mol Biol Cell; reviewed by Cestari I 2020, PLOS Pathogens)

    Overall, this is an excellent study which represents an important step forward in understanding how antigenic variation is controlled in T. brucei. The possibility that this process could be controlled via a signalling pathway has been speculated for a long time, and this study provides the first mechanistic evidence for that possibility.

    Reviewer #1 (Recommendations For The Authors):

    Please see the public review for recommendations.1. It is critical to clearly state whether the observations are made for the endogenous WT protein or the tagged protein. It is good that the authors currently clearly indicate the results observed in vivo are for the RAP1-HA protein. However, this is not as clearly stated for in vitro EMSA analyses. In addition, in discussion, the authors simply assumed that the c-terminally tagged RAP1 behaves the same as WT RAP1 and all conclusions were made about WT RAP1.

    There are two choices here. The authors can validate that RAP1-HA still retains RAP1's essential function as a sole allele in T. brucei cells (as was recommended previously). Indeed, HA-tagged RAP1 has been studied before, but it is the N-terminally HA-tagged RAP1 that has been shown to retain its essential functions. Adding the HA tag to the C-terminus of RAP1 may well cause certain defects to RAP1. For example, N-terminally HA-tagged TERT does not complement the telomere shortening phenotype in TERT null T. brucei cells, while C-terminally GFP-tagged TERT does, indicating that HA-TERT is not fully functional while TERT-GFP likely has its essential functions (Dreesen, RU thesis). Although RAP1-HA behaves similar to WT RAP1 in many ways, it is still not fully validated that this protein retains essential functions of RAP1. By the way, it has been published that cells lacking one allele of RAP1 behaves as WT cells for cell growth and VSG silencing (Yang et al. 2009, Cell; Afrin et al. 2020, mSphere). In addition, although RAP1 may interact with TRF weakly, the interaction is direct, as shown in yeast 2-hybrid analysis in (Yang et al. 2009, Cell).

    Alternatively, if the authors do not wish to validate the functionality of RAP1-HA, they need to add one paragraph at the beginning of the discussion to clearly state that RAP1-HA may not behave exactly as WT RAP1. This is important for readers to better interpret the results. In addition, the authors need to tune down the current conclusions dramatically, as all described observations are made on RAP1-HA but not the WT RAP1.

    The results with RAP1-HA are consistent with previous knowledge of RAP1 interactions with telomeric proteins and DNA. Hence, the C-terminal HA-tagged RAP1 seems, by all measures, functional. Nevertheless, to make it clear for the reader, we added a note in the discussion, lines 244-246: “Although we showed that C-terminal HA-tagged RAP1 protein has telomeric localization (Cestari et al. 2015, PNAS) and interactions with other telomeric proteins (Cestari et al. 2019 Mol Cell Biol); we cannot rule out potential differences between HA-tagged and non tagged RAP1.”

    For a similar reason, the current EMSA results truly reflect how C-terminally His6-tagged RAP1 and RAP1 fragments behave. If the authors choose not to remove the His6 tag, it is essential that they use "RAP1-His6" to refer to these recombinant proteins. It is also essential for the authors to clearly state in the discussion that the tagged RAP1 fragments bind DNA, but the current data do not reveal whether WT RAP1 binds DNA. In addition, the authors incorrectly stated that "disruption of the MybL domain sequence did not eliminate RAP1-telomere binding in vivo" (lines 165-166). In ref 29, deletion of Myb domain did not abolish RAP1-telomere association. However, point mutations in MybL domain that abolish RAP1's DNA binding activities clearly disrupted RAP1's association with the telomere chromatin. Therefore, the current observation is not completely consistent with that published in ref 29.

    We stated in lines 149-150 “…we expressed and purified from E. coli recombinant 6xHistagged T. brucei RAP1 (rRAP1)”. To clarify to the authors, we replaced rRAP1 with rRAP1-His throughout the manuscript text. As for the statement that “disruption of the MybL domain sequence did not eliminate RAP1telomere binding in vivo" (lines 165-166).”. We removed the statement from the manuscript.

    1. There is no evidence, in vitro or in vivo, that binding PI(3,4,5)P3 to RAP1 causes conformational change in RAP1. The BRCT domain of RAP1 is known for its ability to homodimerize (Afrin et al. 2020, mSphere). It is therefore possible that binding of PI(3,4,5)P3 to RAP1 simply disrupts its homodimerization function. The authors clearly have extrapolated their conclusions based on available data. It is therefore important to revise the discussion and make appropriate statements.

    We did not state that PI(3,4,5)P3 causes RAP1 conformational changes. We discussed the possibility. We stated in lines 199-201: “PI(3,4,5)P3 inhibition of RAP1-DNA binding might be due to its association with RAP1 N-terminus causing conformational changes that affect Myb and MybL domains association with DNA.” This is a reasonable discussion, given the data presented in the manuscript.

  8. eLife

    eLife assessment

    Trypanosoma brucei evades mammalian humoral immunity through the expression of different variant surface glycoprotein genes. In this fundamental paper, the authors extend previous observations that TbRAP1 both interacts with PIP5Pase and binds PI(3,4,5)P3, indicating a role for PI(3,4,5)P3 binding. They therefore suggest that antigen switching might have a signal-dependent component. The evidence is mostly compelling, but with some caveats because tagged proteins were used.

  9. eLife

    Reviewer #1 (Public Review):

    Trypanosoma brucei undergoes antigenic variation to evade the mammalian host's immune response. To achieve this, T. brucei regularly expresses different VSGs as its major surface antigen. VSG expression sites are exclusively subtelomeric, and VSG transcription by RNA polymerase I is strictly monoallelic. It has been shown that T. brucei RAP1, a telomeric protein, and the phosphoinositol pathway are essential for VSG monoallelic expression. In previous studies, Cestari et al. (ref. 24) has shown that PIP5pase interacts with RAP1 and that RAP1 binds PI(3,4,5)P3. RNAseq and ChIPseq analyses have been performed previously in PIP5pase conditional knockout cells, too (ref. 24). In the current study, Touray et al. did similar analyses except that catalytic dead PIP5pase mutant was used and the DNA and PI(3,4,5)P3 binding activities of RAP1 fragments were examined. Specifically, the authors examined the transcriptome profile and did RAP1 ChIPseq in PIP5pase catalytic dead mutant. The authors also expressed several C-terminal His6-tagged RAP1 recombinant proteins (full-length, aa1-300, aa301-560, and aa 561-855). These fragments' DNA binding activities were examined by EMSA analysis and their phosphoinositides binding activities were examined by affinity pulldown of biotin-conjugated phosphoinositides. As a result, the authors confirmed that VSG silencing (both BES-linked and MES-linked VSGs) depends on PIP5pase catalytic activity, but the overall knowledge improvement is incremental. The most convincing data come from the phosphoinositide binding assay as it clearly shows that N-terminus of RAP1 binds PI(3,4,5)P3 but not PI(4,5)P2, although this is only assayed in vitro, while the in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al (ref. 24) already. Considering that many phosphoinositides exert their regulatory role by modulating the subcellular localization of their bound proteins, it is reasonable to hypothesize that binding to PI(3,4,5)P3 can remove RAP1 from the chromatin.

  10. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Touray et al investigate the mechanisms by which PIP5Pase and RAP1 control VSG expression in T. brucei and demonstrate an important role for this enzyme in a signalling pathway that likely plays a role in antigenic variation in T. brucei. While these data do not definitively show a role for this pathway in antigenic variation, the data are critical for establishing this pathway as a potential way the parasite could control antigenic variation and thus represent a fundamental discovery.

    The methods used in the study are generally well-controlled. The authors provide evidence that RAP1 binds to PI(3,4,5)P3 through its N-terminus and that this binding regulates RAP1 binding to VSG expression sites, which in turn regulates VSG silencing. Overall their results support the conclusions made in the manuscript. Readers should take into consideration that the epitope tags on RAP1 could alter its function, however.

    There are a few small caveats that are worth noting. First, the analysis of VSG derepression and switching in Figure 1 relies on a genome which does not contain minichromosomal (MC) VSG sequences. This means that MC VSGs could theoretically be mis-assigned as coming from another genomic location in the absence of an MC reference. As the origin of the VSGs in these clones isn't a major point in the paper, I do not think this is a major concern, but I would not over-interpret the particular details of switching outcomes in these experiments.

    Another aspect of this work that is perhaps important, but not discussed much by the authors, is the fact that signalling is extremely poorly understood in T. brucei. In Figure 1B, the RNA-seq data show many genes upregulated after expression of the Mut PIP5Pase (not just VSGs). The authors rightly avoid claiming that this pathway is exclusive to VSGs, but I wonder if these data could provide insight into the other biological processes that might be controlled by this signaling pathway in T. brucei.

    Overall, this is an excellent study which represents an important step forward in understanding how antigenic variation is controlled in T. brucei. The possibility that this process could be controlled via a signalling pathway has been speculated for a long time, and this study provides the first mechanistic evidence for that possibility.

  11. Version published to 10.1101/2023.05.11.540368v3 on bioRxiv
  12. Version published to 10.7554/elife.89331.2 on eLife
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    Author Response

    The following is the authors’ response to the original reviews.

    Reviewer #1 (Recommendations For The Authors):

    1. The authors need to validate that RAP1-HA still retains its essential function. As indicated above, if RAP1-HA still retains its essential functions, cells carrying one RAP1-HA allele and one deleted allele are expected to grow the same as WT cells. These cells should also have the WT VSG expression pattern, and RAP1-HA should still interact with TRF.

    We demonstrated that C-terminally HA-tagged RAP1 co-localizes with telomeres by a combination of immunofluorescence and fluorescence in situ hybridization (Cestari and Stuart, 2015, PNAS), and co-immunoprecipitate telomeric and 70 bp repeats (Cestari et al. 2019 Mol Cell Biol). We also showed by immunoprecipitation and mass spectrometry that HA-tagged RAP1 interacts with nuclear and telomeric proteins, including PIP5Pase (Cestari et al. 2019). Others have also tagged T. brucei RAP1 with HA without disrupting its nuclear localization (Yang et al. 2009, Cell), all of which indicate that the HA-tag does not affect protein function. As for the suggested experiment, there is no guarantee that cells lacking one allele of RAP1 will behave as wildtype, i.e., normal growth and repression of VSGs genes. Also, less than 90% of T. brucei TRF was reported to interact with RAP1 (Yang et al. 2009, Cell), which might be indirect via their binding to telomeric repeats rather than direct protein-protein interactions.

    1. The authors need to remove the His6 tag from the recombinant RAP1 fragments before the EMSA analysis. This is essential to avoid any artifacts generated by the His6-tagged proteins.

    Our controls show that the His-tag is not interfering with RAP1-DNA binding. We show in Fig 3CG by EMSA and in Fig S5 by EMSA and microscale thermophoresis that His-tagged full-length rRAP1 does not bind to scrambled telomeric dsDNA sequences, which demonstrates that His-tagged rRAP1 does not bind unspecifically to DNA. Moreover, in Fig 3G and Fig S5, we show that His-tagged rRAP11-300 also does not bind to 70 bp or telomeric repeats. In contrast, the full-length His-tagged rRAP1, rRAP1301-560, or rRAP1561-855 bind to 70 bp or telomeric repeats (Fig 3C-G). Since all proteins were His-tagged, the His tag cannot be responsible for the DNA binding. We have worked with many different His-tagged proteins for nucleic acid binding and enzymatic assays without any interference from the tag (Cestari and Stuart, 2013; JBC; Cestari et al; 2013, Mol Cell Biol; Cestari and Stuart, 2015, PNAS; Cestari et al. 2016; Cell Chem Biol; Cestari et al. 2019 Mol Biol Cell).

    1. More details need to be provided for ChIPseq and RNAseq analysis regarding the read numbers per sample, mapping quality, etc.

    Table S3 includes information on sequencing throughput and read length. Mapping quality was included in the Methods section “Computational analysis of RNA-seq and ChIP-seq”, starting at line 499. In summary, we filtered reads to keep primary alignment (eliminate supplementary and secondary alignments). We also analyzed ChIP-seq with MAPQ ≥20 (99% probability of correct alignment) to distinguish RAP1 binding to specific ESs, including silent vs active ES (ChIP-seq). We included Fig S4 to show the effect of filtering alignments on the active vs silent ESs. We used MAPQ ≥30 to analyze RNA-seq mapping to VSG genes, including those in subtelomeric regions. Our scripts are available at https://github.com/cestari-lab/lab_scripts. We also included in the Methods, lines 522-524: “Scripts used for ChIP-seq, RNA-seq, and VSG-seq analysis are available at https://github.com/cestari-lab/lab_scripts. A specific pipeline was developed for clonal VSG-seq analysis, available at https://github.com/cestarilab/VSG-Bar-seq.”

    1. The authors should revise the Discussion section to clearly state the authors' speculations and their working models (the latter of which need solid supporting evidence). Specifically, statements in lines 218 - 219 and lines 224-226 need to be revised.

    The statement “likely due to RAP1 conformational changes” in line 228 discusses how binding of PI(3,4,5)3 could affect RAP1 Myb and MybL domains binding to DNA. We did not make a strong statement but discussed a possibility. We believe that it is beneficial to the reader to have the data discussed, and we do not feel this point is overly speculative. For lines 224-226 (now 234-235), the statement refers to the finding of RAP1 binding to centromeric regions by ChIP-seq, which is a new finding but not the focus of this work. To make it clear that it does not refer to telomeric ESs, we edited: “The finding of RAP1 binding to subtelomeric regions other than ESs, including centromeres, requires further validation.” Since RAP1 binding to centromeres is not the focus of the work, future studies are necessary to follow up, and we believe it is appropriate in the Discussion to be upfront and highlight this point to the readers.

    Our model is based on the data presented here but also on scientific literature. We have reviewed the Discussion to prevent broad speculations. When discussing a model, we stated (line 245): “The scenario suggests a model in which …”, to state that this is a working model. Similarly, in Results (line 201) we included: “Our data suggest a model in which…”.

    1. The authors should revise the title to reflect a more reasonable conclusion of the study.

    We agree that the title should be changed to imply a direct role of PI(3,4,5)P3 regulation of RAP1, which is not captured in the original title. This will provide more specific information to the readers, especially those broadly interested in telomeric gene regulation and RAP1. The new title is: PI(3,4,5)P3 allosteric regulation of repressor activator protein 1 controls antigenic variation in trypanosomes

    1. The authors are recommended to provide an estimation of the expression level of the V5-tagged PIP5pase from the tubulin array in reference to the endogenous protein level.

    The relative mRNA levels of the exclusive expression of PIP5Pase mutant compared to the wildtype is available in the Data S1, RNA-seq. The Mut PIP5Pase allele’s relative expression level is 0.85fold to the WT allele (both from tubulin loci). We also showed by Western blot the WT and Mut PIP5Pase protein expression (Cestari et al. 2019, Mol Cell Biol). Concerning PIP5Pase endogenous alleles, we compared normalized RNA-seq counts per million from the conditional null PIP5Pase cells exclusively expressing WT or the Mut PIP5Pase alleles (Data S1, this work) to our previous RNA-seq of single-marker 427 strain (Cestari et al. 2019, Mol Cell Biol). We used the single-maker 427 because the conditional null cells were generated in this strain background. The PIP5Pase WT and Mut mRNAs expressed from tubulin loci are 1.6 and 1.3-fold the endogenous PIP5Pase levels in single-marker 427, respectively. We included a statement in the Methods, lines 275-278: “The WT or Mut PIP5Pase mRNAs exclusively expressed from tubulin loci are 1.6 and 1.3-fold the WT PIP5Pase mRNA levels expressed from endogenous alleles in the single marker 427 strain. The fold-changes were calculated from RNA-seq counts per million from this work (WT and Mut PIP5Pase, Data S1) and our previous RNA-seq from single marker 427 strain (24).”

    1. The authors are recommended to provide more detailed EMSA conditions such as protein and substrate concentrations. Better quality EMSA gels are preferred.

    All concentrations were already provided in the Methods section. See line 356, in topic Electrophoretic mobility shift assays: “100 nM of annealed DNA were mixed with 1 μg of recombinant protein…”. For microscale thermophoresis, also see lines 375-376 in topic Microscale thermophoresis binding kinetics: “1 μM rRAP1 was diluted in 16 two-fold serial dilutions in 250 mM HEPES pH 7.4, 25 mM MgCl2, 500 mM NaCl, and 0.25% (v/v) N P-40 and incubated with 20 nM telomeric or 70 bp repeats…”. Note that two different biochemical approaches, EMSA and microscale thermophoresis, were used to assess rRAP1-His binding to DNA. Both show agreeable results (Fig 3 and 5, and Fig S5. Microscale thermophoresis shows the binding kinetics, data available in Table 1). The EMSA images clearly show the binding of RAP1 to 70 bp or telomeric repeats but not to scramble telomeric repeat DNA.

    Reviewer #2 (Recommendations For The Authors):

    Major comments:

    Figures

    All figures should have their axes properly labeled and units should be indicated. For many of the ChIPseq datasets it is not clear whether the authors show a fold enrichment or RPM and whether they used all reads or only uniquely mapping reads. Especially the latter is a very important piece of information when analyzing expression sites and should always be reported. The authors write, that all RNA-seq and ChIP-seq experiments were performed in triplicate. What is shown in the figures, one of the replicates? Or the average?

    ChIP-seq is shown as fold enrichment; we clarified this in the figures by including in the y-axis RAP1-HA ChIP/Input (log 2). We included in figure legends, see line 710: “Data show fold-change comparing ChIP vs Input.”. For quantitative graphs (Fig 2B, D, and E, and Fig 5F and G), data are shown as the mean of biological replicates. Graphs generated in the integrated genome viewer (IGV, qualitative graphs) is a representative data (Fig 2A, C, and F, and Fig 5D-E). All statistical analyses were calculated from the three biological replicates. Uniquely mapped reads were used. We also included ChIP-seq analysis with MAPQ ≥10 and 20 (90% and 99% probability of correct alignment, respectively) to distinguish RAP1 binding to ESs. Fig S4 shows the various mapping stringency and demonstrates the enrichment of RAP1-HA to silent vs active ES.

    Figure 1 is very important for the main argument of the manuscript, but very difficult (impossible for me) to fully understand. It would be great if the author could make an effort to clarify the figure and improve the labels. Panel Fig 1E. Here it is impossible to read the names of the genes that are activated and therefore it is impossible to verify the statements made about the activation of VSGs and the switching.

    We have edited Fig 1E to include the most abundant VSGs, which decreased the amount of information in the graph and increased the label font. We also re-labeled each VSG with chromosome or ES name and common VSG name when known (e.g., VSG2). We included Table S1 in the supplementary information with the data used to generate Fig 1E. In Table S1, the reader will be able to check the VSG gene IDs and evaluate the data in detail. We included in the legend, line 700: “See Table S1 for data and gene IDs of VSGs.”

    Figure 1F: This panel is important and should be shown in more detail as it distinguishes VSG switching from a general VSG de-repression phenotype. VSG-seq is performed in a clonal manner here after PIP5Pase KD and re-expression. To show that proper switching has occurred place in the different clones, instead of a persistent VSG de-repression, the expression level of more VSGs should be shown (e.g. as in panel E) to show that there is really only one VSG detected per clone. For example, it is not clear what the authors 'called' the dominant VSG gene.

    We showed in supplementary information Fig S1 B-C examples of reads mapping to the VSGs. Now we included a graph (Fig S1 D) that quantifies reads mapped to the VSG selected as expressed compared to other VSG genes considered not expressed). The data show an average of several clones analyzed. Other VSGs (not selected) are at the noise level (about 4 normalized counts) compared to >250 normalized counts to the selected as expressed VSGs.

    As mentioned in the public comments, I don't see how the data from Fig 1E and 1F fit together. Based on Fig 1E VSG2 is the dominant VSG, based on Fig 1F VSG2 is almost never the dominant VSG, but the VSG from BES 12.

    In Fig 1E, the VSG2 predominates in cells expressing WT PIP5Pase, however, in cells expressing Mut PIP5Pase, this is not the case anymore. Many other VSGs are detected, and other VSG mRNAs are more abundant than VSG2 (see color intensity in the heat map). The Mut cells may also have remaining VSG2 mRNAs (from before switching) rather than continuous VSG2 expression. This is the reason we performed the clonal analysis shown in Fig 1F, to be certain about the switching. While Fig 1F shows potential switchers in the population, Fig 1E confirms VSG switching in clones.

    Many potential switchers were detected in the VSG-seq (Fig 1F, the whole cell population is over 107 parasites), but not all potential switchers were detected in the clonal analysis because we analyzed 212 clones total, a fraction of the over 107 cells analyzed by VSG-seq (Fig 1E). Also, it is possible that not all potential switchers are viable. A preference for switching to specific ESs has been observed in T. brucei (Morrison et al. 2005, Int J Parasitol; Cestari and Stuart, 2015, PNAS), which may explain several clones switching to BES12.

    Note that in Fig 1F, tet + cells did not switch VSGs at all; all 118 clones expressed VSG2. We relabeled Fig 1F for clarity and included the VSG names. We added gene IDs in the Figure legends, see line 702 “ BES1_VSG2 (Tb427_000016000), BES12_VSG (Tb427_000008000)…”

    Statements in Introduction / Discussion

    The statement in lines 82/83 is very strong and gives the impression that the PIP5Pase-Rap1 circuit has been proven to regulate antigenic variation in the host. However, I don't think this is the case. The paper shows that the pathway can indeed turn expression sites on and off, but there is no evidence (yet) that this is what happens in the host and regulates antigenic variation during infection. The same goes for lines 214/215 in the discussion.

    We agree with the reviewer, and we edited these statements. The statement lines 82-83: “The data provide a molecular mechanism…” to “The data indicates a molecular mechanism…” For lines 224225: “and provides a mechanism to control…” to “and indicates a mechanism to control…”. We also included in lines 261-262: “It is unknown if a signaling system regulates antigenic variation in vivo.” Also edited lines 262-263: “…the data indicate that trypanosomes may have evolved a sophisticated mechanism to regulate antigenic variation...”.

    New vs old data

    In general, for Figures 1 - 4, it was a bit difficult to understand which panels showed new findings, and which panels confirmed previous findings (see below for specific examples). In the text and in the figure design, the new results should be clearly highlighted. Authors: All data presented is new, detailed below.

    Figure 1: A similar RNA-seq after PIP5Pase deletion was performed in citation 24. Perhaps the focus of this figure should be more on the (clone-specific) VSG-seq experiment after PIP5Pase re-introduction.

    This is the first time we show RNA-seq of T. brucei expressing catalytic inactive PIP5Pase, which establishes that the regulation of VSG expression and switching, and repression of subtelomeric regions, is dependent on PIP5Pase enzyme catalysis, i.e., PI(3,4,5)P3 dephosphorylation. Hence, the relevance and difference of the RNA-seq here vs the previous RNA-seq of PIP5Pase knockdown.

    Figure 2: A similar ChIP-seq of RAP1 was performed in citation 24, with and without PIP5Pase deletion. Could new findings be highlighted more clearly?

    Our and others’ previous work showed ChIP-qPCR, which analyses specific loci. Here we performed ChIP-seq, which shows genome-wide binding sites of RAP1, and new findings are shown here, including binding sites in the BES, MESs, and other genome loci such as centromeres. We also identified DNA sequence bias defining RAP1 binding sites (Fig 2A). We also show by ChIP-seq how RAP1-binding to these loci changes upon expression of catalytic inactive PIP5Pase. To improve clarity in the manuscript, we edited lines 129-130: “We showed that RAP1 binds telomeric or 70 bp repeats (24), but it is unknown if it binds to other ES sequences or genomic loci.”

    Figure 4: Binding of Rap1 to PI(3,4,5)P3, but not to other similar molecules, was previously shown in citation 24. Could new findings be highlighted more clearly?

    We published in reference 24 (Cestari et al. Mol Cell Biol) that RAP1-HA can bind agarose beadsconjugated synthetic PI(3,4,5)P3. Here, we were able to measure T. brucei endogenous PI(3,4,5)P3 associated with RAP1-HA (Fig 4F). Moreover, we showed that the endogenous RAP1-HA and PI(3,4,5)P3 binding is about 100-fold higher when PIP5Pase is catalytic inactive than WT PIP5Pase. The data establish that in vivo endogenous PI(3,4,5)P3 binds to RAP1-HA and how the binding changes in cells expressing mutant PIP5Pase; this data is new and relevant to our conclusions. To clarify, we edited the manuscript in lines 180-182: “To determine if RAP1 binds to PI(3,4,5)P3 in vivo, we in-situ HA-tagged RAP1 in cells that express the WT or Mut PIP5Pase and analyzed endogenous PI(3,4,5)P3 levels associated with immunoprecipitated RAP1-HA”.

    Sequencing.
    I really appreciate the amount of detail the authors provide in the methods section. The authors do an excellent job of describing how different experiments were performed. However, it would be important that the authors also provide the basic statistics on the sequencing data. How many sequencing reads were generated per run (each replicate of the ChIP-seq and RNA-seq assays)? How long were the reads? How many reads could be aligned?

    The sequencing metrics for RNA-seq and ChIP-seq for all biological replicates were included in Table S3 (supplementary information). The details of the analysis and sequencing quality were described in the Methods section “Computational analysis of RNA-seq and ChIP-seq”. To be clearer about the analysis, we also included in Methods, lines 522-524: “Scripts used for ChIP-seq, RNA-seq, and VSG-seq analysis are available at https://github.com/cestari-lab/lab_scripts. A specific pipeline was developed for clonal VSG-seq analysis, available at https://github.com/cestari-lab/VSG-Bar-seq.”.

    Minor comments:

    Figure 1B: I would recommend highlighting the non-ES VSGs and housekeeping genes with two more colors in the volcano plot, to show that it is mostly the antigen repertoire that is deregulated, and not the Pol ll transcribed housekeeping genes. This is not entirely clear from the panel as it is right now.

    The suggestion was incorporated in Fig 1B. We color-coded the figure to include BES VSGs, MES VSGs, ESAGs, subtelomeric genes, core genes (typically Pol II and Pol III transcribed genes), and Unitig genes, those genes not assembled in the 427-2018 reference genome.

    Were the reads in Figure 2a filtered in the same way as those in Figure 2C? To support the statements, only unique reads should be used.

    Yes, we also added Fig S4 to make more clear the comparison between read mapping to silent vs active ES.

    It would be good if the authors could add a supplementary figure showing the RAP1 ChIP-seq (WT and cells lacking a functional PIP5Pase) for all silent expression sites.

    We had RAP1 ChIP-seq from cells expressing WT PIP5Pase already. We have it modified to include data from the Mutant PIP5Pase. See Fig S3 and S5.

    In Figure 5D, after depletion of PIP5Pase, RAP1 binding appears to decrease across ESAGs, but ESAG expression appears to increase. How can this be explained with the model of RAP1 repressing transcription?

    We included in the Results, lines 208-212: “The increased level of VSG and ESAG mRNAs detected in cells expressing Mut PIP5Pase (Fig 5D) may reflect increased Pol I transcription. It is possible that the low levels of RAP1-HA at the 50 bp repeats affect Pol I accessibility to the BES promoter; alternatively, RAP1 association to telomeric or 70 bp repeats may affect chromatin compaction or folding impairing VSG and ESAG genes transcription.”.

    Reviewer #3 (Recommendations For The Authors):

    Line 114 - typo? Procyclic instead of procyclics:

    Fixed, thanks.

    Line 233 - the phrasing here is confusing, may want to replace "whose" with "which" (if I am interpreting correctly):

    Thanks, no changes were needed. I have had the sentence reviewed by a Ph.D.-level scientific writer.

    Methods - there is no description of VSG-seq analysis in the methods. Is it done the same way as the RNA-seq analysis? Is the code for analysis/generating figures available online?

    The procedure is similar. We included an explanation in Methods, lines 503-504: “RNA-seq and VSG-seq (including clonal VSG-seq) mapped reads were quantified…”. Also, in lines 522-54: “Scripts used for ChIP-seq, RNA-seq, and VSG-seq analysis are available at https://github.com/cestari-lab/lab_scripts. A specific pipeline was developed for clonal VSG-seq analysis, available at https://github.com/cestarilab/VSG-Bar-seq.”.

    Fig 1H - Is this from RNA-seq or VSG-seq analysis of procyclics?

    The procyclic forms VSG expression analysis was done by real-time PCR. To clarify it, we included it in the legend “Expression analysis of ES VSG genes after knockdown of PIP5Pase in procyclic forms by real-time PCR”. We also amended the Methods, under the topic RNA-seq and real-time PCR, line 402-407: “For procyclic forms, total RNAs were extracted from 5.0x108 T. brucei CN PIP5Pase growing in Tet + (0.5 µg/mL, no knockdown) or Tet – (knockdown) at 5h, 11h, 24h, 48h, and 72h using TRIzol (Thermo Fisher Scientific) according to manufacturer's instructions. The isolated mRNA samples were used to synthesize cDNA using ProtoScript II Reverse Transcriptase (New England Biolabs) according to the manufacturer's instructions. Real-time PCRs were performed using VSG primers as previously described (23).”

    Fig 2 A - Where it says "downstream VSG genes" I assume "downstream of VSG genes" is meant? the regions described in this figure might be more clearly laid out in the text or the legend

    Fixed, thanks. We included in the text in Results, line 140: “… and Ts and G/Ts rich sequences downstream of VSG genes”.

    Fig 2E - what does "Flanking VSGs" mean in this context?

    We added to line 705, figure legends: “Flanking VSGs, DNA sequences upstream or downstream of VSG genes in MESs. “

    Fig 2H - Why is the PIP5Pase Mutant excluded from the Chr_1 core visualization?

    We did not notice it. We included it now; thanks.

  14. eLife

    eLife assessment

    Trypanosoma brucei evades mammalian humoral immunity through the expression of different variant surface glycoprotein genes. In this fundamental paper, the authors extend previous observations that TbRAP1 both interacts with PIP5Pase and binds PI(3,4,5)P3, indicating a role for PI(3,4,5)P3 binding and suggesting that antigen switching is signal dependent. While much of the evidence is compelling, the work would benefit from further controls to rule out that any of the observed effects come from the protein tags used.

  15. eLife

    Reviewer #1 (Public Review):

    Comments on the original submission:

    Trypanosoma brucei undergoes antigenic variation to evade the mammalian host's immune response. To achieve this, T. brucei regularly expresses different VSGs as its major surface antigen. VSG expression sites are exclusively subtelomeric, and VSG transcription by RNA polymerase I is strictly monoallelic. It has been shown that T. brucei RAP1, a telomeric protein, and the phosphoinositol pathway are essential for VSG monoallelic expression. In previous studies, Cestari et al. (ref. 24) has shown that PIP5pase interacts with RAP1 and that RAP1 binds PI(3,4,5)P3. RNAseq and ChIPseq analyses have been performed previously in PIP5pase conditional knockout cells, too (ref. 24). In the current study, Touray et al. did similar analyses except that catalytic dead PIP5pase mutant was used and the DNA and PI(3,4,5)P3 binding activities of RAP1 fragments were examined. Specifically, the authors examined the transcriptome profile and did RAP1 ChIPseq in PIP5pase catalytic dead mutant. The authors also expressed several C-terminal His6-tagged RAP1 recombinant proteins (full-length, aa1-300, aa301-560, and aa 561-855). These fragments' DNA binding activities were examined by EMSA analysis and their phosphoinositides binding activities were examined by affinity pulldown of biotin-conjugated phosphoinositides. As a result, the authors confirmed that VSG silencing (both BES-linked and MES-linked VSGs) depends on PIP5pase catalytic activity, but the overall knowledge improvement is incremental. The most convincing data come from the phosphoinositide binding assay as it clearly shows that N-terminus of RAP1 binds PI(3,4,5)P3 but not PI(4,5)P2, although this is only assayed in vitro, while the in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al (ref. 24) already. Considering that many phosphoinositides exert their regulatory role by modulate the subcellular localization of their bound proteins, it is reasonable to hypothesize that binding to PI(3,4,5)P3 can remove RAP1 from the chromatin. However, no convincing data have been shown to support the author's hypothesis that this regulation is through an "allosteric switch".

    Comments on revised manuscript:

    In this revised manuscript, Touray et al. have responded to reviewers' comments with some revisions satisfactorily. However, the authors still haven't addressed some key scientific rigor issues, which are listed below:

    1. It is critical to clearly state whether the observations are made for the endogenous WT protein or the tagged protein. It is good that the authors currently clearly indicate the results observed in vivo are for the RAP1-HA protein. However, this is not as clearly stated for in vitro EMSA analyses. In addition, in discussion, the authors simply assumed that the c-terminally tagged RAP1 behaves the same as WT RAP1 and all conclusions were made about WT RAP1.

    There are two choices here. The authors can validate that RAP1-HA still retains RAP1's essential function as a sole allele in T. brucei cells (as was recommended previously). Indeed, HA-tagged RAP1 has been studied before, but it is the N-terminally HA-tagged RAP1 that has been shown to retain its essential functions. Adding the HA tag to the C-terminus of RAP1 may well cause certain defects to RAP1. For example, N-terminally HA-tagged TERT does not complement the telomere shortening phenotype in TERT null T. brucei cells, while C-terminally GFP-tagged TERT does, indicating that HA-TERT is not fully functional while TERT-GFP likely has its essential functions (Dreesen, RU thesis). Although RAP1-HA behaves similar to WT RAP1 in many ways, it is still not fully validated that this protein retains essential functions of RAP1. By the way, it has been published that cells lacking one allele of RAP1 behave as WT cells for cell growth and VSG silencing (Yang et al. 2009, Cell; Afrin et al. 2020, mSphere). In addition, although RAP1 may interact with TRF weakly, the interaction is direct, as shown in yeast 2-hybrid analysis in (Yang et al. 2009, Cell).

    Alternatively, if the authors do not wish to validate the functionality of RAP1-HA, they need to add one paragraph at the beginning of the discussion to clearly state that RAP1-HA may not behave exactly as WT RAP1. This is important for readers to better interpret the results. In addition, the authors need to tune down the current conclusions dramatically, as all described observations are made on RAP1-HA but not the WT RAP1.

    For a similar reason, the current EMSA results truly reflect how C-terminally His6-tagged RAP1 and RAP1 fragments behave. If the authors choose not to remove the His6 tag, it is essential that they use "RAP1-His6" to refer to these recombinant proteins. It is also essential for the authors to clearly state in the discussion that the tagged RAP1 fragments bind DNA, but the current data do not reveal whether WT RAP1 binds DNA. In addition, the authors incorrectly stated that "disruption of the MybL domain sequence did not eliminate RAP1-telomere binding in vivo" (lines 165-166). In ref 29, deletion of Myb domain did not abolish RAP1-telomere association. However, point mutations in MybL domain that abolish RAP1's DNA binding activities clearly disrupted RAP1's association with the telomere chromatin. Therefore, the current observation is not completely consistent with that published in ref 29.

    2. There is no evidence, in vitro or in vivo, that binding PI(3,4,5)P3 to RAP1 causes conformational change in RAP1. The BRCT domain of RAP1 is known for its ability to homodimerize (Afrin et al. 2020, mSphere). It is therefore possible that binding of PI(3,4,5)P3 to RAP1 simply disrupts its homodimerization function. The authors clearly have extrapolated their conclusions based on available data. It is therefore important to revise the discussion and make appropriate statements.

  16. eLife

    Reviewer #2 (Public Review):

    In this manuscript, Touray et al investigate the mechanisms by which PIP5Pase and RAP1 control VSG expression in T. brucei and demonstrate an important role for this enzyme in a signalling pathway that likely plays a role in antigenic variation in T. brucei. While these data do not definitively show a role for this pathway in antigenic variation, the data are critical for establishing this pathway as a potential way the parasite could control antigenic variation and thus represent a fundamental discovery.

    The methods used in the study are generally well-controlled. The authors provide evidence that RAP1 binds to PI(3,4,5)P3 through its N-terminus and that this binding regulates RAP1 binding to VSG expression sites, which in turn regulates VSG silencing. Overall their results support the conclusions made in the manuscript. Readers should take into consideration that the epitope tags on RAP1 could alter its function, however.

    There are a few small caveats that are worth noting. First, the analysis of VSG derepression and switching in Figure 1 relies on a genome which does not contain minichromosomal (MC) VSG sequences. This means that MC VSGs could theoretically be mis-assigned as coming from another genomic location in the absence of an MC reference. As the origin of the VSGs in these clones isn't a major point in the paper, I do not think this is a major concern, but I would not over-interpret the particular details of switching outcomes in these experiments.

    Another aspect of this work that is perhaps important, but not discussed much by the authors, is the fact that signalling is extremely poorly understood in T. brucei. In Figure 1B, the RNA-seq data show many genes upregulated after expression of the Mut PIP5Pase (not just VSGs). The authors rightly avoid claiming that this pathway is exclusive to VSGs, but I wonder if these data could provide insight into the other biological processes that might be controlled by this signaling pathway in T. brucei.

    Overall, this is an excellent study which represents an important step forward in understanding how antigenic variation is controlled in T. brucei. The possibility that this process could be controlled via a signalling pathway has been speculated for a long time, and this study provides the first mechanistic evidence for that possibility.

  17. Version published to 10.1101/2023.05.11.540368v2 on bioRxiv
  18. Version published to 10.7554/elife.89331.1 on eLife
  19. eLife

    Author Response

    eLife assessment:

    Trypanosoma brucei evades mammalian humoral immunity through the expression of different variant surface glycoprotein genes. In this fundamental paper, the authors extend previous observations that TbRAP1 both interacts with PIP5pase and binds PI(3,4,5)P3, indicating a role for PI(3,4,5)P3 binding and suggesting that antigen switching is signal dependent. While much of the evidence is compelling, one reviewer suggested that the work would benefit from further controls.

    We appreciate the evaluation of the work and agree that the findings substantially advance our understanding of antigenic variation. A detailed response to the public review is included below, which addresses and clarifies the issues raised by the reviewers, including those concerning controls. We also want to highlight the comment by Reviewer #3 “The methods used in the study are rigorous and well-controlled…. their results support the conclusions made in the manuscript.”. We hope this and our comments will help address the issue of controls in this eLife statement.

    Reviewer #1 (Public Review):

    Trypanosoma brucei undergoes antigenic variation to evade the mammalian host’s immune response. To achieve this, T. brucei regularly expresses different VSGs as its major surface antigen. VSG expression sites are exclusively subtelomeric, and VSG transcription by RNA polymerase I is strictly monoallelic. It has been shown that T. brucei RAP1, a telomeric protein, and the phosphoinositol pathway are essential for VSG monoallelic expression. In previous studies, Cestari et al. (ref. 24) have shown that PIP5pase interacts with RAP1 and that RAP1 binds PI(3,4,5)P3. RNAseq and ChIPseq analyses have been performed previously in PIP5pase conditional knockout cells, too (ref. 24). In the current study, Touray et al. did similar analyses except that catalytic dead PIP5pase mutant was used and the DNA and PI(3,4,5)P3 binding activities of RAP1 fragments were examined. Specifically, the authors examined the transcriptome profile and did RAP1 ChIPseq in PIP5pase catalytic dead mutant. The authors also expressed several C-terminal His6-tagged RAP1 recombinant proteins (full-length, aa1-300, aa301-560, and aa 561-855). These fragments’ DNA binding activities were examined by EMSA analysis and their phosphoinositides binding activities were examined by affinity pulldown of biotin-conjugated phosphoinositides. As a result, the authors confirmed that VSG silencing (both BES-linked and MES-linked VSGs) depends on PIP5pase catalytic activity, but the overall knowledge improvement is incremental. The most convincing data come from the phosphoinositide binding assay as it clearly shows that N-terminus of RAP1 binds PI(3,4,5)P3 but not PI(4,5)P2, although this is only assayed in vitro, while the in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al (ref. 24) already. Considering that many phosphoinositides exert their regulatory role by modulating the subcellular localization of their bound proteins, it is reasonable to hypothesize that binding to PI(3,4,5)P3 can remove RAP1 from the chromatin. However, no convincing data have been shown to support the author’s hypothesis that this regulation is through an “allosteric switch”. Therefore, the title should be revised.

    We appreciate the reviewer’s detailed evaluation of our work. There are a few general comments that we would like to clarify. We will break them into three points. All data included here are new and were not previously published.

    i) “RNAseq and ChIPseq analyses have been performed previously …(ref. 24).” Reference 24 is Cestari et al. 2019, Mol Cell Biol. We, or others, have not published ChIP-seq of RAP1 in T. brucei. Previous work showed ChIP-qPCR, which analyses specific loci. The ChIP-seq shows genome-wide binding sites of RAP1, and new findings are shown here, including binding sites in the BES, MESs, and other genome loci such as centromeres. We also identified DNA sequence bias defining RAP1 binding sites (Fig 2A). We also show by ChIP-seq how RAP1-binding to these loci changes upon expression of catalytic inactive PIP5Pase. As for the RNA-seq, this is also the first time we show RNA-seq of T. brucei expressing catalytic inactive PIP5Pase, which establishes that the regulation of VSG silencing and switching is dependent on PIP5Pase enzyme catalysis, i.e., PI(3,4,5)P3 dephosphorylation. To improve clarity in the manuscript, we edited page 4, line 122, as follows: “We showed that RAP1 binds telomeric or 70 bp repeats (24), but it is unknown if it binds to other ES sequences or genomic loci.”

    ii) “The in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al. (ref. 24) already.”. We published in reference 24 that RAP1-HA can bind agarose beads-conjugated synthetic PI(3,4,5)P3. Here, we were able to measure T. brucei endogenous PI(3,4,5)P3 associated with RAP1-HA (Fig 4F). Moreover, we showed that the endogenous RAP1-HA and PI(3,4,5)P3 binding is about 100-fold higher when PIP5Pase is catalytic inactive than WT PIP5Pase. The data establish that in vivo endogenous PI(3,4,5)P3 binds to RAP1-HA and how the binding changes in cells expressing mutant PIP5Pase; this data is new and relevant to our conclusions.

    iii) “no convincing data have been shown to support the author’s hypothesis that this regulation is through an “allosteric switch””. We show here in vitro and in vivo data supporting the conclusion. We show that PI(3,4,5)P3 binds to the N-terminus of rRAP1-His with a calculated Kd of about 20 µM (Fig 4B-E, Table 1). In contrast, we show by EMSA and binding kinetics by microscale thermophoresis that rRAP1-His binds to 70 bp and telomeric repeats via protein regions encompassing the Myb (central) or Myb-L domains (C-terminal) but not the N-terminus containing the VHP domain (Fig 3C-G, and Fig S5). Using microscale thermophoresis, we also show that rRAP1-His binds to 70 bp and telomeric repeats with Kd of 10 and 24 nM, respectively (Fig 3 and Table 1). Notably, we show that 30 µM of PI(3,4,5)P3, but not PI(4,5,)P2 – used as a control – disrupts rRAP1-His binding to 70 bp and telomeric repeats, changing Kds to about 188 and 155 nM, respectively (Fig 5A-C). We also show that PI(3,4,5)P3 does not disrupt the binding of rRAP1-His fragments (Myb or MybL) without the N-terminus domain (Fig S5), implying binding of PI(3,4,5)P3 to RAP1 N-terminus is required for displacement of RAP1 DNA binding domains (Myb and MybL) from telomeric and 70 bp repeats, and that PI(3,4,5)P3 is not competing for Myb or Myb-L binding to DNA. Moreover, we show that RAP1-HA binding to 70 bp and telomeric repeats in vivo is displaced in T. brucei cells expressing catalytic inactive PIP5Pase (Fig 5D-G), which we show results in RAP1-HA binding about 100-fold more endogenous PI(3,4,5)P3 than in T. brucei expressing WT PIP5Pase (Fig 4F). The in vivo data agrees with the in vitro data. The data show a typical allosteric regulator system, in which binding of a ligand to one site of the protein, here PI(3,4,5)P3 binding to RAP1 N-terminus, affects other domains (RAP1 Myb and Myb-L domains) binding to DNA. To improve the clarity of the title, we will change it in the revised version to imply a direct role of PI(3,4,5)P3 regulation of RAP1 in the process. This will provide more specific information to the readers and addresses the concern of the reviewer related to the “allosteric switch”. The new title will be: PI(3,4,5)P3 allosteric regulation of RAP1 controls antigenic switching in trypanosomes

    There are serious concerns about many conclusions made by Touray et al., according to their experimental approaches:

    1. The authors have been studying RAP1’s chromatin association pattern by ChIPseq in cells expressing a C-terminal HA tagged RAP1. According to data from tryptag.org, RAP1 with an N-terminal or a C-terminal tag does not seem to have identical subcellular localization patterns, suggesting that adding tags at different positions of RAP1 may affect its function. It is therefore essential to validate that the C-terminally HA-tagged RAP1 still has its essential functions. However, this data is not available in the current study. RAP1 is essential. If RAP1-HA still retains its essential functions, cells carrying one RAP1-HA allele and one deleted allele are expected to grow the same as WT cells. In addition, these cells should have the WT VSG expression pattern, and RAP1-HA should still interact with TRF. Without these validations, it is impossible to judge whether the ChIPseq data obtained on RAP1-HA reflect the true chromatin association profile of RAP1.

    Tryptag data show both N- and C-terminus RAP1 with nuclear localization in procyclic forms, although there are differences in signal intensities in the images (http://tryptag.org/?id=Tb927.11.370). It is important to note that Tryptag data is from procyclic forms, and DNA constructs are not validated for their integration in the correct locus. As for the RAP1-HA localization in bloodstream forms, we demonstrated that C-terminally HA-tagged RAP1 co-localizes with telomeres by a combination of immunofluorescence and fluorescence in situ hybridization (Cestari and Stuart, 2015, PNAS), and RAP1-HA co-immunoprecipitate telomeric and 70 bp repeats (Cestari et al. 2019 Mol Cell Biol). We also showed by immunoprecipitation and mass spectrometry that HA-tagged RAP1 interacts with nuclear and telomeric proteins, including PIP5Pase (Cestari et al. 2019). Others have also tagged T. brucei RAP1 in bloodstream forms with HA without disrupting its nuclear localization (Yang et al. 2009, Cell; Afrin et al. 2020, Science Advances). As for the experiment suggested by the reviewer, there is no guarantee that cells lacking one allele of RAP1 will behave as wildtype, i.e., normal growth and repression of VSGs genes. Also, less than 90% of T. brucei TRF was reported to interact with RAP1 (Yang et al. 2009, Cell), which might be indirect via their binding to telomeric DNA repeats rather than direct protein-protein interactions.

    1. Touray et al. expressed and purified His6-tagged recombinant RAP1 fragments from E. coli and used these recombinant proteins for EMSA analysis: The His6 tag has been used for purifying various recombinant proteins. It is most likely that the His6 tag itself does not convey any DNA binding activities. However, using His6-tagged RAP1 fragments for EMSA analysis has a serious concern. It has been shown that His6-tagged human RAP1 protein can bind dsDNA, but hRAP1 without the His6 tag does not. It is possible that RAP1 proteins in combination with the His6 tag can exhibit certain unnatural DNA binding activities. To be rigorous, the authors need to remove the His6 tag from their recombinant proteins before the in vitro DNA binding analyses are performed. This is a standard procedure for many in vitro assays using recombinant proteins.

    We show in Fig 3C-G that His-tagged full-length rRAP1 does not bind to scrambled telomeric dsDNA sequences, which indicates that His-tagged rRAP1 does not bind unspecifically to DNA. Moreover, in Fig 3G, we show that His-tagged rRAP11-300 also does not bind to 70 bp or telomeric repeats. In contrast, full-length His-tagged rRAP1, rRAP1301-560, or rRAP1561-855 bind to 70 bp or telomeric repeats (Fig 3C-G). Since all proteins were His-tagged, the His tag cannot be responsible for the DNA binding.

    As for the statement that human rRAP1-His has unspecific DNA binding properties, we could not find a reference to this statement; we cannot compare it without knowing the details of the experiment. Biochemical assays can result in unspecific binding depending on binding/buffer conditions. Also, humans and T. brucei RAP1 share only 15% of amino acid identity; unspecific binding to DNA could be specific to human RAP1.

    1. It is unclear why Nanopore sequencing was used for RNAseq and ChIPseq experiments. The greatest benefit of Nanopore sequencing is that it can sequence long reads, which usually helps with mapping, particularly at genome loci with repetitive sequences. This seems beneficial for RAP1 ChIPseq analysis as RAP1 is expected to bind telomere repeats. However, for ChIPseq, the chromatin needs to be fragmented. Larger DNA fragments from ChIPseq experiments will decrease the accuracy of the final calculated binding sites. Therefore, ChIPseq experiments are not supposed to have long reads to start with, so Nanopore sequencing does not seem to bring any advantage. In addition, compared to Illumina sequencing, Nanopore sequencing usually yields smaller numbers of reads, and the sequencing accuracy rate is lower. The Nanopore sequencing accuracy may be a serious concern in the current study. All telomeres have the perfect TTAGGG repeats, all VSG genes have a very similar 3’ UTR, and all 70 bp repeats have very similar sequences. In fact, the active and silent ESs have 90% sequence identity. Are sequence reads accurately mapped to different ESs? How is the sequencing and mapping quality controlled? Furthermore, it is unclear whether the read depth for RNAseq is deep enough.

    The mean sequence length for the ChIP-seq was about 500 bp (see Table S3), which helps to align reads to ESs and distinguish the different ESs, and it is a reasonable size range to define RAP1 binding sites. Although sequencing depths are usually higher in Illumina than in nanopore (all depending on the amount of sequencing), most Illumina short reads map to multiple genomic sequences, making it difficult to distinguish ESs. This is particularly important for RAP1 because it binds to repeats such as 70 bp and telomeric repeats. Mapping short reads to those regions would be virtually impossible; hence, our choice of nanopore sequencing. For RNA-seq, the ~500 bp read length help sequence alignment to the subtelomeric regions containing many VSG genes. The nanopore reads obtained here had an average sequencing score 12 (i.e., base call accuracy of 94%). Filtering reads with MAPQ ≥ 20 (99% probability of correct alignment) helped us to distinguish RAP1 binding to specific ESs, including silent vs active ES (ChIP-seq) or VSG sequences (RNA-seq). The details of the analysis and sequencing metrics (i.e., sequencing depth and read length) were described in the Methods section “Computational analysis of RNA-seq and ChIP-seq” and Table S3, respectively.

    1. Many statements in the discussion section are speculations without any solid evidence. For example, lines 218 - 219 “likely due to RAP1 conformational changes”, no data have been shown to support this at all. In lines 224-226, the authors acknowledged that more experiments are necessary to validate their observations, so it is important for the authors to first validate their findings before they draw any solid conclusions. Importantly, RAP1 has been shown to help compact telomeric and subtelomeric chromatin a long time ago by Pandya et al. (2013. NAR 41:7673), who actually examined the chromatin structure by MNase digestion and FAIRE. The authors should acknowledge previous findings. In addition, the authors need to revise the discussion to clearly indicate what they “speculate” rather than make statements as if it is a solid conclusion.

    The statement “likely due to RAP1 conformational changes” in lines 218-219 (page 6) is part of the Discussion. We did not make a strong statement but discussed a possibility. We believe that it is beneficial to the reader to have the data discussed, and we do not feel this point is overly speculative.

    For lines 224-226 (page 6), the statement refers to the finding of RAP1 binding to centromeric regions by ChIP-seq, which is a new finding but not the focus of this work. Hence, future studies are necessary for this finding, and we believe it is appropriate in the Discussion to be upfront and highlight this point to the readers. However, for the RAP1 binding to telomeric ES sites, e.g., 70 bp repeats and telomeric repeats (the focus of this work), we validated the binding by EMSA and by performing binding kinetics using microscale thermophoresis.

    We did not include Pandya et al. 2013 NAR because the authors demonstrated RAP1 compaction of chromatin to occur in procyclic forms only. Pandya et al. stated in their abstract: “no significant chromatin structure changes were detected on depletion of TbRAP1 in BF cells”. Hence, the suggested reference is not relevant to the context of our conclusions in bloodstream forms. Nevertheless, we have reviewed the Discussion to avoid broad speculations in the revised version of the manuscript.

    There are also minor concerns:

    1. In the PIP5Pase conditional knockout system, the WT or mutant PIP5Pase with a V5 tag is constitutively expressed from the tubulin array. What’s the relative expression level of this allele and the endogenous PIP5Pase? Without a clear knowledge of the mutant expression level, it is hard to conclude whether the mutant has any dominant negative effects or whether the mutant phenotype is simply due to a lower than WT PIP5pase expression level.

    The relative mRNA levels of the exclusive expression of PIP5Pase Mut compared to the WT is available in the Data S1, RNA-seq. The Mut allele’s relative expression level is 0.85-fold to the WT allele (both from tubulin loci). We also showed by Western blot the WT and Mut PIP5Pase protein expression (Cestari et al. 2019, Mol Cell Biol). Concerning PIP5Pase endogenous alleles, we compared RNA-seq reads counts per million from the conditional null PIP5Pase cells exclusively expressing WT or the Mut PIP5Pase alleles (Data S1, this work) to our previous RNA-seq of single-marker 427 strain (Cestari et al. 2019, Mol Cell Biol). We used the single-maker 427 because the conditional null cells were generated in this strain background. The PIP5Pase WT and Mut mRNAs expressed from tubulin loci are 1.6 and 1.3-fold the endogenous PIP5Pase levels in single-marker 427, respectively. We include a statement in the Methods, page 7, lines 265-268: “The WT or Mut PIP5Pase mRNAs exclusively expressed from tubulin loci are 1.6 and 1.3-fold the WT PIP5Pase mRNA levels expressed from endogenous alleles in the single marker 427 strain. The fold-changes were calculated from RNA-seq reads counts per million from this work (WT and Mut PIP5Pase, Data S1) and our previous RNA-seq from single marker 427 strain (24).”

    1. In EMSA analysis, what are the concentrations of the protein and the probe used in each reaction? The amount of protein used in the binding assay appears to be very high, and this can contribute to the observation that many complexes are stuck in the well. Better quality EMSA data need to be shown to support the authors’ claims.

    All concentrations were provided in the Methods section. See page 9 Electrophoretic mobility shift assays: “100 nM of annealed DNA were mixed with 1 μg of recombinant protein…”. For microscale thermophoresis, also see page 9, Microscale thermophoresis binding kinetics: “1 μM rRAP1 was diluted in 16 two-fold serial dilutions in 250 mM HEPES pH 7.4, 25 mM MgCl2, 500 mM NaCl, and 0.25% (v/v) N P-40 and incubated with 20 nM telomeric or 70 bp repeats…”. Note that two different biochemical approaches, EMSA and microscale thermophoresis, were used to assess rRAP1-His binding to DNA. Both show similar results (Fig 3 and 5, and Fig S5; microscale thermophoresis shows the binding kinetics, data available in Table 1). The EMSA images clearly show the binding of RAP1 to 70 bp or telomeric repeats but not to scramble telomeric repeat DNA.

    Reviewer #2 (Public Review):

    This manuscript by Touray, et al. provides a significant new twist to our understanding of how antigenic variation may be regulated in T. brucei. Key aspects of antigenic variation are the mutually exclusive expression of a single antigen per cell and the periodic switching from expression of one antigen isoform to another. In this manuscript, the authors show, as they have previously shown, that depletion of the nuclear phosphatidylinositol 5-phosphatase (PIP5Pase) results in a loss of mutually exclusive VSG expression. Furthermore, using ChIP-seq, the authors show that the repressor/activator protein 1 (RAP1) binds to regions upstream and downstream of VSG genes located in transcriptionally repressed expression sites and that this binding is lost in the absence of a functional PIP5Pase. Importantly, the authors decided to further investigate this link between PIP5Pase and RAP1, a protein that has previously been implicated in antigenic variation in T. brucei, and found that inactivation of PIP5Pase results in the accumulation of PI(3,4,5)P3 bound to the RAP1 N-terminus and that this binding impairs the ability of RAP1 to bind DNA. Based on these observations, the authors suggest that the levels of PI(3,4,5)P3 may determine the cellular function of RAP1, either by binding upstream of VSG genes and repressing their function, or by not binding DNA and allowing the simultaneous expression of multiple VSG genes in a single parasite.

    While I find most of the data presented in this manuscript compelling, there are aspects of Figure 1 that are not clear to me. Based on Figure 1F, the authors claim that transient inactivation of PIP5Pase results in a switch from the expression of one VSG isoform to another. However, I am not exactly sure what the authors are showing in this panel, nor do the data in Figure 1F seem to be consistent with those shown in Figure 1C. Based on Figure 1F, a transient inactivation of PIP5Pase appears to result in an almost exclusive switch to a VSG located in BES12. However, based on Figure 1E, the VSG transcripts most commonly found after a transient inactivation of PIP5Pase are those from the previously active VSG (BES1) and VSGs located on chr 1 and 6 (I believe). The small font and the low resolution make it impossible to infer the location of the expressed VSG genes, nor to confirm that ALL VSG genes located in expression sites are activated, as the authors claim. Also, I was not able to access the raw ChIP-seq and RNA-seq reads. Thus, could not evaluate the quality of the sequencing data.

    We appreciate the reviewer’s comments and evaluation of our work. Fig 1E shows VSG-seq of a population after transient (24h) exclusive expression of the PIP5Pase mutant, followed by re-expression of the WT PIP5Pase allele for 60 hours (multiple VSGs are detected). As a control, it also shows VSG-seq in cells continuously expressing WT PIP5Pase (mostly VSG2, BES1 is detected). Fig 1F and Fig S1 show the sequencing of VSGs expressed by clones isolated (5-6 days of growth) after a temporary knockdown (24h) of PIP5Pase (tet -), followed by its re-expression. For comparison, no knockdown (tet +) was included. Fig 1F shows potential switchers in the population, the Fig 1E confirms VSG switching in clones.

    To clarify the difference between Fig 1E and 1F, we edited the manuscript on page 3, lines 103-110: “To verify PIP5Pase role in VSG switching, we knocked down PIP5Pase for 24h (Tet -), then restored its expression (Tet +) and isolated clones by limiting dilution and growth for 5-6 days. Analysis of isolated clones after temporary PIP5Pase knockdown (Tet -/+) confirmed VSG switching in 93 out of 94 (99%) of the analyzed clones (Fig 1F, Fig S1). The cells switched to express VSGs from silent ESs or subtelomeric regions, indicating switching by transcription or recombination mechanisms. Moreover, no switching was detected in 118 isolated clones from cells continuously expressing WT PIP5Pase (Tet +, Fig 1F).”. We also edited Fig 1F to indicate temporary knockdown (Tet -/+) vs no knockdown (Tet -). The modifications will be available in the resubmitted version of the manuscript.

    We agree that the heat map is difficult to read due to the amount of information. We will include in the revised version of the manuscript a table with the data in the supplementary information; the reader will be able to evaluate the data in detail.

    A preference for switching to specific ESs has been observed in T. brucei (Morrison et al. 2005, Int J Parasitol; Cestari and Stuart, 2015, PNAS), which may explain several clones switching to BES12. Many potential switchers were detected in the VSG-seq (Fig 1F, the whole cell population is over 107 parasites), but not all potential switchers were detected in the clonal analysis because we analyzed 212 clones total, a fraction of the over 107 cells analyzed by VSG-seq (Fig 1E). Also, it is possible that not all potential switchers are viable. However, the point of the clonal analysis is to validate the VSG switching after genetic perturbation of PIP5Pase.

    Fig 1C shows examples of ES derepression by RNA-seq after 24h exclusive expression of the mutant compared to WT PIP5Pase. The RNA-seq shows that all ESs are derepressed (Fig 1B). This can be visualized in the volcano plot (Fig 1B, BES and MES VSGs are labelled) and on the spreadsheet Data S1. Although all ESs are derepressed after PIP5Pase mutant expression, not all ESs are selected during switching, as observed in Fig 1E-F. This agrees with our previous observations in switching assays with proteins that control VSG switching (Cestari and Stuart, 2015, PNAS).

    As for metrics of sequencing and raw sequencing data. See Methods section, page 13, lines 483-485: “Sequencing information is available in Table S3 and fastq data is available in the Sequence Read Archive (SRA) with the BioProject identification PRJNA934938.” Table S3 has a summary of sequencing data. Metrics information such as sequencing quality and analysis can be found in the Methods section “Computational analysis of RNA-seq and ChIP-seq”. The latter includes information about nanopore reads, i.e., mean Q-score of 12.

    Reviewer #3 (Public Review):

    In this manuscript, Touray et al investigate the mechanisms by which PIP5Pase and RAP1 control VSG expression in T. brucei and demonstrate an important role for this enzyme in a signalling pathway that likely plays a role in antigenic variation in T. brucei.

    The methods used in the study are rigorous and well-controlled. The authors convincingly demonstrate that RAP1 binds to PI(3,4,5)P3 through its N-terminus and that this binding regulates RAP1 binding to VSG expression sites, which in turn regulates VSG silencing. Overall their results support the conclusions made in the manuscript.

    There are a few small caveats that are worth noting. First, the analysis of VSG derepression and switching in Figure 1 relies on a genome that does not contain minichromosomal (MC) VSG sequences. This means that MC VSGs could theoretically be misassigned as coming from another genomic location in the absence of an MC reference. As the origin of the VSGs in these clones isn’t a major point in the paper, I do not think this is a major concern, but I would not over-interpret the particular details of switching outcomes in these experiments.

    The authors state that “our data imply that antigenic variation is not exclusively stochastic.” I am not sure this is true. While I also favor the idea that switching is not exclusively stochastic, evidence for a signaling pathway does not necessarily imply that antigenic variation is not stochastic. This pathway could be important solely for lifecycle-related control of VSG expression, rather than antigenic variation during infection. Nevertheless, these data are critical for establishing a potential pathway that could control antigenic variation and thus represent a fundamental discovery.

    Another aspect of this work that is perhaps important, but not discussed much by the authors, is the fact that signalling is extremely poorly understood in T. brucei. In Figure 1B, the RNA-seq data show many genes upregulated after expression of the Mut PIP5Pase (not just VSGs). The authors rightly avoid claiming that this pathway is exclusive to VSGs, but I wonder if these data could provide insight into the other biological processes that might be controlled by this signaling pathway in T. brucei.

    Overall, this is an excellent study that represents an important step forward in understanding how antigenic variation is controlled in T. brucei. The possibility that this process could be controlled via a signalling pathway has been speculated for a long time, and this study provides the first mechanistic evidence for that possibility.

    We thank the reviewer for the evaluation of our work. We agree that it is difficult to ensure the origin of all VSG genes not having minichromosome sequences; hence we did not emphasize this point in the manuscript. We used the 427-2018 reference genome assembled by PacBio and Hi-C (Muller et al. 2018, Nature), which we believe is the best assembly for the 427 strain, especially related to the VSG genes.

    We also agree that having signaling controlling switching in vitro does not mean the switching necessarily occurs by signaling in vivo. Nevertheless, stochastic switching is an accepted model; but it has not been proved, whereas we provide molecular evidence that signaling can cause switching. To express this reviewer’s suggestion, we edited the Discussion, page 7, line 250: from “our data imply that antigenic variation is not exclusively stochastic” to “our data suggest that antigenic variation is not exclusively stochastic”.

    Most of the RNA-seq data were VSGs genes/pseudogenes. Other genes upregulated included retrotransposons and DNA/RNA processing enzymes such as endonucleases and polymerases. We included in the Results, page 3, line 100: “Other genes upregulated include primarily retrotransposons, endonucleases, and polymerase proteins.”.

  20. eLife

    eLife assessment:

    Trypanosoma brucei evades mammalian humoral immunity through the expression of different variant surface glycoprotein genes. In this fundamental paper, the authors extend previous observations that TbRAP1 both interacts with PIP5Pase and binds PI(3,4,5)P3, indicating a role for PI(3,4,5)P3 binding and suggesting that antigen switching is signal dependent. While much of the evidence is compelling, one reviewer suggested that the work would benefit from further controls.

  21. eLife

    Reviewer #1 (Public Review):

    Trypanosoma brucei undergoes antigenic variation to evade the mammalian host's immune response. To achieve this, T. brucei regularly expresses different VSGs as its major surface antigen. VSG expression sites are exclusively subtelomeric, and VSG transcription by RNA polymerase I is strictly monoallelic. It has been shown that T. brucei RAP1, a telomeric protein, and the phosphoinositol pathway are essential for VSG monoallelic expression. In previous studies, Cestari et al. (ref. 24) have shown that PIP5Pase interacts with RAP1 and that RAP1 binds PI(3,4,5)P3. RNAseq and ChIPseq analyses have been performed previously in PIP5Pase conditional knockout cells, too (ref. 24). In the current study, Touray et al. did similar analyses except that catalytic dead PIP5Pase mutant was used and the DNA and PI(3,4,5)P3 binding activities of RAP1 fragments were examined. Specifically, the authors examined the transcriptome profile and did RAP1 ChIPseq in PIP5Pase catalytic dead mutant. The authors also expressed several C-terminal His6-tagged RAP1 recombinant proteins (full-length, aa1-300, aa301-560, and aa 561-855). These fragments' DNA binding activities were examined by EMSA analysis and their phosphoinositides binding activities were examined by affinity pulldown of biotin-conjugated phosphoinositides. As a result, the authors confirmed that VSG silencing (both BES-linked and MES-linked VSGs) depends on PIP5Pase catalytic activity, but the overall knowledge improvement is incremental. The most convincing data come from the phosphoinositide binding assay as it clearly shows that N-terminus of RAP1 binds PI(3,4,5)P3 but not PI(4,5)P2, although this is only assayed in vitro, while the in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al (ref. 24) already. Considering that many phosphoinositides exert their regulatory role by modulating the subcellular localization of their bound proteins, it is reasonable to hypothesize that binding to PI(3,4,5)P3 can remove RAP1 from the chromatin. However, no convincing data have been shown to support the author's hypothesis that this regulation is through an "allosteric switch". Therefore, the title should be revised.

    There are serious concerns about many conclusions made by Touray et al., according to their experimental approaches:
    1. The authors have been studying RAP1's chromatin association pattern by ChIPseq in cells expressing a C-terminal HA tagged RAP1. According to data from tryptag.org, RAP1 with an N-terminal or a C-terminal tag does not seem to have identical subcellular localization patterns, suggesting that adding tags at different positions of RAP1 may affect its function. It is therefore essential to validate that the C-terminally HA-tagged RAP1 still has its essential functions. However, this data is not available in the current study. RAP1 is essential. If RAP1-HA still retains its essential functions, cells carrying one RAP1-HA allele and one deleted allele are expected to grow the same as WT cells. In addition, these cells should have the WT VSG expression pattern, and RAP1-HA should still interact with TRF. Without these validations, it is impossible to judge whether the ChIPseq data obtained on RAP1-HA reflect the true chromatin association profile of RAP1.

    2. Touray et al. expressed and purified His6-tagged recombinant RAP1 fragments from E. coli and used these recombinant proteins for EMSA analysis: The His6 tag has been used for purifying various recombinant proteins. It is most likely that the His6 tag itself does not convey any DNA binding activities. However, using His6-tagged RAP1 fragments for EMSA analysis has a serious concern. It has been shown that His6-tagged human RAP1 protein can bind dsDNA, but hRAP1 without the His6 tag does not. It is possible that RAP1 proteins in combination with the His6 tag can exhibit certain unnatural DNA binding activities. To be rigorous, the authors need to remove the His6 tag from their recombinant proteins before the in vitro DNA binding analyses are performed. This is a standard procedure for many in vitro assays using recombinant proteins.

    3. It is unclear why Nanopore sequencing was used for RNAseq and ChIPseq experiments. The greatest benefit of Nanopore sequencing is that it can sequence long reads, which usually helps with mapping, particularly at genome loci with repetitive sequences. This seems beneficial for RAP1 ChIPseq analysis as RAP1 is expected to bind telomere repeats. However, for ChIPseq, the chromatin needs to be fragmented. Larger DNA fragments from ChIPseq experiments will decrease the accuracy of the final calculated binding sites. Therefore, ChIPseq experiments are not supposed to have long reads to start with, so Nanopore sequencing does not seem to bring any advantage. In addition, compared to Illumina sequencing, Nanopore sequencing usually yields smaller numbers of reads, and the sequencing accuracy rate is lower. The Nanopore sequencing accuracy may be a serious concern in the current study. All telomeres have the perfect TTAGGG repeats, all VSG genes have a very similar 3' UTR, and all 70 bp repeats have very similar sequences. In fact, the active and silent ESs have 90% sequence identity. Are sequence reads accurately mapped to different ESs? How is the sequencing and mapping quality controlled? Furthermore, it is unclear whether the read depth for RNAseq is deep enough.

    4. Many statements in the discussion section are speculations without any solid evidence. For example, lines 218 - 219 "likely due to RAP1 conformational changes", no data have been shown to support this at all. In lines 224-226, the authors acknowledged that more experiments are necessary to validate their observations, so it is important for the authors to first validate their findings before they draw any solid conclusions. Importantly, RAP1 has been shown to help compact telomeric and subtelomeric chromatin a long time ago by Pandya et al. (2013. NAR 41:7673), who actually examined the chromatin structure by MNase digestion and FAIRE. The authors should acknowledge previous findings. In addition, the authors need to revise the discussion to clearly indicate what they "speculate" rather than make statements as if it is a solid conclusion.

    There are also minor concerns:

    1. In the PIP5Pase conditional knockout system, the WT or mutant PIP5Pase with a V5 tag is constitutively expressed from the tubulin array. What's the relative expression level of this allele and the endogenous PIP5Pase? Without a clear knowledge of the mutant expression level, it is hard to conclude whether the mutant has any dominant negative effects or whether the mutant phenotype is simply due to a lower than WT PIP5pase expression level.

    2. In EMSA analysis, what are the concentrations of the protein and the probe used in each reaction? The amount of protein used in the binding assay appears to be very high, and this can contribute to the observation that many complexes are stuck in the well. Better quality EMSA data need to be shown to support the authors' claims.

  22. eLife

    Reviewer #2 (Public Review):

    This manuscript by Touray, et al. provides a significant new twist to our understanding of how antigenic variation may be regulated in T. brucei. Key aspects of antigenic variation are the mutually exclusive expression of a single antigen per cell and the periodic switching from expression of one antigen isoform to another. In this manuscript, the authors show, as they have previously shown, that depletion of the nuclear phosphatidylinositol 5-phosphatase (PIP5Pase) results in a loss of mutually exclusive VSG expression. Furthermore, using ChIP-seq, the authors show that the repressor/activator protein 1 (RAP1) binds to regions upstream and downstream of VSG genes located in transcriptionally repressed expression sites and that this binding is lost in the absence of a functional PIP5Pase. Importantly, the authors decided to further investigate this link between PIP5Pase and RAP1, a protein that has previously been implicated in antigenic variation in T. brucei, and found that inactivation of PIP5Pase results in the accumulation of PI(3,4,5)P3 bound to the RAP1 N-terminus and that this binding impairs the ability of RAP1 to bind DNA. Based on these observations, the authors suggest that the levels of PI(3,4,5)P3 may determine the cellular function of RAP1, either by binding upstream of VSG genes and repressing their function, or by not binding DNA and allowing the simultaneous expression of multiple VSG genes in a single parasite.

    While I find most of the data presented in this manuscript compelling, there are aspects of Figure 1 that are not clear to me. Based on Figure 1F, the authors claim that transient inactivation of PIP5Pase results in a switch from the expression of one VSG isoform to another. However, I am not exactly sure what the authors are showing in this panel, nor do the data in Figure 1F seem to be consistent with those shown in Figure 1C. Based on Figure 1F, a transient inactivation of PIP5Pase appears to result in an almost exclusive switch to a VSG located in BES12. However, based on Figure 1E, the VSG transcripts most commonly found after a transient inactivation of PIP5Pase are those from the previously active VSG (BES1) and VSGs located on chr 1 and 6 (I believe). The small font and the low resolution make it impossible to infer the location of the expressed VSG genes, nor to confirm that ALL VSG genes located in expression sites are activated, as the authors claim. Also, I was not able to access the raw ChIP-seq and RNA-seq reads. Thus, could not evaluate the quality of the sequencing data.

  23. eLife

    Reviewer #3 (Public Review):

    In this manuscript, Touray et al investigate the mechanisms by which PIP5Pase and RAP1 control VSG expression in T. brucei and demonstrate an important role for this enzyme in a signalling pathway that likely plays a role in antigenic variation in T. brucei.

    The methods used in the study are rigorous and well-controlled. The authors convincingly demonstrate that RAP1 binds to PI(3,4,5)P3 through its N-terminus and that this binding regulates RAP1 binding to VSG expression sites, which in turn regulates VSG silencing. Overall their results support the conclusions made in the manuscript.

    There are a few small caveats that are worth noting. First, the analysis of VSG derepression and switching in Figure 1 relies on a genome that does not contain minichromosomal (MC) VSG sequences. This means that MC VSGs could theoretically be misassigned as coming from another genomic location in the absence of an MC reference. As the origin of the VSGs in these clones isn't a major point in the paper, I do not think this is a major concern, but I would not over-interpret the particular details of switching outcomes in these experiments.

    The authors state that "our data imply that antigenic variation is not exclusively stochastic." I am not sure this is true. While I also favor the idea that switching is not exclusively stochastic, evidence for a signaling pathway does not necessarily imply that antigenic variation is not stochastic. This pathway could be important solely for lifecycle-related control of VSG expression, rather than antigenic variation during infection. Nevertheless, these data are critical for establishing a potential pathway that could control antigenic variation and thus represent a fundamental discovery.

    Another aspect of this work that is perhaps important, but not discussed much by the authors, is the fact that signalling is extremely poorly understood in T. brucei. In Figure 1B, the RNA-seq data show many genes upregulated after expression of the Mut PIP5Pase (not just VSGs). The authors rightly avoid claiming that this pathway is exclusive to VSGs, but I wonder if these data could provide insight into the other biological processes that might be controlled by this signaling pathway in T. brucei.

    Overall, this is an excellent study that represents an important step forward in understanding how antigenic variation is controlled in T. brucei. The possibility that this process could be controlled via a signalling pathway has been speculated for a long time, and this study provides the first mechanistic evidence for that possibility.

  24. Version published to 10.1101/2023.05.11.540368v1 on bioRxiv