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

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.

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.

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?

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.

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.

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.

Author Response:

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.

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, mouse 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 position in Figure 2A. Examples of identified LEDGF-binding motifs will be presented.

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, DNMT6, we conducted separate BLAST searches using those proteins as baits as described in Methods. The 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 (Figure S6). 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, this precluded us from adding this information to Figure 7. However, we have included the published reports of DNA methylation status for these species in Supplementary Table S2 (see column ‘5mC’; species for which 5mC is detected are marked with Y and the relevant PMID). As indicated, DNA methylation was detected in most tested species except for Microplitis demolitor. Many of these data are based on Bewick et al. 2017 (PMID 28025279). During the preparation of this response, we realized that the DNA methylation status reported for some species in Bewick et al. was inferred from the CpG frequency instead of the direct experimental detection of methylated cytosines. Therefore, we have amended Table S2 to indicate the presence of DNA methylation 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. In addition, we realized that Bewick et al. reported that DNA methylation is absent in Aphidius ervi. We originally conducted synteny analysis on Aphidius gifuensis, which lacks DNMT1 and CDCA7, since Aphidius ervi protein data were not available in NCBI. By conducting tBLASTn search against the Aphidius ervi genome, we confirmed that the presence and absence pattern of CDCA7, HELLS, DNMT1, DNMT3 and UHRF1 in Aphidius ervi is identical to that of Aphidius gifuensis. In other words, DNA methylation is known to be absent in Aphidius ervi, which has lost DNMT1 and CDCA7. Altogether, among the 17 Hymenoptera species that we analyzed (listed in the amended Table S2), the 6 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.

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.

This point was discussed in the third paragraph of the Discussion, but we will better highlight this. 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.

[...]

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, and we would like to explore this. Having anticipated this weakness, we specifically conducted a CoPAP analysis exclusively for Ecdysozoa species, where orthology assignment is straightforward, which supported our major conclusion. (For example, if we conduct BLAST search the clonal raider ant Oocerea biroi 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, top BLAST hit from Oocerea biroi 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, regardless of the alternative methods employed for orthology assignment, this will never be perfect (particularly in Excavata and SAR). 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 erroneously missed DNMT5 orthologs of Leucosporidium creatinivorum, Postia placenta, Armillaria gallica and Saitoella complicata., and DNMT6 ortholog from Fragilariopsis cylindrus. We also had 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 (Figure S6). Due to this ambiguity, we did not count them as DNMT1 or DNMT4 in our CoPAP analysis. These minor errors and ambiguity should not affect our presence-absence pattern in our original CoPAP analysis, and thus we feel that further refinement is unlikely to significantly affect our major conclusion.