Banp regulates DNA damage response and chromosome segregation during the cell cycle in zebrafish retina

Article activity feed

1. 

   Version published to 10.7554/elife.74611 on eLife

   Aug 9, 2022
2. 

   Version published to 10.1101/2021.11.03.467208v4 on bioRxiv

   Jun 24, 2022
3. 

   Version published to 10.1101/2021.11.03.467208v3 on bioRxiv

   Apr 29, 2022
4. 

   eLife

   Apr 4, 2022

   Author Response:

   Reviewer #1 (Public Review):

   In this manuscript, the authors exploit retinal cell proliferation and neurogenesis in zebrafish to study banp, a protein that is essential in humans and embryonic lethal in mice. The authors performed large-scale mutagenesis and identified a mutant known as "rw337" that compared to WT cells the mutant zebrafish have smaller eyes and optic tectum. They found that the retinas of these mutants have mitotic-like round cells that accumulate indicating mitotic arrest. Sequencing of these mutants identified that the rw337 mutant gene encodes a truncated banp protein. Expression of WT Banp occurs primarily in retinal and neuronal cells in Zebrafish. Interestingly, rw337 showed significant decrease in retinal photoreceptors number and neuronal formation within the OPL and IPL were morphologically …

   Author Response:

   Reviewer #1 (Public Review):

   In this manuscript, the authors exploit retinal cell proliferation and neurogenesis in zebrafish to study banp, a protein that is essential in humans and embryonic lethal in mice. The authors performed large-scale mutagenesis and identified a mutant known as "rw337" that compared to WT cells the mutant zebrafish have smaller eyes and optic tectum. They found that the retinas of these mutants have mitotic-like round cells that accumulate indicating mitotic arrest. Sequencing of these mutants identified that the rw337 mutant gene encodes a truncated banp protein. Expression of WT Banp occurs primarily in retinal and neuronal cells in Zebrafish. Interestingly, rw337 showed significant decrease in retinal photoreceptors number and neuronal formation within the OPL and IPL were morphologically disrupted and had fewer cells. The authors found that rw337 cells have increased numbers of DSBs in the retina over time (via TUNEL) assays. They found that mitotic defects and apoptosis are spatially and temporally occurring in distinct regions of the retina as prolonged phosphorylation of histone H3, which indicates an issue in exit of mitosis, occurred in apical surface of the neural retina whereas apoptosis occurred in retinal progenitor cells (via Caspase 3 staining). The authors then went on to examine the role of replication stress regulators like p53, atm, and atr and showed that protein and RNA levels of banprw337 were increased and upregulated. As p53 binds banp in zebrafish, it was not surprising that regulators of p53 were enhanced in banprw337 mutants. Intriguingly, the authors found that two genes which are essential for chromatin segregation were downregulated in banprw337 mutants and banp morphants as a result of chromatin accessability decreases near the TSS of resulting in decreased transcriptional activity of cenpt and ncapg genes. Finally, the authors temporally monitored mitosis in mitosis of banprw337 mutants and found that chromosomal segregation is abnormal and takes longer. The authors have performed a thorough analysis of the impact of the banp gene on retinal biology and its importance regulating replication stress response and cenpt and ncapg expression. This paper is important to retinal biology, genome stability, and replication stress response fields and requires minor revision.

   Strengths:
   • These studies exploit zebrafish retinal development and its cell-cycle regulation as knockout of Banp/ SMAR1 is an essential gene in human cells and embryonic lethal in mice.
   • The authors show that this gene is involved in replication stress responses involving p53, atm, and atr signaling.
   • The authors show that banp is required for chromatin segregation factors and chromatin accessability by binding to banp sequences (TCTCGCGAGA) upstream of specifically cenpt and ncapg. Interestigly the mutant rw337 had decreased chromatin accessability near the transcript start sites of these genes. This is an elegant study of how a gene is regulating the transcription of two genes essential for chromatin segregation.
   •
   Weaknesses:
   • The authors could highlight the protein names of both zebrafish and humans throughout the text using standard nomenclature description with humans proteins all capitalized etc... This will enable the reader to understand their findings in the context of fascinating biology and human disease/cancer.

   We have revised nomenclature of genes and proteins throughout the text, consistent with nomenclature conventions as follows.

   species /gene/ protein zebrafish / banp / Banp mouse / Banp / BANP human / BANP / BANP

   In the revised manuscript, we have used human/mouse/zebrafish nomenclature in sentences relating findings that were achieved using human/mouse/zebrafish samples, respectively.

   • As banprw337 mutants show such severe morphological disruption a discussion on the impact of this work for the vision community could strengthen the importance of understanding how this gene functions.

   We appreciate this suggestion. In response to comments from the editor and reviewer #2, we have revised the Introduction to mention that vertebrate retina is an excellent model system to dissect mechanisms of cell-cycle regulation and DNA damage response-mediated neuronal cell death. We believe that our banp paper will have an impact on the retinal community. Furthermore, in addition to the role of Banp in cell-cycle regulation, most photoreceptors fail to differentiate in banp mutants, whose phenotypes are more severe than other retinal cell-types. Nuclear architecture, especially heterochromatin and euchromatin patterns, are quite differently organized in photoreceptor neurons and dynamically changed during rod photoreceptor differentiation, so we suspect that Banp may be important for photoreceptor differentiation through regulation of its nuclear organization. In the future, we will investigate this underlying mechanism. There are very interesting perspectives on retinal phenotypes in banp mutants, which may attract retinal and vision community researchers. However, these are diverse topics. So, in the current manuscript, we have limited the discussion to within cell-cycle regulation.

   • Gamma H2AX phosphorylation is a global marker of DSBs and stalled forks. The authors did not note that H2AX phorylation is present and a marker of stalled replications forks.
   o PMID: 11673449, PMID: 20053681, doi:10.1101/gad.2053211, https://doi.org/10.1016/j.cell.2013.10.043 etc.

   We appreciate this suggestion. We have added a statement on gamma-H2AX and cited appropriate references.

   • As gamma H2AX phosphorylation recruits DNA repair factors like BRCA2, speculation of importance of these genes may be of interest to the DNA repair community.

   We agree that to clarify which step or steps of DNA replication stress and the DNA repair mechanism are direct targets of Banp, it is important to consider how DNA repair factors are affected in banp mutants. Among Banp transcriptional target genes, we found that wrnip1 mRNA expression is significantly reduced in banp mutants. We have added these data to a new Figure 6-figure supplement 2. wrnip1 protects stalled replication forks from degradation and promotes fork restart during replication stress by cooperating with BRCA2. It was recently reported that WRNIP1 functions in translesion synthesis (TLS) and template switching (TS) at stalled forks, and also interstrand crosslink repair (ICR). It is possible that the loss of Wrnip1 causes defects in fork stabilization for restart, and ICR, leading to genomic instability. We have added this material to the Discussion and have revised a summary figure (Figure 7).

   Reviewer #2 (Public Review):

   Babu et al report the role of the zebrafish banp gene in the developing retina. They find that banp is required for faithful S-phase as well as mitosis.

   Manuscript strengths: 1- The authors performed a large-scale mutagenesis screen and successfully identified a causative banp gene mutation from these efforts, which represent a significant amount of work. 2- The authors provide a substantial amount of cellular-level analysis of a host of cell cycle-related phenotypes in the banp mutant retina. The data are of high technical quality and the experiments are well-executed. For the most part, the data support the conclusions.

   We are grateful for the reviewer’s high estimation of our work.

   Manuscript weaknesses: 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."

   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 #2 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 replicationstress 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 (Public Review):

   Babu and colleagues demonstrate that banp is expressed in the retina progenitor cells among other locations, and mutational loss of it results in increased mitosis, increased apoptosis, increased DNA damage, and the failure to differentiate photoreceptors. Importantly, these phenotypes are seen at a time period when retina progenitors undergo rapid cell cycles and differentiate into multiple cell types that make up the fully developed retina. Rescue with the wild type and phenocopy with another mutant allele provide strong support that the phenotypes results from loss of banp. Mutant animals show elevated p53 protein and reduction of p53 delays the onset of apoptosis by 24 hours. Mutant animals show altered transcriptional profile, with increased p53 expression and decreased expression of two genes that encode proteins needed for chromosome segregation. The authors propose that loss of banp results in defective DNA replication and DNA damage as well as mitotic chromosome segregation failures, all of which contribute to p53-dependent apoptosis to reduce cell number and cause developmental defects.

   Banp is a very interesting protein. Also known as Scaffold/matrix attachment region binding protein 1, it is known to regulate the transcription of a number of genes including those important in oncogenesis. In vivo function of Banp, especially in the context of normal development, remains to be better understood. The current study fills this knowledge gap but I have some concerns about the interpretation of the data, the presentation and the potential impact. Specifically:

   We are very pleased that reviewer #3 understood and appreciated the significance of our study.

   Increased expression of atm and atr is observed and the authors suggest that replication stress and DNA damage activate the checkpoints to cause cell cycle arrest. There are several problems with this conclusion, which is depicted in Fig. 4G. Checkpoint activation occurs via phosphorylation changes in ATM/ATR and not through their transcriptional upregulation, which would take too long for a response that occurs within minutes.

   We agree with the referee that upregulation of ATR/ATM mRNA expression may represent chronical activation of DNA replication stress and DNA damage response. In addition to ATR/ATM mRNA upregulation, RNA-seq analysis revealed that exo5 is one of the TOP15 upregulated genes in banp mutants (Fig. 3B). exo5 plays a critical role in ATR-dependent replication restart (Hambarde et al., 2021), suggesting that chronic replication stress occurs in banp mutants. We have mentioned exo5 upregulation in the Results section. As Referee 1 suggested, phosphorylation of H2AX is induced by ATR prior to DSBs, indicating that gammaH2AX is a marker of DNA replication stalling as well as of DSBs. We showed that gamma-H2AX+ cells are more numerous in banp mutants (Figure 4CF) and morphants (Figure 4-figure supplement 1AB) and in S phase banp mutant cells (Figure 4-figure supplement 1CDEFF’), suggesting that DNA replication stress and subsequent DNA damage linked to fork breakage are induced in banp mutants. We have revised the text by adding this statement in the Results section. In addition, we have revised Fig. 4G and its legend, in order to more clearly show the role of ATR and ATM in DNA replication fork repair and HR-mediated DNA repair in response to DSBs, and tp53-mediated regulation of cell survival and death.

   ATM/ATR-dependent checkpoints arrest cells in G1 or G2 so you would expect reduced S and M phases. Yet, the authors saw increased M and no change in S.

   It is puzzling that BrdU+ cell number does not change because if cells are indeed arrested in mitosis, they should be prevented from going into S phase and BrdU+ cell numbers should decrease.

   There is no significant difference in the BrdU+ fraction of total retinal cells between wild-type and banp mutants at 48 hpf (Fig. 2-figure supplement 1AC), suggesting that cell-cycle arrest in S phase does not occur at significant levels in banp mutants at 48 hpf. At present, we have no good tool to detect G1 phase in zebrafish developing retina, because the Cdt1 fluorescent protein of the FUCCI zebrafish line cannot be stably driven in highly proliferating tissues such as zebrafish retina due to its very short G1 duration. Thus, we cannot determine whether G1 arrest occurs in banp mutant retina. However, we found that mRNA expression of p21 cdk inhibitor is upregulated in banp mutants, using bulk RNA-seq (Figure 3AB) and RT-PCR (Figure C), so it is still possible that banp mutant retinal cells are (probably partially) arrested in G1 phase. We have added this possibility to the Discussion. Further study is necessary to evaluate this point.

   It is not addressed whether cenpt and ncapg expressed in the retina and whether are their expressions decreased in banp mutants. The RNAseq data is from whole animals.

   RNA-seq data (Fig 3AB) were obtained from embryonic heads, but not whole bodies (see Materials and Methods). In accordance with this suggestion, to examine whether cenpt and ncapg mRNAs are expressed in retina, we performed in situ hybridization. We confirmed that these mRNAs are expressed in proliferative cells in zebrafish retina and have added these data to new Figure 5-figure supplement 1. In addition, we also confirmed that cenpt and ncapg mRNA expression is absent in banp mutants (see panels at 48 hpf in Fig. 5-figure supplement 1).

   The rescue by banp-EGFP in Fig.1G is very nice. But it looks like there is partial rescue also with EGFP-banp(rw337) in the same panel. The defects the last panel do not seem as severe as in non inj controls. There are fewer pyknotic nuclei and the cell layers lack gaps. Quantification of the extent or reproducibility of the rescue is lacking.

   We conducted acridine orange (AO) staining of retinas of wild-type, banp mutants, and banp mutants injected with banp(wt)EGFP and with EGFP-banp(rw337). We confirmed that banp(wt)EGFP significantly suppressed apoptosis in banp mutant retinas, whereas EGFP-banp(rw337) did not. We have added these data to new Figure 1-figure supplement 5. So, there is no partial rescue by EGFP-banp(rw337).

   Some of the conclusions lack supporting data. For example, line 99: "Thus, Banp is required for integrity of DNA replication and DNA damage repair." There are no data for the integrity (meaning 'fidelity'?) of DNA replication and there are no DNA repair assays.

   Thank are grateful for this suggestion. We understand that the term “integrity” could be too strong and changed it to “regulation.”

   In another example, non-overlap of pH3 (M phase) and caspase+ cells is interpreted to mean that cells are dying in S phase (Figure 2 supplement 1). But the data are equally consistent with cells dying in G1 and G2.

   In addition to non-overlap of the pH3+ and caspase+ areas along the apico-basal axis of the retina (Fig.2-figure supplement 1DG), we did not observe mitotic death in our live imaging of mitosis in banp morphant retinas. Considering the very short G2 phase of retinal cells in zebrafish, we conclude that apoptosis occurs mostly in retinal progenitor cells undergoing G1 or S phase, or differentiating neurons. However, we cannot exclude the possibility that apoptosis occurs in G2 phase. So, we have revised the text. Furthermore, caspase 3+ cells were mostly located in the intermediate zone of the neural retina along the apico-basal axis, whereas pH3+ cells were localized at the apical surface of the neural retina (Fig. 2-figure supplement 1G), suggesting that apoptosis occurs mostly in retinal progenitor cells during G1, S or G2 phase, or in differentiating neurons. Accordingly, we have revised Fig. 2-figure supplement 1L, to suggest that apoptosis may be induced in G1, S, or G2 phase.

   The model in Figure 7 includes components without accompanying supportive data. For example, the arrow from Banp to DNA repair that indicates a direct role and the arrow from tp53 to delta113 tp53 that indicates direct activation.

   Thank appreciate this suggestion. We have revised Figure 7 and its legend. In new Figure 7, we used solid arrows for regulatory pathways confirmed by us and previous other groups, and dotted arrows for proposed regulatory pathways. We already cited a reference (Chen et al., 2009), indicating direct activation of ∆113 tp53 by FL tp53.

   The data that together support a single point are often split up among figures. For example, increased pH3+ cells shown in Fig. 2 and is interpreted as mitotic arrest. But it is equally possible that cells are undergoing extra divisions (and then dying). Support for mitotic arrest is provided by live imaging of mitosis, which is not presented until the last figure (Fig. 6). There are many such instances in the manuscript.

   A similar concern was raised by reviewer #2. Please see our response.

   Banp is already known for roles in p53-dependent transcription and in apoptosis (e.g. Sinha et al papers cited in the manuscript). Banp is also known to bind to the promoter regions of cenpt and ncapg (Grand et al and Mathai et al papers cited in the manuscript). These genes are known to be involved in mitosis in zebrafish (Hung et al and Seipold et al papers cited in the manuscript). In terms of what is new about banp function in this report, the requirement for banp in a critical phase of retina development and spontaneous induction of DNA damage come to mind. Unfortunately, how loss of banp leads to this defect remains to be addressed.

   A related concern was raised by the editors and also by reviewer #2. Please see our responses. We found that wrnip1 mRNA expression is drastically reduced in banp mutants, which may cause DNA replication stalling and abnormal phenotypes.

   Read the original source
5. 

   eLife

   Apr 4, 2022

   Evaluation Summary:

   This study reports the phenotype of a zebrafish banp mutant that was identified in a screen for eye defects. Banp is known to regulate the transcription of a number of genes including those important in oncogenesis. In vivo function of Banp, especially in the context of normal development, remains to be better understood. The current study fills this knowledge gap and identifies the roles of banp during replication stress responses and mitosis. With somewhat more careful interpretation of the data and a clearer presentation of the results and their potential impact, this study will be of interest to scientists studying development, DNA damage responses and apoptosis.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with …

   Evaluation Summary:

   This study reports the phenotype of a zebrafish banp mutant that was identified in a screen for eye defects. Banp is known to regulate the transcription of a number of genes including those important in oncogenesis. In vivo function of Banp, especially in the context of normal development, remains to be better understood. The current study fills this knowledge gap and identifies the roles of banp during replication stress responses and mitosis. With somewhat more careful interpretation of the data and a clearer presentation of the results and their potential impact, this study will be of interest to scientists studying development, DNA damage responses and apoptosis.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

   Read the original source
6. 

   eLife

   Apr 4, 2022

   Reviewer #1 (Public Review):

   In this manuscript, the authors exploit retinal cell proliferation and neurogenesis in zebrafish to study banp, a protein that is essential in humans and embryonic lethal in mice. The authors performed large-scale mutagenesis and identified a mutant known as "rw337" that compared to WT cells the mutant zebrafish have smaller eyes and optic tectum. They found that the retinas of these mutants have mitotic-like round cells that accumulate indicating mitotic arrest. Sequencing of these mutants identified that the rw337 mutant gene encodes a truncated banp protein. Expression of WT Banp occurs primarily in retinal and neuronal cells in Zebrafish. Interestingly, rw337 showed significant decrease in retinal photoreceptors number and neuronal formation within the OPL and IPL were morphologically disrupted and had …

   Reviewer #1 (Public Review):

   In this manuscript, the authors exploit retinal cell proliferation and neurogenesis in zebrafish to study banp, a protein that is essential in humans and embryonic lethal in mice. The authors performed large-scale mutagenesis and identified a mutant known as "rw337" that compared to WT cells the mutant zebrafish have smaller eyes and optic tectum. They found that the retinas of these mutants have mitotic-like round cells that accumulate indicating mitotic arrest. Sequencing of these mutants identified that the rw337 mutant gene encodes a truncated banp protein. Expression of WT Banp occurs primarily in retinal and neuronal cells in Zebrafish. Interestingly, rw337 showed significant decrease in retinal photoreceptors number and neuronal formation within the OPL and IPL were morphologically disrupted and had fewer cells. The authors found that rw337 cells have increased numbers of DSBs in the retina over time (via TUNEL) assays. They found that mitotic defects and apoptosis are spatially and temporally occurring in distinct regions of the retina as prolonged phosphorylation of histone H3, which indicates an issue in exit of mitosis, occurred in apical surface of the neural retina whereas apoptosis occurred in retinal progenitor cells (via Caspase 3 staining). The authors then went on to examine the role of replication stress regulators like p53, atm, and atr and showed that protein and RNA levels of banprw337 were increased and upregulated. As p53 binds banp in zebrafish, it was not surprising that regulators of p53 were enhanced in banprw337 mutants. Intriguingly, the authors found that two genes which are essential for chromatin segregation were downregulated in banprw337 mutants and banp morphants as a result of chromatin accessability decreases near the TSS of resulting in decreased transcriptional activity of cenpt and ncapg genes. Finally, the authors temporally monitored mitosis in mitosis of banprw337 mutants and found that chromosomal segregation is abnormal and takes longer. The authors have performed a thorough analysis of the impact of the banp gene on retinal biology and its importance regulating replication stress response and cenpt and ncapg expression. This paper is important to retinal biology, genome stability, and replication stress response fields and requires minor revision.

   Strengths:
   • These studies exploit zebrafish retinal development and its cell-cycle regulation as knockout of Banp/ SMAR1 is an essential gene in human cells and embryonic lethal in mice.
   • The authors show that this gene is involved in replication stress responses involving p53, atm, and atr signaling.
   • The authors show that banp is required for chromatin segregation factors and chromatin accessability by binding to banp sequences (TCTCGCGAGA) upstream of specifically cenpt and ncapg. Interestigly the mutant rw337 had decreased chromatin accessability near the transcript start sites of these genes. This is an elegant study of how a gene is regulating the transcription of two genes essential for chromatin segregation.
   •
   Weaknesses:
   • The authors could highlight the protein names of both zebrafish and humans throughout the text using standard nomenclature description with humans proteins all capitalized etc... This will enable the reader to understand their findings in the context of fascinating biology and human disease/cancer.
   • As banprw337 mutants show such severe morphological disruption a discussion on the impact of this work for the vision community could strengthen the importance of understanding how this gene functions.
   • Gamma H2AX phosphorylation is a global marker of DSBs and stalled forks. The authors did not note that H2AX phorylation is present and a marker of stalled replications forks.
   o PMID: 11673449, PMID: 20053681, doi:10.1101/gad.2053211, https://doi.org/10.1016/j.cell.2013.10.043 etc.
   • As gamma H2AX phosphorylation recruits DNA repair factors like BRCA2, speculation of importance of these genes may be of interest to the DNA repair community.

   Read the original source
7. 

   eLife

   Apr 4, 2022

   Reviewer #2 (Public Review):

   Babu et al report the role of the zebrafish banp gene in the developing retina. They find that banp is required for faithful S-phase as well as mitosis.

   Manuscript strengths:
   1- The authors performed a large-scale mutagenesis screen and successfully identified a causative banp gene mutation from these efforts, which represent a significant amount of work.
   2- The authors provide a substantial amount of cellular-level analysis of a host of cell cycle-related phenotypes in the banp mutant retina. The data are of high technical quality and the experiments are well-executed. For the most part, the data support the conclusions.

   Manuscript weaknesses:
   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 …

   Reviewer #2 (Public Review):

   Babu et al report the role of the zebrafish banp gene in the developing retina. They find that banp is required for faithful S-phase as well as mitosis.

   Manuscript strengths:
   1- The authors performed a large-scale mutagenesis screen and successfully identified a causative banp gene mutation from these efforts, which represent a significant amount of work.
   2- The authors provide a substantial amount of cellular-level analysis of a host of cell cycle-related phenotypes in the banp mutant retina. The data are of high technical quality and the experiments are well-executed. For the most part, the data support the conclusions.

   Manuscript weaknesses:
   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?

   Read the original source
8. 

   eLife

   Apr 4, 2022

   Reviewer #3 (Public Review):

   Babu and colleagues demonstrate that banp is expressed in the retina progenitor cells among other locations, and mutational loss of it results in increased mitosis, increased apoptosis, increased DNA damage, and the failure to differentiate photoreceptors. Importantly, these phenotypes are seen at a time period when retina progenitors undergo rapid cell cycles and differentiate into multiple cell types that make up the fully developed retina. Rescue with the wild type and phenocopy with another mutant allele provide strong support that the phenotypes results from loss of banp. Mutant animals show elevated p53 protein and reduction of p53 delays the onset of apoptosis by 24 hours. Mutant animals show altered transcriptional profile, with increased p53 expression and decreased expression of two genes that …

   Reviewer #3 (Public Review):

   Babu and colleagues demonstrate that banp is expressed in the retina progenitor cells among other locations, and mutational loss of it results in increased mitosis, increased apoptosis, increased DNA damage, and the failure to differentiate photoreceptors. Importantly, these phenotypes are seen at a time period when retina progenitors undergo rapid cell cycles and differentiate into multiple cell types that make up the fully developed retina. Rescue with the wild type and phenocopy with another mutant allele provide strong support that the phenotypes results from loss of banp. Mutant animals show elevated p53 protein and reduction of p53 delays the onset of apoptosis by 24 hours. Mutant animals show altered transcriptional profile, with increased p53 expression and decreased expression of two genes that encode proteins needed for chromosome segregation. The authors propose that loss of banp results in defective DNA replication and DNA damage as well as mitotic chromosome segregation failures, all of which contribute to p53-dependent apoptosis to reduce cell number and cause developmental defects.

   Banp is a very interesting protein. Also known as Scaffold/matrix attachment region binding protein 1, it is known to regulate the transcription of a number of genes including those important in oncogenesis. In vivo function of Banp, especially in the context of normal development, remains to be better understood. The current study fills this knowledge gap but I have some concerns about the interpretation of the data, the presentation and the potential impact. Specifically:

   Increased expression of atm and atr is observed and the authors suggest that replication stress and DNA damage activate the checkpoints to cause cell cycle arrest. There are several problems with this conclusion, which is depicted in Fig. 4G. Checkpoint activation occurs via phosphorylation changes in ATM/ATR and not through their transcriptional upregulation, which would take too long for a response that occurs within minutes. ATM/ATR-dependent checkpoints arrest cells in G1 or G2 so you would expect reduced S and M phases. Yet, the authors saw increased M and no change in S.

   It is puzzling that BrdU+ cell number does not change because if cells are indeed arrested in mitosis, they should be prevented from going into S phase and BrdU+ cell numbers should decrease.

   It is not addressed whether cenpt and ncapg expressed in the retina and whether are their expressions decreased in banp mutants. The RNAseq data is from whole animals.

   The rescue by banp-EGFP in Fig.1G is very nice. But it looks like there is partial rescue also with EGFP-banp(rw337) in the same panel. The defects the last panel do not seem as severe as in non inj controls. There are fewer pyknotic nuclei and the cell layers lack gaps. Quantification of the extent or reproducibility of the rescue is lacking.

   Some of the conclusions lack supporting data. For example, line 99: "Thus, Banp is required for integrity of DNA replication and DNA damage repair." There are no data for the integrity (meaning 'fidelity'?) of DNA replication and there are no DNA repair assays. In another example, non-overlap of pH3 (M phase) and caspase+ cells is interpreted to mean that cells are dying in S phase (Figure 2 supplement 1). But the data are equally consistent with cells dying in G1 and G2.

   The model in Figure 7 includes components without accompanying supportive data. For example, the arrow from Banp to DNA repair that indicates a direct role and the arrow from tp53 to delta113 tp53 that indicates direct activation.

   The data that together support a single point are often split up among figures. For example, increased pH3+ cells shown in Fig. 2 and is interpreted as mitotic arrest. But it is equally possible that cells are undergoing extra divisions (and then dying). Support for mitotic arrest is provided by live imaging of mitosis, which is not presented until the last figure (Fig. 6). There are many such instances in the manuscript.

   Banp is already known for roles in p53-dependent transcription and in apoptosis (e.g. Sinha et al papers cited in the manuscript). Banp is also known to bind to the promoter regions of cenpt and ncapg (Grand et al and Mathai et al papers cited in the manuscript). These genes are known to be involved in mitosis in zebrafish (Hung et al and Seipold et al papers cited in the manuscript). In terms of what is new about banp function in this report, the requirement for banp in a critical phase of retina development and spontaneous induction of DNA damage come to mind. Unfortunately, how loss of banp leads to this defect remains to be addressed.

   Read the original source
9. 

   Version published to 10.1101/2021.11.03.467208v2 on bioRxiv

   Apr 3, 2022
10. 

    Version published to 10.1101/2021.11.03.467208v1 on bioRxiv

    Nov 4, 2021