Coevolution of the CDCA7-HELLS ICF-related nucleosome remodeling complex and DNA methyltransferases

Reviewer #1 (Public Review):

Funabiki et al, performed a co-evolutionary analysis of Lsh/HELLS and CDCA7, two factors with links to DNA methylation pathways in mammals, amphibia and fish. The authors suggest that conserved roles for the two factors in DNA methylation maintenance pathways can be traced back to the last eukaryotic common ancestor. Overall, the findings are important and the results could be useful for researchers studying DNA methylation pathways in many different organisms.

Comments on current version:

In the revised version of this manuscript the authors addressed all previously raised issues. I would like to thank them for that. The data is now clearly presented and interpreted and more experimental detail has been added. Thus, the manuscript is much improved and provides an interesting basis for experimental follow-up and further functional investigations.

Reviewer #2 (Public Review):

In this manuscript, Funabiki and colleagues investigated the co-evolution of DNA methylation and nucleosome remolding in eukaryotes. This study is motivated by several observations: (1) despite being ancestrally derived, many eukaryotes lost DNA methylation and/or DNA methyltransferases; (2) over many genomic loci, the establishment and maintenance of DNA methylation relies on a conserved nucleosome remodeling complex composed of CDCA7 and HELLS; (3) it remains unknown if/how this functional link influenced the evolution of DNA methylation. The authors hypothesize that if CDCA7-HELLS function was required for DNA methylation in the last eukaryote common ancestor, this should be accompanied by signatures of co-evolution during eukaryote radiation.

To test this hypothesis, they first set out to investigate the presence/absence of putative functional orthologs of CDCA7, HELLS and DNMTs across major eukaryotic clades. They succeed in identifying homologs of these genes in all clades spanning 180 species. To annotate putative functional orthologs, they use similarity over key functional domains and residues - such as ICF related mutations for CDCA7 and SNF2 domains for HELLS - as well as maximum likelihood phylogenetic analyses. Using established eukaryote phylogenies, the authors conclude that the CDCA7-HELLS-DNMT axis arose in the last common ancestor to all eukaryotes. Importantly, they found recurrent loss events of CDCA7-HELLS-DNMT in at least 40 eukaryotic species, most of them lacking DNA methylation.

Having identified these factors, they successfully identify signatures of co-evolution between DNMTs, CDCA7 and HELLS using CoPAP analysis - a probabilistic model inferring the likelihood of interactions between genes given a set of presence/absence patterns. As a control, such interactions are not detected with other remodelers or chromatin modifying pathways also found across eukaryotes. Expanding on this analysis, the authors found that CDCA7 was more likely to be lost in species without DNA methylation.

In conclusion, the authors suggest that the CDCA7-HELLS-DNMT axis is ancestral in eukaryotes and raise the hypothesis that CDCA7 becomes quickly dispensable upon the loss of DNA methylation and/or that CDCA7 might be the first step toward the switch from DNA methylation-based genome regulation to other modes.

The data and analyses reported are significant and solid. Overall, this work is a conceptual advance in our understanding of the evolutionary coupling between nucleosome remolding and DNA methylation. It also provides a useful resource to study the early origins of DNA methylation related molecular process. Finally, it brings forward the interesting hypothesis that since eukaryotes are faced with the challenge of performing DNA methylation in the context of nucleosome packed DNA, loosing factors such as CDCA7-HELLS likely led to recurrent innovations in chromatin-based genome regulation.

Strengths:
- The hypothesis linking nucleosome remodeling and the evolution of DNA methylation.
- Deep mapping of DNA methylation related process in eukaryotes.
- Identification and evolutionary trajectories of novel homologs/orthologs of CDCA7.
- Identification of CDCA7-HELLS-DNMT co-evolution across eukaryotes.

Author Response

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

eLife assessment

This important manuscript reveals signatures of co-evolution of two nucleosome remodeling factors, Lsh/HELLS and CDCA7, which are involved in the regulation of eukaryotic DNA methylation. The results suggest that the roles for the two factors in DNA methylation maintenance pathways can be traced back to the last eukaryotic common ancestor and that the CDC7A-HELLS-DNMT axis shaped the evolutionary retention of DNA methylation in eukaryotes. The evolutionary analyses are solid, although more refined phylogenetic approaches could have strengthened some of the claims. Overall, this study should be useful for researchers studying DNA methylation pathways in different organisms, and it should be of general interest to colleagues in the fields of evolutionary biology, chromatin biology and genome biology.

We sincerely appreciate constructive comments and suggestions by the reviewers and a fair and accurate summary by the monitoring editor. Below we made point-by-point responses to reviewers’ comments.

Reviewer #1 (Public Review):

Overall, I find the work performed by the authors very interesting. However, the authors have not always included literature that seems relevant to their study. For instance, I do not understand why two papers Dunican et al 2013 and Dunican et al 2015, which provide important insight into Lsh/HELLS function in mouse, frog and fish were not cited. It is also important that the authors are specific about what is known and in particular about what is not known about CDCA7 function in DNA methylation regulation. Unless I am mistaken, there is currently only one study (Velasco et al 2018) investigating the effect of CDCA7 disruption on DNA methylation levels (in ICF3 patient lymphoblastoid cell lines) on a genome-wide scale (Illumina 450K arrays). Unoki et al 2019 report that CDCA7 and HELLS gene knockout in human HEK293T cells moderately and extremely reduces DNA methylation levels at pericentromeric satellite-2 and centromeric alpha-satellite repeats, respectively. No other loci were investigated, and it is therefore not known whether a CDCA7-associated maintenance methylation phenotype extends beyond (peri)centromeric satellites. Thijssen et al performed siRNA- mediated knockdown experiments in mouse embryonic fibroblasts (differentiated cells) and showed that lower levels of Zbtb24, Cdca7 and Hells protein correlate with reduced minor satellite repeat methylation, thereby implicating these factors in mouse minor satellite repeat DNA methylation maintenance. Furthermore, studies that demonstrate a HELLS-CDCA7 interaction are currently limited to Xenopus egg extract (Jenness et al 2018) and the human HEK293 cell line (Unoki et al 2019). Whether such an interaction exists in any other organism and is of relevance to DNA methylation mechanisms remains to be determined. Therefore, in my opinion, the conclusion that "Our co- evolution analysis suggests that DNA methylation-related functionalities of CDCA7 and HELLS are inherited from LECA" should be softened, as the evidence for this scenario is not very compelling and seems premature in the absence of molecular data from more species.

We appreciate this reviewer’s thorough reading of our manuscript.

Regarding the citation issues, we will cite Dunican 2013 and Dunican 2015. In addition, we went through the manuscript to update the citations.

As pointed out by the reviewer, the role of CDCA7 in genome DNA methylation was extensively studied in Velasco et al 2018. The result, together with Thijssen et al (2015), and Unoki et al. (2018), supports the idea that ZBTB24, CDCA7 and HELLS act within the same pathway to promote DNA methylation, the pattern of which is overlapping but distinct from DNMT3B-mediated methylation. This observation suggests that a ZBTB24- CDCA7-HELLS mechanism for DNA methylation may involve an alternative DNMT. Interestingly, our analysis of the gene presence-absence pattern revealed that the presence of CDCA7 coincides with DNMT1 more than DNMT3 genes. Indeed, while CDCA7 is lost from diverse branches of eukaryote species, genomes encoding CDCA7 always encode HELLS, and almost always encode DNMT1. Based on this observation, we speculate the role of CDCA7 is tightly linked to HELLS and DNA methylation throughout evolution.

As pointed out by Reviewer 1, the link between CDCA7, HELLS and DNA methylation has not been determined experimentally across these species. However, based on our previously published and unpublished data, we are confident about the functional interaction between CDCA7 and HELLS in Xenopus laevis and Homo sapiens.

Furthermore, the importance of HELLS homologs in DNA methylation has been extensively studied in human, mice and plants. We hope our current study will motivate the field to experimentally test the evolutionary conservation of HELLS-CDCA7 interaction, as well as their importance in DNA methylation, in other species.

The authors used BLAST searches to characterize the evolutionary conservation of CDCA7 family proteins in vertebrates. From Figure 2A, it seems that they identify a LEDGF binding motif in CDCA7/JPO1. Is this correct and if yes, could you please elaborate and show this result? This is interesting and important to clarify because previous literature (Tesina et al 2015) reports a LEDGF binding motif only in CDCA7L/JPO2.

We searched for a LEDGF binding motif ({E/D}-X-E-X-F-X-G-F, also known as IBM described in Tesina et al 2015) in vertebrate CDCA7 proteins, and reported their positions in Figure 2A. Examples of identified LEDGF-binding motifs are now presented in Fig. 2C.

To provide evidence for a potential evolutionary co-selection of CDCA7, HELLS and the DNA methyltransferases (DNMTs) the authors performed CoPAP analysis. Throughout the manuscript, it is unclear to me what the authors mean when referring to "DNMT3". In the Material and Methods section, the authors mention that human DNMT3A was used in BLAST searches to identify proteins with DNA methyltransferase domains. Does this mean that "DNMT3" should be DNMT3A? And if yes, should "DNMT3" be corrected to "DNMT3A"? Is there a reason that "DNMT3A" was chosen for the BLAST searches?

As described in the Methods section, both Human DNMT1 and DNMT3A were used to initially identify any proteins containing a domain homologous to the DNA methyltransferase catalytic domain. Within Metazoa, if their orthologs exist, the top hit from BLAST search using human DNMT1 and DNMT3A show E-value 0.0, and thus their orthology is robust. This is even true for DNMT1 and DNMT3 homologs in the sponge Amphimedon queenslandica, which is one of the earliest-branching metazoan species. For other DNMTs, such as DNMT2, DNMT4, DNMT5, DNMT6, we conducted separate BLAST searches using those proteins as baits as described in Methods. The methyltransferase domain was then isolated using the NCBI conserved domains search. The selected DNMT domain sequences were aligned with CLUSTALW to generate a phylogenetic tree to further classify DNMTs. In response to reviewer #2’s comments, we also generated another multi-sequence alignment of DNMTs using MUSCLE v5 and conducted maximum-likelihood-based phylogenetic tree assembly using IQ-TREE (new Fig. S6). The overall topology of these trees is consistent except for orphan DNMTs. It has been suggested that vertebrate DNMT3A and DNMT3B are derived from duplication of a DNMT3 gene of chordates ancestor (e.g., Liu et al 2020, PMID 31969623). As such many invertebrates encode only one DNMT3. As previously shown (Yaari et al., 2019, PMID 30962443), plants have two distinct DNMT3-like protein family, the ‘true DNMT3’ and DRM, the plant specific de novo DNMT that is often considered to be a DNMT3 homolog (see Reviewer 2’s comment). Our phylogenetic analysis successfully deviated the clade of DNMT3 and DRM from the rest of DNMTs (Figure S6). Yaari et al noted that PpDNMT3a and PpDNMT3b, the two DNMT3 orthologs encoded by the basal plant Physcomitrella patens, are not orthologs of mammalian DNMT3A and DNMT3B, respectively. Therefore, to minimize such nomenclature confusions, any DNMTs that belong to either the DNMT3 or DRM clades indicated in Figure S6 are collectively referred to as ‘DNMT3’ throughout the paper (see Figure S2 for overview).

CoPAP analysis revealed that CDCA7 and HELLS are dynamically lost in the Hymenoptera clade and either co-occurs with DNMT3 or DNMT1/UHRF1 loss, which seems important. Unfortunately, the authors do not provide sufficient information in their figures or supplementary data about what is already known regarding DNA methylation levels in the different Hymenoptera species to further consider a potential impact of this observation. What is "the DNA methylation status" of all these organisms? This information cannot be easily retrieved from Table S2. A clearer presentation of what is actually known already would improve this paragraph.

As the DNA methylation status of the species in the Hymenoptera clade has not been comprehensively tested, we initially did not include this information to Figure 7. However, during the course of the revision, we realized that Bewick et al.2017 (PMID 28025279) reported that DNA methylation is absent from the braconid wasp Aphidius ervi. We originally conducted synteny analysis on Aphidius gifuensis, which has a chromosome-level genome assembly with annotated proteins available in NCBI, whereas annotated proteins for Aphidius ervi protein are not available in NCBI. By conducting tBLASTn search against the Aphidius ervi genome, we now found that the presence/absence pattern of CDCA7, HELLS, DNMT1, DNMT3 and UHRF1 in Aphidius ervi is identical to that of Aphidius gifuensis, with a caveat that genome assembly of Aphidius ervi is at scaffold-level. In other words, DNA methylation, DNMT1 and CDCA7 are absent in Aphidius ervi, where 5mC is undetectable. Additionally, we also realized that the DNA methylation status reported for some species in Bewick et al. 2017 was inferred from the CpG frequency instead of the direct experimental detection of methylated cytosines. Therefore, we have amended Table S3 to indicate the presence of 5mC only for those species where this was experimentally tested. As such, we now consider the DNA methylation status of Fopius arisanus, which lacks DNMT1 and CDCA7, to be unknown.

Altogether, among the 17 Hymenoptera species that we analyzed (listed in the amended Table S3), the 8 species that have detectable DNA methylation all encode CDCA7, whereas the 2 species that do not have detectable DNA methylation lack CDCA7. We will note this finding in the revised text, and include the known 5mC status in the new Figure 7.

Furthermore, A. thaliana DDM1, and mouse and human Lsh/Hells are known to preferably promote DNA methylation at satellite repeats, transposable elements and repetitive regions of the genome. On the other hand, DNA methylation in insects and other invertebrates occurs in genic rather than intergenic regions and transposable elements (e.g. Bewick et al 2017; Werren JH PlosGenetics 2013). It would be helpful to elaborate on these differences.

We were aware of this interesting point, which was discussed in the third paragraph of the Discussion. To better illustrate this point, we now expanded the Discussion (page 14) to speculate about the role of DNA methylation in insects, where emerging evidence indicates the importance of DNMT1 in meiosis. It should be noted that, in the Arabidopsis ddm1 mutant, reduction of CG methylation of gene bodies is common (50% of all methylated euchromatic genes) (Zemach et al, 2013). In addition, hypomethylation is not limited to satellite repeats and transposable elements in ICF patients defective in HELLS or CDCA7 (Velasco et al., 2018).

Reviewer #2 (Public Review):

In this manuscript, Funabiki and colleagues investigated the co-evolution of DNA methylation and nucleosome remolding in eukaryotes. This study is motivated by several observations: (1) despite being ancestrally derived, many eukaryotes lost DNA methylation and/or DNA methyltransferases; (2) over many genomic loci, the establishment and maintenance of DNA methylation relies on a conserved nucleosome remodeling complex composed of CDCA7 and HELLS; (3) it remains unknown if/how this functional link influenced the evolution of DNA methylation. The authors hypothesize that if CDCA7-HELLS function was required for DNA methylation in the last eukaryote common ancestor, this should be accompanied by signatures of co-evolution during eukaryote radiation.

To test this hypothesis, they first set out to investigate the presence/absence of putative functional orthologs of CDCA7, HELLS and DNMTs across major eukaryotic clades. They succeed in identifying homologs of these genes in all clades spanning 180 species. To annotate putative functional orthologs, they use similarity over key functional domains and residues such as ICF related mutations for CDCA7 and SNF2 domains for HELLS. Using established eukaryote phylogenies, the authors conclude that the CDCA7-HELLS-DNMT axis arose in the last common ancestor to all eukaryotes. Importantly, they found recurrent loss events of CDCA7-HELLS-DNMT in at least 40 eukaryotic species, most of them lacking DNA methylation.

Having identified these factors, they successfully identify signatures of co-evolution between DNMTs, CDCA7 and HELLS using CoPAP analysis - a probabilistic model inferring the likelihood of interactions between genes given a set of presence/absence patterns. As a control, such interactions are not detected with other remodelers or chromatin modifying pathways also found across eukaryotes. Expanding on this analysis, the authors found that CDCA7 was more likely to be lost in species without DNA methylation.

In conclusion, the authors suggest that the CDCA7-HELLS-DNMT axis is ancestral in eukaryotes and raise the hypothesis that CDCA7 becomes quickly dispensable upon the loss of DNA methylation and/or that CDCA7 might be the first step toward the switch from DNA methylation-based genome regulation to other modes.

The data and analyses reported are significant and solid. However, using more refined phylogenetic approaches could have strengthened the orthologous relationships presented. Overall, this work is a conceptual advance in our understanding of the evolutionary coupling between nucleosome remolding and DNA methylation. It also provides a useful resource to study the early origins of DNA methylation related molecular process. Finally, it brings forward the interesting hypothesis that since eukaryotes are faced with the challenge of performing DNA methylation in the context of nucleosome packed DNA, loosing factors such as CDCA7-HELLS likely led to recurrent innovations in chromatin-based genome regulation.

Strengths:

• The hypothesis linking nucleosome remodeling and the evolution of DNA methylation.

• Deep mapping of DNA methylation related process in eukaryotes.

• Identification and evolutionary trajectories of novel homologs/orthologs of CDCA7.

• Identification of CDCA7-HELLS-DNMT co-evolution across eukaryotes.

Weaknesses:

• Orthology assignment based on protein similarity.

• No statistical support for the topologies of gene/proteins trees (figure S1, S3, S4, S6) which could have strengthened the hypothesis of shared ancestry.

We appreciate the reviewers’ accurate summary, nicely emphasizing the importance of the our study. We agree that better phylogenetic analysis for orthology assignment will strengthen our conclusion. Having anticipated this weakness, however, we specifically conducted a CoPAP analysis exclusively for Ecdysozoa specieswhich supported our major conclusion, as orthology assignment is straightforward in these species. For example, if we conduct BLAST search against the clonal raider ant Oocerea biroi protein dataset using human HELLS as a query, top 1 hit is a protein sequence annotated as one of three isoforms of ‘lymphoid-specific helicase” (i.e., HELLS), with E value 0.0. Similarly, the top BLAST hit from the Oocerea biroi dataset using human DNMT1 as a query also returns with isoforms of DNMT1 with E value 0.0. As such, there are little disputes in orthology assignment in Ecdysozoa. Outside of Chordata, classification of DNMTs, particularly in Excavata and SAR, require more extensive identification in these supergroups. Our current orthology assignment for the major targets in this study (HELLS, DNMT1, DNMT3, DNMT5) is largely consistent with published results (Ponger et al., 2005 PMID 15689527; Huff et al, 2014 PMID 24630728; Yaari et al., 2019 PMID 30962443; Bewick et al., 2019 PMID 30778188). However, while we are preparing this response and re-crosschecking our assignments with these references, we realized that we had erroneously missed DNMT5 orthologs in Leucosporidium creatinivorum, Postia placenta, Armillaria gallica and Saitoella complicata, and a DNMT6 ortholog in Fragilariopsis cylindrus. We also recognized that DNMT4 orthologs were identified in Fragilariopsis cylindrus and Thalassiosira pseudonana in Huff et al 2014 (PMID 24630728), but in our phylogenetic analysis, these proteins form a distinct clade between DNMT1/Dim-2 and DNMT4 (original Figure S6), although the confidence level of this classification by Huff et al was not strong. To resolve this potential confusion in DNMT annotations, we generated new multiple sequence alignments with MUSCLE v5 and IQ-TREE 2 (maximum likelihood-based method, coupled with selection of optimal substitution model and bootstrapping). The tree topology was not significantly altered between the two methods, except for the unambiguous location of orphan DNMTs and DNMT4-related proteins. To avoid unnecessary confusion in the DNMT annotations, we decided to present MUSCLE-IQ- TREE for the DNMT phylogenetic tree and classification (new Fig. S6). The raw results of IQ-TREE analysis for CDCA7/zf-4CXXC_R1, HELLS SNF2 domain, and DNMTs are included as Dataset S1-S3. We then conducted CoPAP analysis using the corrected classification. As it is not clear a priori if fungal specific CDCA7-like proteins (now referred to as CDCA7F with class II zf-4CXXC_R1) should be considered CDCA7 orthologs, we conducted CoPAP against two lists; the first list includes CDCA7F in the CDCA7 group, whereas the second list includes a separate category of class II zn-4CXXC_R1, which includes CDCA7F. Both results show slightly different topology in the coevolutionary linkages but support our major conclusion that CDCA7 coevolved with DNMT1-UHRF1 and HELLS. These new CoPAP results are shown in Fig. S7.

Reviewer #1 (Recommendations For The Authors):

Summary

Last sentence: "...a unique specialized role of CDCA7 in HELLS-dependent DNA methylation maintenance...". What do the authors mean?

Our analysis strongly indicates that CDCA7 is dispensable in systems lacking HELLS and DNMT (particularly DNMT1). In other words, species preserve CDCA7 only if it has both HELLS and DNMT1 (or in some cases DNMT5). The importance of HELLS homologs in DNA methylation has been extensively studied in human, mouse and plants. However, in these studies, substantial DNA methylation remains despite the defective HELLS/DDM1 (especially in euchromatic regions). Additionally, there are species (e.g., Bombyx mori) that have DNMT1 and detectable DNA methylation but lacks HELLS and CDCA7. These observations suggest that the role of CDCA7 must be unique and specialized in a way that it is strongly coupled to HELLS-dependent DNA methylation (but not HELLS-independent DNA methylation), and that this function of CDCA7 seems to be inherited from the last eukaryotic common ancestor.

Introduction

• page 3: "DNMTs are largely subdivided into maintenance and de novo DNMTs" - Which species are the authors referring to?

As described in the cited reference (Lyko 2018), maintenance DNA methylation and de novo DNA methylation are well accepted functional classification of DNA methylation. It is also currently accepted that distinct DNMTs execute maintenance DNA methylation or de novo DNA methylation, although crosstalk between these processes has been reported. Therefore, we stated, “DNMTs are largely subdivided into maintenance DNMTs and de novo DNMTs”, and this subdivision is species independent.

• page 3" "Maintenance DNMTs recognize hemimethylated CpGs. " - Can the authors please define the species and/or literature they are referring to? This seems important to clarify. For instance, mammalian DNMT1 requires a co-factor, UHRF1, which recognizes hemimethylated DNA and H3K9me3 (Bostick et al 2007).

We meant to describe, “Maintenance DNMTs directly or indirectly recognize hemimethylated CpGs…”. The specific requirement of UHRF1 for DNMT1-mediated maintenance DNA methylation is explained in the subsequent sentence “In animals…”. In the case of Cryptococcus neoformans, DNMT5 recognizes hemimethylated DNA independently of UHRF1 in vitro to execute maintenance methylation.

• page 3: The authors may want to mention that A. thaliana also has a de novo DNA methyltransferase, DRM2, a homolog of the mammalian DNMT3 methyltransferases. This seems important, since they show in Figure 1 that a de novo methyltransferase is found in A. thaliana. Also, later in their manuscript they mention plant de novo DNA methylation.

Thanks for pointing this out. As shown in Figure 5, we classified plant DRMs as DNMT3-like proteins, but we now note this in the Introduction.

• page 3: Sentence starting "In about 50% of ICF patients,..." - Why is DNMT3B referred to as "de novo", is it not a de novo DNA methyltransferase?

You are correct. Quotation marks are now removed to avoid unnecessary confusion.

• page 4: Sentence starting "Indeed, the importance of HELLS/CDCA7 in DNA methylation maintenance...", - Which references (Han et al., 2020; Ming et al., 2021; Unoki, 2021; Unoki et al., 2020) provide experimental evidence for a role of CDCA7 in DNA methylation maintenance by DNMT1?

Thanks for pointing out the typo. “/CDCA7” is now removed.

• page 5: Sentence starting "Indeed, it has been shown that DNMT3A..." - Should DNMTB be DNMT3B?

Yes. This is now corrected.

Results

• Page 5: Sentence starting "However, we identified a protein..." - No A. thaliana reference?

We added Zemach et al 2010, and Chan et al 2005.

• Figure 2B: "ICF4 mutations" should this be "ICF3 mutations"?

• Figure 3: "ICF4 mutations" should this be "ICF3 mutations"?

• Figure 4: "ICF4 mutations" should this be "ICF3 mutations"?

• Figure S1: Orange colored "CDC7L (fish), CDC7e, CDC7, CDC7L" is there an "A" missing?

• Figure S5: "ICF4 mutations" should this be "ICF3 mutations"?

These typos are now corrected. Thank you.

• Figure S7: What is "CDCA7(II)" referring to, "zf-4CXXC_R1 class II (plants)"?

The original CDCA7 (II) included proteins with class II zf-4CXXC_R1, which are found in plants, fungi, Acanthamoeba castellanii and Amphimedon. Among those species, the prototypical CDCA7 orthologs are absent only in fungi. It has been a priori unclear if fungal proteins with class II zf-4CXXC_R1 (now we term CDCA7F) should be included in CDCA7 for CoPAP analysis. Although we originally included CDCA7F in CDCA7, we now show the results of two analyses. In the first one (Fig. S7A) CDCA7F was included in CDCA7, whereas in in the second one (Fig. S7B) CDCA7F was included in the separate category of class II zf-4CXXC_R1. Topologies of two results are slightly different, but they both show coevolutionary linkage between the CDCA7 and DNMT1- UHRF1 cluster.

• Figure 4 and 5: In the case of preliminary genome assemblies what is the difference between empty squares with dotted lines and filled squares without dotted lines?

As it is difficult to be certain of a gene’s absence (did the species lose the gene or is it simply not annotated due to incomplete genome coverage?), we illustrated the absence of a gene in preliminary genome assemblies with an empty square with dotted outline. Since the presence of a gene is evident regardless of the level of genome assembly, the presence of a gene is represented with filled squares with solid lines, even for preliminary genome assemblies.

• Figure 1: Why was Mus musculus - one of the main model organisms used for many DNA methylation studies not included? Also what are empty and filled squares?

Filled and empty squares indicate the presence and absence of the indicated genes, respectively. Clarifying statement is now added in the figure legends. Mus musculus is now included in the figure.

• Figure S2: Adding the existence of DNA methylation and DNMT3 in the bottom right part of the figure (overall no of species) would make this panel more informative

We included this overview to summarize the co-retention of CDCA7, HELLS and maintenance DNMTs across the analyzed species. We decided not to include DNA methylation, since DNA methylation status is known for only a fraction of the listed species. Inclusion of DNMT3 will introduce too many possible gene presence-absence combinations to convey a clear message. However, we now mention in the revised text (page 11, second paragraph) that unlike the prevalent co-retention of DNMT1 in species with CDCA7, we identified several species that possess CDCA7, HELLS and DNMT1 but lack DNMT3. These examples include insects such as the bed bug Cimex lectularius and the red paper wasp Polistes canadensis.

• Page 6: Sentence starting "This leucine zipper sequence is highly conserved..." - Figure/Reference missing?

The sequence alignment of the leucine zipper is now shown in Fig. 2C.

• page 6: Sentence starting "In contrast to zf-4CXXC_R1 motif-containing proteins..." - The authors may want to mention the role of the CXXC zf domain in KDM2A/B, DNMT1, MLL1/2 and TET1/3 and what the CDCA7 CXXC zf domain is/could be required for.

The notion that zf-CXXC binds to nonmethylated CpG is now included. Due to the substantial difference between zf-CXXC and zf-4CXXC_R1, we hesitated to relate the function of zf-4CXXC_R1 with zf-CXXC, but we now discuss a potential role of zf- 4CXXC_R1 in sensing DNA methylation status in Discussion (Page 13).

• page 7: Sentence starting "Second, the fifth cysteine is replaced..."- Zoopagomycota" - Figure 4A does not have this labeling, one has to deduce this from Figure 4B.

We fixed this by including the list of Zoopagomycota species in the main text.

• page 7: Sentence containing "Neurospora crassa DMM-1 does not directly regulate DNA methylation or demethylation but rather..." - How does the information about DMM- 1 relate to what is shown in Figure 4B, to CDCA7, HELLS and DNMTs? Please clarify.

Both Neurospora DMM-1 and Arabidopsis IBM1 contain the JmjC domain and are implicated in an indirect control mechanism of DNA methylation. Since it has never been pointed out that they have a divergent zf-4CXXC_R1 domain, which clearly shares the origin with CDCA7 proteins, we thought that this is important to note. We realized that we did not clearly mark Neurospora XP-956257 as DMM-1 in Fig. 4B. This is now fixed.

• Heading "Systematic identification of CDCA7, HELLS and DNMT homologs in eukaryotes". When mentioning CDCA7 the authors may want to decide on the use of one consistent definition of "prototypical (Class I) CDCA7-like proteins (i.e. CDCA7 orthologs)" "Class I CDCA7 proteins". Constantly changing the way how they refer to these proteins is very confusing.

We now make it clear that we call proteins with class I zf-CXXC_R1 motif CDCA7 orthologs. We also define class II zf-4CXXC_R1 (as those with a substitution at ICF- associated glycine residue). Since no clear CDCA7 orthologs can be found in fungi, we now call fungi proteins with class II zf-4CXXC_R1 “CDCA7F”, implying its ambiguous orthology assignment.

Under this heading there is also no mention of DNMTs. Instead, the authors introduce DNMTs under the heading "Classification of DNMTs in eukaryotes" - Please clarify.

This is now corrected.

• page 9: Sentence containing "... presence of DNMT1, UHRF1 and CDCA7 outside of Viridiplantae and Opisthokonta is rare". What does "rare" mean? How is UHRF1 relevant here?

Among the 32 species outside of Viridiplantae and Opisthokonta, only the Acanthamoeba castellanii genome encodes clear orthologs of DNMT1, UHRF1 and CDCA7. Although it is often difficult to deduce if the selected panel of species is a reasonable representation, we think that it is not unreasonable to state that Acanthamoeba is a rare case to encode this set of proteins outside of Viridiplantae and Opisthokonta. We include UHRF1 since it is a well-established activator of DNMT1, and indeed our CoPAP analysis showed a tight coevolution of UHRF1 with DNMT1. Outside of Viridiplantae and Opisthokonta, only Acanthamoeba castellanii and Naegleria gruberi encode UHRF1. Interestingly, these two species also encode CDCA7 and HELLS.

Having said that, we rephrased this sentence, which reads; “Species that encode a set of DNMT1, UHRF1, CDCA7 and HELLS are particularly enriched in Viridiplantae and Metazoa.”

• page 11: Sentence containing "..., that the function of CDCA7-like proteins is strongly linked to HELLS and DNMT1,..." What do the authors mean with "the function of CDCA7-like proteins"? And what happened to DNMT3?

Our observation that almost all species that contain CDCA7 (including fungal CDCA7F) also have DNMT1 and HELLS, despite the frequent loss of these genes in species that do not contain CDCA7, indicates “that the function of CDCA7-like proteins is strongly linked to HELLS and DNMT1”. We found only 2 species that possesses CDCA7 (class I or class II) but not DNMT1 among the panel of 180 species. These 2 exceptional species, Naegleria gruberi and Taphrina deformans, do encode UHRF1-like proteins and a DNMT (an orphan DNMT in N. gruberi and DNMT4 in T. deformans). In contrast, we found 26 species that possess CDCA7 (or CDCA7F) but not DNMT3 (Table S1), so the linkage between CDCA7 and DNMT3 is weaker.

• page 11: Sentence containing "..., CDCA7 is lost from this gene cluster in parasitoid wasps, including Ichneumonoidea wasps and chalcid wasps". This sentence is confusing because already in an earlier paragraph the authors say that "Microplitis demolitor lost CDCA7" and in the following sentence they say "...among Ichneumonoidea wasps, CDCA7 appears to be lost in the Braconidae clade, ...". It would greatly help this reader if the authors could streamline these sentences and also decide on whether CDCA7 is lost in M. demolitor or CDCA7 appears to be lost in M.demolitor.

The confusion was in part due to the difficulty in differentiating between the true loss of a gene versus its apparent absence in a species due to an incomplete genome assembly, including for of M. demolitor. To verify that the loss of CDCA7 was not due to gaps in the genome assembly, we executed the synteny analysis. However, we edited this section to improve the readability (Page 12-13).

What could be the role for HELLS/CDCA7 in insect DNA methylation? In several cases, the authors analyses reveal co-evolutionary links between DNMT3 (DNMT3A?) and CDCA7/HELLS. I do not understand why this finding is not really discussed by the authors. Instead there is a strong focus on replication-uncoupled DNA methylation maintenance. Could the authors elaborate why?

The role of DNA methylation in insects is largely unclear, so discussion must be highly speculative. A recent finding in the clonal raider ant, showing that DNMT1 is not essential for development but is critical for oogenesis, pointed toward a possible more universal role of DNA methylation in meiosis. Stimulated from a finding in Neurospora, where DNA methylation is required for homolog pairing during meiosis, we discuss a speculative model that DNA methylation status acts as a hallmark to distinguish between healthy/young DNA and old/mutated (or competitive/pathogenic) DNA at homolog pairing during meiosis (page 14).

Regarding the cases where CDCA7 and DNMT3 are co-lost, we had discussed about this phenomenon at the last section of Result, stating, “This co-loss of CDCA7 and DNA methylation (together with either DNMT1-UHRF1or DNMT3) in braconid wasps suggests that evolutionary preservation of CDCA7 is more sensitive to DNA methylation status per se than to the presence or absence of a particular DNMT subtype.” Please note that we found several lineages that lacks CDCA7 but has DNMT1 (and DNMT3), whereas almost all species that has CDCA7 also has DNMT1 (but not necessarily DNMT3). Supported with our CoPAP analyses, our results indicate the tight functional link between CDCA7 and DNMT1, but it does not necessarily mean that CDCA7 does not play any role related to DNMT3 or de novo methylation. Clarification of this point and our speculation of how CDCA7 loss is linked to reduced requirement of DNA methylation are discussed in page 13 and 14 with additional texts.

Discussion

• page 12: Where is the data supporting. "... the red flour beetle Tribolium castaneum possesses DNMT1 and HELLS, but lost DNMT3 and CDCA7"?

Figure 5, Figure S2 and Table S1. This is now noted in the text.

• page 14: Based on which parts of their analyses or evidence from the literature can the authors speculate that "...the evolutionary arrival of HELLS-CDCA7 in eukaryotes might have been required to transmit the original immunity-related role of DNA methylation from prokaryotes to nucleosome-containing (eukaryotic) genomes"? Please clarify.

This is inferred from the well-known role of DNA methylation in bacteria for defending against phage viruses. However, it was not correct to state that such a function was inherited from prokaryotes. It should be stated that it was inherited from the last universal common ancestor (LUCA). We also admit that it is not clear if such an immunity-related role was inherited from LUCA, or if it emerged through convergent evolution. Therefore, we amended this description to emphasize our hypothesis that the advent of CDCA7 was “a key step to transmit the DNA methylation system from the LUCA to the eukaryotic ancestor with nucleosome-containing genomes”.

Supplementary Figures/Tables

• page 26: Table S2 and Table S3, it seems that these tables show data that supports what is shown in Figure 7 and not Figure 5.

You are correct. Thank you for pointing out the typos.

Has the methylation status been assessed in C. glomerata, C. typhae, Chelonus insularis, Diachasma alloeum or Aphidius gifuensis? Please clarify in Table S2.

Not to our knowledge. However, as we realized that absence of DNA methylation in Aphidius ervi was previously reported (Bewick et al 2017), we now included this data together with presence/absence analysis of DNMT1, UHRF1, DNMT3, CDCA7 and HELLS. Known presence/absence of DNA methylation is now shown in Fig.7.

Reviewer #2 (Recommendations For The Authors):

Recommendation to strengthen the paper:

1. Phylogenetics:

• Test and report the appropriateness of the substitution model used in protein alignments/trees.

• Use Maximum likelihood methods and/or MCM Bayesian inference to build and report trees with well supported topologies. This is required to properly assign orthology (shared ancestry). This will avoid false interpretation due to technical limitation of similarity-based phylogenies (without statistical support). Figure S1, S3, S4 and S6.

To address these points, we made new multisequence alignments using MUSCLE v6 and generated phylogenetic trees using the maximum likelihood-based IQ-TREE 2, where multiple models were screened. A consensus tree was generated after 1000 bootstrap replicates from the best alignment and model. The topology and assignment of these new trees were largely consistent with the original trees, except for some corrections in DNMT assignment as discussed below.

1. We realized that we erroneously missed DNMT5 orthologs of Leucosporidium creatinivorum, Postia placenta, Armillaria gallica and Saitoella complicata., and DNMT6 orthologs from Fragilariopsis cylindrus reported in Huff et al 2014 (PMID 24630728). They are now included in the new list and CoPAP analysis.

2. DNMT4 orthologs were identified in Fragilariopsis cylindrus and Thalassiosira pseudonana by Huff et al 2014 (PMID 24630728), but in our original phylogenetic analysis, these proteins form a distinct clade between DNMT1/Dim-2 and DNMT4. The new tree and classification are more consistent with Huff et al, so we present the new tree in Fig. S6 and conducted the classification based on this tree.

Beside Fig. S6, we decided to maintain original Fig. S1, S3 and S4 (with a few adjustments) for better visibility, but we included the results of IQ-TREE analysis as Dataset S1-S3.

The CoPAP analysis based on the revised assignment slightly changed the topology of coevolutionary linkages. In addition, we obtained a slightly different result depending on whether fungal specific CDCA7 with class II zn-4CXXC_R1 (now referred to as CDCA7F) is included as a CDCA7 ortholog or not. Despite this difference, we reproducibly observed the coevolutionary linkage between CDCA7 and DNMT1- UHRF1.

• Be more careful with wording: RBH is not sufficient to call gene/proteins orthologs (e.g. Page 8). The above mentioned method will help you support this claim (+ synteny when you can).

We were aware of this issue. This is why we conducted phylogenetic tree building based on sequence alignment of full-length HELLS (Fig. S3) and SNF2 domain only (Fig. S4), as explained in the text. We found that the RBH criterion is robust in Metazoa; orthologs are easily recognizable with very low E-value (0.0) and extensive homology over the full length of the protein, while synteny is not practical to employ in the diverse set of species.

• Also, use "co-retention" or "co-evolution" but not "co-selection" when describing CoPAP results - as CoPAP does not test for signature of natural selection.

This is a good point and is now corrected.

• The statistics (p-val...) underlying the CoPAP analyses should be explained.

The explanation is now added in Methods section.

“A method to calculate p-value for CoPAP was described previously (Cohen et al., 2012, PMID 22962457). Briefly, for each pair of tested genes, Pearson's correlation coefficient was computed. Parametric bootstrapping was used to compute a p-value by comparing it with a simulated correlation coefficient calculated based on a null distribution of independently evolving pairs with a comparable exchangeability (a value reporting the likelihood of gene gain and loss events across the tree).”

1. Figure S2 and S3 could be improved for readability

After consideration of this criticism, we decided to keep their original formats for following reasons.

Figure S2. The purpose of this list is to better visualize the comprehensive list shown in Table S2. A consolidated list is already shown in Figure 5. An alternative choice is to make a diagram where individual species names are unreadable. This kind of presentation is seen in many published papers, but we found that they are not helpful to check the details. As this is a supplementary figure, we prefer to show the detailed data that can be visible without a specialized software.

Figure S3. This figure is included to show which SNF2 family proteins are more likely to be misassigned as HELLS/DDM1 orthologs. We believe that the figure serves this purpose.

1. What is the meaning of the coloring patterns of ICF residues in znf?

ICF residues are highlighted as light blue in the schematics to indicate its conservation. In the alignment, the coloring reflects the level of conservation within the shown set of proteins, and the choice of coloring was set by Jalview.

1. To improve clarity: the introduction could be more focused on evolutionary considerations and functional link between CDCA7-HELLS and DNMTs.

We revised the first paragraph of the introduction to illustrate this point.

1. Could indicate the CDC7A loss / DNA methylation hypothesis in the abstract.

We now included this hypothesis in the Abstract.