The sensitivity of human immune system cells to gravity changes has been investigated in numerous studies. Human macrophages mediate innate and thus rapid immune defense on the one hand and activate T- and B-cell-based adaptive immune response on the other hand. In this process they finally act as immunoeffector cells, and are essential for tissue regeneration and remodeling. Recently, we demonstrated in the human Jurkat T cell line that genes are differentially regulated in cluster structures under altered gravity. In order to study an in vivo near system of immunologically relevant human cells under physically real microgravity, we performed parabolic flight experiments with primary human M1 macrophages under highly standardized conditions and performed chromatin immunoprecipitation DNA sequencing (ChIP-Seq) for whole-genome epigenetic detection of the DNA-binding loci of the main transcription complex RNA polymerase II and the transcription-associated epigenetic chromatin modification H3K4me3. We identified an overall downregulation of H3K4me3 binding loci in altered gravity, which were unequally distributed inter- and intrachromosomally throughout the genome. Three-quarters of all affected loci were located on the p arm of the chromosomes chr5, chr6, chr9, and chr19. The genomic distribution of the downregulated H3K4me3 loci corresponds to a substantial extent to immunoregulatory genes. In microgravity, analysis of RNA polymerase II binding showed increased binding to multiple loci at coding sequences but decreased binding to central noncoding regions. Detection of altered DNA binding of RNA polymerase II provided direct evidence that gravity changes can lead to altered transcription. Based on this study, we hypothesize that the rapid transcriptional response to changing gravitational forces is specifically encoded in the epigenetic organization of chromatin. The immune system is one of the systems critically affected under spaceflight conditions ,2,3, andThe underlying causes how gravity effects or regulates mammalian cells are still largely unknown. However, there are considerations of a direct mechanical effect on cell shape and geometry  that is Then, we performed comparative studies of complete transcriptome analyses after different periods of altered gravity on parabolic flights and ballistic suborbital research rocket missions. Here, we found rapid and pronounced transcriptome changes in human Jurkat T cells after as little as 20s of altered gravity (microgravity as well as hypergravity), which was followed by a rapid and nearly complete adaptation response after 5 min ,15. In pHowever, our studies to date have not yet been able to show whether, in addition to the chromatin structure-specific transcript changes found, there are also changes in RNA polymerase II DNA binding or epigenetic changes. Additionally, so far, we have studied only T cell lines but not primary cells. We therefore chose the model system of isolated primary human macrophages not only because of its immunological relevance, but also to investigate whether basic structural transcriptional and chromatin-modifying effects are also detectable in other cell types.All histones can be post-translationally modified, especially at histone tails, directly regulating chromatin structure and recruiting non-histone proteins to chromatin. High levels of H3K4me3 are found at the promoter sequences of active genes . TrimethBased on these findings, we could postulate a general model of cellular gravisensing through changes in nuclear 3D structure . FurtherWhole-genome epigenetic detection of DNA-protein binding at different loci is possible using chromatin immunoprecipitation (ChIP) techniques. Since its invention in 2007, the ChIP-seq technique has rapidly become the leading technology for analyzing DNA-binding proteins and their binding sites . In contThe experimental environment of a parabolic flight also imposes operational and thus methodological–experimental constraints. Essentially, this is the physically defined maximum time of the microgravity phase due to the parabolic maneuver performed in the Earth’s atmosphere. Therefore, also here it had to be judged whether the experiments should be better performed in “simulated microgravity” under laboratory conditions or under “real microgravity” under flight conditions. While operational constraints in real microgravity flight samples limits the level of maximum standardization, more and significant methodological limitations exist for ground-based simulations : PreviouIn order to study an in vivo near system of immunologically relevant human cells under physically real microgravity, we performed parabolic flight experiments with primary human macrophages. The experiments were standardized to the highest possible degree, as far as the experimental limitations of the flight experiment and the ChIP-Seq method allowed.The aim of this study was to assess the binding patterns of the regulatory histone H3K4me3 and of RNA polymerase II in human immune cells during short phases of microgravity to test if epigenetic reactions towards hyper-/microgravity already appear after a few seconds of stimulation. Therefore, primary human cells have been exposed to 20 s of hypergravity (BL hypg) and consecutive 20 s of microgravity (μg) during the 28th DLR parabolic flight campaign (PFC), and fixed at different points in time to yield different gravity condition groups . SamplesTo analyze the binding behavior of RNA Polymerase II and histone H3K4me3, areas in the genome were identified that were enriched for reads after performing ChIP sequencing and therefore representing areas of protein–DNA interaction. These ChIP-seq peaks have been called out of raw RNA fragment reads, consensus peak sets have been defined, and consecutively, ChIP peaks with significant change in binding strength between different conditions of gravity have been identified and classified.To analyze the protein–DNA binding behavior under different gravity conditions, fixed, immunoprecipitated and processed samples (three per gravity condition per protein of interest) have been sequenced. From raw ChIP-seq reads (filtered for ENCODE blacklist regions) , bindingp values of 0.001 to 0.01 for H3K4me3 and 5 × 10−6 to 1.6 × 10−3 for RNA Pol II). For H3K4me3 1g ground control versus altered g and 1g in-flight versus altered g for RNA Pol II, no significantly altered peaks could be identified.The experimental flight hardware design did not allow an in-flight quenching procedure so that lower formaldehyde concentrations were required to avoid the risk of over-fixation. Considering potential fixation “smearing”, carryover of conditions that lie close to each other timewise, a first differential binding analysis was performed of normal gravity versus a joint altered gravity condition. Therefore, μg and BL hypg samples were merged, since BL hypg was fixed only 20 s before µg and therefore potentially “smeared” into this consecutive flight phase . A diffeRemarkably, for H3K4me3, only peaks that showed weaker binding in altered gravity could be identified. Not a single peak with increased binding affinity was detectable A. AdditiFor RNA Pol II, only 16 seemingly uncorrelated peaks could be identified as differential, with no observable patterns in terms of increased / decreased binding A. Peak cAs a negative control, the contrast 1g GC versus 1g IF was assessed for significantly differential peaks for both antibodies, which should not contain any gravity-related peaks if fixation smearing would not affect the first parabola for the 1g IF condition. No significant peaks could be detected for H3K4me3 for a FDR of <0.1 (first peaks at FDR = 1.0) and only 2 significant peaks appeared for RNA Pol II for a FDR of <0.1 (8 peaks for a FDR < 0.5), one on chr2 and one on chr8. Therefore, the assay setup demonstrated stability against experimental noise induced by random biological effects and by the flight setup, which displayed a major source of technical effects for transcriptomics studies .Since crosslinking kinetics of formaldehyde are largely unknown and likely differ for every protein–DNA interaction, a split analysis was performed, where the two gravity conditions BL hypg and μg were analyzed separately, even though they had a temporal distance of only 20 s. Depending on the crosslinking behavior, this could improve the results if the hypergravity and the microgravity samples were so different, that an averaging, as performed above, would blur out singular effects. However, this could also compromise results if the samples from the two phases would be very similar, because then statistical power would be reduced by moving from six samples to three for the altered gravity condition.p values of 10−28 to 0.01) increased by an order of magnitude for comparisons to 1g ground control (284 peaks for 1g GC vs. μg and 32 peaks for 1g GC vs. BL-hypg), additionally the comparison of 1g in-flight to μg resulted in 207 significantly different peaks, where no differential binding sites could be identified for the merged comparison. A large fraction of peaks for 1g IF vs. μg was also present in 1g GC vs. μg with 1g GC vs. μg bearing additional peaks that were unique to this comparison. This behavior was expected, since under ideal conditions 1g IF vs. μg should contain only peaks that are specific to altered gravity, 1g GC vs. μg should contain the same peaks but bear additional ones associated with effects from the overall flight  that should be cancelled out in the 1g IF vs. μg comparison. Since the crosslinking kinetics might cover slowly crosslinking loci that are related to altered gravity in the 1g IF vs. μg comparison due to crosslinking carryover, it was still worthwhile to analyze both the 1g IF and the 1g GC comparison; 1g GC vs. BL-hypg showed 32 differential peaks, most of them shared with 1g GC vs. μg. These could be hypergravity-specific or carryovers from the microgravity phase; 1g IF vs. BL-hypg did not show any differential peaks at all.For RNA Pol II, this analysis yielded a surprise A. The nuThe peaks for RNA Pol II were non-randomly distributed: sites of decreased binding strength localized close to the centromeres for most chromosomes, e.g., very prominently on chr2 or chr7. Sites with increased binding were spread out more evenly with a certain accumulation at the flanking regions of chromosomes. This observed behavior was quantified for all three comparisons B. The diRNA Pol II peak classifications C did notp values of 0.004 to 0.11 with a mean of 0.06), 2804 differential peaks emerged, among them only three peaks with increased binding strength could be identified . Considering that each of the peaks taken on its own had a 30% chance of being a false positive this would qualify the separate results as a weak finding, but taken altogether, as for the aggregated analysis, an overwhelming fraction of sites showed decreased binding. Local clusters could again be identified but were more evenly distributed among all chromosomes -based calling algorithm. Since RNA Pol II peaks have a wide distribution in size, calling such peaks as sharp and broad is both legitimate. The previous results could be confirmed for peak type distribution , differeDriven by the observation that peaks with significantly increased and decreased binding strength for RNA Pol II showed a fundamentally different distribution along chromosomes, separate analysis of such peaks was performed A,B. SuchTherefore, under microgravity, RNA Polymerase II showed increased binding to several loci that correspond to genes but decreased binding to central regions that do not correspond to genes.For the histone, additionally to lowered binding strength, the overall number of consensus binding peaks within gravity conditions was investigated C. A Vennp value cutoff of <0.01 was applied. For this dataset, out of the 10 most enriched pathways, 4 immune-associated pathways have been identified. The pathway with the highest gene ratio was “Neutrophil degranulation” with 111 genes out of 2804 total peaks downregulated in the dataset, out of the 480 genes that are part of this pathway. These pathways have been assessed for overlaps, only “Class I MHC mediated antigen processing & presentation” and “Antigen processing: Ubiquitination & Proteasome degradation” had a significant overlap of included genes. These four pathways contained 220 unique hits from the sites of interest derived from the significantly different peaks, out of the 2804 total peaks. They contained 952 unique protein-coding genes. With a Fisher’s exact test p value of 4.7 × 10−16, this displayed a highly non-random distribution. Since multiple independent immune system pathways displayed the largest group of associated pathways of H3K4me3 binding changes under microgravity and represented almost 10% of all hits, a functional association with the immune system seems likely.The large number of sites with differentially bound peaks for H3K4me3 under microgravity almost exclusively consisted of genic regions. To test if these hits were randomly distributed or followed a certain systematic, an enrichment analysis exploiting the Reactome database was performed . Since tRegarding the validity of the methods used, the experimental system was shown to be robust to random biological effects due to the flight experiment, which is typically a major source of technical confounding effects in transcriptomics studies .Due to the operational and technical limitations of the flight experiment, quenching had to be performed after landing. Therefore, an experimental approach with a relatively low formaldehyde concentration had to be chosen. Since little data are known to date about the specific timing of the biochemical crosslinking reaction in different environments, it is possible that the crosslinking processes are not completely finished at the end of the respective flight phase. Such an effect could be present in the H3K4me3 detection, as no differences could be measured between the 1g vs. BL hypg groups. In contrast, when the BL hypg and μg groups, which were only 20 s apart, were merged in comparison to the 1g group, significant differences could be detected. In the case of RNA Pol II detection, the “carryover” effect seems to have less impact, as separation into a hypergravity and a microgravity phase increased statistical significance, and clear differences between all comparisons could be observed. The complex kinetics of formaldehyde cross-linking poses an inherent experimental challenge that must therefore be considered when interpreting the results.In this study, primary human M1 macrophages from blood donors were used, which a priori suggests some biological variance. In fact, the sample differences in H3K4me3 detection were clearly due to the different donors and were still present when the cells had been cultured under reproducible standard conditions for 10 days before the start of the experiment. In contrast, the samples showed no such interindividual differences in RNA-Pol II detection, indicating clear epigenetic differences but fewer differences in RNA-Pol II-dependent transcription between individual donors.Interestingly, only downregulation of DNA-H3K4me3 binding loci could be identified in microgravity, which were chromosomally and structurally unequally distributed: Three-quarters of all affected loci were located on the four chromosomes chr5, chr6, chr9 and chr19, and there on the p arm of the chromosome. Here, the density for the relatively short chr19 was exceptionally high, with 3.21 differential peak marks per 10 million base pairs (bp), compared to an average of 0.24 across the entire genome. Most of the affected loci were localized near transcription start sites, with most of them located near both exon and intron sequences C.The near-universal chromatin modification tri-methylation of lysine 4 on histone H3 (H3K4me3) at the transcription start site of active genes in eukaryotes reflect the amount of transcription ,20,32 anRNA polymerase II is the main complex of the transcription apparatus . In micrThe detected whole-genome effects of gravity-induced changes on DNA-H3K4me3 and DNA-RNA polymerase II binding loci are in agreement with the gravitational genomic model developed on Jurkat T cells , accordiWe recently found in real-time experiments on a suborbital ballistic research rocket that the cytoskeleton of primary human M1 macrophages responded to gravity changes within seconds and re-adapted within minutes . In addiBased on this study, we hypothesize that the rapid transcriptional response to changing gravitational forces is specifically encoded in the epigenetic organization of chromatin. Whether and how this gravitational-force-responsive coding is to be understood in terms of a coordinated adaptive response to altered gravity must be revealed by further functional studies.® method C-28055, where cells were subjected to a cytokine-driven differentiation process that lasted 10 days and comprises regular medium changes of M1 Macrophage generation medium DXF. Cells of 112.5 × 106 were seeded per bottom of a standard 75 cm2 cell culture flasks, which matches the flight hardware dimensions. For the parabolic flight culture flask bottoms with the attached× M1 macrophages were put into 200 mL Nutrimix-bags  with 70 mL of medium, and subsequently integrated into custom made hard plastic containers as secondary containment. Samples were cultured and prepared for the flight in the laboratory at University of Zurich and transported to Bordeaux providing constant incubation at 36.5 °C before each flight day.Peripheral blood monocytic cell (PBMCs) were isolated from buffy coats from six different donors, provided by the blood donation Zurich service  via Ficoll density gradient centrifugation. M1-Macrophages were differentiated according to the standardized PromocellExperiments under real altered gravity were performed during the 28th DLR Parabolic Flight Campaign (PFC) onboard the Airbus A310 ZERO g (F-WNOV) starting from Bordeaux airport (BOD). Cell culture flask bottoms inside their respective containers were stored inside a 36.5 °C incubator installed inside the aircraft. Samples were flown on campaign flight day 1 (1 March 2016), flight day 2 (2 March 2016), and flight day 3 (3 March 2016).The parabolic flight included 31 consecutive parabolas, each consisting of a steady 1g flight phase, a hypergravity pullup phase of ~20 s, a microgravity phase of ~20 s and a second hypergravity phase of ~20 s. The experimental setup consisted of four different conditions, each with 6 replicate samples, one per donor number. The 1g inflight samples were fixed five minutes before the first parabola by addition of formaldehyde. The baseline hypergravity (BL hypg) samples were fixed at the end of the first hypergravity phase of the first parabola, and the microgravity sample (μg) was fixed at the end of the microgravity phase of the first parabola. Ground control samples did not board the plane but were stored under the same conditions and fixed right after the landing of the aircraft. A detailed experimental fixation scheme can be found in Formaldehyde fixation was performed by injecting 7 mL of 0.5% formaldehyde via a syringe and connecting tubes, to the cells cultivated in 70 mL cell culture medium. To avoid over-fixation, Glycine/PBS in molar excess was added as a quencher after 2 h. The concentration had to be chosen lower than typical ChIP crosslinking concentrations to avoid over-fixation since the addition of a quencher was not possible in-flight due to hardware/regulatory constraints. Feasibility of chosen concentrations were determined by ChIP pre-studies in cooperation with ActiveMotif. Due to the largely unknown crosslinking kinetics of formaldehyde, a carryover effect from the first seconds of the subsequent parabolic flight phases cannot be excluded  according to manufacturer’s protocol using 100 μL chromatin and 3 μg antibody. H3K4me3  and RNA Pol II  were selected as targets. Sequencing of DNA fragments was performed following a standard Illumina sequencer protocol. All 24 samples where sequenced on the same flow cell with a read depth of 25 million reads and 75 cycles.Sequence reads were demultiplexed by the proprietary Illumina bcl2fastq software. Raw FASTQ files where adapter-trimmed and quality-trimmed by TrimGalore  utilizinTo account for the property of RNA Pol II to display both broad and sharp peak behavior, MOSAiCS peak caller was utilized  MappabilPeaks were associated with specific genes if the peak maximum lied within 3000 bp upstream or downstream of a transcription start site (TSS). Since RNA Pol II does not behave like a TSS-acting transcription factor, a peak was also associated with a gene if its maximum lied within any exon/intron of such gene even if this was more than 3000 bp downstream of the TSS. Based on these settings, annotation was performed using the ChIPSeeker function annotatePeak .Significantly bound differential peaks were identified using the DiffBind package . Raw pea


Amongst the most important outputs of the biopharmaceutical industry are recombinant proteins, many of which are produced by integrating transgenes into the genomes of mammalian cells. However, expression is highly variable and can be unstable during prolonged culture. This is often due to epigenetic mechanisms silencing the transgenes. To combat this problem, vectors have been engineered to include ubiquitous chromatin opening elements (UCOEs) that protect against silencing. Here, we recount the evidence that UCOEs can modify chromatin environments and benefit biomanufacturing. Multiple types of gene regulatory elements have been tested for their ability to sustain transgene expression, including ubiquitous chromatin opening elements (UCOE), stabilising anti-repressor elements (STAR), Scaffold or matrix attachment regions (S/MAR) and the DNase I hypersensitive site 4 insulator of the chicken beta-globin locus control region (cHS4) 2Four fragments of DNA have been classified as UCOEs to date: three human  and SURF1/SURF2 UCOEs) and the murine Rps3 UCOE. Of these, the first to be described as a ubiquitous chromatin opening element was the human TBP/PSMB1 fragment; this contains three genes within a ∼ 50 kb window, of which two housekeeping genes, encoding TATA-binding protein (TBP) and the proteasomal subunit C5 (PSMB1), are divergently transcribed from promoters spaced ∼ 1 kb apart A. The prLike the TBP/PSMB1 UCOE, the A2UCOE also contains three genes, two of which are divergently transcribed housekeeping genes, encoding heterologous nuclear riboprotein A2/B1 (HNRPA2B1) and heterochromatin protein 1Hs-y (CBX3) B. Both tRudina and Smolke The mouse Rps3 UCOE has a single gene promoter, unlike the three human UCOEs, which all contain two divergent promoters. Divergent transcription may therefore not be a requirement for UCOE function. The Rps3 UCOE fragment is 3.2 kb in length and includes a comparatively small CpG island of 358 bp; nevertheless, it was able to provide high and homogenous eGFP expression over 28 days of continuous culture in CHO-K1 cells 3The epigenetic signature of UCOEs was first investigated at the endogenous A2UCOE locus in human peripheral blood mononuclear cells. A 2,644 bp CpG island spans the divergently transcribed promoters of HNRPA2B1 and CBX3. Bisulfite conversion confirmed a ∼ 5 kb methylation-free region that extends beyond the CpG island from ∼ 3 kb downstream of the HNRPA2B1 promoter to 1.5 kb into the CBX3 gene  [40]. NaA 1.2 kb A2UCOE fragment maintained its endogenous epigenetic characteristics at an ectopic genomic location in P19 cells The ability of A2UCOE to resist epigenetic silencing depends on the promoter it is linked to. Alongside a tissue-specific myeloid MRP8 promoter, the HNRPA2B1 region of A2UCOE became significantly methylated after 21 days in P19 cells, as did 87 % of CpG dinucleotides in the linked MRP8 promoter, similar to the endogenous MRP8 promoter An 8 kb A2UCOE fragment reduced DNA methylation of an adjacent CMV promoter in the majority of CHO-K1 clonal cell lines over 120 generations in culture To date, mechanistic research into how UCOEs negate silencing has been limited to the A2UCOE. However, a large body of ChIP-Seq datasets detailing chromatin environment has become publicly available in recent years, especially for the human genome. Such data provide an opportunity to analyse chromatin modifications surrounding endogenous UCOE loci in multiple cell lines, including the lymphoblastoid cell line GM12878 , HeLa ceAll three human UCOEs have an extensive CpG island which includes the dual promoter regions . The A2UAlthough the organisational similarities and common epigenetic signatures of the UCOEs point toward shared mechanisms of action, the defining characteristics remain uncertain. The Rps3 UCOE demonstrates that a bidirectional promoter is not essential for activity. The four UCOEs all have CpG islands, but these are found in 60–70 % of vertebrate promoters 4Biopharmaceutical manufacturing commonly involves the random integration of transgene DNA into the genome of a host cell, such as CHO. Expression from different integration sites is highly variable due to differing epigenetic environments. To control for population heterogeneity and production instability caused by varying epigenetic environments, vectors can be engineered to include gene regulatory elements, such as UCOEs The first use of a UCOE to improve recombinant protein titre was reported in 2002 4.1UCOEs’ ability to confer stable expression during long-term culture has been demonstrated in multiple industrially-relevant cell lines, including several lineages of CHO cells UCOEs have also been tested following site-specific integration into a predetermined genomic site. The Rps3 UCOE and 1.5 kb A2UCOE flanking an antibody light chain transgene stabilised its expression over six weeks of continuous culture at two distinct chromosomal locations Gene amplification methods can be valuable for producing high-expressing clones 4.2The ability of UCOEs to increase recombinant protein titre irrespective of stability has been widely reported. Inclusion of a 1.5 kb A2UCOE fragment doubled production of an anti-IL-2Ra mAb from 370 μg/L to 746 μg/L in a pool of CHO-K1 cells without significant changes in copy number Expression of adalimumab from a monocistronic pFUSE expression vector was significantly enhanced by placing the Rps3 UCOE upstream of a CMV promoter The use of UCOEs to increase recombinant protein production is not limited to full-sized mAbs. A 4 kb A2UCOE fragment upstream of a hCMV promoter increased yield of anti-CD55 and anti-C5 scFv-Fc fusion proteins in a CELLine flask system 4.3To add further complexity, the efficiency of UCOEs is highly dependent on the UCOE-promoter combination. Nair and colleagues investigated the effect of the 3.2 kb Rps3 UCOE or the 1.5 kb A2UCOE on expression of a large protein, B domain-deleted factor VIII (BDD-VIII), driven by 5 different promoters in stably transfected baby hamster kidney 21 (BHK21) cells The effect of UCOEs has also been tested in cells harbouring separate light chain and heavy chain antibody vectors; the highest titre was obtained when A2UCOE was placed only in the heavy chain vector, increasing the titre 5- to 8-fold over alternative combinations Increasing the number of UCOEs relative to the GOI can boost expression. Thus, two UCOE copies allowed higher yields of anti-TNFa than a single UCOE copy, dependent on the choice of promoter 5The past 20 years have witnessed impressive progress in the development of recombinant cell lines and biomanufacturing technologies, with far-reaching implications for the availability of pharmaceuticals. Here, we have assessed the UCOE class of DNA elements for their ability to promote enhanced and sustained transgene expression in cell lines that are commonly used for biomanufacturing. There is evidence that UCOEs encourage a favourable epigenetic environment that is depleted of DNA methylation and repressive histone marks. Incorporation of UCOEs into vectors can be advantageous, generating a more homogeneous population of expressing cells and a greater number of high-expressing clones. As such, UCOE-based gene expression systems offer an attractive strategy to reduce the time and cost associated with upstream biomanufacturing stages. The A2UCOE is the most extensively studied and epigenetic characterisation has been limited to this UCOE. By mining published ChIP-Seq datasets bioinformatically, we have found that all four known UCOEs possess similar patterns of histone modifications, suggesting shared molecular mechanisms of action , Fig. 7.Rebecca E. Sizer: Data curation, Investigation, Formal analysis, Writing – original draft. Robert J. White: Conceptualization, Supervision, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Vibrio cholerae, quorum sensing signals are transduced by phosphorelay to the transcription factor LuxO. Unphosphorylated LuxO permits expression of HapR, which alters global gene expression patterns. In this work, we have mapped the genome-wide distribution of LuxO and HapR in V. cholerae. Whilst LuxO has a small regulon, HapR targets 32 loci. Many HapR targets coincide with sites for the cAMP receptor protein (CRP) that regulates the transcriptional response to carbon starvation. This overlap, also evident in other Vibrio species, results from similarities in the DNA sequence bound by each factor. At shared sites, HapR and CRP simultaneously contact the double helix and binding is stabilised by direct interaction of the two factors. Importantly, this involves a CRP surface that usually contacts RNA polymerase to stimulate transcription. As a result, HapR can block transcription activation by CRP. Thus, by interacting at shared sites, HapR and CRP integrate information from quorum sensing and cAMP signalling to control gene expression. This likely allows V. cholerae to regulate subsets of genes during the transition between aquatic environments and the human host.Many bacteria use quorum sensing to control changes in lifestyle. The process is regulated by microbially derived “autoinducer” signalling molecules, that accumulate in the local environment. Individual cells sense autoinducer abundance, to infer population density, and alter their behaviour accordingly. In  Vibrio cholerae is a Gram-negative bacterium responsible for the human disease cholera1. Estimates suggest 3 million annual infections, of which 100 thousand are fatal2. Most disease instances are attributed to the El Tor V. cholerae biotype, which is responsible for the ongoing 7th cholera pandemic3. Globally, over 1 billion people inhabit areas of endemicity and future climatic change is likely to exacerbate the risk of illness4. The success of V. cholerae as a pathogen is underpinned by an ability to colonise both aquatic ecosystems and the human intestinal tract1. In waterways, V. cholerae prospers by forming biofilms on arthropod exoskeletons. Degradation of these chitinous surfaces ultimately liberates N-acetylglucosamine (GlcNAc) for metabolism by the microbe5. Upon ingestion by a human host, V. cholerae express genetic determinants for acid tolerance, intestinal colonisation, and virulence. Diverse transcription factors regulate the transition and respond to signals including bile6, temperature7, nucleotide second messengers9, and chitin availability5. Understanding these regulatory networks is important to determine how V. cholerae can switch between environments to cause disease outbreaks11.V. cholerae between ecological niches12. Briefly, V. cholerae produce at least 3 autoinducer (AI) signalling molecules: cholera AI-1 (CAI-1), AI-2, and 3,5-dimethylpyrazin-2-ol (DPO)13. In the environment, these compounds are detected by receptors in neighbouring cells and indicate population density. Importantly, whilst AI-2 and DPO are produced by multiple bacterial species, CAI-1 is only made by other members of the Vibrio genus14. Thus, V. cholerae can determine the crude composition of bacterial populations. In the absence of their cognate AIs, when population density is low, the receptors for CAI-I and AI-2 target the transcription factor LuxO for phosphorylation via a phosphorelay system16. When phosphorylated, LuxO upregulates the production of four small quorum regulatory RNAs (Qrrs)17. In turn, the Qrrs control expression of two global transcription factors: AphA and HapR19. Importantly, whilst AphA production is activated by Qrrs, synthesis of HapR is repressed. Hence, AphA and HapR control gene expression at low and high cell density respectively19. A simplified outline of the LuxO dependent regulatory pathway for HapR is illustrated in Quorum sensing is key for the transition of hapA, required for V. cholerae migration through intestinal mucosa, HapR is a TetR-family member that binds DNA as a homodimer via a N-terminal helix-turn-helix motif21. Many clinical isolates of pandemic V. cholerae have lost the ability to properly express HapR and this may indicate adaptation to a more pathogenic lifestyle22. In V. cholerae, HapR regulates the expression of ~100 genes to promote ‘group behaviours’ including natural competence, repression of virulence genes, and escape from the host intestinal mucosa23. In other Vibrio spp., equivalent regulons are larger. For example, LuxR in Vibrio harveyi regulates over 600 genes24. Expression of HapR can be influenced by other factors. In particular, cAMP receptor protein (CRP), a regulator that controls metabolism of alternative carbon sources, including chitin, upregulates HapR25. In this study, we used chromatin immunoprecipitation and DNA sequencing (ChIP-seq) to identify direct DNA binding targets of HapR and its upstream regulator, LuxO. We show that the degenerate DNA consensus bound by HapR frequently overlaps targets for CRP. At such sites, HapR and CRP co-operatively bind offset faces of the double helix. Strikingly, this occludes a key CRP surface required to activate transcription. This simple mechanism allows V. cholerae species to integrate quorum sensing, and cAMP signalling, in the control of gene expression.Identified as a regulator of V. cholerae has been investigated, it is not known which HapR responsive genes are directly controlled by the protein23. Similarly, the extent of the direct LuxO regulon is unknown. Hence, we sought to map the binding of LuxO and HapR across the V. cholerae genome. To facilitate this, luxO and hapR were cloned in plasmids pAMCF an pAMNF respectively. The resulting constructs, encoding LuxO-3xFLAG or 3xFLAG-HapR, were used to transform V. cholerae strain E7946. In subsequent ChIP-seq experiments, anti-FLAG antibodies were used to select fragments of the V. cholerae genome bound with either LuxO or HapR. The derived binding profiles are shown in VC1142 and VC1143. These divergent genes encode cold shock-like protein CspD, and the Clp protease adaptor protein, ClpS, respectively. Note that the LuxO binding signal at this locus is small, compared to the qrr1-4 targets, but may still be involved in transcription regulation . However, some known targets had weak or poorly reproducible binding signals  is not known32. Of the 7 promoters repressed by HapR in vivo, we observed repression in 6 cases in vitro  (lanes 7–42). Conversely, the murQP promoter (PmurQP) subject to repression by HapR in vivo, generated no transcript in vitro (lanes 1–6). Full gel images are shown in We focused our attention on new HapR target promoters where adjacent coding sequence could be used to predict encoded protein function. For these 24 targets, regulatory DNA was cloned upstream of  in vivo . ConsistmurQP operon (VC0206-VC0207) encodes functions important for recycling of peptidoglycan33. Briefly, cell wall derived N-acetylmuramic acid (MurNAc) is transported across the inner membrane, and simultaneously phosphorylated, by the phosphotransferase system dependent permease MurP. Resulting MurNAc-6P is hydrolysed by MurQ to generate N-acetylglucosamine 6-phosphate (GlcNAc-6P). Intriguingly, GlcNAc-6P can also be derived from chitin break down and this coincides with expression of HapR. Hence, we focused on understanding the role of HapR bound upstream of murPQ. The HapR ChIP-seq binding signal at the murQP locus is shown in murQP in vitro was likely because an undefined transcriptional activator is absent (murQP identified a close match to the consensus binding site for CRP (5’-TGTGA-N6-TCACA-3’). Furthermore, this sequence was located 41.5 bp upstream, of the murQP TSS (34. To measure binding of CRP to the murQP regulatory region we used electrophoretic mobility shift assays (EMSAs). Consistent with our prediction, CRP bound to the murQP regulatory DNA  and this was blocked by addition of HapR (lanes 5–8). We also repeated our prior lacZ fusion experiments, using the Δ211 PmurQP derivative, and V. cholerae E7946 lacking crp and/or hapR. The result is shown in hapR caused increased transcription from PmurQP only when CRP was present. Hence, HapR also represses CRP dependent murQP transcription in vivo.Recall that, in the absence of CRP, Pin vitro  and 3d. (in vivo . An expln assays . As expemurPQ. This suggests similar nucleic acid sequences are recognised by each factor. 9 (top) and HapR (bottom) ChIP-seq targets. The two motifs have features in common that align best when the logo centres are offset by 1 base pair. This is consistent with the arrangement of binding sites upstream of murPQ . The number of overlapping targets was reduced 7-fold. An equivalent analysis of the V. harveyi genome produced similar results  is in close proximity to positively charged HapR residue R123 . As expected, HapR bound tightly to the wild type CRP:DNA complex (lanes 5–9). Conversely, HapR had a lower affinity for DNA in complex with CRPE55A (lanes 14–18). This suggests that the E55A mutation in CRP destabilises the interaction with HapR.Co-operative DNA binding by transcription factors can result from their direct interactiondue R123 . In initng EMSAs . Whilst 38. Hence, AR3 is likely to be important for activation of PmurQP . Crucially, whilst HapR reduced transcription dependent on wild type CRP by 50-fold (compare lane 4 with lanes 6–9) only a 2-fold effect of HapR was observed with CRPE55A (compare lane 13 with lanes 15–18). In the presence of CRPE55R, HapR was even less effective (compare lane 22 with lanes 24–27).Residue E55 locates to a negatively charged surface of CRP called Activating Region 3 (AR3). This determinant aids recruitment of RNA polymerase when CRP binds close to the promoter −35 elementf PmurQP . We infehapR expression at low cell density . Cells lacking hapR, alone or in combination with luxO, had a similar growth defect in all conditions. Strikingly, the luxO mutant (high cell density locked), exhibited a growth defect only when MurNAc was the sole carbon source (compare red data points). Specifically, these cells exhibited an extended lag phase in MurNAc. This extended lag phase was not apparent when both luxO and hapR were deleted, consistent with the effect of luxO being mediated by HapR-dependent repression of murQP.Phosphorylated LuxO activates expression of the Qrr sRNAs that inhibit  density . Consequ9) and HapR  either HapR or CRP bound poorly to DNA in the absence of the other protein. However, when both factors were added together, all DNA shifted into a distinct low mobility complex. We conclude that co-operative binding of HapR and CRP to shared targets is common.In a final set of experiments, we turned our attention to other sites shared by CRP  and Protein A sepharose beads. Immunoprecipitated DNA was blunt-ended, A- tailed, and ligated to barcoded adaptors before elution and de-crosslinking. ChIP-seq libraries were then amplified by PCR and purified. Library quality was assessed using an Agilent Tapestation 4200 instrument and quantity determined by qPCR using an NEBnext library quantification kit (NEB). Libraries were sequenced as described previously46 and reads are available from ArrayExpress using accession code E-MTAB-11906. Single-end reads, from two independent ChIP-seq experiments for each strain, were mapped to the reference V. cholerae N16961 genome (chromosome I: NC_002505.1 and chromosome II: NC_002506.1) with Bowtie 247. The read depth at each position of the genome was determined for each BAM file using multibamsummary. Each binding profile was then normalised to an average genome-wide read depth of 1 read per base. Following normalisation, the average read depth per base for each pair of replicates was calculated. The resulting files were used to generate the circular plots in 48. For peak selection, the files were viewed as graphs using the Artemis genome browser49. After visually identifying an appropriate cut-off, peaks were selected using the “create features from graph” tool. Note that our cut-off was selected to identify only completely unambiguous binding peaks. Hence, weak or less reproducible binding signals, even if representing known targets, were excluded  and lysed by sonication. Inclusion bodies, recovered by centrifugation, were resuspended with 40 ml of buffer 2 (25 mM Tris-HCl pH 8.5 and 4 M urea) before the remaining solid material was again recovered and then solubilised using 40 ml of buffer 3 (25 mM Tris-HCl pH 8.5 and 6 M guanidine hydrochloride). Cleared supernatant was applied to a HisTrap HP column  equilibrated with buffer A (25 mM Tris-HCl pH 8.5 and 1 M NaCl). To elute His6-HapR, a gradient of buffer B  was used. Fractions containing His6-HapR were pooled and the protein was transferred into buffer X  by dialysis. Finally, we used Vivaspin ultrafiltration columns to reduce sample volume. The concentration of His6-HapR was then determined.We purified 40. Plasmid templates were isolated from E. coli using Qiagen Maxiprep kits. Each in vitro transcription assay contained 16 μg/ml DNA template in 40 mM Tris pH 7.9, 5 mM MgCl2, 500 μM DTT, 50 mM KCl, 100 μg/ml BSA, 200 μM ATP/GTP/CTP, 10 μM UTP and 5 μCi α-P32-UTP. Purified HapR and CRP were added at the indicated concentrations prior to the reaction start point. In experiments where CRP was used, the protein was incubated with cAMP 37 °C prior to addition. Transcription was instigated by addition of RNA polymerase holoenzyme prepared in advance by incubation of the core enzyme with a 4-fold excess of σ70 for 15 minutes at room temperature. After 10 minutes incubation at 37, reactions were stopped by the addition of an equal volume of formamide containing stop buffer. Reactions were resolved on an 8% (w/v) denaturing polyacrylamide gel, exposed on a Bio-Rad phosphor screen then visualised on a Bio-Rad Personal Molecular Imager. The quantify transcript levels, we measured the intensity of bands corresponding to RNAI and the RNA of interest using Quantity One software. After subtracting background lane intensity, we calculated the RNA of interest to RNAI ratio. The maximum ratio was set to 100 % activity with other ratios shown a percentage of this maximum. Experiments were repeated at least twice with similar results.Experiments were done using our prior approach40. Full gel images are shown in Promoter DNA fragments were excised from plasmid pSR and end-labelled with γ32-ATP using T4 PNK (NEB). EMSAs and DNase I footprints were done as previously described 20, with an arginine residue. The double helix of the QacR DNA complex is hidden in the final model.The model of the ternary DNA-CRP-HapR complex was generated in PyMOL by aligning PDB depositions 1jt0 (QacR-DNA complex) and 6pb6 (CRP-DNA complex). Alignments were done manually and guided by the relative two-fold centres of symmetry for each complex. Each structure was positioned so that their DNA base pairs overlapped and binding centres were offset by 1 base pair. The Mutagenesis function of PyMOL was used to replace QacR sidechain K107, equivalent to HapR R123Supplement 1Supplement 2Supplement 3qrr1 and VC1142 loci.Figure 1-figure supplement 1: Binding of LuxO and the a. ChIP-seq coverage plots for individual experimental replicates. Signals above or below the horizontal line correspond to reads mapping to the top or bottom strand respectively.VC1142 and VC1143.b. Sequence of the intergenic region between  The LuxO target site is shown in red. Start codons for the divergent genes VC1142 and VC1143 are in green. Transcription start sites identified by Papenfort et al32 are in uppercase with arrows indicating the direction of transcription. Underlined sequences are potential promoter −10 elements for the housekeeping RNA polymerase assuming a minimal requirement for the sequence 5'-TANNNT-3'.Supplement 4Figure 1-figure supplement 2: Example HapR binding signals.a. Binding peaks for HapR that fall above our cut-off for peak selection. The HapR ChIP-seq binding profiles are shown in green and genes are shown as blue arrows.b. Binding peaks for HapR, at known targets, that fall below our cut-off for peak selection. Binding signals for HapR are shown at known target genes. These peaks for not selected by our analysis because the signal was too weak and/or insufficiently reproducible.Supplement 5hapR promoter region in the presence and absence of CRP.Figure 4-figure supplement 1: Binding of HapR to the  Electrophoretic mobility shift assay showing migration of the hapR regulatory region with different combinations of CRP  and HapR . For incubations with both factors, the same range of HapR concentrations was used with 0.1 μM CRP.Supplement 6Vibrio harveyi.Figure 5-figure supplement 1: Global overlap of CRP and HapR binding sites in  A position weight matrix (PWM), corresponding to each DNA sequence logo shown in V. harveyi genome sequence (strain ATCC 33843) using FIMO. Distances between the identified CRP and HapR sites were calculated. Proximal sites were always overlapping and offset by one base pair (top panel). Overlap was greatly reduced when the analysis was applied to a randomised version of the same genome sequence (bottom panel).Supplement 7Figure 5-figure supplement 2: Models of the DNA-CRP and DNA-CRP-HapR complexes. The models were generated using PDB submissions 6pb6 (E. coli CRP in complex with a class II CRP dependent promoter) and 1jt0 (S. aureus QacR bound to its DNA target). The DNA is shown in grey and positions hypersensitive to DNAse I cleave, in the context of the DNA-CRP complex, are highlighted red  or HapR and derivatives .Supplement 9Figure 5-figure supplement 4: Co-operative DNA binding of HapR and CRP is common. Electrophoretic mobility shift assays showing migration of the indicated regulatory regions with different combinations of CRP (1 μM) and HapR (0.19 μM). For VCA0691 the concentration of HapR was 0.57 μM.Supplement 10Figure 2b source data 1: Gel image TIFF fileSupplement 11Figure 2b source data 2: Gel image TIFF fileSupplement 12Figure 2b source data 3: Gel image TIFF fileSupplement 13Figure 2b source data 4: Gel image TIFF fileSupplement 14Figure 2b source data 5: Gel image TIFF fileSupplement 15Figure 2b source data 6: Gel image TIFF fileSupplement 16Figure 3c source data 1: Gel image TIFF fileSupplement 17Figure 3c source data 2: Gel image TIFF fileSupplement 18Figure 3d source data: Gel image TIFF fileSupplement 19Figure 4a source data: Gel image TIFF fileSupplement 20Figure 4b source data: Gel image TIFF fileSupplement 21Figure 4c source data 1: Gel image TIFF fileSupplement 22Figure 4c source data 2: Gel image TIFF fileSupplement 23Figure 4d source data: Gel image TIFF fileSupplement 24Figure 5c source data: Gel image TIFF fileSupplement 25Figure 5e source data: Gel image TIFF fileSupplement 26Figure 5-figure supplement 3 source data 1: Gel image TIFF fileSupplement 27Figure 5-figure supplement 3 source data 2: Gel image TIFF fileSupplement 28Figure 5-figure supplement 4 source data 1: Gel image TIFF fileSupplement 29Figure 5-figure supplement 4 source data 2: Gel image TIFF fileSupplement 30Figure 5-figure supplement 4 source data 3: Gel image TIFF fileSupplement 31Figure 5-figure supplement 4 source data 4: Gel image TIFF file


DNA is stored in the nucleus of a cell in a folded state; however, only the necessary genetic information is extracted from the required group of genes. The key to extracting genetic information is chromatin ambivalence. Depending on the chromosomal region, chromatin is characterized into low-density “euchromatin” and high-density “heterochromatin”, with various factors being involved in its regulation. Here, we focus on chromatin regulation and gene expression by the yeast FACT complex, which functions in both euchromatin and heterochromatin. FACT is known as a histone H2A/H2B chaperone and was initially reported as an elongation factor associated with RNA polymerase II. In budding yeast, FACT activates promoter chromatin by interacting with the transcriptional activators SBF/MBF via the regulation of G1/S cell cycle genes. In fission yeast, FACT plays an important role in the formation of higher-order chromatin structures and transcriptional repression by binding to Swi6, an HP1 family protein, at heterochromatin. This FACT property, which refers to the alternate chromatin-regulation depending on the binding partner, is an interesting phenomenon. Further analysis of nucleosome regulation within heterochromatin is expected in future studies. Gene expression requires the binding of transcriptional activators that recognize and bind specific upstream activating sequences of DNA on the promoter of each gene or enhancer of distal sites ,2,3. In The FACT complex, a heterodimer of Spt16 and SSRP1, was isolated as an RNA polymerase II transcriptional elongation factor required for efficient chromatin transcription ,34,35,36CDC68, a gene responsible for causing G1 arrest [NHP6 gene and the fission yeast nhp6+ gene are different: there are two copies of NHP6 in budding yeast, NHP6A and NHP6B [nhp6+ in fission yeast [SPT16 and POB3 are essential genes, whereas NHP6A/B are nonessential genes in budding yeast [spt16+ is an essential gene, whereas pob3+ and nhp6+ are nonessential genes [pob3+ and nhp6+, which are functional bipartite analogues of SSRP1 in multicellular organisms, exceptionally through eukaryotic species. Considering that the function of Spt16 is required even in the absence of Pob3 and Nhp6, it is likely that it exerts a certain chromatin regulatory role, which is specific to Spt16 in fission yeast. In suggesting a role for each FACT component, human SSRP1 has Spt16-dependent and independent roles in transcriptional regulation [NHP6A and NHP6B, and strains in which both NHP6A and NHP6B are simultaneously disrupted showed growth defects. Under this condition, the expression of the POB3-NHP6 fusion gene was found to complement the growth defect shown by the nhp6ab∆ strain [C-terminus of Pob3 supported FACT activity without free Nhp6. However, this FACT variant was not efficiently released from nucleosomes, in turn exhibiting toxicity in yeast [In yeast, it consists of an Spt16/Pob3 heterodimer and the high-mobility group box (HMGB) protein Nhp6 ,43,44,451 arrest . Pob3 an1 arrest ,48. The nd NHP6B , whereason yeast . Second,al genes , allowingulation ,53. In bgulation . This mi∆ strain . Moreovein yeast . Recent in yeast . Phosphoin yeast ,58, suggin yeast ,60. FACTin yeast ,62,63. Ain yeast . These rin yeast .Various molecular models have been proposed to explain the means by which FACT transforms the nucleosome ,64,65, bAfter binding of the Spt16/Pob3 heterodimer to the mononucleosome, the acidic tail at the C-terminus of the two proteins competes with the nucleosomal DNA on the surface of histone H2A/H2B. Following this competition, Nhp6 binds to the fluctuated DNA and promotes the divergence of histone H2A/H2B from DNA in the nucleosome, with the tandem PH domain of Spt16 depositing the histone H2A/H2B dimer from the octasome and transforming it to a hexasome or tetrasome . StudiesCLN1 and CLN2 genes [HO) gene promoter is regulated by SFB and FACT  HDAC [HO gene, thereby strongly repressing transcription [HO needs to be strictly regulated in the mother cell. The promoter structure of the HO gene is relatively long and complex compared with that of common yeast genes, and consists of two sequential regions, URS1 and URS2  HDAC ,89 that cription ,92,93,94and URS2 B. The co element . The SAG element ,75,95,96HO promoter and the dynamic changes in chromatin along cell cycle progression is shown in HO gene by RNA polymerase II  at its cleosome ,117. Swicleosome ,119. Hetcleosome ,121, whicleosome ,123,124,cleosome ,126,127.pob3+ (pob3∆) had comparable levels of histone H3K9 methylation and Swi6 localization in the heterochromatic region to those of the wildtype strain, but with high levels of heterochromatic expression of ncRNAs. Phenotypic analysis of the pob3∆ strain indicated that heterochromatic silencing was defective in heterochromatin without a significant loss of levels of histone H3K9 methylation and HP1/Swi6-binding [pob3∆ strain was decreased to half that of the wildtype, suggesting the existence of a Pob3-independent recruitment mechanism of Spt16 onto heterochromatin. Genetic analysis also revealed that pob3∆swi6∆ double disruption exhibited an additive silencing defect compared with that shown in each of the pob3∆ and swi6∆ single-mutant strains [Genetic analysis and ChIP-qPCR showed that fission yeast strains lacking -binding ,128. ChI strains . Therefo strains ,120,129. strains ,131,132,Drosophila melanogaster [pob3∆ strain. Different from the pob3∆ strain, a major decrease in the levels of histone H3K9 methylation and Swi6-binding was observed [To elucidate this binding mode between Spt16 and Swi6, we carefully compared the primary sequences of Swi6 and Chp2 in the CSD and found a difference in the β1–β2 connecting loop, which forms the protrusion in the CSD homodimer . We idennogaster , but phynogaster . Interesobserved . This inOur analysis revealed the molecular mechanism by which FACT is recruited onto heterochromatin for the dynamic regulation of the H2A/H2B dimer and optimal management of nucleosomes in heterochromatin . The H2Apob3∆ strain indicated that reposition of the H2A/H2B dimer was dependent on the recognition of the stable H3/H4 dimer in nucleosomes by the tandem PH domain of Pob3 [Within the heterochromatin, two nucleosomes are bridged by the HP1/Swi6 heterodimer via histone H3K9me A. Struct of Pob3 .FACT is an important transcription stimulating factor that transiently relaxes the chromatin structure through its histone H2A/H2B chaperone activity. The transcription-activating properties of FACT are especially demonstrated in START, the checkpoint of the G1/S phase, through binding to SBF/MBF in budding yeast ,75,76. H


Arabidopsis. Using fluorescence and immunoelectron microscopy, we found that GBPL3 localizes to the nuclear rim and is enriched in the nuclear pore. Proximity labeling proteomics and protein-protein interaction assays revealed that GBPL3 is predominantly distributed at the NPC basket, where it physically associates with NB nucleoporins and recruits chromatin remodelers, transcription apparatus and regulators, and the RNA splicing and processing machinery, suggesting a conserved function of the NB in transcription regulation as reported in yeasts and animals. Moreover, we found that GBPL3 physically interacts with the nucleoskeleton via disordered coiled-coil regions. Simultaneous loss of GBPL3 and one of the 4 Arabidopsis nucleoskeleton genes CRWNs led to distinct development- and stress-related phenotypes, ranging from seedling lethality to lesion development, and aberrant transcription of stress-related genes. Our results indicate that GBPL3 is a bona fide component of the plant NPC and physically and functionally connects the NB with the nucleoskeleton, which is required for the coordination of gene expression during plant development and stress responses.The nuclear basket (NB) is an essential structure of the nuclear pore complex (NPC) and serves as a dynamic and multifunctional platform that participates in various critical nuclear processes, including cargo transport, molecular docking, and gene expression regulation. However, the underlying molecular mechanisms are not completely understood, particularly in plants. Here, we identified a guanylate-binding protein (GBP)-like GTPase (GBPL3) as a novel NPC basket component in  The nuclear basket is an essential structure of the nuclear pore complex and serves as a dynamic and multi-functional platform. This study reveals the guanylate-binding protein GBPL3 as a critical factor that joins the nuclear pore complex and the underlying nucleoskeleton, potentially creating a molecular hub for gene expression regulation during plant development and stress responses. The nuclear pore complex (NPC) is a structurally conserved multiprotein assembly embedded within the nuclear envelope and forms the main gateway to allow the nucleocytoplasmic exchange of macromolecules. The NPC is constructed by 500 to 1,000 nucleoporin proteins (Nups) of approximately 40 different types in multiple copies , which acis-regulatory elements and topologically associating domains [TPR and Mlp1/2p lead to nuclear retention of mis-spliced or aberrant mRNAs [Among the principal NPC modules, the NB protrudes into the nucleoplasm and establishes intimate connections with inner nuclear peripheral structures. Accumulating evidence supports that the NB is a multifunctional platform that plays versatile roles beyond mediating cargo transport . For exa domains . These a domains –29. Suppnt mRNAs –32.Besides interacting with the chromatin, the NB is also tightly associated with the nucleoskeleton distributed underneath the nuclear envelope. Cryo-electron tomography and super-resolution imaging analyses revealed that the NPC basket makes close contact with lamin filaments in animals –35. TPR Arabidopsis TPR homolog, NUA, has been shown essential for the total mRNA export. Compromised NUA function led to pleiotropic developmental phenotypes including early flowering, phyllotaxy defects, and reduced fertility [Arabidopsis FG repeats-containing nucleoporin Nup136 is likely a functional homolog of vertebrate Nup153 and has been reported to be involved in total mRNA export as well as microRNA biogenesis through its direct interaction with the TREX-2 complex [Arabidopsis [Arabidopsis [Compared to animals and yeasts, the function of NB in plants is far less well understood. The ertility ,39. The  complex ,41. Nup8bidopsis . Nup136 bidopsis . This figbpl3 mutants display stunted growth and sterility. More importantly, the combination of different nucleoskeleton mutants with gbpl3 mutant resulted in distinct development- and stress-related phenotypes and aberrant transcription of stress-related genes, suggesting a role of GBPL3 in connection with different nucleoskeleton components to differentially contribute to gene expression regulation at the NPC basket.In this study, we describe GBPL3, a guanylate-binding protein (GBP), as an NB-associated protein in plants. GBPL3 was highly enriched at the NE and probed by multiple NPC basket and nucleoskeleton components in our previous proteomics profiling. Consistently, we showed that GBPL3 confers physical interaction with components of both the NB and the nucleoskeleton. Fluorescence imaging coupled with immunogold-labeling electron microscopy revealed that GBPL3 is predominantly located at the NPC. Proximity labeling proteomics showed that GBPL3 not only interacts with NB and nucleoskeleton proteins but also is associated with a large number of chromatin remodelers, transcription regulators, and components of RNA splicing and processing machinery, potentially facilitating transcription regulation at the NB. The Arabidopsis [p-value = 1.2 × 10−9) with a high peptide-spectrum match (PSM) score -associated proteome in bidopsis ,45. AmonM) score , suggestM) score . In contGBPL3 with genes that encode the nucleoskeleton and nucleoporins in Arabidopsis using ATTED-II [GBPL3 is strongly coexpressed and clustered into a small group with 4 nucleoskeleton genes CRWN1/2/3/4 and the NB gene NUA   and nucleoskeleton protein (AtCRWN1/HsLamin_A)  in bidopsis . Besidesbidopsis  and S1B.asy.org) , and in Lamin_A) , suggestArabidopsis stable transgenic lines expressing mEGFP-GBPL3 fusion protein. Although the construct was driven by the GBPL3 native promoter, we were able to detect a difference in the mEGFP-GBPL3 expression level in independent transgenic lines based on immunoblots , we conclude that GBPL3 is functionally localized to the nuclear periphery but forming large condensates is not essential for GBPL3 function, at least under normal conditions. However, we cannot exclude that LLPS of GBPL3 may still occur at a level beyond detection by fluorescence microscope in low expression lines.To validate this hypothesis, we first verified the subcellular localization of GBPL3. We generated unoblots , potentiunoblots . Howeverunoblots , in agrespecific . BecausemEGFP-GBPL3 transgenic lines with low expression. In nearly all samples examined, we found that gold particles are predominantly associated with the inner nuclear membrane, close to nuclear envelope openings indicative of nuclear pores   . For pro3 itself . Samplesectively . We mergArabidopsis . Remarkably, GBPL3 also specifically probed all reported NB components  but barely labeled other NPC constituents  analysis revealed that GBPL3 proxitome is highly enriched in proteins that function in chromatin modification and organization, transcription initiation and regulation, and RNA splicing and processing , which aARABIDOPSIS RESPONSE REGULATOR 1 (ARR1), ABA-RESPONSIVE KINASE SUBSTRATE 2 (AKS2), C-TERMINAL DOMAIN PHOSPHATASE-LIKE 1 (CPL1), and TOPLESS-RELATED 1 (TPR1), and mRNA binding or processing proteins F OLIGOURIDYLATE BINDING PROTEIN 1C (UBP1C), SUPPRESSORS OF MEC-8 AND UNC-52 1 (SMU1), and SNW/SKI-INTERACTING PROTEIN (SKIP). We fused these proteins with mEGFP and transiently expressed them in N. benthamiana. When expressed alone, 6 of the fusion proteins formed spontaneous nuclear condensates while the other 5 diffused in the nucleus , and SALK_139144 (gbpl3-4). However, none of the 3 alleles exhibited observable developmental defects, nor did they show decreased GPBL3 expression measured by RT-qPCR using the whole seedling or young leaf tissue  that target the coding region of the very N-terminus of GBPL3, its GBP domain, and its CC domain, respectively  at 14 aa and 696 aa, respectively. The gbpl3-7 allele contains a 19-bp deletion that results in a premature stop codon at 116 aa. Lastly, Cas9 caused a 12-nucleotide in-frame deletion at 600 bp downstream from the start codon in the gbpl3-9 mutant, which led to a 4 amino acid deletion in the GBP domain  in 1-week-old gbpl3-7 seedlings compared to WT  in the gbpl3 mutant overlapped considerably with GO terms enriched in the nucleoskeleton mutant crwn1 crwn2  did not show observable growth phenotype, we found that nup82-2 dramatically enhances the stunted growth of gbpl3-7, whereas nup136-3 has a very limited effect on the phenotype , gbpl3-3 (SALK_016366), nup136-3 (SALK_076476), nup82-2 (SALK_024526), were obtained from Arabidopsis Biological Resource Center (ABRC). The gbpl3-6, gbpl3-7, gbpl3-8, and gbpl3-9 mutants were generated by CRISPR/Cas9-mediated gene editing. The crwn1, crwn2, crwn3, crwn4, crwn1 crwn4, and crwn2 crwn3 mutant seeds were kindly provided by Dr. Eric Richard at Boyce Thompson Institute. All double or triple mutants of gbpl3-7 with nup82/136 or crwns were obtained by genetic crossing and confirmed by PCR-based genotyping. pGBPL3: GBPL3-TurboID-3HA transgenic lines are in WT background and pGBPL3: mEGFP-GBPL3 and pGBPL3: mCherrry-GBPL3 transgenic lines are in gbpl3-7 mutant background. Four pGBPL3: mEGFP-GBPL3 lines with lower expression were identified, and 3 lines with higher expression were identified. All transgenic plants were generated by floral dip transformation using Agrobacterium tumefaciens GV3101 and selected using Basta herbicide by spraying. All primers used for genotyping were listed in Arabidopsis and Nicotiana Benthamiana plants were grown under a 16 h light/8 h dark-light cycle at 22°C.All mCherry, mEGFP, n/c-YFP, and TurboID-3HA were cloned into entry vector pBSDONOR p4r-2 or pBSDONOR p1-4, and cDNA fragments of Nup136, Nup82 or genomic DNA fragments of GBPL3, CRWN1, CRWN3 were cloned into entry vector pBSDONOR p1-4 or pBSDONOR p4r-2 using BP reaction (BP clonase II). The native promoter of GBPL3  was amplified and cloned into empty vector pEG100 by In-fusion cloning . All fusion constructs were generated by combining 1 pBSDONOR p1-4 construct, 1 pBSDONOR p4r-2 construct, and an original or modified pEG100 destination vector by LR reaction (LR clonase II). To generate the construct for histochemical staining, the cDNA fragment of GUS was amplified and inserted into entry vector pBSDONOR p1-2 and further cloned into modified pEG100 containing the native promoter of GPBL3 using LR reaction. To generate constructs for components of the GBPL3 proxitome, cDNA fragments of ARP4, APRF1, PWO1, TAF5, ARR1, AKS2, CPL1, TPR1, UBP1C, and SMU1 were cloned into a modified pEG100 vector containing GFP by In-fusion cloning. For PWO1, genomic DNA fragment was cloned into pBSDONOR p1-4, which was subsequently combined with pBSDONOR p4r-2-mEGFP into pEG100 using LR reaction. All binary expression constructs are under 35S promoter except GBPL3, which is driven by its native promoter. To generate constructs for yeast-two hybrid assay, cDNA fragments of GBPL3-C (GBPL3 without the GTPase domain), Nup136, Nup82, CRWN1, CRWN2, CRWN3 were subcloned into pGBKT7 or pGADT7 by In-fusion cloning. CRISPR/Cas9 constructs for gene editing were described previously [GBPL3 were created using the online webserver CRISPOR (http://crispor.tefor.net/). sgRNA scaffold was then amplified using pCBC-DT1T2 vector as the template and inserted into an egg cell promoter-driven pHEE401 binary vector by golden gate assembly. All primers used for cloning were listed in Gateway (Thermo Fisher) and In-fusion (Vazyme) technology were used for cloning unless otherwise specified. To generate constructs for protein localization, coexpression, BiFC, complementation, proximity labeling assay, eviously . BrieflyArabidopsis plants, 5-day-old pGBPL3-mEGFP-GBPL3 transgenic seedlings with low or high expression were used. Coexpression and BiFC assay were performed using agrobacterium-mediated transient protein expression in N. benthamiana as previously described [N. benthamiana plants, and images were taken in leaf epidermal cells 2 days past infiltration. Fluorescence images were obtained using a Zeiss LSM880 inverted confocal microscope with the GaAsP detector.To image escribed . AgrobacArabidopsis seedlings stably expressing mEGFP-GBPL3 were dissected and cryofixed by an HPM100 (Leica Microsystems). The frozen specimens were freeze-substituted at −80°C and embedded in HM20 resin (Electron Microscopy Sciences) at −45°C. After polymerization at −45°C, samples were washed with acetone and embedded in Lowicryl HM20 resin with an increasing gradient of 33%, 66%, and 100% HM20 concentration. After UV polymerization at −45°C, the root tips were sectioned and immunolabeled with a GFP antibody (Santa Cruz) as described previously [Arabidopsis seedlings expressing mGFP-GBPL3 and wild-type seedlings were counted. A total of 40 images were taken for each individual root, and gold particles located on the nuclear membrane , nucleoplasm, and the cytoplasm were counted.Root tips from eviously . Sample GBPL3 with known nucleoporin and nucleoskeleton genes, the averaged logic scores based on both microarray and RNA-seq data were retrieved from ATTED-II version 11.0 (https://atted.jp/) and visualized in a clustering heatmap using R. Protein domain prediction was performed in SMART (http://smart.embl-heidelberg.de/). Nuclear localization signals were predicted by cNLS Mapper (http://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) with a score of more than 5.0. Intrinsically disordered regions (IDRs) were predicted by the Database of Disordered Protein Predictions (D2P2) (https://d2p2.pro/) and Predictor of Natural Disordered Regions (PONDR) (http://www.pondr.com/).Proteomic data of GBPL3 were retrieved from our previous subtractive proteomics and proximity labeling proteomics using SUN1, MAN1, PNET2_A, PNET2_B, KAKU4, CRWN1, Nup82, SINE1, and WIP1 as baits ,45 . The sequence alignment was carried out using ClustalW2, and the phylogenetic tree was built by MEGA-X (https://www.megasoftware.net/) using the maximum likelihood method with 1,000 bootstrap replicates. All sequences of GBPL3 homologs are provided in Phylogenetic analysis of GBPL3 was performed as previously described . BrieflypGBPL3-GUS were used. All fresh tissues were incubated in the darkness overnight at 37°C following vacuum infiltrated in the staining buffer (100 mM sodium phosphate buffer (pH 7.0), 10 mM EDTA, 0.1% Triton X-100, 2 mM potassium ferrocyanide, 2 mM potassium ferricyanide, 1 mM X-gluc (5-bromo-4-chloro-3-indolyl glucuronide)) for 1 h. The stained seedlings or tissues were washed with 70% ethanol to remove chlorophyll before being imaged using Zeiss Lumar Stereoscope.To examine the expression pattern, GUS staining was performed as described previously . Entire Yeast two-hybrid analysis was performed using the Matchmaker GAL4-based Two-Hybrid System 2 (Clontech) according to the manufacturer’s instructions. The activation domain fusion (prey) constructs and the DNA-binding domain fusion (bait) constructs were transformed into yeast strains Y187 and AH109, respectively. The yeast cells of Y187 and AH109 expressing corresponding proteins were mated in 2 × YPDA medium at 30°C for 24 h before plated and selected on double (SD-Leu-Trp) and quadruple (SD-Leu-Trp-Ade-His) dropout media at 30°C for 3 to 5 days.Total protein was extracted from transgenic seedlings using protein extraction buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 0.5% Nonidet P-40, 0.5% Nadeoxycholate, plant protease inhibitor cocktail, 1 mM PMSF, and 40 μm MG132). Protein extracts were separated by SDS-PAGE, followed by immunoblot analysis using an anti-GFP antibody , anti-HA antibody , or streptavidin-HRP .pGBPL3-GBPL3-TurboID-3HA transgenic seedlings and wild-type non-transformants were treated with 50 μm biotin for 4 h at room temperature. For each sample, 0.4 g of treated seedlings were harvested and frozen directly in liquid nitrogen. Two biological replicates were used for each sample. The material was grounded into fine powder, and total protein was extracted with protein extraction buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 0.5% Nonidet P-40, 0.5% Nadeoxycholate, protease inhibitor cocktail, and 40 μm MG132). Protein extraction was subjected to 2 tandem PD-10 desalting columns  to deplete free biotin. The following flow-through was collected for affinity purification. The eluted protein fraction was mixed with 50 μL pre-washed streptavidin-coated magnetic beads . The sample was incubated on a rotor wheel overnight at 4°C, and the beads were separated on a magnetic rack and washed 5 times with the protein extraction buffer.The TurboID-based proximity labeling and affinity purification of biotinylated protein have been described previously with modifications . Brieflyp < 0.01, and normalized PSM > 4. Volcano plot and heatmap were generated by R. To construct the protein–protein interaction networks of GBPL3, interactions were retrieved from the STRING database  (https://string-db.org/) and visualized by Cytoscape (version 3.9.0).For on-beads tryptic digestion, the streptavidin beads were washed with PBS buffer 3 times and incubated with 1 μg trypsin in 100 μL 50 mM triethylammonium bicarbonate buffer (TEAB) solution overnight at room temperature with gentle shaking. The resulting digests were separated from the beads on a magnetic rack and dried down by Speedvac. The peptides were dissolved with 10 μL 0.1% trifluoroacetic acid (TFA) and desalted using 10 μL C18 desalting ZipTips according to the manufacturer’s instructions. The purified peptides were then dried down and re-dissolved with 20 μL formic acid (FA) before being subjected to LC-MS/MS analysis. MS/MS spectra were searched against TAIR 11 database using Scaffold 5 and MSFragger 3.2 software with default criteria to harvest PSM and LFQ intensities, respectively. To analyze proximity labeling data, both PSM and LFQ data were used as input, and DESeq2 and DEP packages in R were applied to enrich GBPL3 proximal candidates with cutoffs: fold-change > 4, ACTIN2 was used as a reference gene, and 2 to 3 biological replicates were used for each sample. All qPCR primers can be found in RNA was extracted using TRIzol reagent  from about 0.1 g of 5-day-old seedlings. The first-strand cDNA was synthesized using the Maxima First Strand cDNA Synthesis with dsDNase Kit . SYBR Green PCR Master Mix  was used for qPCR reaction, which was performed on a CFX96TM Real-Time PCR Detection System (Bio-Rad). gbpl3-7 seedlings were collected. Total RNA was extracted using the Direct-zol RNA Miniprep kit  with a DNase treatment, following the manufacturer’s instructions. Library construction, quality control, and RNA sequencing were performed by Novogene (https://en.novogene.com/) on an Illumina HiSeq 2000. Each sequencing run results in 38 to 50 million raw reads per sample. Sequencing read counts were quality checked and trimmed to get rid of adaptor contaminations and low-quality reads. Cleaned reads were aligned to the reference genome of Arabidopsis (TAIR10) using Hisat2 (v2.1.0). SAM files were sorted and converted to BAM files using SAMtools (v1.14). Read counts of each gene were generated using HTseq-count with default parameters. DESeq2 package (v3.14) was utilized to analyze the difference in gene expression. Differentially expressed genes (DEGs) were determined with criteria of p_value < 0.05 and fold change > 2. Gene ontology annotation of DEGs was conducted using agriGO v2.0 (http://systemsbiology.cau.edu.cn/agriGOv2/). The volcano plot and bubble plot were generated using R package ggplot2 (v3.3.5).For RNA-seq, 1-week-old media-grown Col-0 and S1 Figt test was performed using transformed and averaged PSM values. (B) Phylogenetic analysis of GBPL3 and its homologs from various plant and animal species. The tree and bootstrap values on each branch were generated by MEGA-X using the maximum likelihood method with 100 bootstraps. Sequences used for the phylogenetic analysis can be found in https://swissmodel.expasy.org). (D) Immunoblots with an anti-GFP antibody using total protein extract from wild-type non-transformants and 2 pGBPL3-mEGFP-GBPL3 transgenic seedlings expressing representative higher and lower levels of GBPL3. Ten 1-week-old T3 seedlings were pooled for protein extraction. (E) Fluorescence imaging showing subcellular localization of GBPL3 protein in root cells. One-week-old pGBPL3- mCherry-GBPL3 transgenic plants with higher expression (upper panel) and lower expression (lower panel) levels are used for imaging. Bars = 10 μm. (F) Quantification of GBPL3-GFP immunogold labeling in the low expression line (#6) and WT non-transformant samples. The relative gold density at the nuclear membrane, the nucleoplasm, and the cytoplasm is shown (left). The percentage of gold particles around the nuclear membrane was further quantified in 3 regions: the NPC, the INM, and the ONM (right). For both WT and the GBPL3-GFP line, 3 root samples with 20 cells and approximately 300 gold particles from each were used for the quantification. All underlying data in (A) GBPL3 is significantly enriched in the NE fraction compared to the total MM fraction. Data were retrieved from previously published subtractive proteomics data (PXD015919). Student (PDF)Click here for additional data file.S2 FigArabidopsis plants harboring the pGBPL3:GUS construct. A 5-day-old seedling, the fifth and 10th rosette leaf of a 4-week-old plant, stem, and silique are shown on the upper panel from left to right, and flower and anther tissues are shown on the lower panel. Bars = 1 cm. (B) Immunoblots with HRP-conjugated streptavidin and anti-HA antibody. Wild-type and transgenic seedlings expressing GBPL3-TurboID-3HA were treated with 50 μm free biotin for 4 h before total protein extraction (Input). The total protein was then AP with streptavidin-coated beads to enrich biotinylated protein. Two biological replicates were shown. The asterisks represent naturally biotinylated proteins. AP, affinity purified.(A) GUS staining of transgenic (PDF)Click here for additional data file.S3 Fig2P2 (https://d2p2.pro/) and by PONDR (http://www.pondr.com) are shown in the box and schematic diagram, respectively, for each protein. IDR, intrinsically disordered region; PSM, peptide-spectrum match.(A) Heatmap showing normalized and averaged PSM values of transcription-related and mRNA processing-related GBPL3 proximal proteins from proximity labeling proteomics using GBPL3 and Nup82 as bait. Underlying data can be found in (PDF)Click here for additional data file.S4 Figgbpl3 T-DNA lines. T-DNA insertions in SAIL_635_G05 and gbpl3-3 were located at the 5′ UTR of GBPL3 gene, and gbpl3-4 mutant contains a T-DNA insertion at the 3′ UTR. Arrows indicate qPCR primers. (B) Three-week-old soil-grown WT and gbpl3 T-DNA insertion mutant  plants. (C) Relative expression level of GBPL3 in WT and the 3 gbpl3 T-DNA insertion mutant lines. RT-qPCR was performed using both 5-day-old whole seedlings and the 10th leaves from 4-week-old plants. Data were presented as means ± SD . Actin was used as the reference gene. Student t test was performed using WT as control. **p-value < 0.01; ns stands for not significant. (D) PAM sequences of gRNAs and sequencing results showing CRISPR/Cas9-directed editing in gbpl3 mutant lines. (E) Complementation of the gbpl3-7 mutant by expressing native promoter-driven mEGFP-GBPL3 or mCherry-GBPL3 (T3 generation). One-week-old seedlings are shown. Bars = 1 cm. (F) Relative expression levels of immune-related and ion/nutrient-related maker gene in WT and gbpl3-6 measured by RT-qPCR . Expression level was normalized to that in WT. Student t tests were performed . ns indicates not significant. (G) The overlaps of up-regulated and down-regulated DEGs of crwn1 crwn2 (GSE106615) and gbpl3-7 (GSE199667). The significance level of the overlap was measured using hypergeometric tests. The list of overlapping DEGs was provided in (A) The insertion position of the 3 (PDF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.S5 Data(XLSX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file. 16 Jun 2022Dear Dr Gu, Thank you for submitting your manuscript entitled "GBPL3 is a novel component of the nuclear pore complex in plants and functionally connects the nuclear basket with the nucleoskeleton" for consideration as a Research Article by PLOS Biology.Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to send your submission out for external peer review.However, before we can send your manuscript to reviewers, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.https://www.editorialmanager.com/pbiology) within two working days, i.e. by Jun 20 2022 11:59PM.Once your full submission is complete, your paper will undergo a series of checks in preparation for peer review. After your manuscript has passed the checks it will be sent out for review. To provide the metadata for your submission, please Login to Editorial Manager - a track-changes file indicating any changes that you have made to the manuscript. NOTE: If Supporting Information files are included with your article, note that these are not copyedited and will be published as they are submitted. Please ensure that these files are legible and of high quality (at least 300 dpi) in an easily accessible file format. For this reason, please be aware that any references listed in an SI file will not be indexed. For more information, see our Supporting Information guidelines:https://journals.plos.org/plosbiology/s/supporting-information*Published Peer Review History*Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/*Press*Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.*Protocols deposition*https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocolsTo enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at Please do not hesitate to contact me should you have any questions.Sincerely,InesInes Alvarez-Garcia, PhDSenior EditorPLOS Biologyialvarez-garcia@plos.org------------------------------------------------------------------------DATA POLICY: IMPORTANT - PLEASE READ CAREFULLYhttp://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:1) Supplementary files . Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to  using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).2) Deposition in a publicly available repository. Please also provide the accession code or a reviewer link so that we may view your data before publication. Regardless of the method selected, please ensure that you provide the individual numerical values that underlie the summary data displayed in the following figure panels as they are essential for readers to assess your analysis and to reproduce it:Fig. 1A, B; Fig. 2A; Fig. 3A; Fig. 4D, H, I; Fig. 5B, E, H; Fig. S1A, E; Fig. S3A and Fig. S4C, FNOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived .Please also ensure that figure legends in your manuscript include information on where the underlying data can be found, and ensure your supplemental data file/s has a legend.Please ensure that your Data Statement in the submission system accurately describes where your data can be found.**In addition, you should make now publicly available the data you have deposited in the in ProteomeXchange database (ID: PXD032906).-------------------------------------------------------------------------BLOT AND GEL REPORTING REQUIREMENTS:https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirementsWe require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: ------------------------------------------------------------------------BLURBhttps://journals.plos.org/plosbiology/s/revising-your-manuscript#loc-blurbPlease also provide a blurb which (if accepted) will be included in our weekly and monthly Electronic Table of Contents, sent out to readers of PLOS Biology, and may be used to promote your article in social media. The blurb should be about 30-40 words long and is subject to editorial changes. It should, without exaggeration, entice people to read your manuscript. It should not be redundant with the title and should not contain acronyms or abbreviations. For examples, view our author guidelines: ------------------------------------------------------------------------Reviewers’ commentsRev. 1:This works builds from the author's previous work , identifying components of the Arabidopsis nuclear pore complex by proteomics. In these studies, the protein GBPL3 was discovered; characterization of this newly discovered component of the Arabidopsis nuclear pore complex is the subject of this current manuscript. GBPL3 recruits chromatin remodelers to the NPC, suggesting a role for transcriptional regulation. GBPL3 further interacts with the nucleoskeleton. This work is in contrast with a recent publication  suggesting that GBPL3 acts as a transcriptional regulator of immune response within the nucleus.Overall, I found the data to be compelling, claims appropriately backed with data, and I particularly appreciate the caveats and alternative explanations provided by the authors. Other than the two typos listed below, I have no concerns about the data presented in this manuscript and feel that this makes a valuable addition to our understanding of the plant nucleopore and further provides fodder for future studies.Line 53 - "In metazoan" should be "In metazoans".Line 76 - "CROWED" should be "CROWDED".Rev. 2:This is a detailed and carefully executed study which significantly enhances knowledge of the plant nuclear pore complex, by adding a new and functionally significant protein.15 However, the functional mechanisms behind- consider rewording; 'behind' is unclear as to meaning18 we found that GBPL3 is localizes to the nuclear rim and is enriched in the nuclear pore.26 bona fide - Latin; italicise.74 Nup136 and Nup82 are thought to share a common evolutionary history with a plant75 specific nucleoskeleton protein KAKU4, and conserved motifs in these three proteins mediate physical 76 interaction with CROWED NUCLEIs (CRWNs), filamentous proteins that compose the nucleoskeleton in 77 Arabidopsis (39). Please provide a reference for the common evolutionary history of KAKU4.227 formed spontaneous nuclear condensates while the rest remaining five diffuse in the nucleus231 under the transient overexpression condition better: in transient overexpression336- Adding more complexity, 337 although the four CRWN homologs have been shown to function redundantly in building the 338 nucleoskeleton and in regulating chromatin organization and genome activity (58-60), their double mutant 339 with gbpl3 led to distinct phenotypes, raising an intriguing hypothesis that different types of NB 340 nucleoskeleton joints can be formed by unique combinations between GBPL3 with CRWN proteins, which 341 may differentially contribute to gene expression regulation. Consider rewording the above to simplify for the reader.Figure 1 E) Three-dimensional reconstruction of root cells expressing mEGFP-GBPL3 in a line with lower expression. Please reword for greater clarity; this is not a three dimensional reconstruction…Rev. 3:The manuscript from Yu Tang, Man Ip Ho, Byung-Ho Kang and Yangnan Gu entitled "GBPL3 is a novel component of the nuclear pore complex in plants and functionally connects the nuclear basket with the nucleoskeleton" is an original contribution deciphering the plant NPC structure and function. Starting from the innovative proximity labeling proteomics technique recently developed in plants by the authors, the authors here focused on a specific component called GBPL3 identified in their initial experiments on the nuclear envelope. They clearly show using bio-informatics, molecular biology techniques and genetics that GBPL3 is enriched at the NPC basket, physically interacts with lamina and NUP components, is connected to the transcription machinery and that its mutation affects plant development in an additive/synergistic genetic effect with components of the plant lamina. This manuscript provides a strong validation of the previous observations made by proximity labeling proteomics and describes a new component connected to the NPC basket. For these reasons, I strongly recommend to consider this manuscript for publication in PLOS Biology with the following minor revisions.Minor revisions:Title: Although there is no doubt about the fact that GBPL3 is located at the NPC basket and interacts with the lamina and NPC, it is still questionable whether it is a true component of the nuclear pore basket or rather a protein interacting preferentially with certain components of the NPC basket and connecting the NPC to the transcription machinery. I would suggest to slightly change the title.l31: is the NPC the only possibility for a protein to access the nucleus as the ER that is in continuity to the NE can provide a way to TM anchored proteins to be imported into the nucleus.l32: This number seems a little too large as to my knowledge the NPC is made an octomeric structure with each monomer containing about 30-40 NUPs (i.e. 240-320 proteins / NPC?)l37-38: I recommend to add a citation describing the Plant NPC. Fiserova and Golberg 2009 used electron microcopy to investigate the NPC structure and gave some insights about the NPC basket. They proposed that filaments at the NPC basket are about 10nm.l47: I suggest to refer to "gene gating" by citing the initial Blobel's paper (1985) or to a review such as Tamura 2020, Journal of Plant Researchl73: Can the author reformulate these two sentences? NUP82 and NUP136 are two paralogs that contain FG-repeats and are located at the NB. NUP82 and NUP136 are two plant specific proteins that are redundantly required for...l89: Can the authors better connect this sentence with the following one?l107-Fig1A: can the authors specify in the Fig1A legend which control is control 1 and control2.Nup93a-BoiID2 (control1?) and YFP-BioID2 (control2?)mock-treated samples were used as controlsYFP-BioID2 mock-treated samples (control1?) and non-transformant plants (NT) (control2?) were used as controls.l123: can this be shown in a supplemental figure?l134-135: The authors described a phenomenon that is likely due to the genomic position of the transgene insertion affecting the level of expression and/or that can be linked to a missing regulatory element in their construct. I feel that this section would need some editing to better explain the observed results.First, the authors proposed that there are two set of lines (i.e. high vs low expression) among their transgeneic lines. Can they provide the number of lines produced and the number of high and low expressing lines in Fig S1C.Second, "Even though"... I guess the authors mean that this kind of position effects are usually observed with a p35S promoter and not/less with an endogenous promoter? can this be reformulated to help the reader to understand this unexpected result?l159: "in vivo" in italicl188-189-Fig 2B: It is surprising that the images showing CRWN1 and CRWN3 show that they are not enriched at the NE as described in other studies. As this has already been described in other studies  I would suggest to remove the images of CRWN1 and CRWN3 expression. If possible, it would also be better to select sections of confocal images showing expression at the NE (i.e. a cross section of the median part of the nucleus).l193-Fig 2D-E-F: For clarity; can the author provide results with CRWN2 in their transient expression assays (even if the result is negative)? CRWN4 is missing in these experiments (can the authors give an explanation?).l197-Fig 2F: Here again, it would be also better to select sections of confocal images showing expression at the NE (i.e. a cross section of the median part of the nucleus).l226-230: As GBPL3 form aggregates, one possibility is that it will have the ability to spontaneously attract other co-expressed proteins. I would strongly recommend to add at least one negative control in this experiment by choosing a nucleoplasmic protein that was not suggested to interact or to be in close proximity to GBPL3.l278-281: can the author provide as a supplementary fig a venn diagram showing the overlapping between gbpl3 and crwn1 crwn2 DEGs?can they also provide a ref to crwn1 crwn2 transcriptomic experiments .l298: CRWN1 was often associated with stronger phenotypes in earlier studies from the Eric Richard's group. These previous observations can be referred here.l311-314: Maybe the authors can also recall here that in Y2H, a clear physical interaction was recorded only with CRWN2?l350: « in vivo » in italic 29 Aug 2022AttachmentResponse to reviewers.pdfSubmitted filename: Click here for additional data file. 14 Sep 2022Dear Dr Gu,Thank you for the submission of your revised Research Article entitled "GBPL3 localizes to the nuclear pore complex and functionally connects the nuclear basket with the nucleoskeleton in plants" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Mark Estelle, I am happy to say that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.Please take a minute to log into Editorial Manager at PRESSbiologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with http://www.plos.org/about/media-inquiries/embargo-policy/.We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit Many congratulations and thanks again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. Sincerely, Ines--Ines Alvarez-Garcia, PhDSenior EditorPLOS Biologyialvarez-garcia@plos.org