In order for a cell to divide, it must progress through a series of carefully controlled steps known as the cell cycle. First, the cell replicates its DNA and both copies get segregated to opposite ends. The cell then splits into two and each new cell receives a copy of the duplicated genetic material. If any of the stages in the cell cycle become disrupted or mis-regulated this can lead to uncontrolled divisions that may result in cancer.

Researchers have often used a structure within the eye known as the retina to study the cell cycle in zebrafish and other animals as cells in the retina rapidly divide in a highly controlled manner. A protein called Banp is known to help stop tumors from growing in humans and mice, but its normal role in the body, particularly the cell cycle, has remained unclear. To investigate, Babu et al. studied the retina of mutant zebrafish that were unable to make the Banp protein.

The experiments revealed that two stress responses indicating DNA damage or defects in copying DNA were active in the retinal cells of the mutant zebrafish. This suggested that Banp allows cell to progress through the cell cycle by repairing any DNA damage that may arise during replication. Banp does this by activating the gene for another protein called Wrnip1. Babu et al. also found that Banp helps segregate the two copies of DNA during cell division by promoting the activation of two other proteins called Cenpt and Ncapg. Further experiments identified 31 genes that were directly regulated by Banp.

These findings demonstrate that Banp is required for zebrafish cells to be able to accurately copy their DNA and divide in to two new cells. In the future, the work of Babu et al. will provide a useful resource to investigate how tumors grow and spread around the body, and may contribute to the development of new treatments for cancer.

BTG3-associated nuclear protein (BANP) (also known as Scaffold/matrix attachment region binding protein 1 (SMAR1)) was identified as a nuclear protein that binds to the matrix-associated region (MAR) of the β-locus of murine T cell receptors (Birot et al., 2000; Chattopadhyay et al., 2000). MARs are often found near gene promoters (Scheuermann and Garrard, 1999) and constitute a periodic loop domain, through which chromosomal DNA is topologically organized inside the nucleus (Blasquez et al., 1989; Hart and Laemmli, 1998). Furthermore, BANP functions as a tumor suppressor (Kaul Ghanekar et al., 2003; Kaul Ghanekar et al., 2005). In humans, the BANP gene is located on chromosome 16q24, the genomic region of which often suffers from loss-of-heterozygosity in cervical and breast cancers (Tsuda et al., 1994; Wang et al., 1999). BANP interacts with tumor suppressor tp53 to modulate its functions (Kaul Ghanekar et al., 2003; Pavithra et al., 2009; Sinha et al., 2012; Sinha et al., 2010). Overexpression of BANP suppresses cancer growth (Bhagat et al., 2018; Liu et al., 2014; Taye et al., 2018). However, these proposed cellular functions of BANP were based mostly on in vitro studies, and genetic deletion of Banp was reportedly unsuccessful (Grand et al., 2021), whereas Banp knockout mice show embryonic lethality (Chemmannur et al., 2015), which makes in vivo analysis difficult. Thus, physiological functions of BANP, especially in cell-cycle regulation and tumor suppression, are still unknown. Very recently, it was reported that BANP binds to the nucleotide sequence TCTCGCGAGA, called the ‘Banp motif’, which is enriched near the transcription initiation site of CpG island promoters, a genomic region in which it is difficult to replicate and repair DNA, and which regulates gene transcription in a DNA methylation-dependent manner. BANP promotes transcription of essential metabolic genes in pluripotent stem cells and terminal differentiated neuronal cells in humans and mice (Grand et al., 2021), indicating that BANP functions as a transcription factor at the molecular level. These findings raise the question, “How is the BANP-mediated transcriptional network involved in cell-cycle regulation and tumor suppression?”.

The vertebrate retina is composed of six major classes of neurons and one type of glia (retinal ganglion cells [RGCs], three types of interneurons [amacrine cells, bipolar cells, and horizontal cells], two types of photoreceptors [rods and cones], and Müller glia). These retinal cells are assembled to form the neural circuit responsible for phototransduction and visual processing (Dowling, 2012). During development, retinal progenitor cells are multipotent and give rise to all seven retinal cell types in response to extrinsic and intrinsic cues (Boije et al., 2014). An important characteristic of retinal development is that retinal neurogenesis is linked to cell-cycle regulation. In zebrafish, cell-cycle length of retinal progenitor cells is around 32–49 hr from 6 to 25 hpf, but shortened to 8–10 hr once retinal neurogenesis starts at 25 hpf (Li et al., 2000), suggesting that activation of cell-cycle progression is associated with neurogenesis. It is generally accepted that active cell proliferation may increase DNA replication errors, which induce DNA replication stress and subsequent DNA damage (Berti et al., 2020; Blackford and Jackson, 2017; Waterman et al., 2020). Thus, factors involved in DNA replication checkpoint and DNA damage response are essential to ensure genomic integrity (Murga et al., 2009). Otherwise, retinal progenitor cells accumulate genetic defects leading to apoptosis. Consistently, in zebrafish, retinal apoptosis occurs in the absence of DNA damage response regulators such as DNA damage-binding protein 1 (DDB1) (Yildirm et al., 2015), Fanconi anemia group D2 (Fancd2) (Liu et al., 2003), and Ku80 (Xrcc80) (Bladen et al., 2005). We also showed that retinal apoptosis occurs in zebrafish prim1 mutants, in which DNA replication stress is chronically elevated. In this mutant, a central regulator of the DNA replication checkpoint, Ataxia telangiectasia-mutated and Rad3-related (ATR), is activated, which subsequently activates DNA damage response regulators, Ataxia telangiectasia-mutated (ATM) and Chk2, leading to tp53-mediated apoptosis. Thus, DNA replication checkpoint and DNA damage response function as safeguards to ensure integrity of retinal cell proliferation and neurogenesis (Yamaguchi et al., 2008). Therefore, developing retina is a highly proliferating tissue, in which a spatiotemporal pattern of neurogenesis is tightly coordinated by cell-cycle regulation. So, vertebrate retina provides a great model for studying how cell-cycle regulation, including DNA damage response, ensures neurogenesis and subsequent cell differentiation.

In this study, we identified zebrafish banp mutants, which show mitotic cell accumulation and apoptosis in the developing retina. DNA replication checkpoint and tp53-dependent DNA damage response are activated to induce apoptosis in banp mutant retinas. Thus, Banp is required for regulation of DNA replication and DNA damage repair. Furthermore, consistent with mitotic cell accumulation in banp mutants, our live imaging revealed that chromosome segregation fails to proceed smoothly during mitosis in banp morphants, resulting in a prolonged M-phase. Our RNA-sequencing and ATAC-sequencing enabled us to identify 31 candidates for direct Banp target genes that carry the Banp motif near their promoters. Interestingly, both zebrafish and human genes encoding two chromosome segregation regulators, Cenpt and Ncapg, and a DNA replication fork regulator, Werner helicase interacting protein 1 (Wrnip1) are included in this list. Thus, Banp regulates transcription of cenpt and ncapg to promote chromosome segregation during mitosis, whereas Banp maintains transcription of wrnip1 for recovery from DNA replication stress during S phase. Taken together, these data suggest that Banp is required for cell-cycle progression and cell survival in zebrafish retinas by regulating DNA damage response and chromosome segregation during mitosis.

BANP was initially identified as a nuclear MAR-binding protein and reported to function as a tumor suppressor (Malonia et al., 2011). Very recently, it was reported that BANP binds to the Banp motif enriched near the TSS of CpG island promoters and regulates transcription of metabolic genes (Grand et al., 2021). However, physiological functions of Banp remained to be discovered. In this study, we identified zebrafish banp mutants, in which retinal progenitor cells show mitotic defects and apoptosis during embryonic development. RNA-seq analysis revealed that expression of tp53-dependent DNA damage response regulators is upregulated in banp mutants. In non-stress conditions, tp53 is degraded through ubiquitination by an E3-ubiqutin ligase, Mdm2, and subsequent actions of proteasomes, keeping its protein level very low (Oliner et al., 1993). On the other hand, under stress, such as DNA replication stalling or DSBs, ATR, and ATM are activated and phosphorylate the N-terminal serine of tp53 (Banin et al., 1998; Canman et al., 1998; Siliciano et al., 1997; Tibbetts et al., 1999). This phosphorylation reduces the binding affinity of Mdm2 for tp53, resulting in stabilization of tp53 protein (Shieh et al., 1997; Yadahalli et al., 2019). Indeed, the tp53 protein level is increased, and tp53 knockdown effectively suppresses retinal apoptosis in banp mutants. Furthermore, the number of γ-H2AX+ cells is significantly increased in banp mutants, suggesting elevation of replicative DNA damage. These data suggest that DNA replication stress is elevated in banp mutants, resulting in activation of tp53-dependent DNA damage response, which subsequently induces apoptosis of retinal progenitor cells.

It is generally accepted that tp53 has dual, but opposing roles: cell survival versus cell death in response to DNA replication stress and DNA damage (Chen, 2016; Lees et al., 2021). In the initial phase of the DNA damage response, tp53 binds to its alternative promoter located in intron 4 and activates transcription of an N-terminal truncated isoform, ∆113-tp53, which preferentially activates transcription of target genes related to cell-cycle arrest to promote DNA repair and cell survival (Chen et al., 2009). However, if DNA damage fails to be repaired, but accumulates chronically, FL tp53, in turn, activates transcription of apoptotic genes to eliminate DNA-damaged cells. Previous in vitro studies of human BANP suggest that BANP interacts with tp53 to promote transcription of cell-cycle arrest genes and to suppress transcription of apoptotic genes, leading to promotion of its cell survival role (Sinha et al., 2010). Indeed, our quantitative RT-PCR showed that transcription of ∆113-tp53 is significantly elevated in banp mutants, whereas the mRNA level of FL tp53 is very low. RNA-seq and ATAC-seq also confirmed a higher level of transcription and chromatin accessibility of ∆113tp53 in banp mutants. These data suggest that ∆113tp53-mediated transcription is selectively activated in banp mutants. Thus, it is important to clarify the role of tp53, including ∆113tp53 in mitotic cell accumulation in banp mutants, because pH3+ cells are markedly accumulated in banp mutant retinas prior to apoptosis. However, tp53-MO mildly inhibited accumulation of pH3+ cells in banp mutants. There is no significant difference in pH3+ cell accumulation between tp53-MO and ∆113tp53-MO, or between wild-type siblings and ∆113tp53-MO. So, these data suggest that the contribution of tp53 to mitotic cell accumulation in banp mutants is limited, and that a tp53-independent pathway is responsible for mitotic phenotypes. Our live imaging of mitosis in banp morphant retinas reveals that mitosis is significantly longer due to chromosome segregation defects; however, importantly, almost all mitoses are eventually completed in banp mutants, suggesting that accumulation of pH3+ cells in banp mutants is transient and depends mostly on prolonged mitosis, which is caused by chromosome segregation defects. At present, we have not observed S-phase arrest in banp mutants, because the fraction of BrdU+ cells in the total retinal region is not increased in banp mutants, although it is still possible that banp mutant retinal cells are partially arrested in G1 phase, because mRNA expression of a cdk inhibitor, cdkn1a (p21), is higher in banp mutants than in wild-type siblings. Taken together, we conclude that tp53 mainly mediates retinal apoptosis, and that its role in mitotic defects is limited in banp mutants.

Labeling with anti-γ-H2AX antibody revealed that replicative DNA damage is increased in retinal progenitor cells in banp mutants, suggesting that DNA lesions that cause stalled DNA replication are abnormally induced, or stalled DNA replication and DSBs fail to be repaired in the absence of Banp. Importantly, the number of γ-H2AX+ cells in banp mutant retinas was not increased by either tp53-MO and ∆113tp53-MO, suggesting that tp53 does not repair replicative DNA damage caused by loss of Banp functions. There are two possible functions of Banp in DNA damage regulation. Banp may maintain DNA replication integrity. BANP functions as a transcription factor to regulate expression of essential metabolic genes in pluripotent stem cells and differentiating neurons (Grand et al., 2021). Banp may promote transcription of DNA replication regulators. Our RNA-seq data identified 81 downregulated genes in banp mutants, compared to wild-type siblings. Among them, 31 genes have a Banp motif near their TSSs and contain eight cell-cycle regulatory genes. Among these eight cell-cycle regulators, interestingly, we identified wrnip1. WRNIP1 protects stalled replication forks, especially fork reversal, from MRE11- and SLX4-mediated degradation and promotes fork restart by cooperating with BRCA2 to stabilize RAD51 on single-stranded DNA of stalled forks (Leuzzi et al., 2016a; Leuzzi et al., 2016b; Porebski et al., 2019). In addition, wrnip1 regulates another fork protection mechanism called damage bypass, consisting of translesion synthesis (TLS) (Yoshimura et al., 2019) and template switching (TS) (Jiménez-Martín et al., 2020). WRNIP1 also functions in interstrand crosslink repair (ICR) by promoting the recruitment of FANCD2/FANCI complex at the damaged site (Socha et al., 2020). Indeed, wrnip1 mRNA expression is markedly reduced in banp mutants, suggesting that Banp is required for maintenance of wrnip1 transcription. In this case, loss of Wrnip1 may cause defects in stalled fork stabilization and ICR repair, leading to genomic instability (Kondratick et al., 2021; Nickoloff et al., 2021; Scully et al., 2021). It will be interesting to examine whether knockdown of zebrafish wrnip1 causes similar defects in DNA replication stress and apoptosis to banp mutants. Another possibility is that Banp regulates DNA repair independent of its role as a transcription factor. A previous study using human in vitro culture cells revealed that BANP promotes HDAC6-mediated deacetylation of a DNA end-joining protein, Ku70, which is required for non-homologous end-joining repair (Chaudhary et al., 2014). Since Ku70 is recruited to DSB sites after γ-H2AX accumulation, Ku70 is an interesting candidate. Since knockdown of Ku70 along with radiation-induced DNA damage markedly induces apoptosis in zebrafish embryos (Bladen et al., 2007), it will be interesting to investigate whether this scenario occurs in zebrafish.

In addition to the role of Banp in DNA replication stress and DNA repair mechanisms, mRNA expression of two important regulators of chromosome segregation, ncapg and cenpt, is significantly downregulated in banp mutants. ATAC-seq and RNA-seq confirmed that chromatin accessibility is decreased near the TSSs of these genes in banp mutants, suppressing transcription of these mRNAs. Our de novo motif enrichment study identified the Banp motif sequence, TCTCGCGAGA, as the most enriched motif of closed chromatin regions of downregulated genes. Thus, it is highly likely that Banp directly binds to the Banp motifs of both ncapg and cenpt, opens their chromatin, and promotes their transcription. Consistently, as reported in zebrafish ncapg and cenpt knockdown embryos (Hung et al., 2017; Seipold et al., 2009), chromosome segregation is compromised in mitosis of retinal progenitor cells in banp mutants, suggesting that Banp is an essential regulator of mitosis. Since a Banp motif is found in promoter regions of Ncapg and Cenpt in mice and humans, and Chip sequencing with BANP antibody validated TSS peaks of both Ncapg and Cenpt genes in mice and humans (Grand et al., 2021; Mathai et al., 2016), it will be interesting to confirm whether BANP regulates chromosome segregation during mitosis in mammals.

Lastly, we identified 26 candidate Banp direct-target genes, including cell-cycle regulators such as ncapg, cenpt and wrnip1, which carry Banp motifs near their TSSs, and show markedly decreased chromatin accessibility and transcription in the absence of Banp. This gene list will provide a useful starting point to investigate the Banp-mediated transcriptional network. It was reported that binding of BANP to Banp motifs is dependent on DNA methylation of Banp motifs (Grand et al., 2021), suggesting that an epigenetic mechanism regulates Banp-mediated transcription. Thus, zebrafish banp mutants provide an excellent new model in which to study how epigenetic factors regulate cellular processes for embryonic development via Banp motifs of CpG island promoter genes. In addition, BANP has been proposed as a tumor suppressor (Malonia et al., 2011). It is generally accepted that DNA methylation of essential genes is altered in cancer cells (Baylin et al., 2001), which is expected to modify the pattern of gene transcription through Banp motifs (Grand et al., 2021). It will be interesting to investigate how the Banp-mediated transcription profile is changed in cancer models of zebrafish. Future research on zebrafish banp mutants will provide more in-depth understanding of cancer growth and metastasis, as well as new insights into development of therapeutics for human tumors.

Raw RNA-seq and ATAC-seq datasets of banprw337 mutant and wild-type siblings are available at DDBJ Sequence Read Archive (DRA012572).

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Decision letter after peer review:

Thank you for submitting your article "Banp regulates DNA damage response and chromosome segregation during the cell cycle in zebrafish retina" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Richard White as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Sarah R Hengel (Reviewer #1).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1) Substantially revise the text. All reviewers agreed that the data could be of potential interest and impact, but raised concerns about the presentation of the results. As such the impact and relevance beyond the biologist in the field of zebrafish eye development was difficult to discern. It needs to be written so that someone outside of the retina field understand the implications.

2) Add additional experimentation to better illustrate the new Banp functions in replication stress and mitosis. As is the reviewers agreed that in particular the proposed function during mitosis appears internally inconsistent and contradictory. As one example, the term mitotic arrest usually reflects a failure to complete mitosis, typically as a metaphase arrest. Once anaphase is committed to (chromosome segregation), there is no arrest that the reviewers are aware of e.g. in late anaphase.

Reviewer #2 (Recommendations for the authors):

1. Banp mutants have numerous defects, and perhaps this is not unexpected for a nuclear matrix protein. I'm left wondering what insights are gained from the study beyond that the nuclear matrix is required for numerous cell cycle events?

2. Why did the authors focus on the eye? It is unclear whether this study revealed a sensitivity to eye development regarding nuclear matrix function specifically, or it was just a convenient place in the animal to look.

3. I found the conclusions regarding mitosis to be contradictory. The authors at first emphasize mitotic arrest, but then characterize chromosome segregation defects. How can chromosomes segregate if cells are arrested in mitosis?

4. It would be important to know whether the authors can rule out that S-phase defects cause the M phase defects, or vice versa. Could there be a primary defect, rather than multiple independent defects as the authors conclude?

Reviewer #3 (Recommendations for the authors):

To increase the impact of the work, provide a better understanding of how loss of banp leads to induction of DNA damage (currently presented as a question mark in Figure 7).

Reorganize the figures, the text or both so that the reader can easily recognize all the data that support the same point.

Make sure that all conclusions are supported by data, either new or published.

Line 229: 'Histone H3 (pH3) antibody, which labels mitotic cells in pro-metaphase…' Actually, pH3 is seen also in other stages of mitosis and not just pro-metaphase, so the statement should be corrected.

Line 375: If 'mitotic arrest in banprw337 mutants is mostly mediated by a tp53-independent mechanism.' Provide an explanation for the reduction in pH3 cells with FL tp53 MO in Figure 4E.

Line 319: why do the authors suggest that 'FL tp53 triggers Δ113tp53 transcription in banprw337 mutants'? Is full length p53 known to promote its own transcription from an alternate promoter? If so, please provide data or a citation.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Banp regulates DNA damage response and chromosome segregation during the cell cycle in zebrafish retina" for further consideration by eLife. Your revised article has been evaluated by Richard White (Senior Editor), Katharina Schlacher (Reviewing Editor) and reviewers.

The reviewers noted that the manuscript has been improved but there are some remaining issues that need to be addressed before publication, as outlined below:

1) Line 288-289: The authors changed 'prometaphase' to 'metaphase', but this is inaccurate. The correct statement should indicate that pH3 is seen at many stages of mitosis including prometaphase, metaphase and early anaphase.

2) A major concern from the previous version remains to be clarified better, which is the relationship between the DNA damage in S-phase and the mitotic defects. In particular it needs to be stated clearly if these two defects are truly independent or if one precedes the other. If the S phase defects are independent of the mitotic defects, then few PH3+ cells will have foci. The authors state line 419-425 "There is no significant difference in the pH3+ cell fraction between in the total retinal cells and γ-H2AX+ retinal cells (Figure 4—figure supplement 1J). While there is no significant difference it is slightly decreased so this data could potentially address this issue, but should be clearly presented and interpreted. E.g. if S-phase defects are independent of M-phase defects but S-phase defects precede mitosis, then what happens to those damaged S-phase cells? A concise clarification in the text can address this issue.

3) The cartoons/models should be revised into multiple model panels that are each simplified and concentrate on the essential information, as well as a temporal progression be added (e.g. currently it appears that the BrdU⁺ cells are derived from M phase cells, is this correct?

An additional suggestion but not a requirement is to consider adding wrnip1 data to the abstract. The link to DNA damage is one of the key findings of the work.

Essential revisions:

1) Substantially revise the text. All reviewers agreed that the data could be of potential interest and impact, but raised concerns about the presentation of the results. As such the impact and relevance beyond the biologist in the field of zebrafish eye development was difficult to discern. It needs to be written so that someone outside of the retina field understand the implications.

To emphasize the impact of our work beyond zebrafish eye development, we have revised the text as follows.

1) In the introduction, we have moved the explanation of Banp protein to the first paragraph. Then we introduce the zebrafish retina and its contribution to research on cell-cycle regulation and DNA damage response. We also trimmed some details from the description of mechanisms of retinal development. We believe that these arrangements better emphasize the impact of our findings on Banp and will enable readers to more easily understand the research background of our studies about the role of Banp in cell-cycle and DNA damage response.

2) In the results, we have combined the paragraph on reduced expression of chromosome segregation regulators ncapg and cenpt in banp mutants with the paragraph on live imaging of chromosome segregation phenotypes in banp morphants. These data are shown in a new Figure 5. Accordingly, the impact of Banp motifs on Banp downstream target genes is summarized in an independent section and shown in a new Figure 6. These rearrangements make the role of Banp in cell-cycle regulation more clear, and should increase impact of our findings.

To address the concerns above, we have revised the manuscript as follows.

1) To better illustrate the new Banp functions in replication stress and DNA damage repair, among potential direct targets of Banp, we examined wrnip1, which reportedly protects stalled forks from degradation and promotes fork restart after replication stress. We show that loss of Banp compromise expression of wrnip1, potentially leading to one cause of replication stress (Figure 6 and Figure 6—figure supplement 2).

2) For data consistency, we have changed “mitotic arrest” into “mitotic cell accumulation” or simply “mitotic defects” in descriptions of banp mutant phenotypes, because “mitotic arrest” may mislead the readers to think that M phase progression is permanently arrested during mitosis, which is contradictory to our live imaging observations that mitosis is markedly prolonged, but eventually completed in banp morphants.

We have provided more detail about these additional experiments in a point-by-point response to each referee’s comments. Line number in this letter indicates the position of our corrections in the revised manuscript. Please access the line number in the manuscript with the option “Show all Revisions inline” in Microsoft word. The line number may change if track changes are partially or completely masked.

2) Add additional experimentation to better illustrate the new Banp functions in replication stress and mitosis. As is the reviewers agreed that in particular the proposed function during mitosis appears internally inconsistent and contradictory. As one example, the term mitotic arrest usually reflects a failure to complete mitosis, typically as a metaphase arrest. Once anaphase is committed to (chromosome segregation), there is no arrest that the reviewers are aware of e.g. in late anaphase.

Reviewer #2 (Recommendations for the authors):

1. Banp mutants have numerous defects, and perhaps this is not unexpected for a nuclear matrix protein. I'm left wondering what insights are gained from the study beyond that the nuclear matrix is required for numerous cell cycle events?

As we mentioned in the Introduction, BANP was originally identified as a nuclear protein that binds matrix-associated regions (MARs). MARs are regulatory DNA sequences mostly present upstream of various promoters. MAR-binding proteins interact with numerous chromatin-modifying factors and regulate gene transcription. In addition, it was reported that BANP suppresses tumor growth, and that loss of BANP heterozygosity is associated with several cancers in humans. So, before we started this banp mutant analysis, we expected that loss of Banp might cause defects in the cell cycle. However, because the majority of prior studies on BANP have been done using in vitro systems, its physiological function was still ambiguous. Very recently, it was reported that BANP functions as a transcription factor that binds to Banp motifs and regulates essential metabolic genes. In this study, rather than focusing on the MAR domain, we used this Banp motif to search for direct transcriptional targets of Banp that may function in cell proliferation and differentiation in zebrafish retina. Our study provides the first in vivo evidence that Banp serves as an essential transcription activator of cell cycle genes, including cenpt, ncapg, and wrnip1 via Banp motifs. We believe that such a list of Banp direct target genes provides a new research avenue to discover more precisely how Banp functions in tumor suppression and that it will contribute to medical research on cancer therapy.

Our study did not investigate how the nuclear matrix itself is involved in Banp mutant phenotypes. However, since it is likely that the interaction between MAR domains and nuclear matrix may influence chromatin organization in the nucleus, BANP functions must depend on nuclear matrix configuration. So, while this question is interesting, we think it is beyond the scope of our current study. In addition, we are afraid that the term “matrix-associated nuclear protein” might mislead people to think that Banp is a regulator of nuclear matrix. To better clarify the relationship between Banp and nuclear matrix, we have revised “nuclear matrix-associated protein” -> “nuclear matrix associated region-binding protein” in the text.

2. Why did the authors focus on the eye? It is unclear whether this study revealed a sensitivity to eye development regarding nuclear matrix function specifically, or it was just a convenient place in the animal to look.

Historically, molecular and cellular mechanisms that regulate cell proliferation and differentiation in the nervous system has been intensively studied using the vertebrate retina, because retinal neuronal cell types are fewer than those of other brain regions and its neural circuits are also simpler than those of other brain regions. Furthermore, many research groups, including us, have identified zebrafish retinal mutants, including mutants that show defects in cell-cycle regulation and DNA damage response. Indeed, our group has investigated this topic using retinal apoptotic mutants for the last 20 years. Thus, we focus on the zebrafish retina, because the retina is an excellent in vivo model system to dissect mechanisms of cell-cycle regulation and DNA damage response.

To emphasize the importance of this excellent in vivo model system to researchers beyond the retinal community, we have revised in the Introduction as follows.

The developing retina is a highly proliferating tissue, in which a spatiotemporal pattern of neurogenesis is tightly coordinated by cell-cycle regulation. So, vertebrate retina provides a great model for studying how cell-cycle regulation, including DNA damage response ensures neurogenesis and subsequent cell differentiation. (Line 109-113).

3. I found the conclusions regarding mitosis to be contradictory. The authors at first emphasize mitotic arrest, but then characterize chromosome segregation defects. How can chromosomes segregate if cells are arrested in mitosis?

We apologize for the confusion due to our incorrect usage of the term “mitotic arrest.” Mitotic arrest was one of possibilities that we considered when first examining banp mutant phenotypes, in which we just observed accumulation of mitotic (pH3+) cells. However, when we examined mitosis in Banp morphants using live imaging, we found that mitosis duration is significantly prolonged because of chromosome segregation defects in Banp morphants, but that all 28 mitoses we examined eventually completed cytokinesis. Thus, we finally concluded that mitotic cells are not permanently arrested in M phase, but that mitosis is prolonged. To prevent confusion, we have changed “mitotic arrest” to “mitotic cell accumulation” or simply “mitotic defects” in the Results section on banp mutant phenotype analysis (shown in Figures 2 and 4).

4. It would be important to know whether the authors can rule out that S-phase defects cause the M phase defects, or vice versa. Could there be a primary defect, rather than multiple independent defects as the authors conclude?

We thank reviewer #3 for this suggestion. Interdependence between S phase defects and M phase defects is important to correctly interpret the data on cell-cycle regulation, especially cell-cycle checkpoint and DNA damage response. Indeed, there are interesting reports using in vitro cell culture systems indicating that replication stress induces mitotic death, through specific pathways for example, Masamsetti et al., 2019, Nat. Comm. 10.4224. However, this topic is still challenging to dissect in vivo.

In terms of our findings on Banp functions in zebrafish, we found that two chromosome segregation regulators, ncapg and cenpt, are direct transcription targets of Banp, and that it is likely that loss of Banp causes mitotic defects through downregulation of cenpt and ncapg. From this point, we conclude that mitotic defects are primary effects of the loss of Banp. The next question is how the loss of Banp stalls DNA replication forks and causes subsequent cell death. To address this question, we examined whether Banp direct targets include cell-cycle regulators, especially in S phase. We found that wrnip1 is an interesting candidate, because Wrnip1 reportedly protects stalled replication forks and promotes fork restart after DNA replication stress. In addition, Wrnip1 functions in interstrand crosslink repair (ICR). We found that the mRNA expression level of wrnip1 is markedly decreased in banp mutants, suggesting the possibility that DNA replication stress may be caused by reduction of wrnip1 expression in banp mutants. We present these data in new Figure 6—figure supplement 2. We have revised the possible role of Banp in cell-cycle regulation in new Figure7. Under this scenario, we consider it likely that loss of Banp may cause DNA replication stress through downregulation of S phase regulators, independent of mitotic defects.

However, we cannot exclude the possibility that DNA replication stress causes mitotic defects in banp mutants. Masamsetti et al., 2019, Nat. Comm. 10.4224. revealed that replication stress induces spindle assembly checkpoint (SAC)-dependent mitotic arrest and subsequent mitotic death when tp53 activity is inhibited. We showed that cell death in zebrafish banp mutant retinas was fully suppressed by tp53-MO at 48 hpf, but still occurred at 72 hpf, although there was no significant difference between wildtype and banp mutants (Figure 3GH). In the manuscript, we mentioned the possibility that some tp53-independent mechanism induces retinal apoptosis in banp mutants after 48 hpf. An alternative possibility is that most cell death in banp mutants depends on tp53; however, replication stress persisting in banp mutants injected with MO-tp53 may cause SAC-mediated mitotic death, as reported by Masamsetti et al., 2019. Future studies will be necessary to clarify this possibility.

Reviewer #3 (Recommendations for the authors):

To increase the impact of the work, provide a better understanding of how loss of banp leads to induction of DNA damage (currently presented as a question mark in Figure 7).

Please see our response to Essential revision #2 and Reviewer 2 point #3. We found that wrnip1 mRNA expression is drastically reduced in banp mutants, which may cause DNA replication stalling and abnormal phenotypes.

Reorganize the figures, the text or both so that the reader can easily recognize all the data that support the same point.

Make sure that all conclusions are supported by data, either new or published.

A similar concern was raised by the editors (Essential revision #1). Please see our response.

Line 229: 'Histone H3 (pH3) antibody, which labels mitotic cells in pro-metaphase…' Actually, pH3 is seen also in other stages of mitosis and not just pro-metaphase, so the statement should be corrected.

We thank the reviewer for this correction. We changed “pro-metaphase” to “metaphase” (Line 282-283).

Line 375: If 'mitotic arrest in banprw337 mutants is mostly mediated by a tp53-independent mechanism.' Provide an explanation for the reduction in pH3 cells with FL tp53 MO in Figure 4E.

We are grateful for these comments. Since mitotic cell accumulation is significantly reduced by FL tp53-MO, there is a substantial contribution of FL tp53 to mitotic cell accumulation. So, we have revised the sentences (Line 438-448).

Compared with Standard-MO injection controls (n=376.6±74.8/section), the number of pH3+ cells in banp^rw337 mutants was significantly decreased by FL tp53-MO (n=262.4±68.3/section) (Figure 4C, 4E). However, the number of pH3+ cells was still higher in banp^rw337 mutant retinas injected with FL tp53-MO than in wild-type control retinas injected with FL tp53-MO, indicating that there is a substantial tp53independent fraction. Furthermore, injection of ∆113tp53-MO did not significantly rescue mitotic phenotypes in banp^rw337 mutants (n=303.8±46.6/section) (Figure 4C, 4F). These data suggest a tp53-independent mechanism that causes mitotic cell accumulation in banp^rw337 mutants.

Line 319: why do the authors suggest that 'FL tp53 triggers Δ113tp53 transcription in banprw337 mutants'? Is full length p53 known to promote its own transcription from an alternate promoter? If so, please provide data or a citation.

We have cited a paper reporting the evidence (Line 360-362). The authors claim that FL tp53 initially activates transcription of ∆113tp53 after tp53 protein is stabilized in response to cellular stress (Chen et al., 2009).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The reviewers noted that the manuscript has been improved but there are some remaining issues that need to be addressed before publication, as outlined below:

1) Line 288-289: The authors changed 'prometaphase' to 'metaphase', but this is inaccurate. The correct statement should indicate that pH3 is seen at many stages of mitosis including prometaphase, metaphase and early anaphase.

We have revised accordingly.

Line 227: mitotic cells in prometaphase, metaphase and early anaphase (Prigent and Domitrov, 2013)

2) A major concern from the previous version remains to be clarified better, which is the relationship between the DNA damage in S-phase and the mitotic defects. In particular it needs to be stated clearly if these two defects are truly independent or if one precedes the other. If the S phase defects are independent of the mitotic defects, then few PH3+ cells will have foci. The authors state line 419-425 "There is no significant difference in the pH3+ cell fraction between in the total retinal cells and γ-H2AX+ retinal cells (Figure 4—figure supplement 1J). While there is no significant difference it is slightly decreased so this data could potentially address this issue, but should be clearly presented and interpreted. E.g. if S-phase defects are independent of M-phase defects but S-phase defects precede mitosis, then what happens to those damaged S-phase cells? A concise clarification in the text can address this issue.

Thank you for your suggestions. We agree that it is important to clarify how S phase defects and M phase defects influence each other. As you suggested, it is interesting that pH3+ cell fraction in g-H2AX+ retinal cells is lower than that of total retinal cells, although there is no statistical difference. At 48 hpf, banp mutants show increases in

1

g-H2AX+ cells, pH3 + cells, and apoptotic cells, compared with wild-type control. The reduction of pH3+ cell fraction in g-H2AX+ retinal cells suggest that g-H2AX+ retinal cells are eliminated by apoptosis in S phase prior to mitosis. In addition, M phase defects are likely to be independent of replicative damages mostly introduced in S phase.

We have added the sentences below in the Results section (Line 364-369).

“Despite of no statistical significance, interestingly, averaged fraction of pH3+ cells in γ-H2AX+ retinal cells was lower than that of total retinal cells. Since banp^rw337 mutants show increase in γ-H2AX + cells, pH3 + cells and apoptotic cells at 48hpf, it is likely that DNA damaged cells are eliminated by apoptosis in S phase prior to mitosis. This also suggest that M phase defects may be independent of replicative DNA damage introduced in S phase.”

Furthermore, regarding the question on, “if S-phase defects are independent of Mphase defects but S-phase defects precede mitosis, then what happens to those damaged S-phase cells?”, it is difficult to examine S phase defects and M phase defects separately because both defects appear at 48 hpf. However, our observations indicate that mitosis in banp mutants is eventually completed, and that apoptosis is not overlapped with accumulated pH3+ cell area, suggesting that M phase defects are not associated with apoptosis but only prolonged mitosis duration. So, DNA damage is likely to be inherited by daughter cells through the mitosis, resulting in apoptosis in immature neurons. To clarify how S- and M-phase defects influence each other in banp mutants, we have added the description on the summary of banp mutant phenotype in the Results section (Line 502-509) in Figure 7B and its legends (Line 1470-1480), which we hope guide readers to understand how S- and M-phase defects influence each other during cell-cycle progression in banp mutants.

3) The cartoons/models should be revised into multiple model panels that are each simplified and concentrate on the essential information, as well as a temporal progression be added (e.g. currently it appears that the BrdU⁺ cells are derived from M phase cells, is this correct?

We consider that the cartoons/models pointed by editors above are Figures 2EF. Since the original Figures 2EF globally describe retinal phenotypes of banp mutants, especially only TUNEL and pH3+ cells at 53 to 77 hpf, this explanation does not clearly represent the interpretation drawn from the data of Figure 2 and Figure 2—figure supplement 1. We have revised these two figure panels to clarify what happens at a cellular level: cell-cycle progression and neurogenesis along the apico-basal axis of the neural retina as well as developmental stages. We replaced new cartoons in Figure 2EF and also revised the text of Results section (Line 277-284) and their legends (Line 1288-1311).

An additional suggestion but not a requirement is to consider adding wrnip1 data to the abstract. The link to DNA damage is one of the key findings of the work.

We have added wrnip1 data to the abstract.

Author details

1. Swathy Babu

   Developmental Neurobiology Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Japan

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0612-9484

2. Yuki Takeuchi

   Developmental Neurobiology Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Japan

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8480-5206

3. Ichiro Masai

   Developmental Neurobiology Unit, Okinawa Institute of Science and Technology Graduate University, Onna, Japan

   Contribution

   Conceptualization, Resources, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   masai@oist.jp

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6626-6595

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