Expanded CAG/CTG repeats resist gene silencing mediated by targeted epigenome editing
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (Review Commons)
Abstract
Expanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Previous studies have implicated the histone deacetylase HDAC5 and the DNA methyltransferase DNMT1 as modulators of repeat instability via mechanisms that are not fully understood. Using PInT, we found no evidence that HDAC5 or DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by targeting HDAC5 and DNMT1. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.
Article activity feed
-
-
-
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The manuscript describes two advances. First is the technical development for a protein targeting system called PInT that brings a target protein close to (~320 bp) a DNA sequence of interest. The idea is that localisation of the target protein allows one to distinguish its effects on the DNA sequence either in cis (when targeted) or in trans (when not targeted but expressed at the same level). Since targeting is conveyed by simply adding the small molecule ABA to the experiment, it is easy to compare the two situations. This is a clever idea and it is substantiated by data showing that the …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
Reviewer #1 (Evidence, reproducibility and clarity (Required)):
The manuscript describes two advances. First is the technical development for a protein targeting system called PInT that brings a target protein close to (~320 bp) a DNA sequence of interest. The idea is that localisation of the target protein allows one to distinguish its effects on the DNA sequence either in cis (when targeted) or in trans (when not targeted but expressed at the same level). Since targeting is conveyed by simply adding the small molecule ABA to the experiment, it is easy to compare the two situations. This is a clever idea and it is substantiated by data showing that the components of PInT do not affect triplet repeat instability or gene expression of GFP, into whose gene the PInT system is placed. Moreover, targeting is shown to enable enzymatic activity in the targeted region. Using the DNA methylase DNMT1, there are local increases in DNA methylation. Similarly, targeting the histone deacetylase HDAC5 results in local decreases in histone H3 acetylation.
We thank the reviewer for a thoughtful and helpful review.
What is not clear from these experiments, however, is whether the targeted proteins can interact normally with partner proteins to form functional complexes. One necessary control is to add ChIP for at least one interacting protein each for DNMT1 and for HDAC5 and show that targeting permits normal protein-protein interactions. This experiment is straightforward as specific interacting proteins are known and good antibodies to precipitate those proteins are available.
This is a good suggestion and we plan on doing this experiment in our 59B-Y-HDAC5 and 89B-Y-DNMT1 lines with and without ABA using interacting proteins. The exact interacting protein to be used will depend on the antibodies availability and quality, which we will test. We will start with UHRF1 and HDAC3 for PYL-Dnmt1 and PYL-HDAC5, respectively.
Overall, PInT would likely be useful for many groups studying the effects of chromatin modifiers on a DNA sequence of interest.
The second advance is conceptual and is focused more specifically on triplet repeat expansions. The manuscript describes experiments that measure genetic instability of long CAG-CTG repeats with and without protein targeting. The results show that allele size distributions are not significantly affected by targeting either DNMT1 or HDAC5. One curious outcome that is not discussed is contraction frequency in the HDAC5 experiment. Zero contractions are reported compared to 10-20% contractions in the other two experiments. Authors need to provide an explanation.
*Lack of contractions in this experiment is likely due to the lower number of repeats in this line (59 vs 89/91). It is known that longer repeats display higher frequency of contractions, and contractions are rarely seen in short repeats (Larson et al Neurobiology of Disease 2015, Gomes-Pereira et al PLOS Genet 2007, Morales et al HMG 2020). Albeit, the threshold may be different in our HEK293-derived cells. Of note, we had a clone of 89B-Y-HDAC5 that did not express the expected amount of GFP for unknown reasons and we did not use it here. However, small pool PCRs using this line with 89 repeats showed that contractions were indeed present. Although we cannot rule out that the reason for the contractions is the unknown mutation(s), it suggests that the difference is due to the size of the expansion. We have added a comment in the methods section. *
It reads: “We have noted that cell lines with repeats that are mildly expanded (e.g., 59 CAGs) have fewer contractions than longer ones. This is consistent with several studies in the context of DM1 and HD [82], albeit the size threshold for seeing more contractions may be shorter in HEK293-derived cells than in mice.”
The major issue with this set of experiments is that there is no positive control where instability is shown to be clearly manipulated. A knockdown of FAN1 would be the most likely avenue to pursue for identifying a positive control. This is straightforward to perform since successful FAN1 knockdowns have been described in the literature.
We agree that a positive control to show that the model behaves as expected is necessary. We will add the experiments proposed by the reviewer in the revised version of the manuscript.
The manuscript also looks at effects on gene expression measured by GFP fluorescence intensity. The potential significance is to see if disease-causing genes with expanded triplet repeats can be silenced by targeting chromatin-modifying enzymes. In the examples tested here, the answer seems to be no. Expression of DNMT1 or HDAC5 reduce fluorescence even in the absence of targeting. Upon targeting, there is a small further decrease, but the expanded triplet repeat resists this further decrease. Domain analysis of HDAC5 indicates that protein-protein interactions, not deacetylase activity, are important for silencing. The key interaction may be with HDAC3, since small molecule inhibition of HDAC3 relieved repeat length-dependent silencing by HDAC5. It was very curious that targeting HDAC3 actually increased expression, instead of silencing. The explanation for this observation was inadequate.
We have added the following paragraph to the discussion to address this.
It reads: “We found that targeting of PYL-HDAC3 increases gene expression slightly, independently of repeat size and in the presence of an inhibitor of its catalytic activity. Although this appears counterintuitive, several studies suggest that this is not unexpected. Specifically, HDAC3 has an essential role in gene expression during mouse development that is independent of its catalytic activity [73]. Moreover, HDAC3 binds more readily to genes that are highly expressed in both human and yeast cells [74,75]. The mechanism or function of HDACs binding to highly expressed genes are currently unknown.”
The claim on page 16 final paragraph that the manuscript 'settled a central question for both HDAC5 and DNMT1 and their involvement in CAG/CTG repeat instability' is not supported by the data. Most of the results are negative so it is premature to claim the question is 'settled'.
We have rephrased all the conclusions about this in the text, emphasizing that we find no evidence of a role in cis, rather than stating that there is no role in cis.
Overall, with appropriate modifications described here, these experiments would be of interest with regards to potential therapies of triplet repeat expansion diseases, where silencing the expanded gene is the goal.
**Minor concerns**
P 4, last line. 59 bp should read 59 repeats - This is now fixed.
P 5, line 2. 38 bp of what? This is now amended. It reads: “The CAG/CTG repeats affect splicing of the reporter in a length-dependent manner, with longer repeats leading to more robust insertion of an alternative CAG exon that includes 38 nucleotides downstream of the CAG, creating a frameshift [30].”
P 10, first paragraph. DNA methylation levels rise from ~10% to ~20% with DNMT1 targeting. Is there a good precedent in the literature that the magnitude of this increase can be expected to be biologically meaningful?
To our knowledge, it is the first time that DNMT1 is used for targeted epigenome editing. This is therefore the first evidence that targeting DNMT1 leads to silencing of a reporter construct. Nevertheless, this reviewer’s comment stands: is an increase in DNA methylation of 10 to 20% biologically relevant? The answer to this is yes, changes in 10-20% are known to have functional impact on gene expression in various settings (for example see the recent study in developing oocytes by Li et al Nature 2018). Furthermore, there is evidence that DNMT1 has weak de novo activity (Li et al Nature 2018, Wang et al Nat Genet 2020), consistent with a small increase in CpG methylation upon targeting. We now acknowledge in the discussion that one reason for the lack of effect upon targeting may be that the changes in CpG methylation are not dramatic enough. We also point out more clearly that changes of 10 to 20% are correlated with changes in repeat instability (Dion et al HMG 2008). We have amended the text to reflect this.
The results now reads “To do so, we performed bisulfite sequencing after targeting PYL-DNMT1 for 30 days. This led to changes of 10 to 20% in the levels of CpG methylation, a modest increase(Fig. 3C), which is in line with the weak de novo methyltransferase activity of DNMT1 (for example see [39,40]). Similar changes in levels of CpG methylation in Dnmt1 heterozygous ovaries and testes were seen to correlate with changes in repeat instability in vivo [31].”
The discussion now states: “It should be pointed out that there remains the possibility that DNMT1 targeting did not lead to large enough changes in CpG methylation to affect repeat instability.”
P12 first paragraph. Text describing Fig 5 is confusing. First, GFP expression is referred to in terms of fold decrease, but subsequently in percent. Second, the ABA-induced silencing looks to reduce expression from about 0.6 to 0.5 of control. I presume this is where the claim of 16% comes from but it was not clear. Indeed, this is what we mean.
We now state: “In 16B-Y-DNMT1 cells, ABA treatment decreased GFP expression by 2.2-fold compared to DMSO treatment alone. Surprisingly, ABA-induced silencing was 1.8 fold compared to DMSO alone, or 16% less efficient in 89B-Y-DNMT1 than in 16B-Y-DNMT1 cells.”
P 15 paragraph 2. Where does the P value of 0.78 come from? Fig 7B shows no corresponding value. The P-value in figure 7B has now been corrected.
Reviewer #1 (Significance (Required)):
See above.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
**Summary:**
We still do not know whether epigenetics contributes to repeat instability and/or transcriptional activity in unstable CAG/CTG repeat associated pathologies. The aim of this manuscript is to examine whether induced binding of DNMT1 (CpG methylation) or HDAC5 (histone H3 acetylation) modulates CAG/CTG repeat instability and/or gene silencing upon expansion. For this the authors developed a highly sophisticated reporter system (PlnT) that allows to recruit a specific chromatin modifying enzyme (DNMT1/ HDAC5) to a GFP reporter near a CAG/CTG expansion, in the course of transcription (Dox-inducible promoter). This is to determine whether the CTGs, when lengthened and transcribed, become unstable or impede gene activity via epigenetic modifications.
We appreciate the reviewer highlighting the importance of the question that we address here and the usefulness of PInT.
**Findings:**
1.Binding of DNMT1 to the reporter results in a modest increase (~10%) in local DNA methylation, with no change in repeat instability.
3.Targeting HDAC5 to the reporter results in local reduction in histone H3 acetylation, with no effect on repeat stability.
4.DNMT1/HDAC5 binding reduces GFP intensity differentially, in normal but not expanded alleles.
5.The N-terminal domain of HDAC5, when mutated, abolishes the reduction in GFP expression levels.
6.RGFP966 abolishes the allele-specific effect of HDAC5, resulting in a general decrease in GFP expression regardless of repeat tract size
7.CTG expanded alleles abolish the reduction in GFP repression by HDAC5 via HDAC3 activity
**Conclusions:**
Based on the results using the PlnT reporter assay, the authors claim that:
1.HDAC5 and DNMT1 do not affect repeat instability in cis
2.Expanded CAG/CTGs reduce the efficiency of gene silencing by targeting DNMT1/HDAC5 to the locus
3.Gene silencing that is mediated by HDAC5 recruitment can be abolished by inhibition of HDAC3 activity
Unfortunately, none of the claims in this manuscript are convincing.
We note that in the comments below the reviewer does not include a reason why he/she does not find the claims convincing. We therefore cannot address this criticism.
**General Comments:**
The major drawback of the PlnT experimental approach is that it ignores the importance of the flanking regions and the genomic organization of the endogenous locus. This is a major concern as it makes the conclusions irrelevant to the related loci. In the case of myotonic dystrophy type 1, for example, the reporter should reside within a CpG island, should be positioned immediately next to CTCF binding site(s), and should be transcribed bi-directionally.
HDAC3 and DNMT1 were found to have effects on repeat instability both at reporters, which do not harbour flanking sequences from disease loci, and indeed at endogenous loci in vivo (Dion et al HMG 2008, Debacker et al PLoS Biol 2012, Suelves et al Sci Rep 2017, Williams et al PNAS 2020). This highlights the fact that cis elements from disease loci are not required for chromatin modifiers to affect repeat instability.
The reviewer is suggesting a very interesting set of experiments where specific sequences may be added to our reporter and tested for their influence on gene expression and on repeat instability. PInT is ideally suited for this and we have now added a paragraph highlighting this in the discussion. We have also highlighted that the current study aims to isolate the repeats from its cis-elements to specifically side-step potential locus-specific effects and to look for chromatin modifiers that would be useful for epigenome editing for as many loci as possible.
Furthermore, only large expansions (at least several hundred copies) can trigger heterochromatin at the DM1 locus. None of these features are recapitulated by the PlnT reporter assay, making it difficult to draw any conclusion regarding the role of these chromatin modifying enzymes to the locus.
This is true for DM1 but untrue for other disease loci. For example, we have shown that there are changes in the flanking chromatin marks at the SCA1 locus of a mouse model with 145 repeats (Dion et al HMG 2008), DNA methylation is also affected near a SCA7 transgene with 92 CAG repeats (Libby et al PLoS Genet 2008) and transgenes containing CAG repeats (without the flanking sequences) lead to silencing regardless of where the transgene is integrated in the genome (Saveliev et al Nature 2003). Moreover, HDAC5 had effects on repeat expansion in a cell-based shuttle system containing as few as 22 CAG repeats (Gannon et al NAR 2012), again suggesting that chromatin modifiers affect repeat instability in a wide range of repeat sizes. We have reviewed this in Dion and Wilson TiG 2009.
In fact, the authors state in their Discussion that "targeting a chromatin modifying peptide to different loci can have very different effects"!
This is indeed the case and the reason why we sought to control for locus-specific effects using an exogenous reporter.
To better substantiate their conclusions the authors must set up an improved model system that takes into account the flanking regions and the 3D genomic organization of the locus (TADs). The preferable approach would be to insert a reporter cassette by homologous recombination into the differentially methylated/acetylated regions near the repeats, and compare between normal vs. expanded alleles.
We would like to point out that we have recently published a study where we looked at 3D chromatin folding at the DM1, HD, and the GFP transgene used here. We did not find any evidence for changes in TADs that would underlie changes in repeat instability at these loci (Ruiz Buendia et al Sci Advances 2020). We therefore do not think that it would be important to further manipulate 3D genomic organization in this context.
To be clear, we are not denying that cis elements are likely to have an effect, there is plenty of evidence supporting this. Rather, we are using a reporter assay to disentangle the potential locus-specific (or cis-element specific) effects from the trans-activating factors. In short, we focus on the trans-acting factors rather than on the cis-elements, as suggested by the reviewer.
We believe that the addition of the following paragraph highlights the goal of our study and also bring in the idea that cis acting elements can be studied using PInT.
It now reads:
“We designed PInT specifically to isolate expanded repeats tracts from other potential locus-specific cis elements. This is helpful to identify factors that would affect instability and/or gene expression across several diseases. Moreover, both HDAC3 and DNMT1 were found to impact repeat instability at different loci, including at reporter genes [31,33,36,37,45]. These observations highlight that cis-acting elements from disease loci are not required by chromatin modifiers to affect repeat instability. A potential application of PInT includes cloning in specific cis elements, including CTCF binding sites and CpG islands, next to the repeat tract and evaluate their effects on instability with or without targeting. In fact, PInT can be used to clone any sequence of interest near the targeting site and can be applied for a wide array of applications, beyond the study of expanded CAG/CTG repeats.”
My impression was that there is a lot of data but none of it makes sense.
The focus of the manuscript is not entirely clear: it starts with monitoring the effect of epigenetics on repeat instability and gene activity, then it shifts to the mechanism by which HDAC5 functions, and ends with the allele-specific effect of HDAC5 on gene expression. I lost my train of thought.
We have now improved the transitions in this new version of this manuscript. Specifically, at the core of this manuscript is the development of PInT, which is highly versatile and allowed us to study multiple aspects of expanded CAG/CTG repeat biology. We hope that it is now clearer.
**Other concerns:**
(1)the modest increase in methylation levels following DNMT1 recruitment (10%, reaching a total of 20% at the most) prevents from drawing any conclusions regarding the effect of methylation on stability or expression.
As mentioned in the response to reviewer 1 above, although 10% to 20% of CpG methylation are associated with changes in gene expression in a variety of settings, we now point out that one reason for the lack of effect in cis is that the de novo activity of DNMT1 is too weak to produce an effect.
(2)The effect of protein targeting on GFP levels should be better defined at the RNA/protein level. Does it act by blocking transcription? alternative splicing? or alters steady state levels?
Although the exact mechanism remains unclear, this goes beyond the current scope of this study. All these possibilities remain possible as we pointed out in the discussion.
(3)Fig 5: the scale is different for A vs. B and C. Also, better to compare the effect of targeting on equal sized expansions (either 91, 89 or 58 repeats).
We have fixed the scale on the figures.
Unfortunately, it is not possible to have the same repeat sizes for all the cell lines because by their very nature, repeats are unstable. We have added a note relating to this in the methods.
It reads: “Notably, it is not possible to obtain several stable lines with the exact same repeat size as they are, by their nature, highly unstable. This is why we have lines with different repeat sizes. Furthermore, the sizes can change over time and upon thawing.”
(4)Add asterix for significance in all figures.
This has now been done.
(5)Figure 6: show raw data rather than normalized.
We have now added representative flow cytometry profiles for each construct as a new supplementary figure (S5).
(6)Figure 7: there is a notable difference in GFP expression levels in untreated wild type control (16 CAG repeats) between A vs. B. Why?
Fig. 7a shows PYL targeting only, whereas 7b shows the GFP expression upon PYL-HDAC5 targeting. The values for PYL-HDAC5 targeting are lower because targeting it, unlike targeting PYL alone, silences the reporter.
(7)Avoid redundancy. No need to show schematic representations so many times.
We believe that the schematics make it clearer for the reader.
Reviewer #2 (Significance (Required)):
REFEREES CROSS-COMMENTING
I totally agree with the Reviewer #1 that the PinT targeting system is a potent experimental tool to study the function of specific chromatin binding proteins. However, the significance of the flanking regions is discounted.
We hope it is now clear that we are not discounting the potential significance of flanking regions and that rather we have designed the system to avoid their potentially complicating effects.
The fact that the recruitment of HDAC5 has resulted in a significant reduction in acetylated histones provides evidence for that "the targeted proteins can interact normally with partner proteins to form functional complexes". Still, I agree with that the activity of DNMT1 needs to be better established, considering the minor increase in DNA methylation levels.
We will be using ChIP against interacting proteins of DNMT1 and HDAC5 to address this issue.
The request for a positive control for repeat instability is totally correct.
We will be adding this in the revised manuscript.
It is difficult to discuss the missing effect of HDAC5 on contractions or the unexpected effect of HDAC3 on gene silencing bearing in mind the limits of the experimental system.
There is no expectation for the effect of HDAC5 on contractions as this has not been studied in any system yet. However, we believe that there is no contractions not because of HDAC5 per se but rather because of the shorter repeat size this line has (see comment to reviewer 1 above). We have now addressed the “unexpected effect” of HDAC3 by citing a number of studies finding a similar evolutionary conserved effect (see comment to Reviewer 1 above).
I also agree with the statement that "this manuscript settled a central question for both HDAC5 and DNMT1 and their involvement in CAG/CTG repeat instability", is not supported by the data.
We have now rephrased our conclusions. In this particular case, we changed ‘settled’ to ‘addressed’. We have also rephrased this in the results headings.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary:
We still do not know whether epigenetics contributes to repeat instability and/or transcriptional activity in unstable CAG/CTG repeat associated pathologies. The aim of this manuscript is to examine whether induced binding of DNMT1 (CpG methylation) or HDAC5 (histone H3 acetylation) modulates CAG/CTG repeat instability and/or gene silencing upon expansion. For this the authors developed a highly sophisticated reporter system (PlnT) that allows to recruit a specific chromatin modifying enzyme (DNMT1/ HDAC5) to a GFP reporter near a CAG/CTG expansion, in the course of transcription (Dox-inducible promoter). This is to …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary:
We still do not know whether epigenetics contributes to repeat instability and/or transcriptional activity in unstable CAG/CTG repeat associated pathologies. The aim of this manuscript is to examine whether induced binding of DNMT1 (CpG methylation) or HDAC5 (histone H3 acetylation) modulates CAG/CTG repeat instability and/or gene silencing upon expansion. For this the authors developed a highly sophisticated reporter system (PlnT) that allows to recruit a specific chromatin modifying enzyme (DNMT1/ HDAC5) to a GFP reporter near a CAG/CTG expansion, in the course of transcription (Dox-inducible promoter). This is to determine whether the CTGs, when lengthened and transcribed, become unstable or impede gene activity via epigenetic modifications.
Findings:
1.Binding of DNMT1 to the reporter results in a modest increase (~10%) in local DNA methylation, with no change in repeat instability.
3.Targeting HDAC5 to the reporter results in local reduction in histone H3 acetylation, with no effect on repeat stability.
4.DNMT1/HDAC5 binding reduces GFP intensity differentially, in normal but not expanded alleles.
5.The N-terminal domain of HDAC5, when mutated, abolishes the reduction in GFP expression levels.
6.RGFP966 abolishes the allele-specific effect of HDAC5, resulting in a general decrease in GFP expression regardless of repeat tract size
7.CTG expanded alleles abolish the reduction in GFP repression by HDAC5 via HDAC3 activity
Conclusions:
Based on the results using the PlnT reporter assay, the authors claim that:
1.HDAC5 and DNMT1 do not affect repeat instability in cis
2.Expanded CAG/CTGs reduce the efficiency of gene silencing by targeting DNMT1/HDAC5 to the locus
3.Gene silencing that is mediated by HDAC5 recruitment can be abolished by inhibition of HDAC3 activity
Unfortunately, none of the claims in this manuscript are convincing.
General Comments:
The major drawback of the PlnT experimental approach is that it ignores the importance of the flanking regions and the genomic organization of the endogenous locus. This is a major concern as it makes the conclusions irrelevant to the related loci. In the case of myotonic dystrophy type 1, for example, the reporter should reside within a CpG island, should be positioned immediately next to CTCF binding site(s), and should be transcribed bi-directionally. Furthermore, only large expansions (at least several hundred copies) can trigger heterochromatin at the DM1 locus. None of these features are recapitulated by the PlnT reporter assay, making it difficult to draw any conclusion regarding the role of these chromatin modifying enzymes to the locus. In fact the authors state in their Discussion that "targeting a chromatin modifying peptide to different loci can have very different effects"! To better substantiate their conclusions the authors must set up an improved model system that takes into account the flanking regions and the 3D genomic organization of the locus (TADs). The preferable approach would be to insert a reporter cassette by homologous recombination into the differentially methylated/acetylated regions near the repeats, and compare between normal vs. expanded alleles.
My impression was that there is a lot of data but none of it makes sense.
The focus of the manuscript is not entirely clear: it starts with monitoring the effect of epigenetics on repeat instability and gene activity, then it shifts to the mechanism by which HDAC5 functions, and ends with the allele-specific effect of HDAC5 on gene expression. I lost my train of thought.
Other concerns:
(1)the modest increase in methylation levels following DNMT1 recruitment (10%, reaching a total of 20% at the most) prevents from drawing any conclusions regarding the effect of methylation on stability or expression.
(2)The effect of protein targeting on GFP levels should be better defined at the RNA/protein level. Does it act by blocking transcription? alternative splicing? or alters steady state levels?
(3)Fig 5: the scale is different for A vs. B and C. Also, better to compare the effect of targeting on equal sized expansions (either 91, 89 or 58 repeats).
(4)Add asterix for significance in all figures.
(5)Figure 6: show raw data rather than normalized.
(6)Figure 7: there is a notable difference in GFP expression levels in untreated wild type control (16 CAG repeats) between A vs. B. Why?
(7)Avoid redundancy. No need to show schematic representations so many times.
Significance
REFEREES CROSS-COMMENTING
I totally agree with the Reviewer #1 that the PinT targeting system is a potent experimental tool to study the function of specific chromatin binding proteins. However, the significance of the flanking regions is discounted. The fact that the recruitment of HDAC5 has resulted in a significant reduction in acetylated histones provides evidence for that "the targeted proteins can interact normally with partner proteins to form functional complexes". Still, I agree with that the activity of DNMT1 needs to be better established, considering the minor increase in DNA methylation levels. The request for a positive control for repeat instability is totally correct. It is difficult to discuss the missing effect of HDAC5 on contractions or the unexpected effect of HDAC3 on gene silencing bearing in mind the limits of the experimental system. I also agree with the statement that "this manuscript settled a central question for both HDAC5 and DNMT1 and their involvement in CAG/CTG repeat instability", is not supported by the data.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The manuscript describes two advances. First is the technical development for a protein targeting system called PInT that brings a target protein close to (~320 bp) a DNA sequence of interest. The idea is that localisation of the target protein allows one to distinguish its effects on the DNA sequence either in cis (when targeted) or in trans (when not targeted but expressed at the same level). Since targeting is conveyed by simply adding the small molecule ABA to the experiment, it is easy to compare the two situations. This is a clever idea and it is substantiated by data showing that the components of PInT do not affect …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The manuscript describes two advances. First is the technical development for a protein targeting system called PInT that brings a target protein close to (~320 bp) a DNA sequence of interest. The idea is that localisation of the target protein allows one to distinguish its effects on the DNA sequence either in cis (when targeted) or in trans (when not targeted but expressed at the same level). Since targeting is conveyed by simply adding the small molecule ABA to the experiment, it is easy to compare the two situations. This is a clever idea and it is substantiated by data showing that the components of PInT do not affect triplet repeat instability or gene expression of GFP, into whose gene the PInT system is placed. Moreover, targeting is shown to enable enzymatic activity in the targeted region. Using the DNA methylase DNMT1, there are local increases in DNA methylation. Similarly, targeting the histone deacetylase HDAC5 results in local decreases in histone H3 acetylation. What is not clear from these experiments, however, is whether the targeted proteins can interact normally with partner proteins to form functional complexes. One necessary control is to add ChIP for at least one interacting protein each for DNMT1 and for HDAC5 and show that targeting permits normal protein-protein interactions. This experiment is straightforward as specific interacting proteins are known and good antibodies to precipitate those proteins are available. Overall, PInT would likely be useful for many groups studying the effects of chromatin modifiers on a DNA sequence of interest.
The second advance is conceptual and is focused more specifically on triplet repeat expansions. The manuscript describes experiments that measure genetic instability of long CAG-CTG repeats with and without protein targeting. The results show that allele size distributions are not significantly affected by targeting either DNMT1 or HDAC5. One curious outcome that is not discussed is contraction frequency in the HDAC5 experiment. Zero contractions are reported compared to 10-20% contractions in the other two experiments. Authors need to provide an explanation. The major issue with this set of experiments is that there is no positive control where instability is shown to be clearly manipulated. A knockdown of FAN1 would be the most likely avenue to pursue for identifying a positive control. This is straightforward to perform since successful FAN1 knockdowns have been described in the literature. The manuscript also looks at effects on gene expression measured by GFP fluorescence intensity. The potential significance is to see if disease-causing genes with expanded triplet repeats can be silenced by targeting chromatin-modifying enzymes. In the examples tested here, the answer seems to be no. Expression of DNMT1 or HDAC5 reduce fluorescence even in the absence of targeting. Upon targeting, there is a small further decrease, but the expanded triplet repeat resists this further decrease. Domain analysis of HDAC5 indicates that protein-protein interactions, not deacetylase activity, are important for silencing. The key interaction may be with HDAC3, since small molecule inhibition of HDAC3 relieved repeat length-dependent silencing by HDAC5. It was very curious that targeting HDAC3 actually increased expression, instead of silencing. The explanation for this observation was inadequate. The claim on page 16 final paragraph that the manuscript 'settled a central question for both HDAC5 and DNMT1 and their involvement in CAG/CTG repeat instability' is not supported by the data. Most of the results are negative so it is premature to claim the question is 'settled'. Overall, with appropriate modifications described here, these experiments would be of interest with regards to potential therapies of triplet repeat expansion diseases, where silencing the expanded gene is the goal.
Minor concerns
P 4, last line. 59 bp should read 59 repeats
P 5, line 2. 38 bp of what?
P 10, first paragraph. DNA methylation levels rise from ~10% to ~20% with DNMT1 targeting. Is there a good precedent in the literature that the magnitude of this increase can be expected to be biologically meaningful?
P12 first paragraph. Text describing Fig 5 is confusing. First, GFP expression is referred to in terms of fold decrease, but subsequently in percent. Second, the ABA-induced silencing looks to reduce expression from about 0.6 to 0.5 of control. I presume this is where the claim of 16% comes from but it was not clear.
P 15 paragraph 2. Where does the P value of 0.78 come from? Fig 7B shows no corresponding value.
Significance
See above.
-
-