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  1. Author Response

    Reviewer #3 (Public Review):

    In this manuscript the authors study the consequences in neurons of knocking out the cohesin subunit Rad21. The authors have previously performed a version of chromatin conformation capture called 5C in which they are able to generate very high-resolution chromatin interaction maps across focused regions of the genome. In that study they focused on several activity-inducible genes and showed that there were both pre-existing and activity-inducible interactions of putative enhancers with the promoters of activity-inducible genes. Here to determine if Rad21 is important for those interactions and their functional consequences on gene regulation, they do two different knockouts in postmitotic neurons (cell type cKO and rapid TEV-mediated cleavage). Loss of Rad21 led to impaired expression of many neuronal genes at baseline as well reduced branching and spine density, by comparing against previous HiC maps, the authors show that the most affected genes are those with the largest loops. Then they move on to activity-regulated genes, where they compare the effects of Rad21 deletion on their 5C maps as well as gene expression. These data show that activity-induced genes expression and inducible looping between promoters and putative enhancers proceed largely normally in the absence of Rad21, though large CTCF loops are disrupted.

    Understanding the mechanisms of chromatin organization in the nucleus is important and this group has one of the best methods for studying high resolution chromatin interactions. Knocking out Rad21 is a reasonable strategy to disrupt looping and the 5C data support that the authors did successfully change some aspects of loops in postmitotic neurons that are important for neuronal development. However, the most notable finding in the data is that for the most part, activity-induced gene expression and activity-induced changes in promoter looping to putative enhancers were unaffected in Rad21 knockout neurons. This is rather different from the results of a previously published Rad21 knockout, though the authors don't discuss this.

    Overall this is a well-executed study that presents descriptive data about the functions of cohesin-mediated chromatin architecture in neurons and offers data that suggests that Rad21 is mostly not required for activity-dependent transcription.

    We thank the referee for these thoughtful and constructive comments. However, we note that the interpretation that 'for the most part, activity-induced gene expression and activity-induced changes in promoter looping to putative enhancers were unaffected' appears to overlook that many inducible genes were deregulated at baseline in cohesin-deficient neurons, and a significant proportion of LRGs remained deregulated after activation with KCl or BDNF. We also note that, in contrast to neurons, a sizeable proportion of secondary response genes in macrophages are dependent on interferon expression, which is reduced in the absence of cohesin. Accordingly, exogenous interferon rescues the expression of numerous secondary response genes in cohesin-deficient macrophages (Cuartero et al., 2018). We have addressed these points in detail in the revised manuscript.

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  2. Evaluation Summary:

    Neurons use activity-responsive gene programs to shape cell specific identity and respond appropriately to environmental stimuli. By combining elegant protein degradation and cell-specific knockout approaches with transcriptional profiling and chromatin structure analysis, this manuscript delineates the contributions of cohesin (a key protein responsible for genome structure and organization), in activity-dependent gene expression programs and stimulus-dependent chromatin reorganization. These results demonstrate that cohesin is required for full expression of key genes required for the maturation and activation of cortical excitatory neurons, and reveal a tight correlation between cohesin effects and the genomic distance of higher order chromatin loops.

    (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. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    As an essential subunit of cohesin, RAD21 is a known regulator of large-scale genome organization, acting via a mechanism called loop extrusion to facilitate enhancer-promoter contacts in the genome. Given that enhancers play a key role in cell-specific gene expression patterns and activity-dependent responses, the long-range looping structures enabled by cohesin have been predicted to regulate key aspects of neuronal function. To test this idea, this manuscript from Calderon and colleagues uses both conditional deletion or inducible degradation of RAD21 in cortical neurons (both in vitro and in vivo) to examine resulting effects on gene expression, morphology, and stimulus-dependent transcriptional responses. The major finding of the manuscript is that loss of cohesin in primary neuron cultures results in downregulation of over 1000 genes, with an enrichment for genes involved in synaptic function and neuron differentiation. Not surprisingly, this manipulation also produces robust effects in vivo, impairing cortical layer organization, neuronal complexity, and spine density. Using careful bioinformatic analysis that includes chromatin contact information, the manuscript reports that the predominant effects of RAD21 loss are on long-rage chromatin interactions. Specifically, while RAD21 depletion decreased expression of many genes at baseline, a core set of secondary response genes, characterized by delayed response to depolarizing stimuli or neuronal growth factors, were preferentially affected by the manipulation. Inducibility of a subset of these genes is lost upon RAD21 depletion, and genes with longer chromatin contacts (as measured by HiC interactions) were among the most affected. Notably, RAD21 depletion had few effects on short-range chromatin interactions (or stimulus-dependent loop formation), demonstrating selectivity in its contribution to activity-dependent transcriptional responses in neurons.

    Overall, the results of the manuscript are compellingly presented, the experiments have appropriate controls, and the manuscript is well organized. The strengths of the manuscript are the extensive validation of specific RAD21 knockdown/deletion in cortical excitatory neurons, a systematic characterization of RAD21 depletion effects at the morphological, transcriptional, and chromatin levels, and the use of inducible approaches to manipulate RAD21. The interpretation that cohesin plays distinct roles in regulation of secondary response genes (which tend to be involved in longer chromatin loops) is supported by RNA-seq evidence that stimulus-regulated expression of these genes is lost following RAD21 depletion. In contrast, the evidence that cohesin effects scale with chromatin loop length is largely correlational, and the manuscript lacks a more systematic interrogation of this relationship. Similarly, the manuscript would benefit from a more comprehensive analysis comparing the effects of RAD21 depletion on constitutive and inducible chromatin loops in primary neuron cultures. However, this rigorous manuscript represents a necessary and fundamental first step in identification of the mechanisms by which cohesin (and by extension, chromatin looping) regulates stimulus-dependent gene expression in the nervous system.

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  4. Reviewer #2 (Public Review):

    This is an interesting paper describing neuron-specific chromatin loop regulation, with the main finding centered on role of the cohesion complex in context of activity-dependent gene expression and the observation that 'cohesin dependence of constitutive neuronal genes....scaled with chromatin loop length'. At its core, these are, from the neuroscience perspective, very novel findings.

    Basically, the authors report that Immediate Early genes (IEG) with chromatin loops far shorter than 100Kb are much less affected in conditional mutant neurons with floxed RAD21 cohesin subunit deletion . In contrast, secondary activity-regulated genes (SRGs)with average loop length scaling beyond several hundred Kb are highly sensitive to the loss of RAD21. The authors further strengthen their observation in parallel experiments with Rad21Tev/Tev neurons (this model they recently published, Weiss et al, 2021) with inducible protease digestion of RAD21.

    The strength of the paper is the methodological rigor as it pertains to the study of gene expression changes (incl. two activity induced paradigms, KCL and BDNF) in two types of mutant neurons of the cohesin RAD21 subunit.

    This is an interesting paper describing neuron-specific chromatin loop regulation, with the main finding centered on role of the cohesion complex in context of activity-dependent gene expression and the observation that 'cohesin dependence of constitutive neuronal genes....scaled with chromatin loop length'. At its core, these are , from the neuroscience perspective, very novel findings.

    Basically, the authors report that Immediate Early genes (IEG) with chromatin loops far shorter than 100Kb are much less affected in conditional mutant neurons with floxed RAD21 cohesin subunit deletion. In contrast, secondary activity-regulated genes (SRGs)with average loop length scaling beyond several hundred Kb are highly sensitive to the loss of RAD21. The authors further strengthen their observation in parallel experiments with Rad21Tev/Tev neurons (this model they recently published, Weiss et al, 2021) with inducible protease digestion of RAD21.

    The strength of the paper is the methodological rigor as it pertains to the study of gene expression changes (incl. two activity induced paradigms, KCL and BDNF) in two types of mutant neurons of the cohesin RAD21 subunit.

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  5. Reviewer #3 (Public Review):

    In this manuscript the authors study the consequences in neurons of knocking out the cohesin subunit Rad21. The authors have previously performed a version of chromatin conformation capture called 5C in which they are able to generate very high-resolution chromatin interaction maps across focused regions of the genome. In that study they focused on several activity-inducible genes and showed that there were both pre-existing and activity-inducible interactions of putative enhancers with the promoters of activity-inducible genes. Here to determine if Rad21 is important for those interactions and their functional consequences on gene regulation, they do two different knockouts in postmitotic neurons (cell type cKO and rapid TEV-mediated cleavage). Loss of Rad21 led to impaired expression of many neuronal genes at baseline as well reduced branching and spine density, by comparing against previous HiC maps, the authors show that the most affected genes are those with the largest loops. Then they move on to activity-regulated genes, where they compare the effects of Rad21 deletion on their 5C maps as well as gene expression. These data show that activity-induced genes expression and inducible looping between promoters and putative enhancers proceed largely normally in the absence of Rad21, though large CTCF loops are disrupted.

    Understanding the mechanisms of chromatin organization in the nucleus is important and this group has one of the best methods for studying high resolution chromatin interactions. Knocking out Rad21 is a reasonable strategy to disrupt looping and the 5C data support that the authors did successfully change some aspects of loops in postmitotic neurons that are important for neuronal development. However, the most notable finding in the data is that for the most part, activity-induced gene expression and activity-induced changes in promoter looping to putative enhancers were unaffected in Rad21 knockout neurons. This is rather different from the results of a previously published Rad21 knockout, though the authors don't discuss this.

    Overall this is a well-executed study that presents descriptive data about the functions of cohesin-mediated chromatin architecture in neurons and offers data that suggests that Rad21 is mostly not required for activity-dependent transcription.

    Read the original source
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