The REST remodeling complex protects genomic integrity during embryonic neurogenesis

Thank you for submitting your work entitled "The REST remodeling complex protects genomic integrity during embryonic neurogenesis" for peer review at eLife. Your submission has been favorably evaluated by Fiona Watt (Senior Editor) and two reviewers. One of the two reviewers, Michael Rosenfeld (Reviewer #1), has agreed to reveal his identity.

Below you will find two full reviews of your work. As you can see, the reviewers are highly positive about your findings and request only minor changes. You may want to include some discussion about the relevance of your findings to other tumour types and speculate about inducible REST deletion in adult brain. Please include the quantitation requested in Figure 3 and please consider including some additional markers in Figure 5.

Reviewer #1:

General assessment and major comments:

In the manuscript titled "The REST remodeling complex protects genomic integrity during neurogenesis", the authors reported a very surprising and interesting finding that a previously uncharacterized genome protective role of REST remodeling complex during S-phase of cell cycle in embryonic neurogenesis, which may be the underlying mechanism of a tumor suppressor role of REST in cancer progression and a neuron protective role of REST in age-associated neurodegeneration.

Although REST is a critical transcriptional regulator in neurogenesis, the lack of an obvious neuronal phenotype in the REST knockout model prompted the author "re-open" the case by generating a novel mouse model of REST deficiency. Using a conditional gene-trap strategy, the authors discovered a novel role of REST in genome protection during neurogenesis. Further, they provide in vivo evidence of REST in tumorigenesis using REST and p53 compound knockout mice. In the end, they compared the knockout strategies of two REST mouse models and pinpointed the critical difference between the knockout mouse design strategies that leads to "no obvious neuronal phenotype" conclusion based on the previous REST knockout model.

The role of REST in genome protection during S-phase is a rather surprising finding. The authors revealed an increased number of neurons with positive stains of DNA damage marks such as rH2AX, pATM and p53BP1 in REST KO mice. In addition, they found an increased number of abnormal chromosomal bridges in the REST KO neurons, indicating an important role of REST in S-phase dynamic during neurogenesis. It will be an interesting topic in the future as if REST is involved in DNA damage repair during cancer progression.

There are many links between REST and tumorigenesis previously. However, the causal role of REST deficiency in cancer progression is not yet clearly demonstrated. The authors reported that a significant increase of tumors observed in REST and p53 compound knockout mice, indicating a causal role of REST in tumorigenesis. The author used Nestin Cre in this study; it will be interesting in the future to determine if deletion of REST in the adult brain using inducible Cre on the p53-null background can increase tumorigenesis. In addition, it will be an interesting topic in the future that the tumor suppressor role of REST can be extended to other tumors such as breast cancer observed in human patients.

Mouse models can provide invaluable information in biomedical research. However, it is really rare to "re-open" the investigation if there is a mouse model available since it is considered risky to regenerate a different mouse model with a hope of a different result. The authors' efforts paid off in the end, indicating that micro differences in the experimental design can have quite different outcome. Therefore, a better lesson learned here is to pay more attention to the details of experimental design if the actual results didn't match the predicted based on the hypothesis.

Minor comments:

Overall, the manuscript is well written except a few minor points to be clarified during revision.

1) The authors performed REST ChIP on a few gene targets (Figure 2F). Where are the REST binding sites, on promoters or RE-1 elements of Glrp or Snap25? In addition, what are the expression levels of Glrp or Snap25 in control and REST KO brains?

2) Can the authors clarify CTIP2+ cells (Figure 3E, F, G) between control and REST KO brains? It appears that CTIP2+ cells are increased (Figure 3E), but there are not significant changes reported (Figure 3F, G).

3) What are the percentage of clCasp3/BrdU + or 53BP1/BrdU + cells in control mice (Figure 6G, 6H)?

4) Recently, a few non-coding RNAs or splicing variants have been identified in REST locus from many human cancer cells. Therefore, it is possible that REST locus encodes both REST protein and ncRNAs with distinct biological functions. If the authors can determine the expression of non-coding RNAs or splicing variants in both REST KO strategies, it may help the understanding of the biological roles of REST and the non-coding RNAs in REST locus.

Additional data files and statistical comments:

Can the authors provide transcriptional profiling data comparing control with REST KO brains discussed in the manuscript?

Reviewer #2:

General assessment and major comments:

In this study Nechiporuk et al., uncover an unexpected role for REST in maintaining genomic integrity. They work with a complete loss of function mouse strain (a gene trap reporter) and find that conditional deletion in neural progenitors results in a distinct phenotype from that reported previously. They observe microcephaly in the conditionally deleted mice, and this is consistent with the recent links to human microcephalies. The REST null neural progenitors are also susceptible to malignant transformation when p53 is concomitantly removed. The discrepant findings with the previous knockouts are reconciled by their demonstration that the exon 1 encoded C terminal peptide remains expressed and functional; past mice were therefore not null.

These are interesting and important observations that will be of broad interest to several different research communities. The major novelty is in the realization that REST may protect genomic integrity during cell division.

The REST GT mice have an embryonic lethal phenotype consistent with the previously reported knockout and characterization of this mouse is performed well. They then create mice with a genetic inversion of the GT and breed these to a Nestin Cre driver to achieve conditional deletion. These data are convincing and point to a reduced overall brain size and a thinner cerebral cortex. They go to show that this is due to reduced numbers of cycling cells and increases in apoptotic cells. Figure 5B is particularly convincing and the compelling observation indicating cell death rather than a promiscuous premature neuronal differentiation.

Of particular interest – although unclear how relevant to the human disease – they find PNET/gliomas forming in the mice that harbor the GT allele together with p53. Their observations suggest the role of REST as a tumour suppressor - but related to genetic instability, rather than its function as a repressor of neuronal differentiation.

In Figure 5B it looks like cell death is mainly within the SVZ. It might be worth further defining whether the apoptotic cells are TBR2 basal progenitors or radial glia. So a caspase co-stain with TBR2 and RC2 would distinguish if it were apical progenitors/radial glia that were specifically dying. Higher magnification views of TuJ1, MAP2, RC2, TBR2 and caspase would be useful to show. It would be good to clarify this. Do the progenitors that exit cycle turn on MAP2 prematurely and then die? Of is there some 'proper' progression to neuronal differentiation and then cell death? So morphology and location of the dying cells need better characterization.

There is interest in the hypothesis that neuronal differentiation commitment is linked to extension of G1 times. Would it be useful to discuss their findings in this context? Is this transition something that REST would be involved in or monitoring?

The above are my only minor criticisms. This is an impressive study: rigorous, high quality data, novel insights, beautifully presented and of broad interest. I would recommend publication immediately.