The ToRXRMO arrest was likely a consequence of disrupting a non-RA related role of RXR, since it was so much more severe than the other RA perturbations
Given RXR's role as a promiscuous heterodimer partner, I'm wondering what other NHR orthologs are expressed in Tritia?
The presence of multiple putative components in the shell and dorsal mantle cavity suggest that these structures may be foci of RA signaling
For Tritia, is it possible to perform a pulse-chase with radiolabeled RA and then examine localization?
We find that most treatments cause a very similar spectrum of defects that are centered on the development of the shell
Congratulations on the very intriguing findings! I was wondering if the environmental sources of RA precursors for Tritia obsoleta are known? If so, it may be informative to deplete the precursors from growth media and examine if the phenotypes you've observed in situations where you anticipate RA levels to be reduced are recapitulated in the absence of RA precursors.
Together, these results establish a robust and versatile method for visualizing and quantifying nascent transcription in intact C. elegans tissues.
This is a great, new methodology to study nascent transcription in C. elegans! I'm looking forward to seeing how this approach is extended to incorporate quantitative approaches like EU-RNA-seq or qPCR.
By combining EU labeling with compartment-specific markers, we find that the EU signal localizes to a reticulated network within the fibrillar zone, contrasting with the discrete transcriptional foci often reported in cultured mammalian cells
I'm curious if you have compared this method to approaches like live imaging of MS2 RNA hairpin loops?
We propose that IRF1 brings SPOP to genomic sites occupied by IRF2 thereby promoting IRF2 degradation
Is there any evidence that recruitment of SPOP also targets other promoter-bound factors, like basal transcriptional machinery?
IRF2 limits IRF1 activity under homeostatic conditions but is displaced during an immune response, allowing IRF1-dependent gene programs central to innate immunity and autoinflammation
Congratulations on the elegant and thorough study! I was curious if there are there mutations in IRF2 coding or non-coding regulatory sequences that may link IRF2 with disease states/indications?
Notably, forinhibitors causing the strongest compaction, olaparib and talazoparib, (see Fig. 1), exposingPARP1 to these inhibitors before DNA resulted in a 4 and 6-fold reduction, respectively, inthe subsequent binding to DNA, compared with protocols in which PARP1 was allowed tobind to DNA before exposure to inhibitors (Fig. 3B)
I was curious what is known about how PARP1 finds it's nicked DNA target? Is it possible that the reduced binding to the nicked substrate is due to a reduction in the efficiency of finding the substrate (i.e. sliding or inter- or intra-strand transfer?).
Taken together with Figure 1, thisindicates that upon binding to talazoparib, PARP1 undergoes a large-scale compaction thatstabilises the PARP1 conformation.
This is a really elegant biochemical study--congratulations on the work! I was wondering if you could or have confirmed this compaction using an alternate methodology, like sedimentation velocity analytical ultracentrifugation?
Also, enrichment of the GRE at Pinducible sites is consistent with the known roles of nuclear receptors regulating transcription in response to inflammation26.
Congratulations on the elegant work! I was wondering if you considered treating the cells with glucocorticoid to examine if there is a reversal of alternative splicing and/or expression vs. treatment with IL-beta?
Together, these results demonstrate that functional iPruriceptors can be efficiently generated from hPSCs
Have you tested the neuronal responses of iPruriceptors to other pruritogens, like serotonin or TSLP? If not, does the expression pattern of differentiated iPruriceptors indicate that the receptors for other known pruritogens are present on iPruriceptors?
MEA recordings revealed functional responses to histamine and hIL-31
I'm curious if you have performed the multielectrode array assay on iPruriceptors in parallel with hDRGs for direct comparison?
Representative images showing neuronal morphology in 1-step NGN1, 1-step NGN2, 2-step NGN1, 2-step NGN2 on Day 21.
Congratulations on developing this wonderful resource for studying itch! I have a few questions. Were the two groups of images being compared (2 left panels vs. 2 right panels) captured using similar microscopy techniques? At first glance, the left panels appear to have been captured using possibly DIC microscopy and the right panels captured using brightfield?
For this, N-terminal GST-tag or C-terminal GFP-tag TRPV1 was transiently transfected into human embryonic kidney (HEK) 293 cells.
This is a very intriguing idea linking TRPV1-mediated calpain activation to downregulation of TRPV1! While your engineered HEK and CHO cell systems work well, can you perform this assay in more biologically relevant cells, such as DRGs, or cells more closely related to neurons, like keratinocytes, and examine endogenous proteins?
In the current work, we demonstrated that TRPV1-Ca2+-calpain pathway activated by a regular dose (1 μM) of capsaicin is not associated with cell toxic injury
What do you mean by "regular dose"? It might be helpful to think about this in terms relative to the Kd of capsaicin for TRPV1.
Consequently, TRPV1 Δ820 displayed a similar activation pattern as WT TRPV1 when subjected to voltage stimulation
Do you have a negative control for this experiment, where TRPV1 is absent from the cells?
From these findings, we could conjecture that calpain-mediated cleavage of TRPV1 may promote receptor internalization.
Can you be more direct here and use this assay to show calpain is cleaving TRPV1 and that this leads to receptor internalization? For instance, addition of capsaicin should lead to activation of calpain that should cleave TRPV1, according to your theory. If so, after activation, can you detect the cleaved version of TRPV1 being internalized?
This lack of impact might be caused by the feature of calpain where it recognizes substrates not only by the primary sequence of their amino acids, but also by their overall three-dimensional structure
Given that mutation of residues that were predicted to be critical for cleavage had no effect, it would be important to confirm that this site is cleaved in vivo. This could rule out that the cleavage you observe in vitro is not an artifact due to incubation with high levels of enzyme or overepxression of substrates.
were
Wording-"were" may be omitted?
It may be likely that other species of parasitic helminth species that invade mammalian skin, such as hookworms and Strongyloides spp. have evolved related mechanisms of host modulation.
Can you use information on related, parasitic nematodes which can suppress host immune responses to help elucidate conserved biosynthetic pathways that may give rise to molecules that help evade host immune responses?
These results suggest that exposure to parasitic components during S. mansoni infection suppresses the responsiveness of local TRPV1+ neurons.
This is very elegant work--congratulations! I was wondering if you have any data on whether inhibition of TRPV+ neurons is reversible? If you culture the neurons longer and/or provide additional wash steps, can these neurons again respond normally (similar to control) to capsaicin?
Besides these shell field-marker genes, the transcription factor engrailed was also suppressed in the shell gland region of both species after Ro-41-5253 treatment.
Are there canonical RAR/RXR or RAR/RAR binding site in the promoter of engrailed?
Altogether, we conclude that the molluscan RAR has a property distinct from the vertebrate RAR in response to the RA, and this difference is largely due to the single amino acid substitution at the position 223 with valine in molluscs and phenylalanine in vertebrates respectively.
Performing an equilibrium binding assay between your wild type and mutant receptors and radiolabeled atRA could discriminate between difference in atRA binding affinity rather than potential differences in the receptors’ ability to bind cofactors, or perhaps bind its heterodimer partner.
In contrast, 246 and 47 genes were down- and up-regulated in both Ro-41-5253 and atRA treated larvae, respectively (Fig. 3a), suggesting there may be some level of similarity between atRA and Ro-41-5253 treated embryo
If RAR does not bind atRA, how do you explain the similarity here between treatments with atRA and Ro-41-5253?
If Ro-41-5253 blocks the atRA signal, a common set of genes would be expected to show an inverted response to these treatments.
This is a really intriguing study on the conservation of RAR in guiding developmental patterning across species. Congratulations! I'm curious if cofactors that bind NHRs to activate (coactivators) or repress (corepressors) transcription are conserved in C. gigas? I'm wondering what type of transcriptional responses you anticipate for addition of agonist and antagonist vs. no ligand? Could you see active repression in the absence of ligand, which may complicate your interpretation, especially if, as you suggest later, atRA may not bind RAR in this species.
In AD, initial trigger factors elicit keratinocytes (KCs) to release proinflammatory molecules that184activate immune cells such as ILC2 cells (see “ILC2 cells activation by keratinocytes” in the map
As you note, there are potential drivers of AD and psoriasis. I know this may be asking a lot, but it would be useful to see how these pathways amplify immune signaling by allowing the user to “initiate” an upstream event and, in a temporal fashion, watch as it propagates through the nodes of the system leading to consequences that reinforce the disease state.
The ISD map was built based on the review of 460 AD- and 110 PsO-related articles
This is great resource to help display and condense the complexity of the etiology and maintenance of AD and psoriatic disease states. I may be missing this in your interactive tool, but it would be helpful to include the publications used to develop each node of the model as a link when selecting any particular node. Given the sometimes conflicting information available for association vs. causal factors in the literature, the links would be helpful to place particular weights on any given node.
The data from every time point were then merged to obtain a global table explaining the sex-bias expression for every gene and the DSX mode of action
What is the phenotype of a DSX knockdown in males and in females? I'm just curious if the larva/animals used to examine gene expression changes in DSX knockdowns are sick.
To this end, we compared transcriptomes of dsx knockdown individuals with those of control individuals per time point and sex
This is a very interesting study helping to elucidate the role of DSX in N. vitripennis development! Since there are male- and female-specific isoforms of DSX and DSX can act as both a transcriptional activator and repressor in both females and males, is it possible that a gene knockdown (not isoform specific) could obscure the sex-specific roles of DSX? Can you generate DSX knock ins that can only produce female specific isoforms and then examine the outcome on gene expression? (Presumably, a male-specific isoform can not be propagated.)
However, more research is necessary to understand the role of layilin on non-immune cells, such as gastrointestinal epithelial cells (9, 46) to help build confidence around this therapeutic hypothesis
Is there a complete layilin knockout or other tissue-specific conditional knockouts that provide insight into layilin’s role in other cells/tissues?
Conversely, in pathological situations such as tumors and chronic inflammation, the motility of Tregs may play a larger role in their functionality, allowing them to navigate to various tissue microanatomic niches to attenuate specific cell types and inflammatory mediators.
Congratulations on the elegant study! I'm curious if there are layilin mutations associated with with chronic inflammation or highly metastatic carcinomas?
The deacetylation of RORβ by SIRT1 not only enhances its transcriptional activity but also increases the stability of both RORβ and p300 when in complex.
Could the increased transcriptional activity be due to enhanced stability of RORbeta? The wording suggests that these are independent outcomes.
Acetylation of RORβ by p300 in the presence and absence of the DNA oligomer did not show a significant effect on the extent of acetylation by p300
Do you have any evidence of RORbeta binding in vivo? It would be interesting to examine RORbeta binding to endogenous RORbeta DNA binding sites to see if acetylation changes RORbeta’s propensity to bind target genes promoters.
The acetylated RORβ was subjected to an electrophoretic mobility shift assay (EMSA) in the presence of a 40-bp DNA oligo containing a single RORE sequence.
Do you know if p300 was bound to the ROR-DNA complex (evidence of supershift with a p300 antibody)? I'm curious if p300 binding can alter RORbeta DNA binding after RORbeta is modified?
There were 57 putative RORβ interactors with at least 5-fold increase in abundance relative to the negative control and with a p-value less than 0.05 (n=3), with 18 of these being involved in transcriptional regulation
This is a very interesting study linking the complex interplay of RORbeta posttranslational modifications to RORbeta function-congratulations! Can you provide a list of these interacting proteins? I'm curious if any known interacting proteins were immunoprecipitated, such as co-repressors and other co-activator proteins?
However, comparison of Pin4 mRNA binding andchanges in abundance in pin4 cells revealed generally increased abundance forpreferential Pin4 targets
Can your analysis detect potential changes in alternative splicing that may result from Pin4 binding?
correspond to the mRNAs showing Pin4 association across the CDS (e.g.RPS12)
Is there any correlation between the number of motifs in a particular 3'UTR and the likelihood of being bound by Pin4?
These high-confidence targets showed good reproducibility acrossreplicates in both conditions
How predictive are these motifs for Pin4 binding? Are these motifs enriched on mRNAs bound by Pin4, or are they present but not bound throughout the genome?
Importantly, reversible protein phosphorylation provides a major regulatory mechanism in conformational dynamics and compaction of IDPs
Have you explored whether this phosphorylation-dependent structural rearrangement may play a role in the function of other NHR AF1 or F domains?
The ability of AF1c phosphovariants to adopt multiple structural changes likely creates distinct binding surfaces within the NTD, enabling GR to selectively interact with different regulatory complexes along its signal transduction pathway.
This is a very insightful study! I'm curious if you can model how this newly formed helical structure binds to AF1 interacting domains present on such proteins as TBP?
We repeated these experiments after preincubating our cells in 10μM DHA for 10 minutes
Is 10uM near the physiologic concentration for docosahexaenoic acid?
Overall, the presence of DHA, whether docked to the TM helices of hASIC3 or partitioned into the lipid layer, prevented POPC lipids from accessing and penetrating the pore, ensuring high permeability for water and Na+ ions to flow through the channel.
Based on your model, can you postulate different lipid structures or lipid classes that could alter pore function?
Based on this experimental data, we designed our PUFA-bound simulations where we docked either AG
This is a very interesting study! Using your model, can you predict other channel-specific amino acids that are important for binding to lipid mediators? If so, it would be interesting to mutate these residues and show they have a similar effect as R63 mutants.
In addition, the datademonstrated that the signal most likely originates from the receptor entity and is transmittedthrough the subsequent linker regions to the catalytic dimer
While you're elegant chimera approach suggests lipids are unlikely to have a destabilizing effect, can you rule out an indirect, isoform-specific activating effect?
permanently
Do you mean "stably"?
The action of oleic acid on mAC3 was instantaneous and linear for >25 min
Congratulations on the revealing study! I was curious if you have evidence that the lipids are not generally affecting the biophysical proprieties of the membrane and indirectly activating mAC function?
We analyzed the skin of bitten mice for the presence of B. burgdorferi sensu lato and A. phagocytophilum by PCR in the experiments carried with field-collected ticks. We detected Borrelia and Anaplasma in several but not all the bitten skin
Could you use ticks that are free from zoonotic pathogens to confirm that the pathogens are not causing the inflammatory response?
Following blood feeding for 4 days, all ticks were removed from the back skin and skin was evaluated immediately after and following 6 (day 10) or 10 (day 14) days as depicted in figure 1A
Do you have any evidence that longer feeding times promote a more severe allergic-like response on day 10 or 14, or if a longer feeding time may postpone the onset of inflammation?
To investigate the changes in the skin induced by tick bites, we analyzed both the intact skin and Ixodes ricinus (I. ricinus) bitten skin of C3H/HEN mice by histology
I'm curious why you choose Ixoden ricinus rather than others species of ticks for this study? From my understanding, a preponderance of reported ticks bites that result in a red meat allergy in humans are caused by bites from Amblyomma americanum. I'm just wondering if there is an advantage to using I. ricinus.
We found a significant interaction between time and genotype in rectal temperature measures
The in vivo data is very compelling! I'm curious if there is potential for FosB KO in other cells types--do you know if Mcpt5 is specific only to mast cells?
We found clear bands at the ∼50KdA and ∼37kDA (fig. 1J)
This is a very intriguing paper on the role of FosB in regulating mast cell function! I have a few questions. In figure 1J, are the two lanes replicates of extracts from LAD2 cells, or, like in figure 1B, is one lane vehicle and one lane stimulated? If the latter, the bands in the separate lanes do not look appreciably different. Is it that you see expression but no change in expression with stimulation?
A regulatory role for mitochondrial PA through SLP-2 thus provides new possibilities of therapeutic targeting for diseases with mitochondrial involvement.
LPA signaling has been implicated in promoting mitochondrial homeostasis as well as promoting mitochondrial apoptosis in pathologic conditions. Since STL-1 can bind LPA, I’m wondering if you can comment on how extra and intra-cellular LPA signaling may impact STL-1 function?
Nonetheless, further studies are needed to delineate the exact mechanism of PA-mediated regulation of STL-1/SLP-2
It will be interesting to model how pH changes can change STL-1-lipid interactions and generate point mutations to disrupt binding and observe outcomes.
Interestingly, using animals carrying zcIs13(hsp-6p::GFP), another established reporter of mtUPR activation (17), we observed significantly decreased GFP fluorescence only in the stl-1 mutants with missense mutations R51K and M308I, while the GFP levels in the adult stl-1 null mutants were not significantly altered compared to wild type (WT)
Thanks for posting your great study on linking STL-1 to phosphatidic acid signaling and mtUPR. Can you confirm that tm1544 or other point mutants behave as a true null alleles (e.g. place tm1544 over a larger chromosomal deletion and confirm that tm1544/- phenocopies -/- animals?). Since the hps-6p::GFP reporter behaves differently when crossed with the different stil-1 mutants, is it possible that the mutations are not equivalent and regulate the hsp-6 and hsp-60 reporters differently?
As nuclear receptors share a conserved structure and mechanism, we anticipate that similar ligand-modulated domain rearrangement will be observed in other receptors
Are there any naturally occurring mutations associated with diseases that map to regions that your model predicts would disrupt interdomain interactions? Or conversely, can you generate mutations that your model predicts would disrupt interactions and measure the effects on transcription use simple reporter assays and full-length constructs?
These results suggest an important role for the hinge in mediating contact between LBD and DBD, as well as the ligand-specific nature of this interaction.
This is a really interesting study on the complex interplay between NHR domains! I was wondering if you tested interactions between these domains using in vitro protein-protein interaction assays that can sidestep possible complications of the heterologous cell-based Gal4 system (i.e. “pull-down” assays, etc.)?
These results, along with the observation that rolling behaviors predominate the early behavioral responses to epidermal stimulation (Fig. 2B), suggest that the nervous system prioritizes nocifensive behavioral outputs following epidermal stimulation
While epidermal stimulation can broadly mediate SSN activity, how do you envision epidermal stimulation prioritizing nocifensive behavior? Does ensheathment of C4da and C3da neurons by epidermal cells explain the preferential response?
C4da neurons, elicited nocifensive c-bending and/or rolling behaviors in 73% of larvae
This is an elegant and thorough study mechanistically describing the role of epidermal cells in regulating nociceptive behavior as well as SSN activity more broadly, in Drosophila. Congratulations on your beautiful work! I’m curious why direct optogenetic activation of epidermal cells leads to a similar fraction of larva bending relative to direct activation of nociceptors, versus less than half of larva rolling in response to direct activation of epidermal cells relative to direct activation of nociceptors?
Moreover, although we only tested a single cloxyquin concentration applied 2h before the pruritic challenge, the data demonstrates that TRESK activation can be employed to alleviate pruritus and further underscore the role of this channel modulating pruriceptor excitability
This is a nice proof-of-principle application for TRESK agonists to treat acute and chronic pruritus. It will be interesting to see how long TRESK agonists effects last after initial or repeated treatments--is the effect longer term than 2 hours?
Intraperitoneal injection of cloxyquin (50 mg/Kg) or its vehicle (olive oil), administered two hours before the pruritic test did not induce any significant scratching in wild-type mice
I'm curious if activation of TRESK, though single or repeated injection of cloxyquin, resulted in any phenotypes other than reducing pruritus (concomitant reduction in cold or mechanical sensitivity, or a change in any migraine-like pain-associated behavior.)?
This increased activation of NF-kB-driven transcriptional modules may reflect both the increased accessibility of NF-kB to DNAresponsive elements as well as the heightened recruitment of NF-kB via protein-protein interactions,boosting pathogenic activities in memory astrocytes
Given the potential role of glucocorticoid receptors in antagonizing NF-kB signaling, is there any evidence of discrete or cryptic GR response elements in the promoters of NF-kB-driven transcriptional modules? I'm curious if different GR agonists could reverse promoter-specific p300-dependent acetylation and the associated neurodegenerative phenotypes.
Ep300 inactivation in CNS astrocytes ameliorated EAE, reduceddemyelination, and suppressed pro-inflammatory astrocyte responses as determined by RNA-seqbut did not affect CNS-recruited or peripheral CD4+ T cells
Are there other attributes of astrocyte health that could be assessed to examine if p300 downregulation is not having detrimental effects?
Indeed, we also identified Ep300, which encodes histone acetyltransferase (HAT)p300, as an upstream regulator of the transcriptional response of astrocytes to 2X IL-1β+TNFstimulation
This is a beautiful study showing that epigenic memory in astrocytes can prime cells towards CNS pathologies. From examination of unregulated genes and DNA binding sites in the promotor of p300 or ACLY, are there any candidate transcription factors that could be responsible for the upregulation by IL-1b and TNF?
To exclude the possibility that unc-104(Q94L) might be a gain-of-function allele and to confirm that unc-104(Q94L) is a hypomorphic allele, we crossed unc-104(Q94L) worms and unc-104(lf) to generate transheterozygotes, unc-104(Q94L)/unc-104(lf)
If unc-104(e1265) is a partial loss-of-function allele, is there a deletion, either gene-specific or chromosomal, that one can use to generate a true unc-104(Q94L)/unc-104(-) animal to test for a hypomorphic allele?
Our results indicated that KIF1Bß(Q98L) was unable to rescue the body movement defects
I’m curious if you examined the expression levels of the mutant KIF1Bbeta protein? i.e. does the Q98L mutation alter protein expression levels?
In this paper, we refer to this allele as unc-104(lf).
This is an elegant model to test the function of kinesins conserved across species! I just have a few questions. Is unc-104(e1265) a partial loss-of-function allele – a point mutation that demonstrates reduced PI(4,5)P(2) binding?
A Materials and Methods section may be missing.
I may be missing this, but I don't see a description of how this assay is performed in the figure legend or a materials and methods section in the primary or supplemental text. Could it be that this change in mobility is due to another modification and, if so, could you confirm with mass spectrometry?
This is a really interesting study linking NLRP3 palmitoylation to inflammation. Here, could you use a more sensitive readout for cell cytotoxicity to confirm cell death is not obscuring your results?
Neither mechanical nor thermal sensitivity was altered by compound 6 compared to the vehicle control
Huang, F., et al. 2013 showed that in Slack knockout rats, the knockouts were more sensitive to heat stimuli than control rats at 50oC. Your methods mention a infrared intensity setting of 25. What temperature corresponds to a setting of 25?
These findings are important because they indicate that Slack activators can reduce existing itching.
I'm curious if the antipruritic activity of compound 6 was long lasting. Did you examine if hours or days after administration of compound 6, the mice continued to show a reduction in scratching vs. control in your chronic itch models?
Importantly, we found that systemic treatment of mice with compound 6 (3, 10, or 30 mg/kg i.p.) 15 min prior to subcutaneous (s.c.) injection of chloroquine into the nape of the neck ameliorated scratching behavior in a dose-dependent manner
This is really nice dose-dependent reduction in scratching! I'm curious how you choose the timing for compound 6 administration? And did you try subcutaneous injections of compound 6 as well as systemic administration? I'm wondering if s.c. injections may help reduce potential undesirable off-target effects of compound 6 when compared to systemic administration.
While further work is needed to identify the molecular identity of this cue, our data hint that this may be independent of proximal extra-cellular vesicle release
Are there any mutants that phenocopy ok288 that may point to possible candidates in neuronal cells that cue localization of KCC-3?
First, lack of basolateral-targeting motifs in KCC-3 N-terminal region allows it to be shuttled apically to GAB. Once there, ciliary NRE signals act with the C-terminal 915-997 AA sequence to restrict KCC-3 to a microdomain.
This is a very thorough and detailed study digging into the mechanism of KCC-3’s apical localization in microdomains on AMsh glia. Beautiful work! I'm curious if you’ve looked at versions of KCC proteins that completely lack the N or C terminal variable regions? Assuming they still fold properly and are inserted in the membrane (maybe a stretch), the localization of these KCC mutants might help support these domain’s roles in targeting KCC apically or basolaterally.
Indeed, we note punctate staining in internal vesicular compartments, suggesting that lack of basolateral targeting motifs likely stall KCC-2 membrane targeting
Does this fall into the "other" category? And what alternative types of localization fall into "other"?
We found that nAChRβ3 overexpression in ECs significantly expedited recovery of the intestinal epithelium with ISC proliferation and pdm1 expression reaching levels indistinguishable from unchallenged guts in 2 days, half the normal time
Since the balance and transition from repair to homeostasis likely exists to allow sufficient time for healing from injury, I'm curious if the accelerated recovery with overexpression of nAChRbeta3 results in abnormal repair or healing of the gut epithelium or any other abnormalities?
Overall, we conclude that nAChRβ3 in ECs is essential for gut recovery and recovery-specific enrichment of nAChRβ3 provides an additional level of regulation that likely ensures that ECs are more responsive to ACh while the epithelium transitions to homeostasis
Congratulations!--This is an elegant tour de force investigation into how injured gut epithelium transitions from injury and a state of repair back toward homeostasis. You’ve clearly shown that ECs downregulate Ace and upregulate nAChRbeta3 to become more receptive to ACh. I’m curious if you have any ideas about what signals ECs to downregulate Ace and upregulate nAChRbeta3?
The work here identifies the importance of the CPSF phosphatase module, a regulator of RNA Pol II,307in FLC regulation
This is a beautiful study describing the regulation of the FLC locus. Do you believe APRF1, TOPP4, and LD are specific regulators of only FLC? Or do they play a more general role in regulating repression/activation at many loci? If so, do you have any genome-wide information on how APRF1, TOPP4, and LD mutants effect other genes, particularly those involved in sensing environment cues, like you've examined with flowering? And, if their role is not restricted to regulating FLC, what other phenotypes do you observe with these mutants--do the phenotypes give you insights into other processes they may regulate?
Taken together, the robust202immunoprecipitation of LD and TOPP4 by APRF1, the structural parallels between Ref2-PNUTS-LD203and the similar roles in co-transcriptional processing lead us to propose that LD, APRF1, and TOPP4204may form a functional CPSF phosphatase module in plants
It will be exciting to see if these putative subunits possess phosphatase activity, in vitro, and further dissect how this phosphatase activity might be regulated.
Thus, APRF1 has a role in establishing a silent152chromatin status at FLC (Figure 1H, I)
The evidence that APRF1 is regulating flc expression is strong; however, given APRF1's likely role in regulating a wide range of genes, do you have evidence that APRF1 is directly associating with the flc locus to regulate expression and that the regulation is not indirect?
These results provide proof-of-concept demonstration that TL can be activated by Glucpresented by another cell to enable bioluminescence-mediated transcriptional activation in acell-cell contact/proximity manner..CC-BY-NC 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is madeThe copyright holder for this preprint (whichthis version posted June 1, 2023.;https://doi.org/10.1101/2023.05.31.543150doi:bioRxiv preprint
This is a great tool to help explore and exploit cell-cell specific contacts! I'm curious how providing lower levels of the the receptor fusions by stable integration, rather than transient transfection, would effect the reporter readout? Future studies using transgenic organisms that introduce constructs through single copy integration, would benefit greatly from your tool.
A longer activation time did not further increase the amountof reporter FFluc (Fig. 5B)
In this case, is it possible that TL-betaCat is limiting?
To validate that SNAI1 was indeed released from the cell plasma membrane and translocatedto the nucleus, TL-SNAI1-expressing H1299 cells were fixed before and after light activation,and immunofluorescence staining of SNAI1 was performed. As expected, SNAI1 was foundat the plasma membrane prior to photostimulation, and in the nucleus after (Fig. 2B).
Can you quantify what percentage of SNAI1 was released from the plasma membrane upon photo stimulation? I'm curious if transcription/translation of TL-SNAI1 is rapid enough to ensure a pool of plasma bound SNAI1 that can lead a source of continual transcriptional activation upon constant photostimulation?
24hafter transfection, these cells were either kept in the dark (DARK) or photostimulated(LIGHT) using pulses of light 0.05s/5s break
Your TANGO-light system is a really intriguing adaptation of TANGO and optogenetics! I'm curious in this case, how long did you have to stimulate cells with a 0.05s/5s pulse?
Direct interaction of NHR-49/MDT-15 with HSF-1 or binding to a nearbymotif may thus facilitate the recruitment of the transcription machinery to heat shock genes duringproteotoxic stress
In general, NHR recruitment of coactivators or corepressors is regulated by the position of the terminal helix 12 of the ligand binding domain. Helix 12 is necessary for the proper confirmation of NHR to recruit coactivators, while it is dispensable for recruitment of corepressors. In fact, amputation of NHRs which remove helix 12 convert the receptors into constitutive repressors. While a corepressor for nhr-49 has not been identified, it may be revealing to generate an nhr-49 allele that lacks helix 12. It would be unlikely to bind MDT-15 and may help dissect mdt-15 roles in regulating hsf-1 function, as well the cement the role of mdt-15 in nhr-49 mediated regulation of proteotoxic stress.
Together, the data presented in this study suggests that NHR-49, through its lipid sensing and gene regulatory function, represents a link between lipidmetabolism and regulation of the HSR via modulation of HSF-1 activity (Fig. 5G)
I appreciate the clarity in Figure 5G, but it may be helpful to include all of the genes/proteins involved in this pathway that were mentioned above, to aid in interpreting your conclusions.
The partialsuppression of nhr-49 was likely due incomplete penetrance of the RNAi since the loss-of-functionallele nhr-49(nr2041) nearly completely abrogated the beneficial effects of cbd-1(ok2913) on heatstress survival (Fig. 3F)
It appears that there is a significant difference between the percent survival of wild type worms + control RNAi (Fig 3F) and wild type worms (Fig 3G) as well as cdb-1 + control RNAi (Fig 3F) and cdb-1 (Fig 3G). Is this difference due to the different bacteria used for dsRNA expression and normal growth, for instance, HT115 vs. OP50, or some other variation?
Wefound that RNAi knockdown of mdt-15 completely suppressed and nhr-49 significantly reducedthe prolonged survival of cbd-1(ok2913) following a lethal heat stress (Fig. 3F)
This is an elegant study implicating nhr-49 as an important link tying lipid metabolism to proteotoxic stress. I only had a few questions. Since mediator is a shared coactivator used by numerous transcription factors, could the difference in suppression be due to med-15 targets that are not regulated by nhr-49? And do you know if mdt-15 RNAi recapitulates a null allele of mdt-15?
To identify subpopulations of MuSCs generated by MS023, we performed single-cell RNAsequencing (scRNAseq
A key way PRMTs function is through recruitment to promoters by transcription factors. With your RNAseq data, are there any expression patterns that may help elucidate the primary target promoters regulated by MS023, and therefore the key transcription factor or factors that are targeting the PRMTs? One of better characterized class of transcription factors that can recruit PRMT cofactors are nuclear hormone receptors (NHRs) and NHRs have been shown to be important regulators of stem cell differentiation. If you can identify transcription factor targeting PRMTs, and it is an NHR, use of NHR agonists or antagonists may reinforce the application of MS023 and further increase muscle stem cells’ potential to expand, in vitro.
These findings showthat PRMT1 is the predominant type I PRMT1 in MuSCs.
This is a thorough study demonstrating the utility of the type I PRMT inhibitor, MS023, in allowing muscle stem cells to expand, in vitro. To convincingly show that PRMT1 is the key target mediator of MS023, is there a means to directly target PRMT1 for degradation or downregulation, such as tagging it with an auxin-inducible degron (transgenic mice)?
Expression of the PIWI proteins was further confirmed by Western blotting using antibodies against asymmetric di-methyl-arginine, a post-translational modification that is conserved among PIWI proteins [32].
Antibodies made against asymmetric di-methyl-arginine may cross react and diminish actual changes in PIWI protein expression levels. Are there antibodies made against PIWI proteins in related species that could provide a more specific measure of PIWI protein expression levels in ISE6 cells, revealing what may be a more dramatic change in expression?
Upon knockdown of these genes, we detected 47-84 genes to be differentially expressed compared to the control GFP KD sample (adjusted p-value <0.05, Figure 5A-C and Table S3).
This is an intriguing finding. The number of genes whose expression changes seems small relative to the important and diverse biology processes that are predicted to be regulated by RpRPs and AGOs. When related genes are knocked down in other species, how many genes show significant changes in expression? In addition, is the phenotype of the RpRPs and AGOs knock downs in Ixodes scapularis known?
Therefore, the production of 22nt species from RNAP III transcribed genes was broadly conserved in ticks.
If available, comparison of sRNA conservation across multiple tick species would bolster the idea that these sRNAs are deeply conserved. It may also reveal interesting species-specific biological differences.
We also compared KD efficiency of crRNAs targeting intronic versus exonic regions for the same two RNAs (Fig. 2D).
Has targeting of CRISPR-Csm to 5’ or 3’ UTRs been performed? Could this provide different levels of knock-down and further add your ability to tune RNA levels (in addition to altering spacer length)?
Thus, fluorescently-tagged Csm can be used for easy visualization of RNA in living cells.
Being able to directly target complexes to the native RNA sequences is a significant benefit over the potentially disruptive insertion of RNA aptamer sequences into RNAs to visualize RNA dynamics; however, can binding of multiple CRISPR-Csm complexes to native RNAs disrupt function? Do you have any evidence that CRISPR-Csm complex binding does not disrupt RNA metabolism of ncRNAs or protein coding RNAs or expression of corresponding proteins? Additionally, do you have any evidence that you have the sensitivity to detect single RNA molecules (comparison to smFISH)?
We found that a portion of the initially plated worms die, likely due to lack of array inheritance
TARDIS is a significant step toward generating library scale insertions in animals. I was curious about a few points. Is this observed lethality due to off-target, collateral damage of Cas9 induction? (i.e. In worms that lack arrays and that are not under selective pressure, is there any significant lethality after heat shock?)
Additionally, while split-selection methods allow for direct verification of integration, depending on the downstream use case, integrations should be confirmed by sequencing as errors can still occur, including internal deletions within the insert.
As a means to improve efficiency of homology directed repair using large or difficult to insert templates, have you tried or considered using two Cas9 targeting sequences to promote the insertion of a repair template or library at a single landing site (the repair template flanked by two Cas9 targeting sequences)?
The differences in expression pattern may also reflect differences in the region used as the promotor or the fact that only a single developmental stage (late L4/early adult) was examined
Do these patterns reflect the phenotypes of inserted fluorophore sequences at the endogenous genomic loci, if known? I’m curious how much variation may be due to the expression at this landing site.
The metabolomic data from this study provide a resource for future detailed exploration of sex-biased metabolic pathways and phenotypes.
This elegant study provides a rich data set and a pathway to explore sex-specific metabolomic data in C. elegans and its involvement in a myriad of biological processes.
Furthermore, chromatographic conditions optimized specifically for the detection of very polar and very non-polar metabolites (e.g. lipids) will almost certainly reveal additional male-upregulated compounds.
Given the large expansion of the nuclear hormone receptor (NHR) family in C. elegans, it will be very informative to explore lipid fractions for potential NHR ligands that likely regulate homeostasis, metabolism and reproduction.
To this end, we examined the exo-metabolome of him-5 cultures by collecting the culture media during different time periods throughout larval development and adulthood (Figure 1c).
The decision to choose either male or hermaphroditic fate occurs very early in development, at around the 20 to 100 cell stage. It would be intriguing to examine if sex specific differences in metabolites is evident in the early embryonic stages of development, too. It may provide insights into how cell fate decisions are made and maintained.
Figure 3H
There is no Figure 3H present in the publication. Possibly a typo?
We confirmed that surgical incision leads to a dramatic increase of the substance Pprecursors α- and β-preprotachykinin in the skin about 8 hours after incision
Since substance P precursor levels are substantially elevated in skin after surgical incision (Fig 6A), and substance P has been shown to cause degranulation, releasing a number of factors including histamine, can you postulate why you don't see elevated levels of histamine at the site of incision (Fig 3B)?
It is possible that such mast cell-released BH4 itself contributes to painhypersensitivity by direct action on nociceptors
You elegantly show that neuronally derived BH4 contributes to the increase in BH4 levels after injury but is not involved in pain hypersensitivity. If BH4 can directly activate nociceptors, it seems like you would have noticed a decrease in pain hypersensitivity when you reduced BH4 levels. Do you think there is something chemically unique about mast cell-derived BH4 vs. neurally-derived BH4 that would allow direct activation of nociceptors?
Altogether these experiments reveal that mast cells not only releaseBH4 and serotonin but also the necessary enzymatic machinery required for synthesis of bothmetabolites rapidly after activation.
This is a very intriguing finding-extracellular release of the enzymatic machinery necessary for BH4 and serotonin synthesis could allow signal amplification after degranulation. Is there any evidence of extracellular BH4 synthesis in other contexts from other studies? Or could these enzymes be remnants of the synthesis of serotonin within the granules?
Genotyping of the F3 progeny revealed high rates of recombination within the introgressed region for each of the three guides (gSZ71: 55.9% recombinants [19/35]; gSZ73 52.4% recombinants [33/63]; gSZ74: 10% recombinants [2/20]).
This is s great proof-of-concept example that Cas9-mediated nonhomologous recombination can be a platform in C. elegans to aid in genetically mapping variants.
Numerous studies in C. elegans using co-conversion have achieved a similar efficiency of imprecise repair at a Cas9-induced DSB as you achieved for isolating LOH offspring, which is remarkable. Do you have a sense of the cleavage and repair efficiency of Cas9-induced DSBs you've generated-not just LOH animals?
Given the high rates of Cas9-induced recombination we observed by PCR in a genomic region with undetectable natural recombination
For comparison, from other studies, do you have a sense of the natural recombination efficiency of neighboring loci in this region of chromosome V?
In this study, we show that Cas9-induced double-stranded DNA breaks facilitate targeted nonhomologous recombination in C. elegans
It would satisfying to see this pattern of repair generalize across different regions of different chromosomes. There may be positional effects that influence the rate of Cas9-mediated recombination.
Because of the relative ease of generating targeted recombination events at a high frequency, we anticipate CINR will greatly expedite genetic fine-mapping in C. elegans in regions with low recombination rates, including chromosome centers (Rockman & Kruglyak, 2009) and divergent regions (Lee et al., 2021).
It would interesting to examine if one can recover non-homologous recombination events at a reasonable frequency when targeting highly repetitive regions in the genome that may be prone to favor microhomology-mediated repair from sequences in cis.
Because of the relative ease of generating targeted recombination events at a high frequency, we anticipate CINR will greatly expedite genetic fine-mapping in C. elegans in regions with low recombination rates, including chromosome centers (Rockman & Kruglyak, 2009) and divergent regions (Lee et al., 2021).
It would interesting to examine if one can recover non-homologous recombination events at a reasonable frequency when targeting highly repetitive regions in the genome that may be prone to favor microhomology-mediated repair from sequences in cis.
In this study, we show that Cas9-induced double-stranded DNA breaks facilitate targeted nonhomologous recombination in C. elegans
It would satisfying to see this pattern of repair generalize across different regions of different chromosomes. There may be positional effects that influence the rate of Cas9-mediated recombination.
Given the high rates of Cas9-induced recombination we observed by PCR in a genomic region with undetectable natural recombination
For comparison, from other studies, do you have a sense of the natural recombination efficiency of neighboring loci in this region of chromosome V?
Genotyping of the F3 progeny revealed high rates of recombination within the introgressed region for each of the three guides (gSZ71: 55.9% recombinants [19/35]; gSZ73 52.4% recombinants [33/63]; gSZ74: 10% recombinants [2/20]).
This is s great proof-of-concept example that Cas9-mediated nonhomologous recombination can be a platform in C. elegans to aid in genetically mapping variants.
Numerous studies in C. elegans using co-conversion have achieved a similar efficiency of imprecise repair at a Cas9-induced DSB as you achieved for isolating LOH offspring, which is remarkable. Do you have a sense of the cleavage and repair efficiency of Cas9-induced DSBs you've generated-not just LOH animals?
Altogether these experiments reveal that mast cells not only releaseBH4 and serotonin but also the necessary enzymatic machinery required for synthesis of bothmetabolites rapidly after activation.
This is a very intriguing finding-extracellular release of the enzymatic machinery necessary for BH4 and serotonin synthesis could allow signal amplification after degranulation. Is there any evidence of extracellular BH4 synthesis in other contexts from other studies? Or could these enzymes be remnants of the synthesis of serotonin within the granules?
Figure 3H
There is no Figure 3H present in the publication. Possibly a typo?
It is possible that such mast cell-released BH4 itself contributes to painhypersensitivity by direct action on nociceptors
You elegantly show that neuronally derived BH4 contributes to the increase in BH4 levels after injury but is not involved in pain hypersensitivity. If BH4 can directly activate nociceptors, it seems like you would have noticed a decrease in pain hypersensitivity when you reduced BH4 levels. Do you think there is something chemically unique about mast cell-derived BH4 vs. neurally-derived BH4 that would allow direct activation of nociceptors?
We confirmed that surgical incision leads to a dramatic increase of the substance Pprecursors α- and β-preprotachykinin in the skin about 8 hours after incision
Since substance P precursor levels are substantially elevated in skin after surgical incision (Fig 6A), and substance P has been shown to cause degranulation, releasing a number of factors including histamine, can you postulate why you don't see elevated levels of histamine at the site of incision (Fig 3B)?
Furthermore, chromatographic conditions optimized specifically for the detection of very polar and very non-polar metabolites (e.g. lipids) will almost certainly reveal additional male-upregulated compounds.
Given the large expansion of the nuclear hormone receptor (NHR) family in C. elegans, it will be very informative to explore lipid fractions for potential NHR ligands that likely regulate homeostasis, metabolism and reproduction.
To this end, we examined the exo-metabolome of him-5 cultures by collecting the culture media during different time periods throughout larval development and adulthood (Figure 1c).
The decision to choose either male or hermaphroditic fate occurs very early in development, at around the 20 to 100 cell stage. It would be intriguing to examine if sex specific differences in metabolites is evident in the early embryonic stages of development, too. It may provide insights into how cell fate decisions are made and maintained.
The metabolomic data from this study provide a resource for future detailed exploration of sex-biased metabolic pathways and phenotypes.
This elegant study provides a rich data set and a pathway to explore sex-specific metabolomic data in C. elegans and its involvement in a myriad of biological processes.
Additionally, while split-selection methods allow for direct verification of integration, depending on the downstream use case, integrations should be confirmed by sequencing as errors can still occur, including internal deletions within the insert.
As a means to improve efficiency of homology directed repair using large or difficult to insert templates, have you tried or considered using two Cas9 targeting sequences to promote the insertion of a repair template or library at a single landing site (the repair template flanked by two Cas9 targeting sequences)?
The differences in expression pattern may also reflect differences in the region used as the promotor or the fact that only a single developmental stage (late L4/early adult) was examined
Do these patterns reflect the phenotypes of inserted fluorophore sequences at the endogenous genomic loci, if known? I’m curious how much variation may be due to the expression at this landing site.
We found that a portion of the initially plated worms die, likely due to lack of array inheritance
TARDIS is a significant step toward generating library scale insertions in animals. I was curious about a few points. Is this observed lethality due to off-target, collateral damage of Cas9 induction? (i.e. In worms that lack arrays and that are not under selective pressure, is there any significant lethality after heat shock?)
Upon knockdown of these genes, we detected 47-84 genes to be differentially expressed compared to the control GFP KD sample (adjusted p-value <0.05, Figure 5A-C and Table S3).
This is an intriguing finding. The number of genes whose expression changes seems small relative to the important and diverse biology processes that are predicted to be regulated by RpRPs and AGOs. When related genes are knocked down in other species, how many genes show significant changes in expression? In addition, is the phenotype of the RpRPs and AGOs knock downs in Ixodes scapularis known?
Therefore, the production of 22nt species from RNAP III transcribed genes was broadly conserved in ticks.
If available, comparison of sRNA conservation across multiple tick species would bolster the idea that these sRNAs are deeply conserved. It may also reveal interesting species-specific biological differences.
Expression of the PIWI proteins was further confirmed by Western blotting using antibodies against asymmetric di-methyl-arginine, a post-translational modification that is conserved among PIWI proteins [32].
Antibodies made against asymmetric di-methyl-arginine may cross react and diminish actual changes in PIWI protein expression levels. Are there antibodies made against PIWI proteins in related species that could provide a more specific measure of PIWI protein expression levels in ISE6 cells, revealing what may be a more dramatic change in expression?
We also compared KD efficiency of crRNAs targeting intronic versus exonic regions for the same two RNAs (Fig. 2D).
Has targeting of CRISPR-Csm to 5’ or 3’ UTRs been performed? Could this provide different levels of knock-down and further add your ability to tune RNA levels (in addition to altering spacer length)?
Thus, fluorescently-tagged Csm can be used for easy visualization of RNA in living cells.
Being able to directly target complexes to the native RNA sequences is a significant benefit over the potentially disruptive insertion of RNA aptamer sequences into RNAs to visualize RNA dynamics; however, can binding of multiple CRISPR-Csm complexes to native RNAs disrupt function? Do you have any evidence that CRISPR-Csm complex binding does not disrupt RNA metabolism of ncRNAs or protein coding RNAs or expression of corresponding proteins? Additionally, do you have any evidence that you have the sensitivity to detect single RNA molecules (comparison to smFISH)?