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The EMBO Journal Peer Review Process File - EMBO-2011-77390

Manuscript EMBO-2011-77390

The DNA-binding domain of the Chd1 chromatin remodelling enzyme contains SANT and SLIDE domains Daniel P. Ryan, Ramasubramanian Sundaramoorthy, David Martin, Vijender Singh and Tom OwenHughes Corresponding author: Tom Owen-Hughes, University of Dundee

Review timeline:

Submission date: Editorial Decision: Revision received: Editorial Decision: Accepted:

24 February 2011 30 March 2011 20 April 2011 21 April 2011 21 April 2011

Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.)

1st Editorial Decision

30 March 2011

Thank you for submitting your manuscript for consideration by The EMBO Journal. It has been now been evaluated by three referees and I enclose their reports below. As you will see the referees find the identification of SANT and SLIDE domains in the C-terminus of Chd1 to be interesting and important and they recommend publication in The EMBO Journal pending some minor revision. While the referees suggest a small number of experiments, they do not indicate that they are essential for publication, therefore if you have the data or it can be easily obtained it should be incorporated but they are not absolutely required. Given the interest in the study, should you be able to address the concerns we would be happy to consider a revised version of the manuscript for publication in The EMBO Journal. I should remind you that it is EMBO Journal policy to allow a single round of revision only and that, therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. When you submit a revised version to the EMBO Journal, please make sure you upload a letter of response to the referees' comments. Please note that when preparing your letter of response to the referees' comments that this will form part of the Review Process File, and will therefore be available online to the community. For more details on our Transparent Editorial Process initiative, please visit our website: http://www.nature.com/emboj/about/process.html Thank you for the opportunity to consider your work for publication. I look forward to your revision. Yours sincerely,

© European Molecular Biology Organization

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Editor The EMBO Journal -----------------------------------------------REFEREE COMMENTS Referee #1: The diversity of nucleosome remodelling enzymes fosters the desire to reveal common mechanistic themes in remodelling reactions. In this respect the manuscript by Ryan et al. presents some very satisfying novel findings. They show that the C-terminus of CHD1 contains a major DNA binding domain with striking similarity to the one from the ISWI ATPase, which has been described earlier as consisting of SANT and SLIDE domains from the Becker and Müller labs. The similarity is revealed by the crystal structure that is presented in the current manuscript. The sequence alignments reveal - in hindsight - that this domain is conserved among the CHD relatives. The authors mutate basic residues in the putative DNA binding surface (the DNA binding is only modelled) and confirm their importance for the functioning of CHD1 in vitro and in vivo. This is an important paper that will be much appreciated in the field. The comments listed below may serve to improve certain aspects of the manuscript. Considering the wealth of data presented these are relatively minor issues. 1. It would be interesting to see to what extent the C-terminus of CHD1 contributes to DNA binding in the context of the full length protein. The constructs analysed in figure 1A should be assayed for binding to the 45bp or 20bp DNA fragment. In the corresponding analyses of the ISWI protein, the authors found differences in DNA and nucleosome binding (Grüne, 2003). 2. The two C-terminal deletions (1-1010, 1-860) were inactive in all assays. I assume that they are expressed well and show no sign of global misfolding or aggregation? The expression levels and integrity of the point-mutated CHD derivates is well documented in figure S3A. Is there any difference in solubility between these proteins? 3. Bouazoune et al. (EMBO J. 2002) had shown previously that DNA binding of the Drosophila Mi2 ATPase required N-terminal chromodomains for DNA binding. How does the C-terminus of CHD3/4 family members compare to CHD1? 4. The authors compare the known structures of ISWI and CHD1 in figure 4. The ISWI structure also includes an N-terminal "HAND" domain, which is not included in the CHD1-DBD structure. Do the sequence alignments / secondary structure predictions suggest a HAND-related structure in CHD1? 5. In figure 5D the model for DNA-bound SANT/SLIDE domain with calculated minimal energy is presented. Alternative models with very similar features are presented in the supplements. The main text should include at least one sentence to indicate that all other models are very similar and mainly differ in register or orientation of the DNA. 6. The discussion about nucleosome spacing is entirely inadequate and should be deleted. The authors do not distinguish properly between effects of the ATPase subunits themselves or the effects of associated subunits in remodelling complexes. The literature is wrongly cited (e.g. recombinant ISWI does not space nucleosomes, but leads to close packing) or not cited at all (e.g. recent work from Narlikar's group on the contribution of ACF1 to spacing. The authors should either present a proper discussion or omit the topic entirely. 7. On page 5 (after figure 1D) it is concluded "In concert, the observations suggest a feature within CHD1 required both for engagement with nucleosomes and the catalytic action". This statement could be a bit more specific - at least the C-terminus could be mentioned.


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Referee #2: In this study, Ryan and colleagues investigate the role of the Chd1 C-terminal putative DNA binding region in DNA and nucleosome binding and nucleosome remodeling. Using protein deletion constructs, the authors determine that a minimal domain consisting of residues 1009 to 1274 binds to 20-bp of DNA. Next they further examine this region using a structural approach. This approach reveals that the Chd1 DNA binding domain actually consists of SANT and SLIDE domains, a module that was initially identified in the Drosophila ISWI chromatin remodeling factor. Crystallization of the SANT-SLIDE domains allows the authors to model DNA binding based on the electrostatic surface potential and predict which residues are supposedly important for DNA binding. Ryan and colleagues verify the relevance of their predictions experimentally both in vitro and in vivo using point mutagenesis and show that mutations in the Chd1 SANT-SLIDE domains indeed impact Chd1 functions. Finally, the authors extend their findings by identifying a putative SLIDE domain in proteins belonging to the third subfamily of CHD proteins by using sequence analyses. CHD proteins represent a major family of chromatin remodeling factors and understanding how these proteins regulate genome functions is undoubtedly important. A major strength of this work is the finding that Chd1 proteins harbor SANT and SLIDE domains (features that were missed by conventional primary sequence analyses) and show how critical these domains are in Chd1 functions. Furthermore, the authors provide high-quality crystallographic data of this region and also identify SLIDE domains in the poorly characterized CHD subfamily III. This study is clear, straightforward and convincing and its publication will certainly be of interest to the field. Comments below are for the authors consideration to strengthen an already strong manuscript. Comments: 1) An intriguing experiment would be to measure the nucleosome spacing established by yeast CHD1 and compare it to the ISWI spacing. Have the authors done this? 2) Comments on points of presentation: a) Labeling of the helices (1-10) in figure 4 would help clarify. b) The nomenclature for the groups of mutations in different regions of the protein is confusing. Single site suggests to me a single mutation but in fact often referred in this manuscript to multiple mutations in a single region of the protein. c) Fig. 3 legend: typo "Melanogastor". d) The authors may want to spell the meaning of SANT (SWI3, ADA2, N-CoR and TFIIIB) and SLIDE (SANT-like ISWI domain) once in the text for less specialized readers. Referee #3: This study by Owen-Hughes and colleagues describes a functional and structural characterization of the DNA-binding domain (DBD) of the ATP-dependent chromatin remodeling protein Chd1 from yeast. Structural information on chromatin remodeling enzymes is still very limited. Therefore, analyses addressing this question are of great interest in order to eventually understand the mechanisms of action of chromatin remodeling enzymes. Using EMSA analysis the authors identified a minimal DNA binding motif within the C-terminus of Chd1 (Fig. 1B, Fig. 2B, C) that binds to DNA fragments greater than 20 bp in length as well as to nucleosome core particles containing linker DNA (Fig. 2). Deletion of the DBD abolished ATPase activity and, consequently, nucleosome sliding activity of Chd1 (Fig. 1C,D). The authors resolved the crystal structure of the minimal DBD and uncovered structural similarities to SANT and SLIDE domains of the related remodeler ISWI (Fig. 3, Fig.4). Modeling of the structure suggested a mode of DNA binding via a positively charged surface on this module (Fig. 5B, D). The authors therefore investigated the effects of mutations in several basic residues predicted to make contacts with the DNA backbone. Simultaneous mutations of amino acids located in the different parts of the DBD (SANT, SLIDE) abolished in vitro DNA binding (Fig. 5C) and reduced binding to nucleosome core


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particles (Fig. 6A). Interestingly, combined mutations virtually abolished nucleosome sliding activity (Fig. 6B, Suppl. Fig. 3B) but only modestly affected ATPase activity (Fig. 6C). The same mutations were tested in vivo for their ability to rescue a temperature sensitive growth phenotype of an isw1Δ, isw2Δ, chd1Δ triple mutant. Combined mutations in the SANT and SLIDE domains rendered the respective proteins unable to compensate for the loss of wild-type Chd1 in this assay, whereas mutations in either domain led to a partial rescue of the growth defect (Fig. 6D). Finally, sequence alignments of the Chd1 SANT domains identified related motifs in other protein groups and showed close relation between Chd1- and ISWI-type SANT domains (Fig.7A, Suppl. Fig.4). These analyses also predicted the presence of SLIDE domains in the human CHD6-9 subgroup of proteins (Fig. 7B; Suppl. Fig. 5). The results presented in this paper provide interesting insight into the properties and activities of Chd1 and by inference of other chromatin remodeling factors, such as the CHD6-9 group of remodelers. There are just two points I was wondering about: First, have the authors tested, if the proteins bearing K/R mutations in the DNA-interacting surface can localize to the nucleus or chromatin, respectively, in vivo? Second, similar to the region between helices alpha9 and alpha10, the HL1 region is only poorly conserved among Chd1 proteins and considerably longer in budding yeast thus making it a prominent feature of the yeast DBD structure. Given its apparent positive surface charge - is this part of the DBD also involved in making DNA contacts? Perhaps the authors could discuss the potential function of this sequence part in more detail. 1st Revision - authors' response

20 April 2011

Referee #1: The diversity of nucleosome remodelling enzymes fosters the desire to reveal common mechanistic themes in remodelling reactions. In this respect the manuscript by Ryan et al. presents some very satisfying novel findings. They show that the C-terminus of CHD1 contains a major DNA binding domain with striking similarity to the one from the ISWI ATPase, which has been described earlier as consisting of SANT and SLIDE domains from the Becker and Müller labs. The similarity is revealed by the crystal structure that is presented in the current manuscript. The sequence alignments reveal - in hindsight - that this domain is conserved among the CHD relatives. The authors mutate basic residues in the putative DNA binding surface (the DNA binding is only modelled) and confirm their importance for the functioning of CHD1 in vitro and in vivo. This is an important paper that will be much appreciated in the field. The comments listed below may serve to improve certain aspects of the manuscript. Considering the wealth of data presented these are relatively minor issues. 1.It would be interesting to see to what extent the C-terminus of CHD1 contributes to DNA binding in the context of the full length protein. The constructs analysed in figure 1A should be assayed for binding to the 45bp or 20bp DNA fragment. In the corresponding analyses of the ISWI protein, the authors found differences in DNA and nucleosome binding (Grüne, 2003). This has been added in as a Supplementary Figure S1B. The results mirror that of nucleosome binding, i.e. full-length and 1-1305∆C Chd1 constructs readily bind the 45 bp DNA fragment whereas 1-1010∆C and 1-860∆C show no detectable DNA-binding activity. 2.The two C-terminal deletions (1-1010, 1-860) were inactive in all assays. I assume that they are expressed well and show no sign of global misfolding or aggregation? The expression levels and integrity of the point-mutated CHD derivates is well documented in figure S3A. Is there any difference in solubility between these proteins? A gel showing samples of 1-860∆C, 1-1010∆C, and 1-1305∆C proteins is included in Supplementary Figure S1A. All three constructs expressed better than the full-length protein and behaved well during purification. 1-1010∆C expresses extremely well (an order of magnitude or two better) and was very soluble—we have concentrated this construct to >10 mg/ml without evidence of aggregation. Although 1-1010∆C and 1-860∆C were inactive in our assays, they still retained


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unstimulated ATPase activity (Figure 1D) that was comparable to the wild-type full-length protein, suggesting they are folded correctly but just unresponsive to DNA/nucleosomal stimuli. The full-length point mutants all behaved similar to the wild-type protein; there were no obvious differences in the solubilities. However, this hasn’t been explored extensively as we have not concentrated these proteins beyond low micromolar concentrations as our assays did not require concentration to high levels. 3.Bouazoune et al. (EMBO J. 2002) had shown previously that DNA binding of the Drosophila Mi2 ATPase required N-terminal chromodomains for DNA binding. How does the C-terminus of CHD3/4 family members compare to CHD1? CHD3/4 sequences did appear in our list of hits, but were much lower scoring, and so it is difficult to say whether this is significant or not. So, it is possible SLIDE-like domains may also be present in the C-terminus of CHD3/4, but have diverged significantly from that of Chd1. This will require further analysis to determine if this is the case. This has been reflected in the manuscript by the addition of a supplementary dataset (Supplementary Dataset 1) with the list of results from our final SLIDE-related Hmmer search and the following text on page 12 in the section ‘SLIDE domains are found in other Chd proteins’ “Sequences from CHD3/4 proteins also appeared in our search results but were poor-scoring and ranked alongside a number of general Myb/SANT-containing sequences (Supplementary Dataset 1). Thus, CHD3/4 sequences may also contain a SLIDE-related motif, but this is significantly divergent from that of Chd1 and further functional and structural analysis at will be required to determine the significance of this relationship.” 4.The authors compare the known structures of ISWI and CHD1 in figure 4. The ISWI structure also includes an N-terminal "HAND" domain, which is not included in the CHD1-DBD structure. Do the sequence alignments / secondary structure predictions suggest a HAND-related structure in CHD1? There is no obvious evidence for a HAND-like domain within the Chd1 sequence. The HAND domain of ISWI is directly N-terminal to the SANT domain and is ~100 residues in length. There are only ~90 available residues in yeast Chd1 between the start of the SANT domain (residue 1009) and the end of the C-terminal bridge structure (which spans residues ~860-920) described by Hauk et al. 2010 in their structure of the chromo-ATPase domains of yeast Chd1 (PDB ID 3MWY). Secondary structure predictions indicate the presence of only two helices in this 90-residue region (the HAND domain has four) and the remaining residues (~60) are predicted to contain no secondary structural elements. Sequence alignments of HAND sequences with Chd1 are poor. So it seems that the HAND domain may be a specific feature of ISWI proteins. 5. In figure 5D the model for DNA-bound SANT/SLIDE domain with calculated minimal energy is presented. Alternative models with very similar features are presented in the supplements. The main text should include at least one sentence to indicate that all other models are very similar and mainly differ in register or orientation of the DNA. The section of text regarding our modelling has been modified to cover this. The text in bold below is that which has been added or changed. “Using this mutational data to define ambiguous interaction restraints (AIRs) for the HADDOCK webserver (de Vries et al, 2010) we generated an ensemble of computationally-derived models describing how the SANT and SLIDE domains are likely to interact with DNA—similar models are then clustered and the clusters ranked on the overall HADDOCK score (additional details of the modeling can be found in Supplementary Figure S2 and Supplementary Table SI). The lowest energy model from the top-scoring cluster (RMSD 2.8 ± 1.8 Å for the cluster, n=8) is shown in Figure 5D. The models predict the SANT α1 helix binds across the DNA-phosphate backbone and the SLIDE α10 helix docks into the minor groove with the βL region forming additional contacts with the DNA backbone. Models from the top clusters were highly consistent, with all showing similar modes of binding but just differing in the register or orientation of the DNA (Supplementary Figure S2).” 6. The discussion about nucleosome spacing is entirely inadequate and should be deleted. The


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authors do not distinguish properly between effects of the ATPase subunits themselves or the effects of associated subunits in remodelling complexes. The literature is wrongly cited (e.g. recombinant ISWI does not space nucleosomes, but leads to close packing) or not cited at all (e.g. recent work from Narlikar's group on the contribution of ACF1 to spacing. The authors should either present a proper discussion or omit the topic entirely. The referee’s comments have been taken onboard and we have now changed this part of the discussion to duly include citations from the Narlikar group as well as address the other comments made by the referee. Hopefully, this section is now suitable to be included in the manuscript. 7. On page 5 (after figure 1D) it is concluded "In concert, the observations suggest a feature within CHD1 required both for engagement with nucleosomes and the catalytic action". This statement could be a bit more specific - at least the C-terminus could be mentioned. This has been changed to “In concert, these observations suggest the existence of a DNA-binding feature C-terminal to the Snf2-domain of Chd1 required both for engagement with nucleosomes and the catalytic action.” Referee #2: In this study, Ryan and colleagues investigate the role of the Chd1 C-terminal putative DNA binding region in DNA and nucleosome binding and nucleosome remodeling. Using protein deletion constructs, the authors determine that a minimal domain consisting of residues 1009 to 1274 binds to 20-bp of DNA. Next they further examine this region using a structural approach. This approach reveals that the Chd1 DNA binding domain actually consists of SANT and SLIDE domains, a module that was initially identified in the Drosophila ISWI chromatin remodeling factor. Crystallization of the SANT-SLIDE domains allows the authors to model DNA binding based on the electrostatic surface potential and predict which residues are supposedly important for DNA binding. Ryan and colleagues verify the relevance of their predictions experimentally both in vitro and in vivo using point mutagenesis and show that mutations in the Chd1 SANT-SLIDE domains indeed impact Chd1 functions. Finally, the authors extend their findings by identifying a putative SLIDE domain in proteins belonging to the third subfamily of CHD proteins by using sequence analyses. CHD proteins represent a major family of chromatin remodeling factors and understanding how these proteins regulate genome functions is undoubtedly important. A major strength of this work is the finding that Chd1 proteins harbor SANT and SLIDE domains (features that were missed by conventional primary sequence analyses) and show how critical these domains are in Chd1 functions. Furthermore, the authors provide high-quality crystallographic data of this region and also identify SLIDE domains in the poorly characterized CHD subfamily III. This study is clear, straightforward and convincing and its publication will certainly be of interest to the field. Comments below are for the authors consideration to strengthen an already strong manuscript. Comments: 1) An intriguing experiment would be to measure the nucleosome spacing established by yeast CHD1 and compare it to the ISWI spacing. Have the authors done this? We agree with the referee that this would be an interesting experiment, however, we have not yet done this. To do this systematically would take some time and so we have decided not to wait for these experiments to be completed. 2) Comments on points of presentation: a)Labeling of the helices (1-10) in figure 4 would help clarify. Chd1-DBD in Figure 4 has now been labelled with α1-10. b)The nomenclature for the groups of mutations in different regions of the protein is confusing.


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Single site suggests to me a single mutation but in fact often referred in this manuscript to multiple mutations in a single region of the protein. We have removed any instances where single- or double-site are used to describe the mutations and have just explicitly referred to the mutation. We have also changed the format of the mutants by separating multiple mutations with a “/” between each mutations (e.g. R1016A/K1020A). c)Fig. 3 legend: typo "Melanogastor". This has been corrected d)The authors may want to spell the meaning of SANT (SWI3, ADA2, N-CoR and TFIIIB) and SLIDE (SANT-like ISWI domain) once in the text for less specialized readers. We have added these definitions at the first instance of SANT and SLIDE in the introduction (Page 4). Referee #3: This study by Owen-Hughes and colleagues describes a functional and structural characterization of the DNA-binding domain (DBD) of the ATP-dependent chromatin remodeling protein Chd1 from yeast. Structural information on chromatin remodeling enzymes is still very limited. Therefore, analyses addressing this question are of great interest in order to eventually understand the mechanisms of action of chromatin remodeling enzymes. Using EMSA analysis the authors identified a minimal DNA binding motif within the C-terminus of Chd1 (Fig. 1B, Fig. 2B, C) that binds to DNA fragments greater than 20 bp in length as well as to nucleosome core particles containing linker DNA (Fig. 2). Deletion of the DBD abolished ATPase activity and, consequently, nucleosome sliding activity of Chd1 (Fig. 1C,D). The authors resolved the crystal structure of the minimal DBD and uncovered structural similarities to SANT and SLIDE domains of the related remodeler ISWI (Fig. 3, Fig.4). Modeling of the structure suggested a mode of DNA binding via a positively charged surface on this module (Fig. 5B, D). The authors therefore investigated the effects of mutations in several basic residues predicted to make contacts with the DNA backbone. Simultaneous mutations of amino acids located in the different parts of the DBD (SANT, SLIDE) abolished in vitro DNA binding (Fig. 5C) and reduced binding to nucleosome core particles (Fig. 6A). Interestingly, combined mutations virtually abolished nucleosome sliding activity (Fig. 6B, Suppl. Fig. 3B) but only modestly affected ATPase activity (Fig. 6C). The same mutations were tested in vivo for their ability to rescue a temperature sensitive growth phenotype of an isw1∆, isw2∆, chd1∆ triple mutant. Combined mutations in the SANT and SLIDE domains rendered the respective proteins unable to compensate for the loss of wild-type Chd1 in this assay, whereas mutations in either domain led to a partial rescue of the growth defect (Fig. 6D). Finally, sequence alignments of the Chd1 SANT domains identified related motifs in other protein groups and showed close relation between Chd1- and ISWI-type SANT domains (Fig.7A, Suppl. Fig.4). These analyses also predicted the presence of SLIDE domains in the human CHD6-9 subgroup of proteins (Fig. 7B; Suppl. Fig. S5). The results presented in this paper provide interesting insight into the properties and activities of Chd1 and by inference of other chromatin remodeling factors, such as the CHD6-9 group of remodelers. There are just two points I was wondering about: First, have the authors tested, if the proteins bearing K/R mutations in the DNA-interacting surface can localize to the nucleus or chromatin, respectively, in vivo? Second, similar to the region between helices alpha9 and alpha10, the HL1 region is only poorly conserved among Chd1 proteins and considerably longer in budding yeast thus making it a prominent feature of the yeast DBD structure. Given its apparent positive surface charge - is this part of the DBD also involved in making DNA contacts? Perhaps the authors could discuss the potential function of this sequence part in more detail. Regarding the first point, at present we have not looked at the nuclear/chromatin localisation of these proteins, so we cannot rule out the possibility that mutations affect subcellular localisation.


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The reviewer is correct to point out that HL1 region is longer in budding yeast Chd1, but as this region is poorly conserved amongst Chd1 proteins from different species it is unlikely to have a conserved function. A sentence indicating this has been added to the text on page 6. Although there are some basic residues within this region of Chd1-DBD they are not contacting DNA in our Chd1DBD:DNA models. It is possible this region could contact DNA in other parts of the nucleosome, however, the lack of conservation of this feature (even amongst yeast species) suggests that such interactions are unlikely significant to the remodelling activity. Other changes We have removed a duplicate reference for Tsukiyama et al. 1999 We have corrected a typo regarding the e-value quoted for apis mellifera Chd9 in the final results section, 0.0053 should have been 0.053. Small changes have been made to the text in order to adhere to the 55000 character limit. Note: Due to the insertion of an additional supplementary figure (new Figure S1) references to supplementary figures in the referees comments need to be updated accordingly, e.g. Figure S1 is now S2, Figure S2 is now Figure S3 and so on.

2nd Editorial Decision

21 April 2011

I have read through your revised manuscript and your responses to the referees and find that you have satisfactorily addressed all the initial concerns. I am happy to accept the manuscript for publication in The EMBO Journal, I believe it will make a great contribution. Yours sincerely, Editor The EMBO Journal


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