D. melanogaster gustatory neurons to instruct changes in chromatin accessibility and transcription that underlie sensory adaptations to a high-sugar diet. OGT works synergistically with the Mitogen Activated Kinase/Extracellular signal Regulated Kinase (MAPK/ERK) rolled and its effector stripe  to integrate activity information. OGT also cooperates with the epigenetic silencer Polycomb Repressive Complex 2.1 (PRC2.1) to decrease chromatin accessibility and repress transcription in the high-sugar diet. This integration of nutritional and activity information changes the taste neurons’ responses to sugar and the flies’ ability to sense sweetness. Our findings reveal how nutrigenomic signaling generates neural activity and behavior in response to dietary changes in the sensory neurons.Diet profoundly influences brain physiology, but how metabolic information is transmuted into neural activity and behavior changes remains elusive. Here, we show that the metabolic enzyme O-GlcNAc Transferase (OGT) moonlights on the chromatin of the  The levels and types of dietary nutrients play an essential role in cellular processes such as growth, division, and differentiation by providing fuel and biomass. However, nutrients can also affect these aspects of cell physiology by influencing, and often orchestrating, gene expression programs . These eOGT exclusively in the fly sweet-taste cells prevented the neural and behavioral decrease in sugar responses observed with a high-sugar diet . This cooperation leads to changes in chromatin accessibility and transcription that drive sensory plasticity, and the catalytic activity of OGT plays an instructional role in this process. Thus, our results uncover mechanistic insights into how nutrigenomic signaling translates nutritional information into dietary adaptations in the sensory neurons.Taste sensation changes depending on diet composition. In animals, the levels of bitter, sweet, and salty foods influence how these taste stimuli are perceived, with a general inverse relationship between the amount of a particular food in the diet and the responses of the sensory system to it . For exagar diet . OGT usegar diet . OGT actgar diet . OGT is gar diet . DespiteDNA adenosine methyltransferase Identification (Dam-ID or TaDA) to measure the association of OGT with DNA (Chromatin Accessibility profiling using Targeted DamID (CaTaDA) to assess chromatin accessibility (UAS-LT3-Dam::OGT or UAS-LT3-Dam flies were crossed with Gustatory Receptor 5a GAL4 (Gr5a) flies  and Gr5a>LT3-Dam; tubulin-GAL80ts (yellow) transgenic flies were kept at the permissive temperature and fed a control  or sugar  diet for 3 days  promoter – only expressed in adjacent cells – accessibility was low  flies  to driver 3 days . Dam::OGl design . The nor by diet , and the was low , suggestDam::OGT was associated with chromatin at introns (51%) and transcriptional start sites (TSSs) and promoters (30%) . In flieWe next examined the differential binding of OGT between the two diets. Although the majority of intervals were shared between a CD and SD . To charOur previous work showed that the epigenetic silencer PRC2.1 – specifically its H3K27m activity – was necessary and sufficient to drive sweet-taste plasticity in response to the nutrient environment . In the purple vs. green), which include both Polycomb ‘blue’ and actively transcribed ‘yellow’ chromatin regions (purple vs. pink). We next asked whether the dietary environment changed the association of OGT and Pcl at these loci. There was more OGT and Pcl at the OGT × Pcl shared sites in the SD condition compared to CD, and more OGT than Pcl was present at these sites in both diets  and Dam::OGT (green) revealed a small number of shared intervals . These 1 regions . OGT × Pth diets . Strikinth diets . This nuth diets .GO term analysis of genes shared by OGT/Pcl targets revealed enrichment in regulatory pathways involved in sequence-specific DNA binding, including those implicated in neural differentiation, sodium channel regulator activity, Transforming Growth Factor β and Activin receptor signaling, and dendrite development , left. 3OGT in the Gr5a+ sweet-sensing neurons resulted in flies with similar sweet sensitivity between the two diets (1OGT) and catalytically dead mutants (K872MOGT) to rescue taste plasticity; both of these alleles are homozygous lethal and thus were tested in combination with w1118CS control flies. Neither mutant affected sweet-taste responses on a control diet, but both prevented the lower PER to sucrose observed in SD-fed control flies (O-GlcNAcase (OGA), which removes the GlcNAc moiety from proteins, resulted in lower sweet-taste responses on CD . As shown in wo diets . Thus, Ool flies . This ares on CD . We nextes on CD . Overexped flies . Howevered flies  blocked ed flies , right; ed flies . To finaed flies ; no effeGr5a>LT3-Dam; tubulin-GAL80ts and Gr5a>LT3-Dam::Pcl; tubulin-Gal80ts (Pcl in the Gr5a+ neurons mimics the effects of SD on taste by decreasing responses to sucrose in flies fed a CD (left) – a result dependent on the H3K27 methylation activity of this complex (Pcl overexpression on sucrose responses compared to vehicle-fed flies (right). These results place OGT upstream of PRC2.1 at both the molecular and behavioral levels. Since OGT is an enzyme known to modify proteins, we also asked whether its effects on taste plasticity were mediated, at least in part, through PRC2.1. c429Pcl mutations blocked diet-induced taste plasticity on SD , suggesting that the effects of OGT act largely through PRC2.1. To further confirm these results and link them to the catalytic H3K27 methylation activity of PRC2.1, we treated Gr5a>OGT and control flies with a vehicle or the specific inhibitor of PRC2 (EEDi) while on CD   and the fatty acids taste receptor Ir56D, were enriched in the Gr5a+ fraction compared to the input, while bitter receptor genes (Gr66a and Gr32a) were depleted  or restored  the expression of 52% of the DEGs with SD/CD change  and the transcription factor cabut (cbt). To functionally validate some of these new genes, we knocked down or overexpressed them (depending on their SD/CD log2 fold) in the Gr5a+ neurons and assayed sweet-taste function by PER   in fliesn (TRAP) ; mRNAs sdepleted , indicateriments . Howevereriments . Indeed,mutation . These gmutation . Notableasticity , but alsn by PER . This reTogether, these experiments place OGT and PRC2.1 in the same genetic pathway that directs diet-induced taste plasticity at the chromatin, transcriptional, and behavioral levels. They also argue that the activity of the metabolic sensor OGT may provide the nutrient-dependent context for PRC2.1-mediated changes in chromatin accessibility (but not PRC2.1 recruitment), while PRC2.1 may instead function as an effector of these chromatin and transcriptional changes.PhoRC  and Trx-recruiter Trithorax-like , as well as transcription factors, such as the Zn-finger immediate early gene Stripe , and the nutrient-sensitive factor Sterol-Responsive-Element Binding Protein (SREBP)  loci for enriched cis-regulatory motifs ( (SREBP) . To dete (SREBP) . The onl (SREBP) .Sr cis-regulatory motifs was modest at OGT or Pcl-only loci  but strong at genes bound by both factors   as well as lower PER at both high and low sucrose concentrations (Sr in flies treated with OSMI (green) or with an inhibitor of PRC2  . When weon test) , top, wion test) , bottom.trations . This efD. melanogaster – in sweet-taste and diet-induced taste plasticity.Sr is the downstream transcriptional effector for the Extracellular-signal Regulated Kinase (ERK), a pathway stimulated by neural activity that plays a role in plasticity . We reasSr, the mRNA abundance of rl was higher in the Gr5a+ neurons of flies on SD, but this gene was not a direct target of OGT or PRC2.1 ; however, expression of the active Semrl decreased neuronal responses to sucrose (light blue). These activity phenotypes were reflected in the behavioral taste responses to sucrose, with WTrl flies having identical PER to sucrose as controls and Semrl showing reduced PER across high and low sucrose concentrations . Thus, rl activity, but not higher levels alone, was sufficient to affect sweet-taste plasticity. Not surprisingly, given the known function of rl/ERK in neural activity, we found that this kinase was also necessary for sweet-taste responses, as loss of function mutants and RNAi for rl had lower electrophysiological and behavioral responses to sucrose . At concentrations previously used in flies (15.6 μM) , Trametiesponses ; this haesponses . Treatmecontrols , placingcontrols  and behacontrols  responsecontrols . Of notecontrols , consistcontrols . TogetheD. melanogaster fly to answer these questions.Nutrigenomic signaling plays a role in health and disease by bridging the dietary environment with physiological adaptations. However, the molecular mechanisms and consequences of this type of nutrient sensing are still poorly understood. In particular, how nutrigenomic signals are integrated with cellular contexts has remained hard to define due to the lack of mechanistic nutrigenomic models . In thisHere, we report that the metabolic enzyme OGT is associated with neural chromatin at introns and TSSs. While OGT-associated genes showed subtle but significant changes in chromatin accessibility in response to diet, these dynamics were much more robust at loci co-occupied by both OGT and the epigenetic silencer PRC2.1. At genes decorated by both factors, we observed sizable diet-dependent chromatin variations that were critically dependent on the catalytic activity of OGT. OGT activity was also necessary for the differential transcriptional and taste responses to the high-sugar diet. The OGT- and PRC2.1-bound nutrient-dependent loci were enriched for binding motifs for the activity-dependent transcription factor Sr at TSS, the effector of the ERK pathway. We show that this signaling pathway functions upstream of OGT/PRC2.1 to shape neural and behavioral taste responses to the dietary environment. We thus propose a model where a nutrigenomic pathway composed of OGT and PRC2.1 integrates information from the nutrient and cellular environment via ERK signaling to orchestrate sensory responses to diet . Our datOGT is a conserved enzyme that catalyzes the transfer of UDP-GlcNAc to the serine and threonine residues of proteins . BecauseDrosophila melanogaster. Like in murine embryonic stem cells, OGT was enriched at introns and TSSs and primarily associated with transcriptionally active chromatin. However, we found that half of OGT intervals were also enriched at Polycomb repressive chromatin, consistent with previous reports that the GlcNAc modification is found at PREs, as well as on many Polycomb Group (PcG) proteins; OGT is also associated with PcG complexes to mediate Hox-gene repression  adaptations that underlie behavior or disease? This is one of the central and unresolved questions in nutrigenomics. Our model’s genetic and neural tractability provides a unique opportunity to get some answers. The 162 co-occupied loci identified were enriched for transcription and regulatory factors involved in cell proliferation, differentiation, signaling, and neural activity, as well as pathways implicated in neural plasticity. Many of these DNA-binding factors play essential roles during the development of sensory neurons to set their biophysical properties, such as Ptx1 and cad, but also affect adult taste plasticity. The regulons of these TFs include genes known to affect pre- and postsynaptic branching and structure, as well as synaptic physiology. Thus, our collective data indicate that this nutrigenomic pathway promotes taste adaptations, most likely by re-engaging developmental gene batteries, a mechanism that has been hypothesized to play a role in neural plasticity . WhetherAlthough using sensory plasticity and fly gustatory neurons as a model to study nutrigenomic signaling brings unique advantages, it also has significant limitations. These primarily arise from the small number of cells (60) and the in vivo nature of our model. First, we cannot probe whether OGT, PRC2.1, and Sr/EGR2 physically interact or modify each other in these cells. We also could not probe whether SD enhances the presence of OGT protein in the nucleus compared to a control diet due to the lack of functional antibodies. Thus, evidence for our model arises from the combination of cell-specific molecular, genetic, and physiological data. Second, we only inferred that the loci with Sr/EGR2 motifs integrate activity due to the well-established function of the ERK pathway in activity-dependent plasticity; future studies should address this directly and compare the effects of acute vs. chronic nutrient influx.Further, while inhibitors have allowed us to establish critical epistatic interactions and conduct dietary manipulations while bypassing developmental effects and genetic challenges, we cannot exclude that some of these effects may be non-cell autonomous. Integrating this model with biochemical approaches that preserve the appropriate activity and nutrient context would help address these critical questions. Finally, pathways beyond OGT, ERK, and PRC2.1 may also play a role in sensory plasticity.and future environmental challenges. Future studies in this field will no doubt uncover fascinating insights about the rules of nutrigenomic communication: these discoveries will illuminate how nutrition and gene expression converge to shape cell physiology and provide us with new tools to promote wellness and diminish the burden of disease.In summary, we show that activity and nutrient-sensing mechanisms are integrated at the genomic level to promote neural adaptations to the food environment. In particular, our data reveal a central and instructional role for OGT and meaningful epistatic interactions with sensors (ERK) and effectors (PRC2.1). More generally, we put forth a model where cell and context specificity transforms ‘nutritional data’ – that is, variations in nutrient and metabolite levels – into nutritional information , as show2 anesthesia 1–3 days after eclosion and maintained in a vial that housed 35–40 flies. Flies were acclimated to their new vial environment for an additional 2 days and were moved to fresh food vials every other day. The GAL4/UAS system was used to express transgenes of interest using the Gustatory receptor 5a Gr5a-GAL4 transgene. For each GAL4/UAS cross, transgenic controls were made by crossing the CSw1118  to GAL4 or UAS flies, sex-matched to those used in the GAL4/UAS cross. The fly lines used for this paper are listed in All flies were grown and fed cornmeal food (Bloomington Food B recipe) at 25°C and 45–55% humidity under a 12 hr light/12 hr dark cycle (Zeitgeber time 0 at 9:00 AM) unless otherwise stated. Male flies were collected under COFor all dietary manipulations, the following compounds were mixed into standard cornmeal food (Bloomington Food B recipe)  by melting, mixing, and pouring new vials as in Male flies were food deprived for 18–20 hr in a vial with a Kimwipe dampened with Milli-Q filtered deionized water. PER was carried out as described in Gr5a+ cells) were collected using sieves chilled in liquid nitrogen and dry ice. Frozen tissue was then lysed as previously described  using the NEBuilder HiFi DNA Assembly kit based on the manufacturer’s instructions (New England Biolabs). Transgenic animals were validated by reverse transcription PCR targeting the correct insert. UAS-LT3-Dam::Pcl was as previously described in UAS-LT3-Dam::OGT, UAS-LT3-Dam::Pcl, and UAS-LT3-Dam line were crossed to the tsGr5a-GAL4; tubulin-GAL80. All animals were raised and maintained at 20°C. Expression of Dam::OGT/Pcl and Dam was induced at 28°C for 18 hr. For all experiments, 300 heads of male and female flies were collected per replicate on dry ice by sieving. DNA was extracted following kit instructions (Invitrogen). To identify methylated regions, purified DNA was digested by Dpn I, followed by PCR purification of digested sequences. TaDa adaptors were ligated by T4 DNA ligase (NEB). Adapter ligated DNA was PCR amplified and purified according to the protocol . All reactions included integrated Heat-Labile Double-Strand Specific DNase treatment . The DNA-sequencing libraries for TaDa were generated using the Takara ThruPLEX Kit . For rat RNA-seq, libraries were prepared using the Nugen Ovation Model organism (Rat #0349-32) with 1/10th ERCC spike-in mix. These libraries were run on a NextSeq instrument using a HO 150 cycle cit (75 × 75 bp paired-end reads). All Drosophila libraries were sequenced on the Illumina NextSeq platform (High-output kit v2 75 cycles) at the University of Michigan Genomics Core facility.Generation of RNA sequencing (RNA-seq) libraries was with the Ovation SoLo RNA-seq System for Gr5a+-specific fraction and the input. For comparisons between dietary conditions, DESeq2 was only applied to the Gr5a+-specific IP condition. SD7 and c429Pcl datasets were analyzed from and described in q < 0.1 was used to call DEGs. To identify overlap between datasets GeneOverlap was used  in GATC resolution as described previously . Binding intensity metaplots were made by computing a matrix for specified regions  using the FIMO-binding site discovery tool [cite:doi:10.1093/bioinformatics/btr614]. Hits for each motif were retained as potential binding sites and used to calculate overlaps with other features , as noted. Permutation tests to assess significance were performed through repeated application of the bedtools shuffle [cite: https://doi.org/10.1093/bioinformatics/btq033] command to obtain 100 resamplings  and combining all of the locations that were counted as a potential binding site for any of the Sr motifs available from CIS-BP. Enrichments of overlaps with OGT, Pcl, and PRE sites were calculated by comparing the actual observed count of overlapping features with the mean overlap observed across 1,000 random samplings of the Sr motif locations (preserving the chromosome on which each motif is located during shuffling). For comparison of Sr motif locations with TSSs, we first identified the (strandedness-aware) start location of all ‘gene’, ‘mobile_genetic_element’, or ‘pseudogene’ features from the dmel6 Genbank annotations and then categorized all of these locations as ‘OGT/PRC2’ or ‘Not OGT/PRC2’ based on whether or not the gene was associated with an OGT, Pcl, or PRE location . Defining a region of practical equivalence as a change of no more than 1.5-fold in either direction, we tested the null hypothesis of a change larger than 1.5-fold using the gene-wise estimates of the SE in log2fold change (reported by Deseq2) and the assumption that the actual l2fcs are normally distributed. Rejection of the null hypothesis is evidence that the gene’s expression is not changed substantially between the conditions of interest. Python code for the practical equivalence test is in Statistical tests, sample size, and LT3-Dam::OGT and Pcl flies are available upon request; all other fly lines are available in the BDSC database as shown in All high-throughput data are available at the GEO repository: GSE188757 and GSE146245.  Drosophila gustatory neurons as a model system, the authors provide important mechanistic insight into how nutrigenomic signaling encodes nutritional information into cellular changes. The authors expand previous work by showing that OGT is associated with neural chromatin at introns and transcriptional start sites and that diet-induced changes in chromatin accessibility were amplified at loci with the presence of both OGT and PRC2.1. The work also identifies Mitogen Activated Kinase as a critical mediator in this pathway. This is an elegant group of experiments revealing mechanisms for how nutrigenomic signaling triggers cellular responses to nutrients.Using  public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and K VijayRaghavan as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Allan-Hermann Pool (Reviewer #3).Thank you for submitting your article "Nutrigenomic Regulation of Sensory Plasticity" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:There was relative consensus amongst the reviewers about the interest and potential importance of this study. In addition to the relatively straightforward controls and textual revisions detailed below, there were two major areas in which additional experiments are needed to strengthen and extend the impact of this study.1) Perform additional genetic experiments (detailed below) to confirm and underscore the specificity of the pharmacological experiments. This might include a demonstration that the drug phenocopies the OGT enzymatic loss-of-function and related experiments.2) Presenting analyses in non-sweet sensing neurons to strengthen the impact and specificity of the manipulations.Reviewer #1 (Recommendations for the authors):As the study currently stands, the proposed model is speculative, and a significantly revised approach and design would be needed to support the study's conclusions.1) The genetic interactions of OGT with other genes  rely only on the treatment of the flies with a systemic pharmacological OGT activity inhibitor OSMI. But, it is not possible to draw strong and nuanced conclusions from the datasets that rely heavily of a systemic drug-based manipulation instead of precise genetic alleles. Ideally the authors would want to include the same DAM-ID experiments performed with a OGT enzymatic mutant versus a OGT-WT.The authors are encouraged to ask what happens to PRC occupancy in CD and SD in tissue-specific KD of OGT using RNAi. If the RNAi reagent does not provide the authors a complete loss of function, they should consider using sgRNA-based KD approaches or generate a HA or GFP OGT knock-in line and then utilize degron based tissue-specific KD of OGT.2) For the OSMI experiments, would the authors present evidence that the drug is specific to OGT and has no off-target effects? Alternatively, authors might want to consider presenting results with OGT KD or OGT enzyme specific mutants to correlate OSMI results with. These orthogonal and corroborative datasets would be required to bolster the conclusions.3) This study is measuring the chromatin availability and determined that there was higher accessibility under CD conditions. Since OGT also has critical cytosolic roles, it would be important to determine if this is due to differences in nuclear: cytoplasmic localization of OGT or due to changes in OGT activity. To this end, I would recommend a western blot of Nuclear v. cytoplasmic fractions of OGT for flies on CD vs SD or imaging based approaches would be needed.4) In Figure 2A, the authors describe a high correlation in chromatin binding sites between Pcl and OGT, concluding that the overlapping OGTXPRC2.1 regions are a critical subset for sweet tasting response. However, these overlapping regions are a rather small subset of sites, with the largest subset actually belonging the "yellow" H3K4/H3K36 euchromatin subset. It is unclear from this analysis why the authors chose to pursue the blue chromatin instead. It would be important to rank regulatory regions bound by DAM:OGT alone and DAM:Pcl alone to establish the primary regions of binding for both OGT and Pcl irrespective of each other. These regions can then be compared with the known sweet tasting genes to rank the relevant binding regions. Should there be other regions relevant to sweet taste that are not overlapping between OGT and Pcl that should be reported. This will also give readers a more comprehensive understanding about the role of Sr motifs and if this is a small but significant subset of genes regulated by OGT or comprise the majority of cis-regulatory regions controlled by OGT.5) The authors use PCL as a proxy for PRC2.1 activity as it is the recruiter for PRC2.1. However, why wasn't DAM::PRC2.1 performed to directly compare to OGT? This data should be provided to show the overlap of PRC2.1 to Pcl or it should be explained in the results.6) The authors findings are performed using flies overexpressing Pcl or OGT in Gr5a expressing cells for the TADA, PER experiments, and electrophysiology analysis. Because this analysis is based on an overexpression of the gene of interest, I believe it is important to repeat these experiments in flies overexpressing these genes in neurons unrelated to the sweet taste response- such as in the neighboring bitter taste neurons. This is especially a critical set of experiments to do, as the primary thesis of this study is about how each cell alters its nutrigenomic signaling in different cell types, but the analysis is primarily focused on DAM-ID datasets from overexpressed OGT in Gr5a. The study would be relevant if the authors did comparative analysis of OGT-PRC interactions in neurons that have different sensitivities or taste modalities to sugar.7) The Proboscis extension response needs to be further elaborated on. The data plotted seems to be normalized but this is not explained in the paper. What is actually being measure? What is the N? If the data is normalized, then the authors should present raw data graphed in a supplemental figure at least.8) Figure 4B seems misleading in the way it is plotted. Authors claim a bias of SR genes at the TSS in the PRC2.1 and OGT overlapping regions. However, the y axes are not to the same scale. In fact, the not OGT/PRC2 regions have much higher number of genes with SR motifs at every single position plotted on 4B. Specifically the strong statements findings in the results paragraph on lines 262-283 are not sufficiently supported and the authors are encouraged to revise this or move it to discussion.9) Many of the experiments involved moving flies to OSMI or Trametinib diets. A caveat to this method is that this treatment will influence OGT/ERK in the fly globally. Because both OGT/ERK impact a number of critical biological processes, this makes the results difficult to interpret as impacts of taste sensing or due to the flies being generally unhealthy. It would be helpful to see fly survival on control, OSMI supplemented, and Trametinib diets to determine how detrimental these diet conditions are in general to the fly.10) In general, the figure legends and methods are lacking in detail. For instance, in Figure 1F, are the GO terms for control diet comprised from all peaks listed or just the peaks unique to CD? Same question for the SD GO terms. Abbreviations are not elaborated in the legend or methods  or lacking axis labels , etc. I suggest a thorough review of the methods and legend to add details necessary to interpret the results and make it possible to replicate experimental designs.Reviewer #2 (Recommendations for the authors):1. There are too many instances of non-technical phrases that are unclear/nonspecific. For example, "…OGT moonlights on the chromatin ….", or "… OGT decorates nutrient-sensitive …". These phrases should be replaced with more precise words, unless these phrases are common in the field and I am just unaware.2. The Discussion section is overall too long and delves too much into the previous literature. It should be condensed and there should be more focus on the translational implications of the results.Reviewer #3 (Recommendations for the authors):– Almost all quantitative data in figures lack definition of variance measures (e.g. Panel 3C and E). Are data represented as mean +/I standard deviation, s.e.m.?Drosophila.– Regarding setting up the problem – claiming "how nutritional information is transmuted to neural activity and behavioral changes remains elusive" is inaccurate – we know the receptors, increasingly many cells in the food intake regulatory circuit from sensory input to motor output in – Methods refer to a python code on Github but no github repository is specified.– Please also make the code for analyzing the DaMID and CaTaDa available online. Essential revisions:There was relative consensus amongst the reviewers about the interest and potential importance of this study. In addition to the relatively straightforward controls and textual revisions detailed below, there were two major areas in which additional experiments are needed to strengthen and extend the impact of this study.1) Perform additional genetic experiments (detailed below) to confirm and underscore the specificity of the pharmacological experiments. This might include a demonstration that the drug phenocopies the OGT enzymatic loss-of-function and related experiments.We have carried out several new genetic experiments to address the reviewers' concerns. In the revised manuscript, these experiments are shown in a new Figure 3 and revised Figure 4, and Figure 4-Supplement 1, Figure 5-Supplement 1, and Figure 6-Supplement 1; below, we outlined the experiments and explained the findings for the reviewers’ below before addressing the individual comments.1A. OGT catalytic activity and OSMI-1There are several published OGT mutant alleles, but we were only able to obtain two from the lab that generated them; we were told that all the others, including the UAS-versions which would have been useful for our experiments, were lost due to the mishandling of the fly stocks. We requested these stocks in early 2020 but were unable to get them for nearly a year due to the COVID lab restrictions in the UK; this is why we originally carried out the experiments with OSMI-1 once our own lab reopened at 20% capacity in July 2020-May 2021 since we had previously validated, used, and published with this reagent . In brief, OSMI-1 is a specific inhibitor of OGT developed by Harvard Medical School and the NIH and published in 2015 ; its inhibition is insensitive to UDP-GlcNAc concentration, and it doesn’t affect cell-surface glycosylation.K872MOGT, a full catalytically dead mutant, to the protein null 1 –OGT please note that both of these are homozygous lethal, so here they were tested in heterozygous combination with w1118cs (wcs) control flies . As you can see in Figure 3, the heterozygous catalytically dead mutant, K872M/+OGT , phenocopies the effects of protein null 1OGT allele  in preventing diet-induced taste plasticity on a sugar diet . No effects were observed on a control diet . This provides genetic evidence for OGT activity being essential for taste changes in response to a sugar diet. Please note that as the diagram in Figure 3A now shows, proboscis extension response (PER) is measured from 1 (full extension) to 0 (no extension). Thus, the PER data shown in the manuscript is the raw data (mean +/-SEM) obtained from observing the flies during sucrose stimulation; it is not normalized (a comment of Reviewer 1). Importantly, the phenotype of K872MOGT/+ flies (wild-type taste on sugar diet) is similar to that of OGT RNAi targeted specifically to the Gr5a+ neurons . It is also identical to that of OSMI-1 treatment .In data now added to Figure 3C, we compared O-GlcNAcase (OGA), which removes the GlcNAc moiety. If OGT catalytic activity were important, we would expect that interfering with removing the GlcNAc mark would mimic the effects of a sugar diet (and OGT overexpression) on taste. Figure 3 D shows that the knockdown of OGA in the Gr5a+ neurons resulted in a marked decrease in sucrose taste sensation in control-diet flies . Thus, OGA knockdown recapitulates the effects of OGT overexpression and sugar-diet on sucrose taste, further validating the role of OGT activity in taste plasticity.To further validate the role of OGT catalytic activity, we knocked down the gene for the antagonistic enzyme to OGT, OGT in the Gr5a+ neurons. As a reminder, expression of OGT in the Gr5a+ neurons results in taste depression on a control diet, recapitulating the effects of a sugar diet. Treatment with OSMI-1, however, inhibited the effects of OGT overexpression on sweet taste, as shown in Figure 3E. These results link the OGT overexpression phenotype to its catalytic activity. As reviewer 1 asked, we tested the effects of OSMI-1 and Tretmetinib on survival and found no effect .Finally, to link OSMI-1 to OGT function in our system, we tested the effects of OSMI-1 treatment in flies that overexpress Together, the experiments in the new Figure 3 provide solid evidence for the role of OGT catalytic activity in taste plasticity and support the design of the molecular experiments in Figure 4.1B. Epistasis experimentsReviewers 2 and 3 commented on the “elegant group of experiments” and “the thorough exploration of epistatic relationships between different players in the system.” These epistatic relationships would be very hard – and perhaps impossible– to parse out without the combination of genetic and pharmacological tools we used here. Since Reviewer 1 expressed concern about OGT, Pcl, and PRC2 interactions, we carried out additional genetic and pharmacological epistasis experiments, which we added to Figure 4 in the revised manuscript.OGT overexpression in the Gr5a+ neurons (green) is dependent on the presence of a functioning Pcl (pink and purple). And consistent with the function of Pcl as a PRC2.1 recruiter, Figure 4D shows that the ability of OGT to induce taste plasticity depends on the catalytically active PRC2 complex. These data provide genetic evidence for the data shown in the original version of the manuscript , where we rescued the effects of Pcl overexpression on taste with OSMI; they also suggest that PRC2 H3K27 methylation is likely the effector arm of this nutrigenomic signaling. A note in response to Reviewer 1: we manipulated Pcl instead of Ez because this manipulation is more specific. The PRC2 complex comes in many different “flavors,” all of which contain Ez as the H3K27m. Each of the PRC2 variants is characterized by a different recruiter, which targets the complex to specific regions of chromatin . Thus, by manipulating Pcl, we can target a specific type of PRC2.1; in  we demonstrated that the other PRC2 variants are not involved in taste plasticity; in that manuscript, we also show that Pcl, Ez, Suz12, and Esc mutants phenocopy one another, and that the effects of Pcl on sweet taste are dependent on the catalytic activity of Ez.Figure 4C shows that the taste phenotype caused by Together these new data in Figure 4 place OGT and PRC2.1 in the same genetic pathway, and argue for a strong interaction between OGT, Pcl, and PRC2; these results also support those of the DAM-ID and TRAP molecular experiments, which show synergistic effects between these two players.Additional experimentsrl (we previously only had mutant data) has marked effects on sucrose taste sensitivity We added data showing that Gr5a+-specific knockdown of the kinase We also added a functional characterization as measured by proboscis extension response of some of the sugar diet DEGs that are dependent on OGT/Pcl as suggested by Reviewer 3 .These genetic experiments make the conclusions of the manuscript stronger – we thus hope the reviewers find their concerns addressed.2) Presenting analyses in non-sweet sensing neurons to strengthen the impact and specificity of the manipulations.This study is a cell-specific investigation of nutrigenomic mechanisms in the Gr5a+ sweet-taste neurons; our intention was not to examine mechanisms across different cell types. However, we can understand the reviewers’ comments about this after rereading the abstract and introduction. As such, we have rewritten part of the manuscript to better introduce the rationale behind the study as the integration of metabolic signaling and cellular contexts. We hope this is now an improved framing for the study rationale.eLife. Carrying out this new project would result in an additional 6-7 figures but would not fundamentally change the conclusion of the current work; in fact, it may even take away from the targeted integration of molecular biology and neuroscience we have tried to achieve.About analyzing the effects of diet on other cells, no doubt this is an interesting question. However, this also signifies embarking on a completely separate project that would take, optimistically speaking, at least one year to complete and require a budget of ~ $130,000 (see breakdown). Thus, this suggestion doesn’t seem in line with the peer review and editorial philosophy of Moreover, talking about the practicality of this proposal, we do not have such an unallocated budget, and so this new project would require us first to generate preliminary data on the bitter neurons, then write a grant proposal to fund it; as you can appreciate, this would take longer than a year, especially since we do not even know if the bitter gustatory neurons are affected by a high-sugar diet. Beyond this, looking at the bitter neurons would do little to prove specificity. If we found no effects of this pathway on the activity of the bitter neurons, it wouldn’t establish that the changes in the sweet taste neurons are specific. In fact, the same pathway could be acting in some of the other thousands of fly circuits that were not investigated (Black swan effect). If we did find that OGT/PRC2.1/Sr play a role in the bitter neurons, it would also do little to disprove specificity since their targets would likely be different because the sets of genes expressed in these two sensory neurons are different. By analogy, the protein sensor mTOR is expressed and active in every cell, where it modulates some of the same targets ; however, the effects of the pathway may be different due to the distinct metabolic and genetic idiosyncrasies of cells, as well as cellular compartments. This lack of specificity doesn’t mean that mTOR is not important or unspecific. Finally, we would like to note that we have tested the effects of manipulating OGT levels in other neurons (dopamine and Mushroom Body Output Neurons) without effects on behavior or neural responses ; based on these, OGT doesn’t seem to affect neurons indiscriminately.Budget ~ $129,000Salary and fringe benefits for PD for 10 calendar months: $75,000 DAM ID: $16,0000, Pcl:dam and OGT:dam in CD and SD, with and without OSMI x 4 biological replicates per condition = 32 samples @ $500 per sample (UM Genomics core)TRAP: $16,0000 Pcl mutant and OSMI in CD and SD x 4 biological replicates per condition + sequencing input = 32 samples @ $500 per sample (UM Genomics core)Animals' food and maintenance: $ 5,000, $500 per person/10 monthsReagents: = $17,000, including sequencing kit  x 2 = $12,000, and other reagents such as drugs and plasticNote that this PD would have to be hired and retrained. The first author of the manuscript who carried out the molecular experiments graduated in Dec 2021 but failed to pass on the technical knowledge due to COVID restrictions at the UM: we were completely shut down until July 2020, and at 20% capacity from March 2020 to July 2021 , and no new people joined the lab in 2020-2022 (most of the 2021 grad student class deferred to 2022 and no PD candidates apply).Reviewer #1 (Recommendations for the authors):As the study currently stands, the proposed model is speculative, and a significantly revised approach and design would be needed to support the study's conclusions.1) The genetic interactions of OGT with other genes  rely only on the treatment of the flies with a systemic pharmacological OGT activity inhibitor OSMI. But, it is not possible to draw strong and nuanced conclusions from the datasets that rely heavily of a systemic drug-based manipulation instead of precise genetic alleles. Ideally the authors would want to include the same DAM-ID experiments performed with a OGT enzymatic mutant versus a OGT-WT.The authors are encouraged to ask what happens to PRC occupancy in CD and SD in tissue-specific KD of OGT using RNAi. If the RNAi reagent does not provide the authors a complete loss of function, they should consider using sgRNA-based KD approaches or generate a HA or GFP OGT knock-in line and then utilize degron based tissue-specific KD of OGT.2) For the OSMI experiments, would the authors present evidence that the drug is specific to OGT and has no off-target effects? Alternatively, authors might want to consider presenting results with OGT KD or OGT enzyme specific mutants to correlate OSMI results with. These orthogonal and corroborative datasets would be required to bolster the conclusions.Response to 1 and 2 (as point 1 in the author’s recommendations):K872M/+OGT  100% of OGT but only 50% cat activity, see diagram on the right), phenocopies the effects of protein null 1OGT allele  in preventing diet-induced taste plasticity on a sugar diet (SD). No effects were observed on a control diet (CD). This shows genetic evidence for OGT activity being essential for taste changes in response to a SD. Please note that as the diagram in Figure 3A now shows, proboscis extension response (PER) is measured from 1 (full extension) to 0 (no extension). Thus, the PER data shown in the manuscript is the raw data (mean +/-SEM) obtained from observing the flies during sucrose stimulation; it is not normalized (see below for full response to this comment). Importantly, the phenotype of K872M/+OGT flies (wild-type taste on sugar diet) is similar to that of OGT RNAi targeted specifically to the Gr5a+ neurons . It is also identical to that of the OSMI-1 treatment . OSMI-1 is a specific inhibitor of OGT developed by Harvard Medical School and the NIH and published in 2015 ; its inhibition is insensitive to UDP-GlcNAc concentration, and it doesn’t affect cell-surface glycosylation. We had previously used OGT in .In data now added to Figure 3C and added here for the reviewer’s convenience, we show that heterozygous catalytically dead mutants, OGT overexpression and sugar diet and further validating the role of OGT activity.If OGT catalytic activity were important, we would expect that interfering with the removal of GlcNAcylation would mimic the effects of a sugar diet and OGT overexpression on taste. We tested this possibility by knocking down the antagonistic enzyme to OGT, O-GlcNAcase (OGA), which removes the GlcNAc moiety only in the Gr5a+ neurons. This resulted in a marked decrease in sucrose taste sensation in control-diet flies , mimicking the effects of To further validate these findings, we tested the effects of OSMI-1 treatment in flies that overexpress OGT in the Gr5a+ neurons. As a reminder, expression of OGT in the Gr5a+ neurons results in taste depression on a control diet, mimicking the effects of a sugar diet. Treatment with OSMI-1, however, inhibited the effects of OGT overexpression on taste, as shown in Figure 3 E. These results link the OGT overexpression phenotype to its catalytic activity.Together, the experiments in the new Figure 3 provide further evidence for the role of OGT catalytic activity in taste plasticity.We disagree with the comment that we rely solely on pharmacological manipulations; in fact, we use cell-specific – pharmacological and dietary interventions. Indeed, Reviewers 2 and 3 praised our approaches by saying: “This is an elegant group of experiments revealing mechanisms for how nutrigenomic signaling triggers cellular responses to nutrients” and “Strengths: the thorough exploration of epistatic relationships between different players in the system.” All of our experiments combine genetic manipulations in combination with dietary and/or pharmacological treatments to show that molecular, neural, and behavioral taste phenotypes arise only in specific contexts, so no single phenotype occurs due to nonspecific manipulations. Without this approach, most of these epistatic relationships would be largely inaccessible in this system. We have also used a combination of both genetic and pharmacological tools to implicate not only genes but also their function  to nutrient-specific effects. Third, we established causality and relationship by inducing and rescuing the molecular, behavioral, and electrophysiological phenotypes. Thus, our model is based on a combination of direct and indirect data  obtained from a controlled and careful set of experiments.Pcl. This provides genetic evidence for the data in the original manuscript version , where we rescued the effects of Pcl overexpression with OSMI-1.We could spend the next 6+ months trying to generate cell-specific alleles of OGT, which could bring their own unspecific effect, such as development and compensatory effects. However, this would only marginally add to the story now that we have added additional characterizations. In particular, we carried out additional genetic and pharmacological epistasis experiments, which we added to Figure 4. Here, we show that the taste phenotype caused by OGT overexpression in the Gr5a+ neurons entirely depends on the presence of Pcl instead of Ez because this manipulation is more specific. The PRC2 complex comes in many different “flavors,” all of which contain Ez. Each of the PRC2 variants is characterized by a different recruiter, which targets the complex to specific regions of chromatin . Thus, by manipulating Pcl, we can target a specific type of PRC2.1, and as we showed in, , the other PRC2 variants do not play a role in taste plasticity; in that manuscript, we also show that Pcl, Ez, Suz12, and Esc mutants phenocopy one another, and that the effects of Pcl on taste are indeed dependent on the catalytic activity of Ez. Here we took a similar approach and showed that OGT expression requires not only Pcl, but a catalytically active PRC2 to mediate its effect on taste plasticity .We manipulated Together the new Figure 4 experiments place OGT and PRC2.1 in the same genetic pathway, indicating a strong interaction between OGT, Pcl, and PRC2, and support the results of the DAM-ID and TRAP molecular experiments, which show synergistic effects between these players.ERK (we previously only had mutant data) has marked effects on sucrose taste sensitivity .Finally, we added data showing that Gr5a+-specific knockdown of the kinase The addition of these genetic experiments makes the manuscript stronger and hope the reviewer finds their concerns addressed.3) This study is measuring the chromatin availability and determined that there was higher accessibility under CD conditions. Since OGT also has critical cytosolic roles, it would be important to determine if this is due to differences in nuclear: cytoplasmic localization of OGT or due to changes in OGT activity. To this end, I would recommend a western blot of Nuclear v. cytoplasmic fractions of OGT for flies on CD vs SD or imaging based approaches would be needed.We agree with the reviewer that this is an important possibility. We tried available OGT antibodies for Western blot and immunofluorescence (to retain cell specificity), but they were not functional. In 2019 we attempted to generate rabbit polyclonal antibodies against OGT based on the two peptides below, as recommended by protein chemists:DmOGT(570-592): KPYNFLKKLPTKGRLRIGYLSSDDmOGT(1045-1059): Cys-EKYENGELPDHISAVWe injected 4 different rabbits, which gave rise to two different polyclonals per peptide. To our disappointment, none of the found antibodies showed a specific signal in Western blot and immunofluorescence experiments. We originally mentioned OGT compartmentalization in the discussion, but we agree that this point needs more stress, so we added it to the “limitation” portion of the discussion.4) In Figure 2A, the authors describe a high correlation in chromatin binding sites between Pcl and OGT, concluding that the overlapping OGTXPRC2.1 regions are a critical subset for sweet tasting response. However, these overlapping regions are a rather small subset of sites, with the largest subset actually belonging the "yellow" H3K4/H3K36 euchromatin subset. It is unclear from this analysis why the authors chose to pursue the blue chromatin instead. It would be important to rank regulatory regions bound by DAM:OGT alone and DAM:Pcl alone to establish the primary regions of binding for both OGT and Pcl irrespective of each other. These regions can then be compared with the known sweet tasting genes to rank the relevant binding regions. Should there be other regions relevant to sweet taste that are not overlapping between OGT and Pcl that should be reported. This will also give readers a more comprehensive understanding about the role of Sr motifs and if this is a small but significant subset of genes regulated by OGT or comprise the majority of cis-regulatory regions controlled by OGT.-associated genes have a lower expression than non-PCl-bound genes but also show considerable variation in expression among them. The comparison in Figure 4B with Pcl vs. OGT/Pcl shows that the PCl-only genes have lower expression.The OGT:dam data show that this enzyme is found at about an equal number of genes in yellow and blue chromatin ; only genes in blue chromatin are enriched for neural functions- see new text based on the suggestion to carry out more analyses on these two types of chromatin. The overlap between Pcl and OGT only occurs in a small proportion (162) of these OGT peaks, as shown in Figure 2. As we wrote in the text, the OGT/Pcl peaks are enriched in blue chromatin (permutation test); this is somewhat expected in a way since Pcl occupies blue chromatin, as shown in our previous analyses , and also demonstrated by the lower expression of the OGT x Pcl genes compared to OGT-alone . Intriguingly, in  we observed that PCl-bound, and OGT/Pcl and OGT/PREs regulatory regions in the supplementary excel file; the breakdown for Sr is shown in Figure 4A (now 5A). Very few genes are known as “sweet taste genes” outside of the sweet taste receptors, and the occupancy and expression of these sweet taste receptor genes is unchanged by diet. However, our previous molecular characterization of the sweet sensing neurons did identify two transcription factors, Ptx1, and cad, which we show here are occupied by OGT and Pcl, as mentioned in the manuscript. We took the reviewer’s suggestion and analyzed the GO terms and accessibility for OGT occupancy among yellow and blue chromatin . We found that only blue genes showed enrichment in GO terms, which are a subset of those found in the OGT/Pcl genes. We discuss the results of the analysis in the text.We added the enrichment for cis-regulatory regions among OGT-bound, PCl5) The authors use PCL as a proxy for PRC2.1 activity as it is the recruiter for PRC2.1. However, why wasn't DAM::PRC2.1 performed to directly compare to OGT? This data should be provided to show the overlap of PRC2.1 to Pcl or it should be explained in the results.Pcl binding instead of Ez because this manipulation is more specific. The PRC2 complex comes in many different “flavors,” all of which contain Ez. Each of the PRC2 variants is characterized by a different recruiter, which targets the complex to specific regions of chromatin . Thus, by manipulating and targeting Pcl, we can isolate a specific type of PRC2.1, and as we showed in , the other PRC2 variants do not play a role in taste plasticity; in that manuscript, we also show that Pcl, Ez, Suz12, and Esc mutants phenocopy one another, and that the effects of Pcl on taste are indeed dependent on the catalytic activity of Ez. In the revised version of Figure 4, we now show that OGT expression requires Pcl and a catalytically active PRC2 to mediate its effect on taste plasticity (4D).We manipulated and measured 6) The authors findings are performed using flies overexpressing Pcl or OGT in Gr5a expressing cells for the TADA, PER experiments, and electrophysiology analysis. Because this analysis is based on an overexpression of the gene of interest, I believe it is important to repeat these experiments in flies overexpressing these genes in neurons unrelated to the sweet taste response- such as in the neighboring bitter taste neurons. This is especially a critical set of experiments to do, as the primary thesis of this study is about how each cell alters its nutrigenomic signaling in different cell types, but the analysis is primarily focused on DAM-ID datasets from overexpressed OGT in Gr5a. The study would be relevant if the authors did comparative analysis of OGT-PRC interactions in neurons that have different sensitivities or taste modalities to sugar.The expression of Pcl and OGT for the molecular experiment only occurs for 18 hours. We do not solely use overexpression, but also RNAi, loss of function alleles, and genetic manipulations compared to overexpression. Importantly, we show that our manipulations only have an effect in combination with an environmental variable .(As in the authors’ recommendation response): This study is a cell-specific investigation of nutrigenomic mechanisms in the Gr5a+ sweet taste neurons; it was not our intention to examine mechanisms across different cell types. However, we can understand the reviewers’ comments about this after rereading the abstract and introduction. As such, we have rewritten part of the manuscript to better introduce the rationale behind the study as the integration of metabolic signaling and cellular contexts. We hope this is now an improved framing for the study rationale.eLife. Carrying out this new project would result in an additional 6-7 figures but would not fundamentally change the conclusion of the current work; in fact, it may even take away from the targeted integration of molecular biology and neuroscience we have tried to achieve.About analyzing the effects of diet on other cells, no doubt this is an interesting question. However, this also signifies embarking on a completely separate project that would take, optimistically speaking, at least one year to complete and require a budget of ~ $130,000 (see breakdown). Thus, this suggestion doesn’t seem in line with the peer review and editorial philosophy of Beyond this, we do not have such an unallocated budget, and so this new project would require us first to generate preliminary data on the bitter neurons to write then a grant proposal to fund it; as you can appreciate, this would take longer than a year, especially since we do not even know if the bitter gustatory neurons are affected by a high-sugar diet. Beyond this, looking at the bitter neurons would do little to prove specificity. If we found no effects of this pathway on the activity of the bitter neurons, it wouldn’t establish that the changes in the sweet taste neurons are specific. In fact, the same pathway could be acting in some of the other thousands of fly circuits that were not investigated (Black swan effect). If we did find that OGT/PRC2/Sr play a role in the bitter neurons, it would also do little to disprove specificity since their targets would likely be different because the sets of genes expressed in these two sensory neurons are different. By analogy, the protein sensor mTOR is expressed and active in every cell, where it modulates some of the same targets ; however, the effects of the pathway may be different due to the distinct metabolic and genetic idiosyncrasies of cells, as well as cellular compartments. This lack of specificity doesn’t mean that mTOR is not important. Finally, we would like to note that we have tested the effects of manipulating OGT levels in other neurons (dopamine and Mushroom Body Output Neurons) without effects on behavior or neural responses ; based on these, OGT doesn’t seem to affect neurons indiscriminately.Budget ~ $129,000Salary and fringe benefits for PD for 10 calendar months: $75,000 DAM ID: $16,0000, Pcl:dam and OGT:dam in CD and SD, with and without OSMI x 4 biological replicates per condition = 32 samples @ $500 per sample (UM Genomics core)TRAP: $16,0000 Pcl mutant and OSMI in CD and SD x 4 biological replicates per condition + sequencing input = 32 samples @ $500 per sample (UM Genomics core)Animals' food and maintenance: $ 5,000, $500 per person/10 monthsReagents: = $17,000, including sequencing kit  x 2 = $12,000, and other reagents such as drugs and plasticNote that this PD would have to be hired and retrained. The first author of the manuscript who carried out the molecular experiments graduated in Dec 2021 but failed to pass on the technical knowledge due to COVID restrictions at the UM: we were completely shut down until July 2020, and at 20% capacity from March 2020 to July 2021 , and no new people joined the lab in 2020-2022 (most of the 2021 grad student class deferred to 2022 and no PD candidates apply).7) The Proboscis extension response needs to be further elaborated on. The data plotted seems to be normalized but this is not explained in the paper. What is actually being measure? What is the N? If the data is normalized, then the authors should present raw data graphed in a supplemental figure at least.The data shown are the raw data plotted as mean +/- SEM. In the proboscis extension response, what is measured is the extent of the proboscis extension when the fly labellum is stimulated with different sucrose concentrations. The extent of proboscis extension is measured as 1 = full extension, 0.5=half extension, and 0=no extension. We added this as a diagram in Figure 3A.8) Figure 4B seems misleading in the way it is plotted. Authors claim a bias of SR genes at the TSS in the PRC2.1 and OGT overlapping regions. However, the y axes are not to the same scale. In fact, the not OGT/PRC2 regions have much higher number of genes with SR motifs at every single position plotted on 4B. Specifically the strong statements findings in the results paragraph on lines 262-283 are not sufficiently supported and the authors are encouraged to revise this or move it to discussion.4B (Now 5B) plots the distribution of Sr binding sites along the regions up and downstream of the TSS. The reason for the difference in the Y axis is that far more genes have Sr binding sites and do not have OGT/PRC2/Pcl. What the graph clearly shows, however, is where the position of Sr is relative to the TSS, and the pattern is opposite between OGT/Pcl and non-OGT-Pcl. To address the reviewer’s comment, we have now normalized the counts relative to the genome-wide expectation ; a score of 1.0 indicates the hypothetical genome-wide average overlap with Sr motifs and is shown as a red dashed line. This normalized analysis shows an even stronger enrichment of Sr at the TSS of OGT/PRC2/Pcl genes.9) Many of the experiments involved moving flies to OSMI or Trametinib diets. A caveat to this method is that this treatment will influence OGT/ERK in the fly globally. Because both OGT/ERK impact a number of critical biological processes, this makes the results difficult to interpret as impacts of taste sensing or due to the flies being generally unhealthy. It would be helpful to see fly survival on control, OSMI supplemented, and Trametinib diets to determine how detrimental these diet conditions are in general to the fly.We have now added these experiments to Figure 4-Supplement 1A. There is no effect on survival. Also, OSMI and Trametinib affect the taste system in specific dietary contexts, not indiscriminately.10) In general, the figure legends and methods are lacking in detail. For instance, in Figure 1F, are the GO terms for control diet comprised from all peaks listed or just the peaks unique to CD? Same question for the SD GO terms. Abbreviations are not elaborated in the legend or methods  or lacking axis labels , etc. I suggest a thorough review of the methods and legend to add details necessary to interpret the results and make it possible to replicate experimental designs.In Figure 1, “CD” means the Control diet only GO terms, “SD” means the Sugar diet only go terms, and SD/CD the differential binding go terms. We further clarified this in the legends and text and added the missing abbreviation.Reviewer #2 (Recommendations for the authors):1. There are too many instances of non-technical phrases that are unclear/nonspecific. For example, "…OGT moonlights on the chromatin ….", or "… OGT decorates nutrient-sensitive …". These phrases should be replaced with more precise words, unless these phrases are common in the field and I am just unaware.We were also surprised to see these terms used in the literature. However, “moonlight” is indeed generally used to describe alternative functions of enzymes, especially metabolic enzymes. The term was generated early in the 2000s, but it has been used more in the last 5-10 years; currently, there are 1,700 manuscripts on Pubmed that use “moonlight” as we did in the manuscript. “Decorate” first originated to describe post-translational or non-enzymatic modifications to histones, but more recently, it has also been used to refer to the association of a protein with chromatin or nucleic acids; this is widely used in the epigenetics field, perhaps because some find that using “binding” or “association” may imply direct interactions with DNA or chromatin. Here are some recent examples:Chaoyun Pan, Bo Li, M Celeste Simon. Moonlighting functions of metabolic enzymes and metabolites in cancer. Mol Cell. 2021 Sep 16;81(18):3760-3774. doi: 10.1016/j.molcel.2021.08.031.Boukouris AE, Zervopoulos SD, Michelakis ED. Metabolic Enzymes Moonlighting in the Nucleus: Metabolic Regulation of Gene Transcription. Trends in Biochemical Sciences, 23 Jun 2016, 41(8):712-730 DOI: 10.1016/j.tibs.2016.05.013Youjun Zhang, Arun Sampathkumar, Sandra Mae-Lin Kerber, Corné Swart, Carsten Hille, Kumar Seerangan, Alexander Graf, Lee Sweetlove & Alisdair R. Fernie. A moonlighting role for enzymes of glycolysis in the colocalization of mitochondria and chloroplasts. Nature Communications volume. 11, Article number: 4509 (2020)From Arnould C, Rocher V, Finoux AL, Clouaire T, Li K, Zhou F, Caron P, Mangeot PE, Ricci EP, Mourad R, Haber JE, Noordermeer D, Legube G. Loop extrusion as a mechanism for formation of DNA damage repair foci. Nature. 2021 Feb;590(7847):660-665. “When a DSB forms, the PI3K-related ATM kinase rapidly triggers the establishment of megabase-sized, chromatin domains decorated with phosphorylated histone H2AX (gammaH2AX), which act as seeds for the formation of DNA-damage response foci.”Chul-Hwan Lee, Jia-Ray Yu,1,7 Sunil Kumar, Ying Jin,3 Gary LeRoy, Natarajan Bhanu, Syuzo Kaneko, Benjamin A. Garcia, Andrew D. Hamilton, and Danny Reinberg. Allosteric activation dictates PRC2 activity independent of its recruitment to chromatin. Mol Cell. 2018 May 3; 70(3): 422–434.e6. “Mammalian heterochromatin contains large repressive chromatin domains, including H3K9me2/3-decorated constitutive heterchromatin at pericentromeric and telomeric regions.”2. The Discussion section is overall too long and delves too much into the previous literature. It should be condensed and there should be more focus on the translational implications of the results.We thank the reviewer for the comment; we have edited and shortened the discussion.Reviewer #3 (Recommendations for the authors):– Almost all quantitative data in figures lack definition of variance measures (e.g. Panel 3C and E). Are data represented as mean +/I standard deviation, s.e.m.?We wrote this in the methods but not in each legend; we apologize for the omission and have now added this to each legend.Drosophila.– Regarding setting up the problem – claiming "how nutritional information is transmuted to neural activity and behavioral changes remains elusive" is inaccurate – we know the receptors, increasingly many cells in the food intake regulatory circuit from sensory input to motor output in It was not our intention to downplay previous findings; we used nutritional information to refer to metabolic signaling, but the reviewer's point is correct that the circuitry and molecular perception of sugar are well-established and characterized in flies and thus responses to nutritional information. We have now edited this sentence to make it clearer to what we wanted to refer to.– Methods refer to a python code on Github but no github repository is specified.We apologize for the omission; we have added the python script to the supplementary file 2.– Please also make the code for analyzing the DaMID and CaTaDa available online.The protocol and code we used to analyze these data were published in . We did not generate a new analysis code.ReferencesDrosophila Development.” Open Biology 5 (12): 150234.Mariappa, Daniel, Xiaowei Zheng, Marianne Schimpl, Olawale Raimi, Andrew T. Ferenbach,H-Arno J. Müller, and Daan M. F. van Aalten. 2015. “Dual Functionality of O-GlcNAc Transferase Is Required for Bioinformatics 31 (20): 3371–73.Marshall, Owen J., and Andrea H. Brand. 2015. “Damidseq_pipeline: An Automated Pipeline for Processing DamID Sequencing Datasets.” eLife 9 (June). https://doi.org/10.7554/eLife.54530.May, Christina E., Julia Rosander, Jennifer Gottfried, Evan Dennis, and Monica Dus. 2020. “Dietary Sugar Inhibits Satiation by Decreasing the Central Processing of Sweet Taste.” Ortiz-Meoz, Rodrigo F., Jiaoyang Jiang, Michael B. Lazarus, Marina Orman, John Janetzko, Chenguang Fan, Damien Y. Duveau, Zhi-Wei Tan, Craig J. Thomas, and Suzanne Walker.ACS Chemical Biology 10 (6): 1392–97.2015. “A Small Molecule That Inhibits OGT Activity in Cells.” Science Advances 6 (46). https://doi.org/10.1126/sciadv.abc8492.Vaziri, Anoumid, Morteza Khabiri, Brendan T. Genaw, Christina E. May, Peter L. Freddolino, and Monica Dus. 2020. “Persistent Epigenetic Reprogramming of Sweet Taste by Diet.”


C. geniculata VKM F-3561 (with molecular masses about 1035 and 870 kDa) were purified and characterized for the first time. The ability of the enzymes to oxidize phenylpropanoids and phenolic compounds under neutral environmental conditions with the formation of previously unknown di-, tri-, and tetrameric products of transformation was shown. The possibility to obtain industrially valuable compounds  from caffeic acid using laccase-like oxidases of C. geniculata VKM F-3561 has been shown. Complete nucleotide sequence of the laccase gene, which is expressed at the peak of alkaliphilic laccase activity of the fungus, and its promoter region were determined. Based on the phylogenetic analysis of the nucleotide sequence, the nearest relationship of the isolated laccase gene with similar genes of fungi of the genera Alternaria, Bipolaris, and Cochliobolus was shown. Homologous model of the laccase structure was predicted and a proton channel was found, which was presumably responsible for the accumulation and transport of protons to T2/T3-copper center in the alkaliphilic laccase molecule and providing the functional activity of the enzyme in the neutral alkaline environment conditions.The unique oligomeric alkaliphilic laccase-like oxidases of the ascomycete  In recent years, the interest in a search and study of atypical fungal laccases active in a neutral or slightly alkaline medium is constantly growing, mostly because the new technologies such as cell platforms for the biosynthesis of pharmacologically and industrially valuable compounds, biosensors, biofuel cells for implantable devices, 3D nanodevices for biomedical purposes, synthesis of C-N heteropolymer dyes, and etc., usually use media with pH close to neutral ,5,6,7. HCoprinus cinereus , as, as37], Flanking regions of the laccase gene were obtained by SON-PCR . The reaC. geniculata VKM F-3561 was performed using the gene-specific primers F3561-20d-Lcc-F and F3561-20d-Lcc-R  and final elongation step . PCR amplification of the full laccase gene from the gDNA of 0d-Lcc-R . The reaThe resulting DNA fragments were purified using the Cleanup Standard kit , cloned into pAL2-T vector  according to the manufacturer’s protocol, and sequenced using a service of the Evrogen company .Nucleotide and amino acid sequences were analyzed using the NCBI Blastn and NCBI Blastp web services . The seaThe GenBank accession number for the laccase gene sequence reported in this paper is OR250480.1.https://www.rcsb.org/). The crystal structures of the laccases of Myceliophthora thermophila (RCSB PDB: 6F5K), Thielavia arenaria (RCSB PDB: 3PPS), and Melanocarpus albomyces (RCSB PDB: 1GW0) were selected as templates for homology modeling of the structure of C. geniculata VKM F-3561 laccase by the Modeller 10.1 software package −245, [M+H]+247, [M+H]+261, [M-H]−271, and [M-H]−275, as well as three trimers with [M+H]+369, [M-H]−455, and [M-H]−473 and one tetramer with [M+H]+489. 3-Methoxycatechol was transformed into two dimers (with [M+H]+317 and [M-H]−289) and one product of putative methylation of the parent compound with [M-H]−181. All oligomeric products were isolated and identified for the first time.Applying preparative TLC and mass spectrometry, 18 products of the tested compounds oxidation using laccase-like oxidases of the fungus entified . Ferulicm/z = 140) can be used in industry as precursors for the synthesis of composite materials, molecular sensors, and dendrimers, which are often used as encapsulating agents for the transfer of drug compounds for therapeutic and diagnostic purposes in medicine [m/z = 154) is one of the most powerful known antioxidants, exhibiting anti-inflammatory, antibacterial, and cardioprotective properties [It is known that dihydroxybenzyl alcohols  was about 63 kDa, which corresponds to the mass of the dominant bands of the isolated deglycosylated laccases obtained during their electrophoresis. According to the analysis of the deduced amino acid sequence using the CDD database, the structure of the expected laccase contained three sequentially arranged cupredoxin-like domains, which are typical for the most fungal laccases. In addition, four highly conserved motifs  responsible for copper binding in the active site and typical for the copper-containing laccase family were identified in the resulting sequence . Seven pC. geniculata VKM F-3561 showed the closest relationship with the nucleotide sequences of the genes that presumably encode laccases or laccase-like enzymes of fungi of the genera Cochliobolus, Bipolaris, and Alternaria  of the elements  found inAmong them, 14 HSE sites were observed: nine elements in the reverse direction 5′–3′  and five elements in the forward 3′–5′ .The studied promoter region contained 3 NIT2 GATA sites  and one site in the 3′–5′-direction (−72–67), while bidirectional NIT2 GATA sites were at a distance of 20 bp. Also in the promoter region, two bidirectional NIT2 TATCDH sites (−47–42 and −67–72) were found at a distance of 20 bp between each other. Moreover, two element pairs in areas −42–47 and −67–72 (one NIT2 GATA and one NIT2 TATCDH in each) overlapped at the same location.C. geniculata VKM F-3561 contained two PacC binding sites near the gene in two opposite directions (−54–49 and −77–82) at a distance of 23 nucleotides from each other.The promoter region of the laccase gene of In addition, one creA site  was found in the promoter region and two differently directed XRE elements in the same place of the promoter region not far from gene start (−95–100 and −99–94).MRE  and ARE (antioxidant stress response element) sites were not found in the promoter region of the investigated gene. However, the one STRE element (“stress response element”) was identified in the 5′–3′ direction at the region of −167–171 bp. Also, two ACE sites regulated by protein factor activated in the presence of monovalent Cu+ and Ag+ ions were found in the 3′–5′-direction (−368–359 and −391–382 bp) at a distance of 14 bp from each other. In addition, two differently directed ATATC sequences were located at the distances of −379–383 and −427–423 bp from the laccase gene.C. geniculata VKM F-3561 (461 bp) determined in this work is highly likely a partial sequence, which suggests an incomplete picture of all available regulatory elements of the gene.It should be noted that the promoter regions of laccase genes can include more than 1000 bp . Thus, t2+ ions on the induction of alkaliphilic laccases of the fungus C. geniculata VKM F-3561 is consistent with the absence of the corresponding MRE elements in the studied promoter region of the laccase gene. At the same time, the presence of two ACE elements in the promoter region suggests a possible regulation of transcription of the laccase gene by monovalent copper ions, which opens up the possibility for further optimization of laccase production during submerged cultivation of the fungus C. geniculata VKM F-3561.The absence of a significant effect of CuC. geniculata VKM F-3561 can be induced under conditions of nitrogen restriction, due to the presence of three NIT2-GATA sites, two of which are at a distance of 20 bp, and two NIT2-TATCDH sites at a distance of 20 bp from each other in the promoter region, which can be used in the future to optimize the yield of the target enzyme.It can also be assumed that alkaliphilic laccases of the fungus C. geniculata VKM F-3561 laccase gene to high pH, since such sensitivity, as shown for laccase gene of Fusarium oxysporum, is achieved by the presence of at least three PacC elements in the promoter region [The presence of only two PacC regulatory sites does not give full confidence in the sensitivity of the promoter region of the r region .C. geniculata VKM F-3561, what indicates that temperature stress maybe involved in the regulation of these genes.As in the majority of fungal laccase genes , 14 repeThe presence of XRE elements in the promoter region of the studied gene in the absence of induction of the alkaliphilic laccase activity of the fungus by the tested aromatic inducers may suggest either an inappropriate regulation or regulation by other untested aromatic xenobiotics. Unfortunately, there is not enough information about this regulation, and more studies are required to understand the mechanism of transcription regulation of fungal laccase genes by different xenobiotics.C. geniculata VKM F-3561 laccase homologues with a known crystal structure, which were used as templates for calculating the homological model of the studied laccase structure using the Modeller 10.1 software package.The BLAST web service and the PDB database were applied to search for the C. geniculata VKM F-3561 expressed at the peak of the fungal alkaliphilic laccase activity had a typical fungal laccase structure, consisting of three cupredoxin-like domains  and containing four copper ions in T1- and T2/T3-active sites located between domains a and c  isolated at the peak of fungal alkaliphilic laccase activity.In contrast to the alkaliphilic laccase of M F-3565 , the subtructure  possiblyThe copper ion of the T1 site in the laccase homology model was coordinated by two histidine  and one cysteine (Cys490) residues B. The cyM. roridum VKM F-3565, the bacterial laccase-like protein CotA from Bacillus subtilis, and the laccase of the basidiomycete Trametes versicolor, is represented in C. geniculata VKM F-3561 contains a leucine residue (Leu525), while the M. roridum VKM F-3565 laccase has a phenylalanine residue (Phe508). These residues are typical for fungal laccases and presumably reflect the increased redox potential of their T1-copper [The superposition of the 3D structures of the investigated laccase, the alkaliphilic laccase of 1-copper ,85.C. geniculata VKM F-3561 laccase is the reason of the increased stability of the alkaliphilic laccase-like oxidases isolated at the peak of alkaliphilic laccase activity with 100% retention of the laccase activity after 7 days of storage at room temperature.The presence of a leucine residue in the axial position from the T1-copper atom in the structure of mutant forms of bacterial laccases led to temperature stability . PossiblM. roridum VKM F-3565 laccase, the aspartate residue was located at the same position as the Asp206 residue of the laccase of Trametes versicolor , and Asn507 residues were conserved.The surface of the T2-channel of the laccase of , Tyr463 . Among tC. geniculata VKM F-3561 laccase and M. roridum VKM F-3565 laccase showed the same content of charged amino acids  positively charged both in an acidic and neutral medium, similar to the proton channels found in all alkaliphilic fungal laccases  was close to the Asp489 residue  was found as a structural element presumably participating in the transfer of protons to the T2/T3-center during the catalytic reduction of molecular oxygen.The catalytic triad of aspartate residues (Asp132/Asp118, Asp489/Asp472, and Asp277/Asp268 in the laccases of C. geniculata VKM F-3561 and characterized for the first time. The enzymes were able to catalyze the oxidation of phenylpropanoids and phenolic compounds under neutral environmental conditions with the concomitant formation of potentially pharmacologically and industrially valuable di-, tri-, and tetrameric products. We demonstrated the presence of a proton channel in the C. geniculata VKM F-3561 laccase, which is similar to the proton channel of fungal alkaliphilic laccases of a known structure, providing the functional activity of the enzymes in a neutral-alkaline medium. The preparation and characterization of recombinant alkaliphilic laccase will be the subject of further research.In the present work, the unique oligomeric alkaliphilic laccase-like oxidases with high biotechnological potential were isolated from the ascomycete


Lung adenocarcinoma is one of the most common tumors, and cisplatin is frequently used in treating lung adenocarcinoma patients. This study aimed to look into the roles and mechanisms of HNF4G in cisplatin resistance of lung adenocarcinoma.Cisplatin resistance and gene expression data of 542 cell lines from the CTRP and CCLE databases were analyzed. HNF4G expression was detected in the lung adenocarcinoma cell lines after treatment with various concentrations of cisplatin. Cisplatin sensitivity curves were detected in cells that overexpressed or knocked down HNF4G. The ChIP-Seq data were then analyzed to identify the targets of HNF4G involved in cisplatin resistance. Expression and phosphorylation of the MAPK6/Akt pathway were detected after HNF4G was overexpressed or knocked down. Finally, ChIP-qPCR and dual-luciferase assays were used to investigate the regulation of HNF4G on MAPK6.In cell lines, high expression of HNF4G was significantly positively correlated with cisplatin resistance, and lung adenocarcinoma patients who had high HNF4G expression had a poor prognosis. Cisplatin treatment increased HNF4G expression, and overexpression of HNF4G significantly increased the resistance to cisplatin in A549 and HCC827 cells, whereas knockdown of HNF4G had the opposite effect. HNF4G overexpression increased MAPK6 expression and activated the MAPK6/Akt pathway, while an Akt inhibitor reduced the effects of HNF4G on cisplatin resistance. HNF4G bound to the MAPK6 promoter region, promoting MAPK6 expression, according to ChIP-qPCR and luciferase assays.By binding to the MAPK6 promoter region, HNF4G promotes MAPK6 expression and subsequent Akt phosphorylation, resulting in resistance to cisplatin in lung adenocarcinoma. In recent years, lung cancer has become the second most frequently diagnosed cancer and the leading cause of tumor death . The mosChemotherapy is still one of the most commonly used treatments for lung adenocarcinoma, especially in patients who have advanced lung adenocarcinoma and are resistant to targeted therapy or immunotherapy . UnderstThe Chinese National Collection of Authenticated Cell Cultures  provided two human lung adenocarcinoma cell lines, A549 and HCC827, as well as human embryonic kidney cell line 293T, which were cultured in high-glucose Dulbecco’s modified Eagle’s medium  with 10% fetal bovine serum , 100 U/mL penicillin , and 0.1mg/ml streptomycin (Beyotime).Lentivirus vectors of HNF4G-Flag, sh-HNF4G-1 (5′-GCACCAGAAGAAGCACATTTG-3′), sh-HNF4G-2 (5′-GCACATTTGATGGCAGCAACA-3′), and corresponding negative control were obtained from GENECHEM , transfected into A549, HCC827, and 293T cells, and then screened by puromycin (Beyotime).Cisplatin and MK-2206 were both obtained from TOPSCIENCE .To detect the cisplatin sensitivity of cells, 4,000 cells were seeded into 96-well plates and then treated with 0, 1, 2, 4, 8, and 16 uM cisplatin. After 48 h, alamaBlue  was added, and the fluorescence was monitored at 545 nm excitation and 590 nm emission wavelengths to detect cell viability.β-actin-F: 5′-TGACGTGGACATCCGCAAAG-3′, β-actin-R: 5′-CTGGAAGGTGGACAGCGAGG-3′.As previously described , total RRIPA Lysis Buffer , Protease Inhibitor Cocktail (TOPSCIENCE), and Phosphatase Inhibitor Cocktail III (TOPSCIENCE) were used to extract proteins. SDS-PAGE was used to separate protein samples, which were then transferred to polyvinylidene fluoride membranes. HNF4G , MAPK6 , p-MAPK6 , Akt , p-Akt , GAPDH  antibodies were used to detect the specific protein bands.ChIP was performed using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads)  according to the manufacturer’s instructions. Flag-Tag Antibody  was used to pull down the HNF4G’s binding chromatin in HNF4G-Flag overexpressing cells. The sequences of primers used in ChIP-qPCR were as follows: primer_1F: 5′-GCTAGGATTACAGGCGTGAACCA-3′, primer_1R: 5′-GCTAGGATTACAGGCGTGAACCA-3′, primer_2F: 5′-ACCATCTTGCTGAGAACCAGACA-3′, primer_2R: 5′- CGGAGGAGCGGATATAACCAGGA-3′, primer_3F: 5′-TGTCGCCTTCCAGCCAATCCA-3′, primer_3R: 5′-CCCAGTGCCAGCGTTTACTAAGC-3′.NM 002748.4) and the corresponding mutated sequences on the predicted target sites of HNF4G were cloned into the firefly luciferase reporter plasmids by GENECHEM. These plasmids were then co-transfected with renilla luciferase reporter plasmids into HNF4G overexpressed or knockdown 293T cells. Dual-luciferase reporter assays were performed 48 h after transfection using the Luciferase Reporter Gene Assay Kit (Beyotime).The MAPK6 promoter sequences  and the Cancer Cell Line Encyclopedia (CCLE) website (https://sites.broadinstitute.org/ccle/) (http://gepia.cancer-pku.cn/)  (https://www.encodeproject.org) (https://jaspar.genereg.net)  website (g/ccle/) . The corpku.cn/) . Survivaalysis/) . ChIP-Seect.org)  and the reg.net)  was usedt-test and ANOVA test were performed to compare variables between groups using R software (version 4.0.3). The p values <0.05 were considered significant.Student’s When we examined the relationship between cisplatin resistance and gene expression in 542 cell lines from the CTRP and CCLE databases, we discovered that cells with high HNF4G expression were more resistant to cisplatin. As shown in p-value was not significant, which may be due to the small sample size  of MAPK6 , while pUsing qRT-PCR  and westWe used 1uM MK-2206, an Akt-specific inhibitor, to inhibit the activity of the MAPK6/Akt signaling pathway. When we inhibited Akt activity, there was no longer a significant difference in cisplatin resistance between A549 and HCC827 cells overexpressing HNF4G and controls , indicatWe obtained potential binding sites of HNF4G in the MAPK6 promoter region from the JASPAR website, and designed three pairs of primers for ChIP-qPCR to detect HNF4G binding to the MAPK6 promoter . Our ChITo conduct dual luciferase experiments, we created a firefly luciferase plasmid with the MAPK6 −1000∼200 bp region, as well as a firefly luciferase plasmid with all potential binding sites of HNF4G in this region were randomly mutated . When thThe role of HNF4G in cisplatin resistance has never been reported before, nor has the regulation of MAPK6 expression by HNF4G. Based on the findings, we first discovered that HNF4G promotes MAPK6 expression and subsequent Akt phosphorylation by binding to the MAPK6 promoter region, resulting in cisplatin resistance in lung adenocarcinoma.via the PI3K/AKT pathway by targeting GNG12 and PTK2. All of these findings indicate that the Akt signaling pathway plays an important role in HNF4G action.According to HNF4G primarily promotes tumor progression, according to numerous other studies. In pancreatic cancer, HNF4G was also found to be induced by SMAD4 deficiency, promoting progression and metastasis but being suppressed by the metformin/APMK signaling pathway . Chen etCisplatin is currently the most widely used tumor therapy drug, and it has piqued the interest of many researchers . AlthougOur research has several limitations. First, this study only used data from chemotherapy patients in a public database and did not include our own clinical data. Second, we did not validate the effects of HNF4G on cisplatin resistance in animal models due to limited experimental conditions and funds. Third, in addition to cisplatin, several other platinum-based drugs are widely used in clinical practice, including carboplatin and oxaliplatin. Whether HNF4G increases resistance to other platinum-based drugs remains to be seen. We hope these limitations can be addressed in the future.HNF4G promotes MAPK6 expression and subsequent Akt phosphorylation by binding to the MAPK6 promoter region, resulting in resistance to cisplatin in lung adenocarcinoma.10.7717/peerj.14996/supp-1Figure S1Click here for additional data file.10.7717/peerj.14996/supp-2Figure S2Click here for additional data file.10.7717/peerj.14996/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.14996/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.14996/supp-5Figure S3Click here for additional data file.10.7717/peerj.14996/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.14996/supp-7Figure S4Click here for additional data file.10.7717/peerj.14996/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.14996/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.14996/supp-10Supplemental Information 10Click here for additional data file.


The nucleosome is the fundamental subunit of chromatin. Nucleosome structures are formed by the combination of histone octamers and genomic DNA. Through a systematic and precise process of folding and compression, these structures form a 30‐nm chromatin fibre that is further organized within the nucleus in a hierarchical manner, known as the 3D genome. Understanding the intricacies of chromatin structure and the regulatory mode governing chromatin interactions is essential for unravelling the complexities of cellular architecture and function, particularly in relation to cell fate determination, regeneration, and the development of diseases. Here, we provide a general overview of the hierarchical structure of chromatin as well as of the evolution of chromatin conformation capture techniques. We also discuss the dynamic regulatory changes in higher‐order chromatin structure that occur during stem cell lineage differentiation and somatic cell reprogramming, potential regulatory insights at the chromatin level in organ regeneration, and aberrant chromatin regulation in diseases. The chromatin is folded and assembled into complex hierarchical structures. Precision control of multiple levels of 3D genome structures  is necessary for transcriptional regulation. Cell fate transitions (such as stem cell lineage differentiation and somatic cell reprogramming), organ regeneration, and disease occurrence are all linked to multidimensional chromatin regulation. The chromatin is folded and assembled into a complex hierarchical structure.In this review, we present a summary of the hierarchical organization of the 3D genome, discuss the development of 3C techniques and their derivatives in studying the 3D genome, introduce recent discoveries about higher‐order chromatin structure during stem cell lineage differentiation and somatic reprogramming, review the role of chromatin regulation in organ regeneration and diseases, and provide new insights and perspectives for future investigation of these topics.2cis‐regulatory elements, repetitive elements, and others, interact specifically in dynamic higher‐order structures rather than being arranged in linear order in the spatial organization. The ‘3D genome’, normally refers to the higher‐order chromatin structure, affects numerous biological processes, including gene expression, DNA replication, DNA damage repair, cell differentiation, and embryonic development.Functional elements in eukaryotic genomes, including coding genes, non‐coding genes, 2.1During interphase, chromatin could occupy distinct regions within the nucleus known as CTs Figure .15 It 2.2The genome is further separated into active A compartments and inactive B compartments Figure .19 The2.3Genomic compartments can be further subdivided into topologically associated domains (TADs) at the sub‐megabase scale Figure .25 TAD2.4At the kilobase scale, chromatin fibres condense into loop‐like structures known as chromatin loops Figure .32 TheChromatin loops is closely related with regulation of gene expression.33.1The spatial conformation of chromatin is one of the essential components of transcriptional regulation. Advances in 3C and 3C‐based derivative technologies over the past two decades have made it easier for us to study 3D genome organization Figure .19, 43.2The most notable improvement of the Hi‐C technique is the invention of in situ Hi‐C technology.In addition to the methods described above for capturing whole genome chromatin interactions, the techniques for capturing local chromatin interactions are being developed. Micro‐Capture‐C (MCC)3.3Simultaneously, nonadjacent‐junction approaches have been developed to avoid the inherent linkage bias of standard adjacent‐junction methods; these include genome architecture mapping (GAM)4The advances in 3D genome technology have linked the regulation of chromatin hierarchy to cell fate transitions. A number of reviews have described the contribution of the 3D genome to early embryonic development, cell lineage differentiation, and cell fate transitions.4.1Embryonic stem cells can generally differentiate into intermediate‐state progenitor cells of different lineages and thence into terminally differentiated somatic cells according to established routes.As mentioned earlier, CTCF usually acts as an insulator at the TAD boundary and prevents the interactions between the two sides of the regulatory elements. Simultaneously, CTCF also mediates loop extrusion together with the cohesin complex, which regulates the mutual interaction of distant regulatory elements. Although acute degradation of CTCF in cell lines has little effect on gene expression, prolonged degradation of CTCF leads to morphological abnormalities and cellular differentiation.The critical function of the 3D genome in deciding cell fate transition has been enriched by studies of how chromatin structure is mediated by architectural proteins during cell lineage differentiation. Further investigation is needed to allow us to understand the relationships between architectural proteins and lineage‐specific specification, how these proteins coordinate in the lineage differentiation process, and whether there are distinct regulatory paradigms in chromatin organization during the differentiation of distinct lineages.4.2Terminally differentiated cells undergo cell fate reversal through somatic cell nuclear transfer (SCNT), overexpression of TF sets, or induction by small molecule compounds.Since Takahashi and YamanakaThe interactions between long‐distance enhancers and promoters are crucial in regulating chromatin structure and gene expression. For instance, removing individual KLF4 binding sites inside pluripotent stem cell‐specific enhancers was sufficient to damage EP interactions and decrease the expression of related genes during the reprogramming of MEFs into iPSCs.5Hydra, Drosophila, zebrafish, and mice.Organ regeneration is a major goal of regenerative medicine, which includes the regeneration and repair of damaged tissues as well as the rebuilding of organs using stem cell technology. The essence of regenerative biology at the cellular level is associated with cell fate transition. Therefore, it is crucial to examine how programmes involved in regeneration are affected by changes in the chromatin environment. The regulatory mechanisms that operate at the chromatin level during regeneration events have been extensively studied using model animals such as 5.1By changing the physical spacing of the nucleosomes, ATP‐dependent chromatin remodelling complexes  make DNA more accessible to particular protein factors and thereby change gene expression programmes.5.2The studies related to 3D genome in regulating regeneration are really rare; however, epigenetic modifications are highly connected to 3D genomic regulation.5.3Hydra, Drosophila, zebrafish, and other organisms by integrating omics information on chromatin state and histone modifications.Chromatin regulatory elements  play an essential role in gene expression. With the development of multi‐omics technologies, chromatin regulatory elements associated with regeneration events have been identified in 6Recently, a number of studies have taken advantage of 3C‐derived techologies combined with high‐throughput sequencing to study the relationships between disease‐related genetic variation and chromatin conformation. Abnormal regulation of chromatin structure in diseases has become a focus of current research.6.1Genomic structural variants  and single nucleotide variants are common causes of genetically inherited developmental diseases and cancer.6.1.1WNT6‐IHH‐EPHA4‐PAX3 genes in three TADs separated by two boundary elements characterizes this locus, at which the enhancer element and the EPHA4 gene are in the same TAD. Pathogenic structural variations at these loci in humans include fragment deletions, inversions, and duplications that may cause visible finger malformation phenotypes if the variant involves the boundary element. For example, deletion of EPHA4 at the TAD boundary results in the fusion of two TADs and aberrant contact of the enhancer with the PAX3 gene, leading to misexpression of PAX3 and a brachydactyly phenotype. Inversion of the enhancer leads to abnormal activation of WNT6 and causes syndactyly, while duplication of the enhancer and the IHH sequences leads to misexpression of the IHH gene, causing polydactyly.EPHA4‐PAX3 locus contains tandem CTCF sites at the TAD boundary, which are conserved in mice. Recent study has used animal models to analyse in great detail how each CTCF at the boundary affects gene expression, enhancer insulation, and pathological traits. Studies have revealed that tandem CTCFs serve redundant roles and that insulation of the boundary can have a direct impact on gene expression and on the accompanying phenotype.IGF2 locus has been observed in colorectal cancer patients and shown to lead to new TAD formation and aberrant overexpression of IGF2.SOX9 locus involves the neighbouring KCNJ2 gene, a new TAD will be created, and the KCNJ2 gene will be in aberrant contact with the SOX9 regulatory element and misexpressed, resulting in limb deformity. The generation of aberrant chromatin structures by sequence duplication is also related to copy number and duplication area. If the duplication around the SOX9 locus does not include the KCNJ2 gene, it does not cause misexpression or a disease phenotype.Multiple developmental disorders have been linked to specific loci in chromatin. At the EPHA4‐PAX3 locus, for instance, genomic variation has been associated with several rare limb malformations. A linear distribution of 6.1.2SVs in genomic DNA have been linked to abnormal chromatin structure, leading to disease‐associated dysregulation of gene expression. However, the production of abnormal regulation of gene expression usually requires certain conditions to be met, such as changes in boundary elements and ectopic chromatin contacts. One of the critical phenomena related to ectopic contacts in chromatin is ‘enhancer hijacking’,6.2There is mounting evidence that connects the occurrence of diseases to anomalies in chromatin arrangement. Mutations in the genes that encode architectural proteins are another form of connection between structural changes in chromatin and developmental disorders Figure . AccordiUnderstanding the occurrence and progression of diseases from the standpoint of chromatin structural variation will thus be a future direction for improving disease diagnosis. On the one hand, more sophisticated detection methods and computational tools are required to make it possible to discover relevant chromatin structural alterations. On the other hand, gene editing approaches could be used to create equivalent cell or animal models to explore the possible regulatory processes. Disease simulation and mechanism studies could benefit from using human iPSC‐derived organoid technology, which has shown great promise in disease modelling and regenerative medicine in recent years.7This review summarizes the recent history of 3C and its derived techniques and describes their application and direction for revealing the role of chromatin architecture in stem cell differentiation, somatic cell re‐programming, organ regeneration, and diseases. Examination of the 3D structure of chromatin and the mechanisms that govern chromatin interactions is necessary to gain insight into how cells coordinate their most basic biological functions. Theoretically, understanding how chromatin organization changes dynamically during cell fate transition could help us to create in vitro cell treatment materials that are more effective and safer than currently available. Investigation of chromatin architecture in regeneration and diseases provides theoretical guidelines for in vitro simulation of organ regeneration and disease development with potentially clinical usefulness. However, there are several limitations of the current study: First, the lack of high‐resolution methods to capture 3D chromatin interactions at single cell type or even single cell level. Second, the scarcity of data on 3D genome organization in various cell types, developmental stages, and regeneration processes. Third, the difficulty in correlating and integrating multi‐omics data such as chromatin structure, epigenetic modifications, TF networks in the same biological event, and so forth. Therefore, the utilization of high‐resolution technologies in the future may be necessary to resolve chromatin conformation at the level of single cell types or even single cells. In addition, coordination of multi‐omics sources and functional analysis will be required to comprehensively delineate the rules of chromatin regulation in cell fate transition, regeneration events and disease process.Hongxin Zhong, Jie Zhang, and Yuli Lu wrote the first draft of this paper. Gongcheng Hu and Guangjin Pan helped to prepare for this work. Hongjie Yao supervised and approved the final version.This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16010502), National Key R&D Program of China (2021YFA1100300), and National Natural Science Foundation of China .The authors declare that they have no competing interests.