SATB2‐LEMD2 interaction links nuclear shape plasticity to regulation of cognition‐related genes

Abstract SATB2 is a schizophrenia risk gene and is genetically associated with human intelligence. How it affects cognition at molecular level is currently unknown. Here, we show that interactions between SATB2, a chromosomal scaffolding protein, and the inner nuclear membrane protein LEMD2 orchestrate the response of pyramidal neurons to neuronal activation. Exposure to novel environment in vivo causes changes in nuclear shape of CA1 hippocampal neurons via a SATB2‐dependent mechanism. The activity‐driven plasticity of the nuclear envelope requires not only SATB2, but also its protein interactor LEMD2 and the ESCRT‐III/VPS4 membrane‐remodeling complex. Furthermore, LEMD2 depletion in cortical neurons, similar to SATB2 ablation, affects neuronal activity‐dependent regulation of multiple rapid and delayed primary response genes. In human genetic data, LEMD2‐regulated genes are enriched for de novo mutations reported in intellectual disability and schizophrenia and are, like SATB2‐regulated genes, enriched for common variants associated with schizophrenia and cognitive function. Hence, interactions between SATB2 and the inner nuclear membrane protein LEMD2 influence gene expression programs in pyramidal neurons that are linked to cognitive ability and psychiatric disorder etiology.

1. At least three different studies have investigated the consequences of Satb2 elimination in neurons on mouse behavior and physiology. Two of these studies are referred in the text: Jaitner et al., 2016 and Li et al., 2017a. The third one by Zhang et al. 2019 is, however, not referred. The authors should discuss these results and refer whether the behavioral analyses were preformed in the same combination of floxed strain and cre driver. The article lacks a biological frame to interpret the relevance of the reported changes in nuclear morphology and gene expression. The authors should consider to include their own electrophysiological or behavioral analyses in the neuronalspecific KOs investigated here and strengthen the connection between Satb2, changes in nuclear morphology, and plasticity. For example, are the neurons responding to a given experience (i.e., Fos positive) more likely to present nuclear infoldings? Does this situation change in Stab2-cKOs in correlation with memory impairments? 2. After reporting that the elimination of Satb2 or Lemd2 impairs activity-dependent changes in nuclear morphology, it would be interesting to examine how these deficits affect activity-driven transcription. The decision of restricting the RNA-seq experiments to the stimulated condition, excluding the analysis of transcription in the basal condition, prevents a proper evaluation of gene induction. We do not know if the reported changes correspond to basal differences between genotypes or specifically emerge after stimulation. A 2x2 design (wt/cKO x Sal/Bic) would enable more analyses and might provide much more interesting results, particularly if the main objective is to relate the morphological changes with activity-dependent transcription and plasticity.
3. As summarized by the authors (p. 16), the study provides evidence that cooperation between SATB2 and other proteins of the nuclear envelope/lamina determine plastic changes in nuclear envelope geometry in response to action potential bursting. However, they did not conduct any experiment to explore the relevance and biological role of these changes. They later indicate (p. 18): "Consistent with this hypothesis, we did observe an impaired IEG response upon both SATB2 knockout and LEMD2 depletion in bicuculline-stimulated primary cortical neurons". Unfortunately, this sentence is not accurate the authors did not explore the IEG response, because to do that they would need to compare basal and induced levels in WT and cKO. As indicated above, this is one of the main caveats of the study.
4. The authors should demonstrate the specificity and efficiency of their conditional knockout. What is the percentage of neurons presenting Satb2 ablation? If the percentage is not close to 100% (as it happens in some CamK2a-cre lines), the authors could explore in the same slide the differential response and morphology of neurons expressing or lacking Satb2. 5. To complement the experiments in neuronal cultures, the authors should examine if activitydependent changes of nuclear morphology are also impaired in vivo. For example, inducing status epilepticus with kainic acid or pilocarpine. It would be also very interesting to see if IEG induction (e.g., Fos) is affected in Satb2-KO cells and if the deficit somehow correlates with the impaired change in the nuclear envelope. 6. The experiment using rAAV to overexpress Satb2 could suggest that Satb2 is produced in response to response to activity and the activity-dependent increase in Satb2 levels causes the nuclear morphology change. This view is consistent with the results presented by Li and colleagues (2017). Is Satb2 also upregulated in the stimulation paradigms examined in this study? 7. In the same experiment, as they previously claimed that the infolding is triggered by the action potential, it would be interesting to block the action potential and examine if the overexpression still causes an increase in infolded nuclei. 8. The authors refer in the Discussion to a possible role of SATB2 and LEMD2 in the regulation of epigenetic marks (in particular they mention H3K9me2 ans H3K9me3). Given the current shortcomings of the study, it would be interesting that the authors strengthen their study by directly investigating the proposed connection between these proteins and the histone modifications.
9. It would be interesting to examine the spatial relocation in the nucleus (center to periphery) of some of the candidate genes identified in the transcriptome screen by fluorescence in situ hybridization (FISH) analysis. This would definitively strengthen the proposed model. 10. Availability of the datasets. The authors should indicate that their RNA-seq datasets have been deposited in a public database such as GEO and provide the access number in the manuscript. In addition, they should provide a token for access during peer review to the editor and reviewers. In most journals dealing with genomic data, the deposition of the datasets in a public repository is a mandatory requirement for publication.
Other comments: 11. The Abstract should be re-written to summarize better the objectives, results and conclusions of the study. It is surprising, for example, that the third sentence already states the main conclusion, when the results of the study have not been presented.
12. The results of the IPs and western-blot presented in Figure 1 should be quantified. This is advisable in all cases, but particularly important in the case of Figure 1A(iii) given the very subtle difference between co-IP SATB2 using the LEMD2 IgG antibodies. How do the authors explain the poor recovery compared to the other experiments.
13. In page 6, the authors study BAF, a protein that binds LEM domains. They prove that BAF is not important in the interaction between LEMD2 and SATB2. They should provide some background information about why they thought that BAF could be important in the interaction.
14. In page 6, line 17, the author mention that HeLa cells devoid of endogenous SATB2. They should probably mention this earlier because they also used HeLa in the GST experiment.
18. In page 10 line 9 and 10, the authors mention "a cocktail of three siRNAs targeting distinct parts of the Lemd2 mRNA 3'UTR reproducibly caused a strong reduction of LEMD2 (Fig 3A, I and Suppl. Fig 4A)". However it is not clear if the results presented in the Figures correspond to the cocktail or to an individual siRNA. This should be indicated in the legends. Also, the sequence of the siRNAs targeting Lemd2 mRNA should be provided. 19. In page 11, the authors claim that the "ectopic expression of STAB2 or VPS4 increased the number of infolded nuclei", as well as a dominant negative of Vps4 abolished the increase in infolded nuclei. However in Suppl Fig5 there is not image presenting these differences just a bar plot. Also the meaning of VPS4aDN is not explained (although I suppose it corresponds to the dominant negative mutant). 20. Fig. 4A and Suppl. Fig 7: Why did the authors decide to collapse the values higher than 6 in the Y-axis. These highly significant strongly up-and down-regulated genes re likely the most interesting candidates for further exploration. Are the IEGs located in this area? 21. The authors should probably highlight the axes and their intersection in the heatmap presented in Fig. 4C. The description of the graph is quite obscure, but it seems that a significant number of genes upregulated in cKOs show the opposite behavior in the siLemd2 experiment. 22. In page 13, line 14: Fig 3D does not exist, probably they refer to 4D.
23. In page 13, line 20: they said "62 genes were co-regulated by SATB2 in adult cortex", based in the numbers of Figure 4E, ii, there are 61 genes. Figure 4E, iii some negative genes should be added to illustrate the differences.

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25. Some genome browser images showing representative genes differentially expressed in the RNA-seq analysis could be added to Fig. 4. It would be particularly interesting to present this together with the profile for Satb2 activity-dependent binding.
26. The first sentences of page 14 mention early and late response genes but do not cite the source of these lists and classification.
27. Also in page 14, line 7: the statistic needs a parenthesis to be reported in the same format than below.
28. In page 16, the authors claim that their analysis "reveals a previously unexpected overlap of the human phenotypes that converge on SATB2-and LEMD2-controlled gene-sets". They should test this by comparing the enrichments in the geneset corresponding to the overlap of the SATB2-KO and LEMD2-depleted screens and those exclusive of the SATB2 and LEMD2 screens.
Referee #3: In the submitted manuscript, Feurle et al. describe the Satb2-Lemd2 interactions and their potential role in the nuclear organization and gene expression regulation. Satb2 is an important chromatin regulator and transcription factor involved in the control of multiple aspects of brain development and functions. The authors have recently identified LEMD2 protein as and interacting partner of SATB2. LEMD2 is a nuclear protein implicated in organization of chromatin and nuclear structure. In the current manuscript the authors characterized Satb2-Lemd2 both physical and genetic interactions. They show that Satb2 and LEMD2 interact physically via Cut domain on Satb2 and this interaction does not depend on BAF protein. On the other hand they show that Satb2 can control neuronal activity induced nuclear shape changes. Most importantly, in a series of genetic experiments, they show that Satb2 mediated nuclear folding induction requires Lemd2 as well as its partner, ATPase VPS4. Next the authors compared gene expression changes in Satb2 and Lemd2 depleted neurons after bicucullin-treated neurons. Bicucullin is a GABA blocker that causes high levels of excitation and used in vitro to model neuronal activity. In these experiments the authors detected a number of genes that are regulated by both genes. Finally, the authors carried out computational analysis of Lemd2 regulated genes and found that some of these genes were mutated in human autism, schizophrenia and ID patients To summarize: this is an impressive work that clearly shows a novel role of Satb2 in organizing nuclear shape as well as identifies its interacting partner. Both genetic and biochemical experiments are well designed and conducted. However, there are several week points as outlined below.
Involvement of Lemd2 in shaping neuronal nucleus is shown only in vitro, while for Satb2, the authors used both in vitro and in vivo approaches. I suggest similar in vivo experiment for Lemd2. The authors seem to have all the tools, to transfect cortical or hippocampal neurons in vivo, without necessity of making a null allele. It would be interesting to see if absence of Lemd2 will have a similar phenotype in vivo, and whether it is Satb2 dependent.
Second problem is that the authors use bicucullin treatment as a model for activity induced genes. This is OK to a certain extent, however as any in vitro model has its limitations. On the other hand it is a model of epileptiform activity. Since it is a treatment that can cause robust non physiological changes, it can cause a lot of gene expression changes too that could be artefacts of global network malfunction. I would recommend the authors to be careful in their interpretations and tone down their conclusions about activity induced genes.

Response to reviewers
Referee #1: This is an extremely innovative paper exploring the cooperative function/interaction of SATB2 chromatin organizer and LEMD2 inner nuclear membrane protein and a subset of ESCRT protein complexes as regulator of the shape of neuronal nuclei, in context of activity-regulated paradigms and with implications for neuronal gene expression.
The paper presents a huge amount of work and in general has been conducted state of the art as it pertains to the co-immunoprecipitation experiments, the microscopy, with the hypothesis stringently tested in cell culture and in vivo mouse mutant models.
The paper is innovative and will have significant impact as the regulation of the 3D shape of neuronal nuclei is heavily understudied , with very little knowledge on molecular mechansism s thus far.
My only comment/criticsm is that there is one peculiar piece of evidence missing in this paper, which is the spatial proximity of genes jointly targeted by the SATB2-LEMD2 proteins to the inner nuclear membrane. One would expect that their SATB2-LEMD2 sensitive genes will show a change in their proximity to the inner nuclear membrane. could this be tested for at least some of the genes.
At the very least, this should be better discussed in the paper, or even better, tested experimentally.

11th Sep 2020 1st Authors' Response to Reviewers
Response: First, we would like to thank the reviewer for referring to our manuscript as "extremely innovative". This comment provides very strong motivation for us.
We agree with the referee that studying nuclear localization and mobility relative to the inner nuclear membrane of LEMD2/SATB2-regulated loci upon neuronal activation is important. In order to address this question, we first successfully established immunofluorescence / DNA FISH in cortical cultures for cFos and Gadd45γ using protocol and probes described previously by distances to the nuclear envelope that can be measured in an infolded nucleus. Obviously it becomes really complicated in 3D. We are currently establishing algorithms for clustering 3Dreconstructed neuronal nuclei according to their morphology using UMAP. This should allow us to measure mobility of FISH signals relative to complex shapes of the nuclear envelope in clusters rather than individual nuclei. While these experiments are ongoing, we are currently unable to provide reliable data. Therefore, we refrain from showing results of our preliminary experiments. Instead, we followed the recommendation of the reviewer to explain the situation in the discussion part of our manuscript. We have added the requested behavioral experiment in adult animals. Using Satb2 CamkCre mice, for which we have previously described deficient L-LTP and long-term memory, allowed for the correlation of cFos expression, nuclear infolding and memory deficit. To study the correlation between nuclear infoldings and neuronal activation in vivo, we used exposure to novel environment as an experimental model. In contrast to pharmacologically-triggered neuronal activation, e.g. KA-induced status epilepticus that causes strong and synchronous activation in all hippocampal subfields, the exploration of a novel and rich spatial context is an established behavioral paradigm that activates sparse neuronal assemblies throughout the hippocampus by a natural stimulus, thus allowing for the analysis of both activated (cFos-positive) and inactive (cFos-negative) neurons. As outlined in the modified manuscript as well as in the novel Fig 5, the results show that the deep invaginations of the nuclear membrane are a correlate of neuronal activation triggered by a naturalistic stimulus and that SATB2 is required for both nuclear envelope plasticity and cFos induction in vivo.  (p. 16), the study provides evidence that cooperation between SATB2 and other proteins of the nuclear envelope/lamina determine plastic changes in nuclear envelope geometry in response to action potential bursting. However, they did not conduct any experiment to explore the relevance and biological role of these changes. They later indicate (p. 18): "Consistent with this hypothesis, we did observe an impaired IEG response upon both SATB2 knockout and LEMD2 depletion in bicuculline-stimulated primary cortical neurons". Unfortunately, this sentence is not accurate the authors did not explore the IEG response, because to do that they would need to compare basal and induced levels in WT and cKO. As indicated above, this is one of the main caveats of the study. We are very grateful for the suggestion to analyze a mosaic of Satb2-deficient and Satb2expressing cells in the same histological sections. This was not possible for Satb2 due to the complete nature of the transgenic modification as stated above. However, motivated by the reviewer's comment, we followed such a mosaic-analysis strategy for our in vivo Lemd2 knockdown analysis as will be outlined below.

To complement the experiments in neuronal cultures, the authors should examine if activity-dependent changes of nuclear morphology are also impaired in vivo. For example, inducing status epilepticus with kainic acid or pilocarpine. It would be also very interesting to see if IEG induction (e.g., Fos) is affected in Satb2-KO cells and if the deficit somehow
correlates with the impaired change in the nuclear envelope.

Response:
Here we refer to our response given to point number 1. The requested experiment has been added, however instead of using harsh pharmacological stimulation, we decided to stimulate neurons with natural sensory stimulation by exposure to novel environment.

Response:
We have characterized the effect of neuronal activation and BDNF on SATB2 levels in hippocampal cultures in substantial detail. This work, we would like to point out, was already

Response:
We have tested the effect of overexpression of Satb2 on infoldings in hippocampal neurons in the absence of Bic-stimulation. Since under these conditions neurons are only sparsely active, further pharmacological inhibition will have no or very little effect.

Response:
We agree with the referee that analyses of Satb2-dependent alterations in epigenetic marks are important. However, we fail to see an immediate link to the content of the present study focusing on nuclear shape. These epigenetic analyses are part of a separate study, in which we will describe the effect of Satb2 on 3D chromatin configuration in pyramidal neurons.

Response:
We refer to our response given to the same point raised by Reviewer #1

Availability of the datasets. The authors should indicate that their RNA-seq datasets have been deposited in a public database such as GEO and provide the access number in the
manuscript. In addition, they should provide a token for access during peer review to the editor and reviewers. In most journals dealing with genomic data, the deposition of the datasets in a public repository is a mandatory requirement for publication.

Response:
All transcriptome data sets have been deposited as requested by the policy of EMBO J.
Other comments: 11. The Abstract should be re-written to summarize better the objectives, results and conclusions of the study. It is surprising, for example, that the third sentence already states the main conclusion, when the results of the study have not been presented.

Response:
The abstract has been modified according to this comment. Figure 1 should be quantified. This is advisable in all cases, but particularly important in the case of Figure 1A(iii) given the very subtle difference between co-IP SATB2 using the LEMD2 IgG antibodies. How do the authors explain the poor recovery compared to the other experiments.

Response:
Western blot raw data are provided following EMBO J guidelines. Two different explanations for the comparably weak LEMD2 signal can be put forward. First, the available antibodies for LEMD2 are of poor quality in stark contrast to SATB2 antibodies. Second, it appears likely that only a minor fraction of LEMD2 is bound to SATB2 in primary neurons and can be co-immunoprecipitated with SATB2. Due to these limitations, we deemed it important to corroborate the protein interaction between SATB2 and LEMD2 with recombinant tagged versions of both proteins. As is evident from the results depicted in Fig.1, these experiments provided evidence for very robust and strong interactions between SATB2 and LEMD2.

In page 6, the authors study BAF, a protein that binds LEM domains. They prove that BAF
is not important in the interaction between LEMD2 and SATB2. They should provide some background information about why they thought that BAF could be important in the interaction.

Response:
The manuscript has been modified accordingly.

In page 6, line 17, the author mention that HeLa cells devoid of endogenous SATB2. They
should probably mention this earlier because they also used HeLa in the GST experiment.

Response:
The reviewer is correct and we apologize for the mistake. The manuscript has been corrected. Figure 1 they likely meant to say (616-733).

Response:
Again the reviewer is correct. The manuscript has been corrected.

P. 9: The authors wrote: "Injection with rAAV8-EGFP did not rescue the loss of infoldings in
Satb2-deficient CA1 pyramidal neurons (Fig 2E)". However, Figure 2E does not present this result and, supposedly, correspond to non-infected mice.

Response:
We have used commercially available cocktails of siRNAs (targeting Lemd2, Vps4a, Vps4b and scrambled siRNA) with proprietary sequences not disclosed to the customer. The order numbers of the cocktails are provided in the manuscript. For the revision of the manuscript we have generated AAV-shRNA viruses which we then used to knockdown Lemd2 in vitro and in vivo. We have designed these shRNAs and the sequences are described in the revised manuscript. We found results to be consistent between siRNA and shRNA knockdown in all comparisons.

Response:
We decided against showing these infoldings because their appearance is not principally different from the other infolded nuclei depicted in the manuscript rendering their display redundant information. The reviewer is correct, DN stands for dominant negative. This information has been added to the manuscript. Fig. 4A and Suppl. Fig 7: Why did the authors decide to collapse the values higher than 6 in the Y-axis. These highly significant strongly up-and down-regulated genes re likely the most interesting candidates for further exploration. Are the IEGs located in this area?

Response:
The entire figure 4 has been reorganized. New data have been added in the format of Volcano blots. The effect on IEGs specifically is described in Table EV3. Fig. 4C. The description of the graph is quite obscure, but it seems that a significant number of genes upregulated in cKOs show the opposite behavior in the siLemd2 experiment.

Response:
The reviewer is correct. Some genes are regulated in opposite direction by Satb2 versus Lemd2 loss-of-function. All relevant information about individual genes is available in the deposited GEO dataset. IEG expression in general is affected in the same direction for individual genes, upor down-regulated, by Satb2 or Lemd2 loss-of-function. Fig 3D does not exist, probably they refer to 4D.

Response:
The manuscript has been corrected.
23. In page 13, line 20: they said "62 genes were co-regulated by SATB2 in adult cortex", based in the numbers of Figure 4E, ii, there are 61 genes.

Response:
These figures have changed (increased) due to the addition of more sequencing information. Figure 4E, iii some negative genes should be added to illustrate the differences.

Response:
We are not sure what the referee is referring to with "negative genes".

Response:
The Manuscript has been modified accordingly.

Response:
The primary motivation for the GSA analyses was to characterize the relevance of the novel LEMD2-regulated gene set for human phenotypes. We were surprised to observe that it is associated with in essence the same phenotypes that we have reported before for SATB2dependent genes. Restricting the analysis to the commonly regulated gene sets would drastically reduce statistical power and consequently the quality of the results.

Response:
We agree with the referee that studying the effect of LEMD2 depletion on nuclear morphology is important. Unfortunately, siRNA-mediated knockdown, while very useful in vitro, is practically impossible in vivo due to the unstable nature of the agent. We are not aware of relevant publications applying this method in CNS. Hence, in order to perform the requested experiment, we generated and characterized shRNA expressing AAV viruses which turned out to very effectively reduce LEMD2 protein levels. As additional advantage, these viruses also encode mCherry as fluorescent marker allowing us to discriminate virus-transduced from nontransduced neurons on identical sections. We are happy to report in the revised manuscript that upon stereotactic injection of these viruses into adult hippocampus our analyses of mosaic sections containing both tranduced and non-transduced neurons yielded highly significant results demonstrating that Lemd2 knockdown in fact causes deficit in nuclear infolding of pyramidal neurons in vivo .

Second problem is that the authors use bicucullin treatment as a model for activity induced
genes. This is OK to a certain extent, however as any in vitro model has its limitations. On the other hand it is a model of epileptiform activity. Since it is a treatment that can cause robust non physiological changes, it can cause a lot of gene expression changes too that could be artefacts of global network malfunction. I would recommend the authors to be careful in their interpretations and tone down their conclusions about activity induced genes. common tests, such as t-test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann-Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; are tests one-sided or two-sided? are there adjustments for multiple comparisons? exact statistical test results, e.g., P values = x but not P values < x; definition of 'center values' as median or average; definition of error bars as s.d. or s.e.m.
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Is there an estimate of variation within each group of data?

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