Escherichia coli leucine-responsive regulatory protein (Lrp). Lrp senses the environmental nutrition status and subsequently regulates up to one-third of the genes in E. coli, either directly or indirectly. Lrp exists predominantly as octamers and hexadecamers (16mers), where leucine is believed to shift the equilibrium toward the octameric state. In this study, we analyzed the effects of three oligomerization state mutants of Lrp in terms of their ability to bind to DNA and regulate gene expression in response to exogenous leucine. We find that oligomerization beyond dimers is required for Lrp’s regulatory activity and that, contrary to previous speculation, exogenous leucine modulates Lrp activity at its target promoters exclusively by inhibiting Lrp binding to DNA. We also show evidence that Lrp binding bridges DNA over length scales of multiple kilobases, revealing a new range of mechanisms for Lrp-mediated transcriptional regulation.Feast-famine response proteins are a widely conserved class of global regulators in prokaryotes, the most highly studied of which is the  Escherichia coli has seven classically recognized global regulators: CRP, IHF, FNR, Fis, ArcA, H-NS, and Lrp (leucine-responsive regulatory protein)  . Lrp is protein) . While Lprotein)  and has directly , little directly .E. coli  when l-leucine concentrations are low  but that the oligomeric state switches to predominantly Lrp octamers in the presence of l-leucine, suggesting that leucine modulates Lrp function by altering Lrp’s oligomerization state , basic pI = 9.2), and highly abundant  DNA binding protein in E. coli  and is tordingly . The E.  factors , 8–10, a.8-kDa, bfactors –. To acco.2, and hon state –14. Howel-leucine modulates Lrp gene regulation by analyzing the global effects of leucine on Lrp binding to DNA and its subsequent effects on gene expression via paired Lrp chromatin immunoprecipitation sequencing (ChIP-seq) and RNA polymerase (RNAP) ChIP-seq. In order to further investigate the function of various Lrp oligomerization states, we also analyzed the effects of exogenous leucine on a “dimer-only” Lrp mutant (ΔC11) and two “octamer-only” Lrp mutants, D114E and L136R, which were originally discovered through their inability to respond to leucine at the ilvIH promoter and were later characterized as octamer-only mutants through size exclusion chromatography and dynamic light scattering  and the same medium supplemented with branched-chain amino acids  or stationary phase (Stat) cells). The strain harboring the dimer-only ΔC11 mutant had roughly the same growth rate as that of the lrp::scar (“scar” refers to the FRT recombinase site left after marker excision) strain under both conditions ; however, ΔC11 levels are substantially reduced over the course of the growth curve (Pdiff > 99%). We obtained weaker evidence that D114E Lrp was more highly expressed than WT Lrp under all conditions tested (Pdiff > 76%), while L136R Lrp was expressed at lower levels than WT Lrp (Pdiff > 80%)  , which m10.1128/mbio.02690-22.1TEXT S1lrp mutants in the present study. Download Text S1, PDF file, 0.04 MB.Description of the genetic background used for Copyright © 2023 Ziegler and Freddolino.2023Ziegler and Freddolino.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02690-22.7TABLE S1Table S1, XLSX file, 0.01 MB.Strains used in the present study. Shown for each strain are the name commonly used in the present work, the complete genotype, and origins  was comparable across conditions, confirming that our lrp-thyA constructs mimic WT strains. The dimer-only Lrp mutant (ΔC11) behaved like the lrp::scar mutant at oppA  are clustered into six groups (I to VI). Group I consists of dimer-only Lrp ΔC11 samples and generally exhibits low levels of binding to DNA at most Lrp binding sites, indicating that this dimer-only mutant is deficient in DNA binding. We thus excluded Lrp ΔC11 from further ChIP-seq analyses. Group II is a mixture of WT and D114E samples, both grown in LIV, which also exhibited lower levels of Lrp binding, with the exception of a few highly occupied sites. Group III (L136R-LIV) and Group IV (WT-Min) cluster closely, indicating that at the genome-wide level, L136R often fails to recognize exogenous leucine. Similarly, Group V (L136R-Min) and Group VI (D114E-Min) cluster closely and exhibit the highest levels of Lrp binding of all samples. Given that D114E and L136R were both identified as octamer-only mutants, it was notable that D114E exhibits exogenous leucine sensitivity but that L136R does not (given the similarity of Groups III and IV), potentially explaining the difference in the growth rates between these two mutants (We performed a paired Lrp and RNA polymerase (RNAP) ChIP-seq experiment  to measu mutants . As desc10.1128/mbio.02690-22.2TEXT S2Text S2, PDF file, 0.03 MB.Detailed description of different classes of Lrp-regulated genes. Download Copyright © 2023 Ziegler and Freddolino.2023Ziegler and Freddolino.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 0.01 by approximate permutation tests). For the Lrp peaks overlapping a TSS, we next calculated the number of transcription units (TUs) that are directly regulated by Lrp  and found that 41.92% of WT-, 33.33% of D114E-, and 51.61% of L136R-regulated TUs were under direct Lrp regulation under at least one condition tested. Given that Lrp binding is strongly enriched at promoters, it is likely that the remaining poised Lrp sites could become direct targets under a condition not tested in this experiment, as was previously suggested . The significance for total numbers was calculated by comparing rates using the poisson.test function in R, whereas the significance of percentages was determined using a 2-sample test for the equality of proportions using the prop.test function in R. Download FIG S1, TIF file, 0.9 MB.Heat maps of total Lrp peaks, Lrp peaks at promoters, and direct Lrp targets for each strain and condition. For each plot, the numbers in each box represent the total numbers (or percentages) of unique Lrp peaks or transcription units (TUs) across both biological replicates of both lineage replicates for each strain under each condition. The “Total” column in each heat map represents the total number of unique Lrp peaks or TUs within each strain across all conditions and is not simply the sum across the row. Asterisks represent the significance relative to WT Lrp under that condition  strongly activated ilvIH in Min but not LIV, whereas D114E and L136R strongly activated ilvIH in both Min and LIV (replotted in ilvIH promoter. The change in the average RNAP occupancy (relative to that of the lrp::scar mutant) across the ilvIH transcripts in our experiments , a concerted effect with Lrp on others, and an inhibitory  effect on Lrp for the remainder . To address the discrepancy with previous classifications of Lrp targets into concerted, reciprocal, or independent (see above), we turned to the paired Lrp ChIP-seq and RNA sequencing (RNA-seq) data set reported previously by Kroner et al.  and clasThe clustering of similar Lrp occupancy patterns in aroG region. Here, L136R Lrp occupancy in LIV . While the increased occupancy of L136R at the pepD-gpt promoters did not have a significant differential effect on pepD expression in Min, it caused a significant increase in gpt repression relative to WT Lrp, suggesting that this increased binding to DNA is functional and leads to changes in Lrp-mediated gene regulation. Overall, our findings show that L136R is mostly insensitive to leucine and thus can bind more strongly to its binding sites than WT Lrp, which is inhibited by leucine. It is important to note that while Lrp occupancy is quantitatively higher in L136R samples than in WT samples, there do not appear to be many novel DNA binding sites in L136R relative to WT Lrp during log-phase growth in Min  overlapped an annotated TSS. An example of one such novel D114E Lrp peak is shown in se genes .argA promoter  in Min, but also forms an additional peak within the argA ORF, where neither WT nor L136R Lrp is bound. Intriguingly, this additional D114E peak at argA is associated with significant Lrp-mediated activation of argA, whereas both the WT and L136R, which lack this additional peak, repress argA in Min  and found that the occupancy of WT Lrp had an average of 2.2 local peaks, whereas D114E Lrp showed an average of 3.1 local peaks . For exaA in Min . While tfadR-ycgB-dadAX region, which contains a strong primary Lrp peak (Peak 3) at the divergent ycgB and dadAX promoters and three secondary peaks (two upstream and one downstream) in Min  forms higher-order oligomers along the DNA, especially in Min, where binding to DNA is generally stronger. We thus constructed four strains  on each strain to measure changes in Lrp occupancy in Min-Log relative to two control regions (in the cysG and mdoG open reading frames). Within the primary peak (Peak 3), there are 11 previously characterized Lrp dimer binding sites. Among them, the 17-bp Lrp5 binding site is thought to play a particularly important role; it is essential for the derepression of dadAX in LIV but is typically bound by Lrp in both Min and LIV  in Min , bottom, and LIV .ycgB-dadA intergenic region significantly reduced (but did not eliminate) Lrp binding to Peak 3 while not substantially affecting Lrp binding to the secondary peaks , relative to WT cells. The sign convention is such that positive values indicate higher expression levels of the target gene for the indicated genotype than for the WT. It is notable that the perturbation of the secondary sites (pink bars) is sufficient to substantially alter the expression of both ycgB and dadX, consistent with our inference of interactions between the multiple Lrp binding sites at this locus being important for the regulatory output, even though the effects on binding at Peak 3 are small in magnitude. We also note that the effects on the gene expression of the secondary scramble cannot arise solely due to the modest decrease in binding at Peak 3 because in that case, dadA expression would also be expected to decrease (as in the case of the lrp::scar mutant). For the sake of completeness, we also include effects on transcript levels arising from the Lrp5-scr and Peak3-scr perturbations, but note that these cases should be interpreted with extreme caution because they involve changes to the sequence of the ycgB-dadA intergenic region. Download FIG S2, TIF file, 0.2 MB.Effects of Lrp binding-site perturbations on the expression of genes in the Copyright © 2023 Ziegler and Freddolino.2023Ziegler and Freddolino.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.02690-22.10TABLE S4Table S4, XLSX file, 0.01 MB.Statistical analysis of ChIP-qPCR occupancy data. Occupancy data from Copyright © 2023 Ziegler and Freddolino.2023Ziegler and Freddolino.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ygaP ORF) at the stpA-alaE region  experiments in the absence or presence of l-leucine. As was expected based on our in vivo experiments, Lrp bridges DNA, but the addition of l-leucine abrogates Lrp-mediated DNA bridging  suggested that both mutants also had the same regulatory function  in minimal media that bridge DNA. Our ChIP-qPCR and quantitative reverse transcriptase PCR (qRT-PCR) experiments on strains with mutated sequences at the locations of double and secondary Lrp peaks demonstrate that the Lrp complexes binding nearby peaks likely interact with each other. Indeed, our AFM experiments directly demonstrate that Lrp bridges DNA but that Lrp-mediated DNA bridging is abrogated in the presence of leucine. The bridging behavior and associated effects on Lrp-mediated gene regulation that we observed at the stpA-alaE promoter are likely characteristic of similar mechanisms at play in other regions with multiple Lrp binding peaks.By coupling small-scale experiments with high-throughput ChIP-seq on WT Lrp and three oligomerization mutants under various conditions, we were also able to connect the effects of leucine, the oligomerization state, and DNA binding. Dimer-only Lrp (ΔC11) was effectively unable to bind DNA despite being expressed at only slightly lower levels than WT Lrp at mid-log phase, corroborating previous  results . Previoufunction , 15. DesE. coli Lrp belongs to an ancient and highly conserved class of proteins called feast-famine response proteins (FFRPs), with homologs in almost all sequenced archaea and nearly half of all sequenced bacteria  in orange, with D114 residues highlighted in cyan. The image on the right is rotated 90° along the vertical axis relative to the image on the left. (B) Same as panel A except that the L136 residues are highlighted in cyan. Download FIG S3, TIF file, 2.5 MB.Location of residues D114 and L136 on the Lrp octamer structure. (A) Crystal structure of octameric Lrp (PDB accession number Copyright © 2023 Ziegler and Freddolino.2023Ziegler and Freddolino.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the All experimental procedures are described in detail in 24–30–10.1128/mbio.02690-22.9TABLE S3Table S3, XLSX file, 0.01 MB.Primers and oligonucleotides used in the present study. Download Copyright © 2023 Ziegler and Freddolino.2023Ziegler and Freddolino.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the lrp knockout cells under the same condition was subtracted from the occupancy scores prior to analysis. For RNA polymerase ChIP-seq, we summarized the counts of aligned reads at the level of transcription units, followed by differential expression calling using deseq2 (Sequencing data were analyzed using an updated version of the processing pipeline described previously  to obtaig deseq2 . FurtherGSE198120. The full set of AFM images used in our analysis is available at https://doi.org/10.6084/m9.figshare.21836319.All high-throughput sequencing data related to this study are available at the GEO under accession number


These results lead to the conclusion that increased dopamine signalling in the MBs of flies reared in a poor nutritional environment underlies the behavioural changes observed due to this condition during development.Nutrient scarcity is a frequent adverse condition that organisms face during their development. This condition may lead to long-lasting effects on the metabolism and behaviour of adults due to developmental epigenetic modifications. Here, we show that reducing nutrient availability during larval development affects adult spontaneous activity and sleep behaviour, together with changes in gene expression and epigenetic marks in the mushroom bodies (MBs). We found that open chromatin regions map to 100 of 241 transcriptionally upregulated genes in the adult MBs, these new opening zones are preferentially located in regulatory zones such as promoter-TSS and introns. Importantly, opened chromatin at the Dopamine 1-like receptor 2 regulatory zones correlate with increased expression. In consequence, adult administration of a dopamine antagonist reverses increased spontaneous activity and diminished sleep time observed in response to early-life nutrient restriction. In comparison, reducing  With these cutoff parameters, 280 differentially expressed genes were identified, with 241 of them (86%) being upregulated and the remaining 39 (14%) being downregulated . Most genes that change their expression are related to neuronal processes in Reactome pathways . A group of 45 genes is directly related to the nervous system which are depicted in a heatmap (figure 2c). Taking a closer look at enrichment in Gene Ontology (GO) categories, we found upregulated genes in the category of ‘synapse organization’ , ‘dopamine receptor signalling pathway’ , ‘dopamine catabolic process’ (GO:0042420), ‘cation transport’  and ‘adult locomotory behaviour’ (GO:0008344). These gene clusters suggest that MB neurons could have increased excitability, neurotransmitter reception  and transmission to output neurons . We found that dopamine receptors dop1R2 and dop2R, which are highly expressed in MBs [To isolate Kenyon cell nuclei, first, we expressed a UAS transgene coding for a nuclear membrane-tagged EGFP protein . Open chromatin zones were identified as windows or regions between 10 and 100 bp and compared between the two nutritional conditions to identify peaks exclusive to RF. We identified 7991 exclusive peaks for RF distributed with high percentages within the region  containing the promoter and transcriptional start site (TSS), and the first intron , genomic zones usually associated with transcriptional regulation [We found clear patterns of open chromatin state on promoters and transcription start sites (TSS) on the genome in each diet a. Open cgulation . We annogulation  and, witd, see red dots; full analysis is in electronic supplementary material, file S5). Two TFBS correspond to the Trithorax-like transcription variants (Trl.1 and Trl.2), whose binding sites directed the complexes to favouring open chromatin for transcription, possibly by overcoming the repressive effect of polycomb group complex [figure 3d), a TF required for olfactory neuron development. Interestingly, silencing bab1 gene in MB during development generates smaller alpha/beta lobules in this neuropil [Since most new chromatin open sites locate in transcriptional regulatory zones, we searched for transcription factor binding sites (TFBS) footprints and their changes between nutritional conditions. We performed a binding-site shift analysis with TOBIAS  , using afigure 3 complex ,40. Thusneuropil , supporta). GO analysis from this cross data show significant enrichment in chemotaxis, excitability (expressed as voltage-dependent channels) and dopaminergic transmission  map to ATAC-seq open chromatin sites in TSS, introns, or both a. GO ana. 2.4Drosophila brain [b,c). Also, we confirmed the increment in Dopamine receptor dop1R2 which is up regulated in mRNA-seq (a) with qPCR using the same isolation nuclei method (b), giving a log2(fold change) over 1.5 .Since chromatin accessibility and mRNA-seq reveal dopaminergic signalling as a relevant component of the response to RF, and that KCs have the highest expression of dopamine receptors in the adult la brain ,27,30, wmRNA-seq a with qPi method b, givingb,c. Afterwards we measured spontaneous activity and sleep behaviour. Remarkably, Flupentixol treatment was able to restore the locomotor activity  and the sleep behaviour  of adult flies raised under RF to the levels of CF flies. Of note, the effect of Flupentixol was more evident in kashMB247 > Gal4; UAS-EGFP::msp300 genetic background than in the oregon-rc background . The restoration of the behavioural phenotypes was more potent during the light phase of the day than in the dark phase, supporting the premise of increased excitability to the stimulus occurring in the light phase.To test whether decreasing dopamine signalling could reverse the behavioural effects produced by RF, we use Flupentixol, which is a dopamine antagonist tested previously in insects –44. We tfigure 1dop1R2 in mushroom body neurons could reverses the altered behaviour at some level. This dopamine receptor, whose mRNA expression increased  in RF conditions, also with new chromatin open regions in the regulatory area (intron), is a good candidate to mediate potential changes in the dopamine signalling in MBs, considering in addition that it is almost exclusively expressed in MBs neurons [Tub-Gal80ts; MB247-Gal4 genotype, which makes it possible to activate the Gal4/UAS system exclusively at 29°C. The diagram of this experiment is presented in figure 5e, it is worth mentioning that the control treatment was also subjected to temperature induction, in this case the control RNAi targets GFP. As observed in figure 5f,g, dop1R2 RNAi expression in MB neurons of animals raised in RF produced minor changes in adult behaviour, both in locomotion and sleep, suggesting that dopamine signalling through this receptor, specifically in MB neurons is highly relevant for maintaining these long-term behavioural changes, which are based at the transcriptional and epigenetic level.Considering an alternative and more specific approach to prove the point related to dopamine signalling, we tested if knockdown of ncreased a,b in RF neurons . RNAi exAltogether, the results demonstrate how low-nutrient availability during development, but not fasting, generates adult flies with alterations in locomotion and sleep, even if those individuals were fed with high nutrients during adulthood. Examining transcription profiles and chromatin states in MBs neurons, we were able to associate these behavioural alterations with an increment of dopamine signalling within this circuitry, which is responsible for locomotion, memory and sleep.. 3Drosophila melanogaster could be an excellent model to evaluate the mechanisms by which developmental malnutrition affects adult behaviour leading to mental illness. Here, we show that a restrictive nutrition during the larval stages of Drosophila development results in increased spontaneous activity and in a reduction of sleep time in adults.In humans, malnutrition during prenatal development is a frequent condition in poor and developing countries , generat. 3.1Drosophila larvae [a larvae –49 and aa larvae  increasea larvae  and birda larvae .b; electronic supplementary material, figure S1A and B). During this period, effector neurons and temperature receptors are more sensitive due to the circadian cycle [In our results, the increase in activity is more evident during daylight hours (light phase), when these animals typically feed . Similarly, adult flies maintained under starvation conditions generate shorter sleep times during the day [Our results show that, in accordance with the locomotor alterations, adult  the day . HoweverThese compensatory effects could be associated with food search and feed, which are necessary to gain biomass but not grow after the eclosion. Also, there is evidence that larvae with nutrition low in calories and macronutrients, but with normal nutrition later during adulthood, increase their biomass exhibiting macronutrient preference, without increasing in external body size . This in. 3.2loci, which could be explained by changes in mRNA dynamics, such as rates of degradation/stability [The open chromatin analysis combined with mRNA-seq led us to propose a group of genes whose transcription is regulated directly from the open/close chromatin state. Interestingly, most of those genes have functions related with processes that would explain the behavioural phenotype ; table 2tability . These pin utero malnutrition are triggered in adulthood [Interestingly, we found a greater number of new open chromatin sites in TSS than genes that upregulated their expression. Histone bivalence may explain this result. Equal presence of H3K4me3  and H3K27me3  on these promoters and TSS ,60, in wdulthood  and not Drosophila dopamine biosynthesis pathway involves tyrosine and L-Dopa as intermediaries [We showed that early-life undernutrition impacts chromatin state and gene expression in the adult MBs neurons, affecting excitability and dopamine signalling . Dopaminediaries , and itsediaries , all expediaries .Drosophila, as well as its role in MB neurons [figure 1b,c; electronic supplementary material, figure S1). However, dopamine signalling is complex in the MB; Dop1R1 can reduce motor activity [a,b), the open chromatin configuration in this gene (b) and also considering the fact that its expression is specific in KCs [The modulation of motor behaviour by dopamine has been well documented in  neurons ,66. Sinc neurons ,27,43, c neurons , agreeinactivity . Togethehis gene b and alsc in KCs .. 3.3c,d; electronic supplementary material, figures S6 and S7). These results led us to propose that pharmacological treatment of adults can ameliorate the behavioural alterations generated in response to undernutrition during development. Therefore, dopaminergic disbalance in this context may be considered in treating psychiatric disorders which could have some basis in utero malnutrition. The role of dopamine in maintaining the altered behaviour under developmental nutrition restriction as previously mentioned could be further narrow down to DopR2. The knockdown of dop1R2 in adult MBs neurons, confirmed that reducing the expression of this receptor in these specific neurons is sufficient to generate the partial reversion in the behaviour phenotype, revealing the key role of the epigenetic and transcriptional regulation of this receptor in MBs neurons in mediating the changes derived from poor nutrition in development.Based on the above results, which suggest an important role for dopamine signalling, we pharmacologically inhibit this pathway, supplementing the diet of adults with Flupentixol, a dopamine receptor antagonist. This treatment moderates the effects of early-life undernutrition, decreasing the effects on spontaneous activity and sleep behaviour  with 1/5 of nutrients in CF. CF was composed by beer yeast 10% w/v, sucrose 5% w/v, 1.3% w/v agar, 6% v/v propionic acid. RF was composed by beer yeast 2% w/v, sucrose 1% w/v, 1.3% w/v agar, 6% v/v propionic acid. Activity monitors in vials were maintained in a 12 h:12 h light-dark (LD) cycle. In the first 5 days of life before the behavioural analysis, adult flies were fed in filter paper over agar, with 5% w/v sucrose and yeast extract 10% w/v, and optionally with 1.0–1.5 mM of Flupentixol.Female-virgin flies were analysed on Dam2-Trickinetics system on tubes with 5% sucrose on one side and measured the movement for 5 days, using the last 2.5 days for the main analysis. Data extraction and analyses were performed with re for R , and plo. 4.2KASHUAS-EGFP::Msp-300, Ma and cols. [MB247 > Gal4 (Bloomington stock 50742) for specific MBs expression. Other behaviour analyses were done with flies oregon-rc (Bloomington stock 5) and canton-s (Bloomington stock 64349). For Knockdown experiments, a dop1R2 RNAi from the TRiP collection (Bloomington stock 26018), and a control RNAi against GFP (Bloomington stock 35786) were used. These stocks were crossed with ts; MB247-Gal4+; Tub-Gal80 to perform a temperature inducible expression.For MBs nuclei tag expression, we use nd cols.  under th. 4.3kash per replicate were frozen on liquid nitrogen to detach the heads after a brief vortex. The heads were homogenized at 4°C on isolation nuclei extraction buffer [2; 10 mM KCl), in an eppendorff tube with polypropylene tip, then kept for 15 min on ice, and then 20 pulls with a polypropylene tip. The homogenized was filtered on a cell strainer (40 µm) and then the nuclei were isolated with a centrifuge at 2000 rpm for 5 min. The nuclei were resuspended on 1 ml of IP buffer, then incubated for 1 h with 5 µg of EGFP antibody (abcam ab290) and 50 µl of Dynabeads-Protein A (Thermo scientific). The nuclei were isolated with a magnet to remove supernatant and 500 µl of Trizol was added. Samples were vortexed and 0.1 µl of chloroform was added. The mixture was vortexed and centrifuged at 14 000 rpm . The supernatant was recovered and RNA was precipitated with 0.8 volume of isopropanol and pelleted at 14 000 rpm , washed once with 70% ethanol and pelleted again. Ethanol was removed, and the pellet was resuspended on 20 µl of molecular biology water. RNA concentration was measured for quantity and quality. Libraries for mRNA-seq were generated with CATS mRNA-seq kit from Diagenode, according to instructions of the manufacturer. Libraries were sequenced on Illumina Hiseq 2500. The same isolation procedure was performed for qPCR analysis.For ATAC-seq and transcriptomic analysis, 200 female flies expressing EGFP::Msp-300n buffer  . After peak calling, the significant peaks were annotated with HOMER [mRNA-seq paired-end reads were trimmed with Trimmomatic and analysed with Cuffdiff  and annommomatic  and pairmmomatic  (Drosophth HOMER . To deteth HOMER . To identh HOMER  with the


They constructed a DME transgene under the control of an embryo-specific promoter and introduced it into a triple mutant of ROS1, DML2, and DML3 (termed rdd mutant). They then crossed the transgenic homozygous rdd/rdd mutant with heterozygous dme/DME plants, and obtained transgenic wild type along with dme, rdd, drdd plants in which DME was expressed primarily in the embryo, thus rescuing the seed abortion phenotype of homozygous dme and drdd plants.Cytosine DNA methylation controls cell fate and stress responses by silencing transposable elements and regulating gene expression. The level of DNA methylation results from the balance between methyltransferase and demethylase activities. In Arabidopsis, active DNA demethylation is controlled by four DNA glycosylases/lyases: DEMETER (DME), REPRESSOR OF SILENCING1 (ROS1), DME-LIKE2 (DML2), and DML3. Heterozygous  embryos , precludlleagues  created dme, 2,091 hypermethylated regions in rdd, and 2,601 hypermethylated regions in drdd compared to wild type, suggesting that the four demethylases function redundantly in leaves. By transcriptomic analysis, the scientists observed a correlation between gene hypermethylation and reduced transcript accumulation in drdd, suggesting that the DRDD pathway safeguards gene expression by “cleaning up” excessive DNA methylation. Among hypermethylated genes in drdd, the flowering time regulator FLOWERING LOCUS C was the most down-regulated gene, and drdd plants flowered earlier than the other genotypes. Importantly, natural Arabidopsis accessions with high DNA methylation at FLOWERING LOCUS C tended to flower earlier than low methylation accessions. By bypassing the embryo abortion phenotype of dme, the authors were able to explore how somatic DNA demethylation impacts flowering in Arabidopsis.The authors analyzed leaf DNA methylomes and observed 276 differentially methylated regions in Biological systems possess circadian clocks that enable the coordination of cellular functions with diurnal light/dark cycles. In Arabidopsis, the regulatory component of the circadian clock is composed of numerous transcriptional regulators. Among them, the MYB transcription factor REVEILLE8 (RVE8) interacts with NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED (LNK) proteins to regulate the transcription of dusk-expressed genes . Yuan Malnk plants. LNK physically interacted with RNA polymerase II and a histone chaperone subunit, and the association of RNA polymerase II and the histone chaperone with dusk-expressed genes decreased in lnk plants. The authors also showed that LNK and the histone chaperone are part of the same protein complex and that the chaperone is required for RNA polymerase II occupancy at dusk genes. Finally, the authors demonstrate that nascent RNA accumulation of dusk genes decreased in lnk mutant plants. This study provides a model for LCL action in RVE8 that is required for the interaction with LNK. In turn, LNK interacts with a histone chaperone complex. The results supported a role for this interaction in promoting the progression of RNA polymerase II along dusk-expressed genes.The authors then discovered that the enrichment of RNA polymerase II and a histone modification of active chromatin at dusk genes decreased in 3 photosynthesis occurs in a single cell type: mesophyll cells. In contrast, C4 photosynthesis occurs in two distinct cell types: atmospheric CO2 is fixed in mesophyll cells as C4 acids that are then transferred to Kranz cells where they are decarboxylated to generate CO2 that will be re-fixed by ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco). The partitioning of C4 photosynthesis in two distinct cell types concentrates CO2 at the site of Rubisco activity and reduces competition for oxygen, hence favoring photosynthesis over photorespiration (3 and C4 photosynthesis to envision the biological engineering of photosynthesis.Photosynthesis is the major player in atmospheric carbon fixation on earth and is essential for numerous heterotrophic organisms. Cpiration . It is t3 or C4, but exceptions to the rule exist. The amphibious leafless sedge Eleocharis vivipara develops C4 anatomy and biochemistry when grown in terrestrial conditions, in contrast to C3 anatomy and biochemistry when grown in submerged conditions (see E. vivipara plants with abscisic acid induced a shift from C3 to C4 anatomy and biochemistry (4 to C3 anatomy, but not biochemistry, in terrestrial forms of E. vivipara (3 and C4 biochemistry are genetically uncoupled from anatomical transitions.In general, plant species are either Cions see . Twenty-hemistry . The samvivipara . The Uen


CUX1) has been proven to be a key regulator in sheep hair follicle development. In our previous study, CUX1 was identified as a differential expressed gene between Hu sheep lambskin with small wave patterns (SM) and straight wool patterns (ST); however, the exact molecular mechanism of CUX1 expression has been obscure. As DNA methylation can regulate the gene expression, the potential association between CUX1 core promotor region methylation and lambskin pattern in Hu sheep was explored in the present study. The results show that the core promoter region of CUX1 was present at (−1601–(−1) bp) upstream of the transcription start site. A repressive region (−1151–(−751) bp) was also detected, which had a strong inhibitory effect on CUX1 promoter activity. Bisulfite amplicon sequencing revealed that no significant difference was detected between the methylation levels of CUX1 core promoter region in SM tissues and ST tissues. Although the data demonstrated the differential expression of CUX1 between SM and ST probably has no association with DNA methylation, the identification of the core region and a potential repressive region of CUX1 promoter can enrich the role of CUX1 in Hu sheep hair follicle development.CUT-like homeobox 1 ( CUX1), which is also known as CULT1, belongs to the homeodomain transcription factor family [CUX1 have been reported, the promoter-specific CCAAT-displacement activity of CUX1 can bind to the region surrounding the CCAAT box and repress the expression of the target protein even when the protein is bound at a distance. To date, CUX1 has been proven to participate in many biological processes [CUX1 can lead to altered hair follicle morphology, and the loss of its exon  can disturb the proper growth of the hair fiber and cause multiple hair abnormalities. The mutant CUX1 tm1Ejn can also lead to coat hair in mice, while the abnormal hair phenotype diminishes with age [CUX1 can serve as a positive regulator of the Wnt pathway in cancers [CUX1-binding site exists in wnt5a, a key gene regulating TGFβ and Notch signaling [CUX1 in hair follicle growth, these aforementioned results enlighten us that certain regulation may also exist in the sheep hair follicle functionalities. The effect of CUX1 on sheep lambskin has also been reported in our previous studies; Zhou et al. [CUX1 can drive the proliferation of DPCs and regulate the activation of subsets of genes including WNT10, MMP7, C-JUN, PPARD, FOSL1, etc.  [CUX1 to downregulate its mRNA and protein expression, and consequently affect the proliferation of DPCs [CUX1 expression regulation and hair follicle growth and development. Collectively, this evidence led to our hypothesis that the sheep hair follicle functionalities are mediated by CUX1 expression; however, the specific molecular mechanism is still required to be deciphered.Hu sheep is a native breed of sheep characterized by precocity and high productivity in China. Hu sheep lambskins, known for their unique water-wave pattern, are popular in the clothing industry and the home sector worldwide . The typr family . As a trrocesses ,14,15, iwith age ,17. Rega cancers . A conseignaling , and altu et al.  revealedduction) . In addi of DPCs , which iCSX and SOX genes increased with age before 2 years old in Angora rabbits [CUX1 gene is closely related to early-life growth trajectories and male infertility. Considering that the formation of lambskin is determined during the early-life growth period of lambs, and that CUX1 was found differentially expressed in the hair follicles of lambskin with small waves and straight hair patterns [CUX1 may be the reason for the regulation of CUX1 expression in sheep lambskin. Our goals were thus to investigate the association between DNA methylation in the CUX1 gene and Hu sheep lambskin pattern. In the present study, we first evaluated the expression of the CUX1 gene and Hu sheep lambskin tissue and determined the core promoter region using bioinformatics analyses and dual luciferase assay. Then, the methylation level of the CUX1 core promoter region was detected via bisulfite amplicon sequencing. Our study aims to gain insights into the CUX1-related molecular mechanism underlying hair follicle functionalities, and provide basic knowledge for lambskin production in the sheep industry.DNA methylations are one of the most common epigenetic modifications in promoter regions of eukaryotic genomes. They are involved in gene silencing and genetic stability in mammals ,24,25. R rabbits , and the rabbits . The lnc rabbits . A previ rabbits  found thpatterns , a hypot2 of skin from the dorsal side of full-sib 3-day-old Hu sheep lambs with different lambskin patterns was collected and divided into two groups: small waves , and straight wool . The collected samples were snap-frozen in liquid nitrogen and then stored at −80 °C until use.The sample information was detailed described in our previously published reports . BrieflyTotal RNA was extracted from the collected tissues using Trizol  per the manufacturer’s instructions. The first strand of cDNA was prepared using a FastKing RT Kit according to the manufacturer’s instructions , and cDNA was stored at −20 °C until use.2O, and 2 µL cDNA. PCR amplification was performed in triplicate using the following conditions: initial denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. The dissociation curve was analyzed after amplification. A melting temperature (Tm) peak at 85 °C ± 0.8 on the dissociation curve was used to determine the specificity of PCR amplification.Quantitative real-time PCR (RT-qPCR) was performed in 20 μL of reaction mixture that contained 10 µL FastReal qPCR premix , 0.4 µL of each forward and reverse primer, 6.4 µL RNase-Free ddHGAPDH was selected as the reference gene, the primers were designed using Primer Premier 5 software (−ΔΔCt method [CUX1. The results were shown as relative expression level (log2 Fold Change mean ± standard error) using GraphPad Prism 6 software.software . The 2−Δt method  was used2 at 37 °C.Isolation, culture and identification of Hu lamb dermal papilla cells (DPCs) were carried out according to our laboratory methods . DPCs anOvis aries) CUX1 gene was obtained from the NCBI database (Accession number: XM_004020987.5). The prediction of the CUX1 core promoter region was conducted using Neural Network Promoter Prediction software v2.0 . Based on the prediction results, the 2000-bp upstream sequence was truncated at the 3′ end for the amplification of six promoter fragments including CUX1-U1 (1985 bp), CUX1-U2 (1601 bp), CUX1-U3 (1151 bp), CUX1-U4 (751 bp), CUX1-U5 (499 bp) and CUX1-U6 (149 bp). The detailed information on the designed primers is shown in The 2000-bp upstream sequence of the transcription start site of the sheep .Then, the genomic DNA was isolated from the collected skin tissues using the TIANamp genomic DNA Kit  as per the manufacturer’s instructions. The six promoter fragments were amplified from the isolated genomic DNA using PCR, and then gel-purified, sequenced and ligated into a pGL3-Basic vector using ClonExpressCUX1-U1~U6 were co-transfected with a pRL-TK vector into DPCs and HEK-293T using jetPRIME transfection reagent . The cells were collected at 48 h after transfections for the dual luciferase assay using the Dual Luciferase Reporter Assay Kit  as per the manufacturer’s instructions. The CUX1 core promoter region was determined based on the luciferase activity of each sample normalized to the Renilla luciferase activity.Subsequently, pGL3-http://www.urogene.org/cgi-bin/methprimer2/MethPrimer.cgi, accessed on 1 July 2023). Then, the PCR products were purified and ligated into the pMD19-T vector.The Genomic DNA of skin tissues was transformed using the EpiTect Fast DNA Bisulfite Kit , the transformed DNA was used as a template for PCR amplification via the PyroMark PCR Kit . The PCR primers  were deshttp://quma.cdb.riken.jp/, accessed on 1 July 2023).Ten positive clones of SM and ST groups were randomly chosen for bisulfite amplicon sequencing . The methylation level was evaluated using the QUMA software v1.1.13 . Student’s CUX1 was found to be highly expressed in the hair follicles of the SM group .In the present study, we further evaluated the expression of patterns A. The reCUX1 expression mechanisms underlying lambskin pattern formation.Considering that the promoter regions are the main regulatory elements of gene expression, our study mainly focused on the core promoter-induced CUX1 transcription start site was obtained and assessed using the Neural Network Promoter Prediction software. The results showed that five potential core promoter regions of the CUX1 gene were predicted, which were present at −1789–(−1739) bp (a), −395–(−345) bp (b), −319–(−269) bp (c), −258–(−208) bp (d) and −247–(−197) bp (e) upstream of the transcription start site  bp.Subsequently, pGL3- in DPCs A and HEK in DPCs B, and luCUX1-U3 was extremely significantly lower (p < 0.01) than that of U4 and U6, and given the molecular mechanism of gene promotion, we assume that certain regulatory elements of CUX1 transcription may exist at −1151–(−751) bp, such as the key methylated CpG sites.It is worth noting that the luciferase activity of the pGL3-To further explore the molecular regulatory roles of the CUX1 core promoter region, a methylation analysis was conducted. The prediction results of MethPrimer A showed CUX1 mRNA expression.The results of BAS  showed tIRF2BP2 gene can lead to the regulation of EIF2S2 and IRF2BP2 mRNA expression, and is associated with the formation of hairy fleece and woolly fleece phenotypes [EIF2S2 and IRF2BP2 genes have both proven to be a regulator of transcription factors (EIF2S2 encodes a transcription factor and IRF2BP2 functions as a transcription factor coregulator). Another well-studied transcription factor that plays essential roles in hair follicle functionalities is SOX10. As a member of the Sox gene family, SOX10 can mediate the expression of genes involved in stem cell behavior during embryonic development. During the hair follicle cycle, the expression of SOX10 can control the hair follicle stem cell function and regulate hair follicle morphogenesis and postnatal follicular cycling [HOXC13, JUNB, LHX2, VDR and GATA3 have also been reported participating in hair follicle differentiation [CUX1 is a key member of the homeodomain transcription factors family. According to its specific CCAAT-displacement activity, CUX1 can function as a transcriptional repressor during the cell cycle and has proven to be associated with multiple cellular processes including cell differentiation, proliferation, cell cycle, etc. [CUX1 expression is associated with the proliferation of DPCs, which can lead to the promotion of hair follicle growth and development in sheep. Several studies have reported post-translational modifications of CUX1 including phosphorylation, dephosphorylation, acetylation and proteolytic processing [CUX1; however, there is little knowledge concerning the other transcriptional modifications of CUX1, such as DNA methylation. Here, we analyzed the effects of promoter methylation on CUX1 transcriptional activity and its potential role in the formation of lambskin pattern.The formation of lambskin pattern is a dynamic and complex process which happens in early life and involves delicate molecular regulatory mechanisms. Lambs carrying hairy lambskin (with waves) are of more economic value and have proven to be more adapted to harsh environmental conditions. Although the genetic basis of Hu sheep lambskin pattern formation is still largely unknown, studies on other sheep breeds have identified subsets of candidate genes in wool phenotype; a genome-wide study in Romane and Merinos sheep revealed that the insertion of a polymorphism asEIF2S2) into the 3′ UTR of the S2 into t cycling . In addintiation . Collectle, etc. ,35. Our ocessing , which cCUX1-related researches, the methylation modifications of CUX1 have been proven to be associated with complex biological processes, including amygdala and hippocampus development [CUX1 was observed in preterm ovine cerebrum and cerebellum compared to the normal ovine. CUX1 was also identified as a highly methylated gene in brain tissue of fetal sheep [CUX1 methylation on early nervous system development; however, the role of CUX1 methylation in sheep hair follicle growth and development remains obscure. Our previous study [CUX1 was found to be highly expressed in the hair follicles of the SM group. Consistent with our previous results, the present study also revealed this in the lambskin tissue of the SM group rather than the ST group, which further proves that CUX1 is a potential candidate gene regulating the lambskin pattern of Hu sheep.It is well acknowledged that DNA methylation is a fundamental epigenetic mark that correlates with gene expression. Regarding elopment , chronicelopment  and obeselopment . In sheeal sheep , which hus study  detectedCUX1 expression, the core promoter region of the CUX1 gene was first defined via a dual luciferase reporter assay. Estimating promoter activity is the most commonly used approach to determine the core promoter region. In the present study, we observed that the U2 region (−1601–(−1) bp) had the highest luciferase activity in both the 293T cell line group and the DPCs group. The previous study showed that core promoter motifs can significantly increase promoter activity [CUX1 in sheep. Generally, the luciferase activity of truncated core promoter fragments decreases gradually with increasing truncation length; however, the luciferase activity of the U3 (−1151–(−1) bp) was observed to be significantly lower than that of U2 and U6 (−751–(−1) bp). Similar results [lck promoter which, when deleted, causes an increase in promoter activity and lck expression. Xu et al. also found that deterrent transcription factors exist in cattle; deletion of the PAX7 gene promoter region can lead to a 2.79-fold increase in promoter activity [CUX1 promoter activity; deletion of this region may also have a promoting effect on the CUX1 promoter activity and expression. Of course, in-depth analyses are needed to prove our hypothesis.Promoter methylation is the most obvious epigenetic modification of DNA methylation ,41,42. Tactivity ,45; henc results  were obsactivity . CollectCUX1 core promoter region, which indicates that there was no correlation between the methylation of CpG sites in Island 1 and CUX1 mRNA expression. As previously mentioned, CUX1 was proven to be a highly methylated gene in the lamb nervous system [CUX1 expression remains to be established. It is possible that the CUX1 expression is regulated by other factors, such as miR-143, as we previous reported [Subsequently, DNA methylation analyses were conducted based on the identified core promoter region. The results of the MethPrimer showed that two CpG islands were predicted, which were present at −886–(−429) bp (Island 1) and −427–(−33) bp (Island 2). Interestingly, the majority of Island 1 and Island 2 were present at U4 and U3 regions, not in the core promoter region (U2). Previous research has shown that methylation of key CpG sites can influence the promoter activity of a specific gene ,49,50,51s system ; howeverreported .CUX1 core promoter region. Considering the overlapping region between the predicted CpG Islands and the repressive region, only CpG Island 1 was selected for the BSA and the DNA methylation analyses showed no difference between the two groups; The results could have been different if CpG Island 2 was selected for the BSA. Regarding the determination of the CUX1 core promoter region, to date, no study focused on the CUX1 core promoter has been reported; hence, the required knowledge is lacking to make comparisons between different species, and further study is still required to validate the reliability of the U2 region as a CUX1 core promoter in other species.It is cautionary to mention the limitations of our study regarding the bisulfite amplicon sequencing and CUX1 was significantly higher in SM tissues than in ST tissues. Five potential core promoter regions of the CUX1 gene were predicted based on bioinformatics analysis, and the core promoter region of CUX1 was determined at (−1601–(−1) bp) upstream of the transcription start site via dual luciferase assay. A repressive region (−1151–(−751) bp) with a strong inhibitory effect on CUX1 promoter activity was also identified. Bisulfite amplicon sequencing showed that four and six methylated CpG sites were detected in the CUX1 CpG Island 1 region of SM tissues and ST tissues, respectively. No significant difference was detected between the methylation levels of SM tissues and ST tissues; hence, the differential expression of CUX1 between SM and ST probably has no association with the CUX1 core promoter DNA methylation. Our studies further enrich the role of CUX1 in hair follicle growth and development and can provide basic knowledge for the Hu sheep lambskin industry.In conclusion, the expression level of


By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria.Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive   proVWXoperon of Escherichia coli as a model system to show how the nucleoid associated protein H-NS regulates gene expression in vivo by local chromatin remodelling.Here, the authors use the They do so by DNA bending, lateral filament formation along the DNA, or DNA bridging, the latter of which results in the formation of long- and short-range DNA loops that contribute to the global and local structural organisation of the genome. NAP binding is sensitive to environmental changes such as fluctuations in temperature, pH, and osmolarity. Hence, NAPs organise the bacterial chromosome into a dynamic structure that is re-modelled in response to changes in the cell environment. NAPs also function as transcription factors that coordinate global gene regulation in response to environmental stimuli. Collectively, these characteristics entail a coupled sensor-effector model of NAPs whereby NAPs regulate gene expression by reorganising the local structure of the chromosome at the level of individual operons in response to stimuli that modulate their architectural propertiesEscherichia coli is a 137 amino acid long NAP that functions as a global regulator of gene expression12 and a xenogeneic silencer13. It exists as a dimer in solution14, formed via interaction between a pair of N-terminal dimerisation domains17. The dimer binds to the minor groove of AT-rich DNA with an AT-hook-like motif within its C-terminal DNA binding domain18. The interaction serves as a nucleation point for the co-operative multimerization of H-NS along the DNA to form a protein-DNA filament. Structurally, the H-NS dimers are held together via interactions between central dimer-dimer interaction domains20. H-NS—DNA filaments can repress transcription by occluding the binding of RNA polymerase to promoter and promoter-like elements trapped within the structures23, but the filaments do not exert a detectable roadblocking effect on elongating RNA polymerase in vitro24. The presence of two DNA binding domains per dimer allows the H-NS multimer to recruit a second DNA molecule to form a DNA—H-NS—DNA bridge27. DNA—H-NS—DNA bridges occlude the binding of RNA polymerase (RNAP) to promoters28, block transcription initiation at the promoter clearance step by trapping RNAP in a loop29, and impede transcription elongation in vitro24.Histone-like Nucleoid Structing protein (H-NS) of 11. Its structural conformation and, by extension, its global gene regulation, is sensitive to physicochemical cues. Its response to osmolarity, in particular, is modulated by a K+- and Mg2+-sensitive α-helix, helix α330, that extends between the N-terminal dimerisation domain and central dimer-dimer interaction domain19. In vitro and in silico studies indicate that the stabilisation of the helix α3 by Mg2+ favours the open conformation of H-NS dimers in which both C-terminal DNA-binding domains are available for DNA binding. This promotes the formation of DNA—H-NS—DNA bridges that interfere with transcription initiation and elongation. Destabilization of helix α3 by K+ shifts the equilibrium of the conformation assumed by H-NS dimers towards the closed state where one DNA-binding domain of the dimer is folded onto the body of the protein27. This favours the formation of H-NS—DNA filaments27 – structures that hinder transcription initiation but are conducive to elongation24. In contrast to eukaryotic systems where a causal link between chromatin architecture and transcription has been established31, in vivo evidence highlighting an interplay between local three-dimensional chromatin organisation and gene expression in prokaryotes is lacking. The dual role of H-NS as a transcription factor and an osmoresponsive architectural protein makes it an attractive choice for addressing this knowledge gap. Hence, we examined the H-NS-mediated structural regulation of the proVWX operon in Escherichia coli.H-NS is a coupled sensor-effectorProVWX (proU)33 is an H-NS-regulated, osmoresponsive operon activated by the rapid cytoplasmic accumulation of K+ (counter-ion: glutamate) that ensues as a primary response to hyper-osmotic stress41. The acute increase in cytoplasmic K+ prevents cell dehydration by drawing water back from the environment42. This, however, occurs at the expense of protein stability and function36. K+ also behaves as a second messenger that activates the cell’s secondary long-term responses to osmotic stress, which includes the activation of proVWX. The products of the expression of the three structural genes – proV, proW, and proX – assemble into the ProU transporter – a transmembrane protein complex of the ATP-binding cassette (ABC) superfamily43. ProU imports osmoprotectants such as glycine-betaine, proline-betaine, and proline, among others, into the cell with high affinity45. The osmoprotectants maintain a low osmotic potential in the cytoplasm without an adverse effect on cellular physiology, allowing K+ to be expelled from the cell46.E. coli proU operon is inherent in the sequences of its σ70-dependent promoter (P2), and cryptic48 σS-dependent promoter (P1), positioned 60 and 250 bp upstream of the proV open reading frame (ORF), respectively50  that extends from ~300 bp upstream of the proV ORF to ~1100 bp into the proV ORF50  positioned at −229 to −47 of the P2 transcription start site (TSS), and the downstream regulatory element (DRE) that stretches across P2 from −40 to +17756 , the H-NS—DNA complex organises into a transcriptionally-repressive bridged conformation to silence proVWX56, reminiscent of the role of H-NS at the rrnB P1, hdeAB, and bgl promoters64, and that the cellular influx of K+ upon a switch to a higher osmolarity may drive a K+-mediated reorganisation of the H-NS—DNA complex to relieve proVWX repression51.Owing to Rho-dependent termination of transcription from P1 under ordinary growth conditions, proU68. IHF increases the expression of proU but does not affect the osmo-inducibility of the operon67. IHF mediates its effect via a putative binding site positioned ~450 bp upstream of P267, and the specific binding site of the protein in the −33 to +25 region around the P2 TSS69. Due to limited study, it is unclear whether proVWX activation as a result of upstream IHF binding is affected through P1 or P268, however, the specific binding of IHF at P2 structurally modifies the promoter to partially relieve H-NS-mediated repression at low osmolarity69. An effect of ihf deletion on the expression of the proVWX operon was not observed in genome-wide studies65, likely due to the modest decrease in the expression of the operon at low osmolarity conditions in the absence of IHF67. HU regulates expression from the proU P2 promoter and its osmoinducibility68. ΔhupA, ΔhupB, and ΔhupAhupB mutants show reduced proU-lacZ expression compared to the corresponding wild-type strain, and a reduced fold-change in expression from P2 between low and high osmolarity conditions68. HU plays its role independently of H-NS as evidenced from ΔhupB Δhns double mutants, where expression from P2 is realised as sum of the individual mutations rather than an epistatic output68. The proU-lacZ expression results presented for ΔhupA, ΔhupB, and ΔhupAhupB strains68 conflict with genome-wide transcriptome data66 which indicate that a ΔhupA mutation derepresses proU during the exponential phase of growth, ΔhupB derepresses the operon during transition-to-stationary phase, and ΔhupAhupB in stationary phase66. However, the detection of proU expression at the transcriptional level from its native locus in the chromosome in genome-wide experiments66 and post-translationally from a plasmid-localised proU-lacZ gene in reporter gene expression assays68 may account for the discrepancy. The ectopic placement of a non-native proU-lacZ construct eliminates the effects of relevant regulatory elements and perturbs local topological states that impact proU expression either directly, or indirectly by affecting the binding of regulatory NAPs. StpA, an H-NS paralogue (58% sequence identity)71 that has been proposed to function as a molecular back-up for H-NS72 represses proV-lacZ 9-fold when expressed from multi-copy plasmids in E. coli Δhns73. Chromatin Immunoprecipitation (ChIP) of FLAG-tagged StpA shows that in wild-type E. coli and in E. coli Δhns, StpA binds the proVWX regulatory region and its occupancy spans over P1 and P274. StpA also binds the proV ORF, however, only as a hetero-oligomeric complex with H-NS74. A ChIP signal of FLAG-tagged StpA is not observed at proV in E. coli Δhns74. Conversely, FLAG-tagged H-NS is detected at proV in E. coli ΔstpA74.In addition to H-NS, the NAPs IHF and HU also regulate proVWX operon is also regulated by genetic and chemical factors that modify the supercoiling density of the genome76. Mutations to DNA gyrase, and growth in media supplemented with non-lethal concentrations of novobiocin, a DNA gyrase inhibitor, reduce the levels of negative supercoiling in the cell and the expression of proVWX77. Mutations in topA, and growth in high osmolarity media increase negative supercoiling density and proVWX expression75. However, extremely high levels of negative supercoiling observed in ΔtopA mutants repress the operon75. Furthermore, in a purified in vitro transcription system, transcription from the P2 promoter increases with increasing negative supercoiling density of the template51. Increased negative supercoiling may favour transcription by facilitating the melting of the relatively GC-rich P2 promoter and promoting a structural reorganisation of the H-NS—NRE nucleoprotein complex46.The transcription of the 50, supercoiling density, and architectural proteins – HU68, IHF69, StpA78, and the osmoresponsive H-NS56 – in the activation and repression of the proU operon from the P2 promoter implies that the regulation of proVWX may involve local three-dimensional reorganisation of chromatin structure, analogous to eukaryotic chromatin remodelling. Here, we use RT-qPCR and 3C-qPCR to study the interplay between transcription and local three-dimensional chromatin structure at the proVWX operon in low and high salt conditions, and in response to a hyper-osmotic shock.Collectively, the coordinated role of an inherently osmoresponsive promoterproU operon involved transcriptional and translational fusions of lacZ to truncates of proV and the subsequent detection of the specific activity of β-galactosidase (lacZ) in a variety of genetic backgrounds and osmolarity conditions as a measure of proU expression79. The technique is limited in that it relies on post-translational detection. It requires transcription of the reporter gene construct and translation of the mRNA for a read-out. As such, it is more reflective of intracellular protein levels than transcription. RT-qPCR (reverse transcriptase quantitative PCR) directly measures transcript levels. Therefore, we used RT-qPCR to study the osmolarity-dependent response of proVWX. We designed eleven primer pairs to evaluate the transcriptional profile across the operon and its flanking genes  84 positioned 23 basepairs downstream of the proW2 amplicon. Transcription initiating from this site may account for the increase in the transcript levels of proX. The promoter regulating expression from this site is likely a non-canonical promoter rather than a classical H-NS-repressed spurious promoter since H-NS ChIP does not show a reproducible H-NS signal at proW  compared to its upstream end (amplicon proW1)  in the E. coli transcriptome detected the presence of rG4 structures within the proW coding sequence87, occurring between the proW1 and proW2 amplicons  as a model, rG4s were shown to play a role in the stabilisation of transcripts87. K+ stabilises rG4s when the ion occupies the centre of the quartet88. Extrapolation of these findings to rG4s at proW suggests that at higher intracellular concentrations of K+, the increase in the relative transcript level of proW2 may be detected due to the stabilisation of rG4s at the 5’ region of the transcript.The increased transcript level in the downstream region of W1) Fig.  implies proVWX operon is repressed in NT331 cells growing exponentially in a medium with an NaCl concentration of 0.08 M. Upon a hyper-osmotic shock that raises [NaCl] from 0.08 M to 0.3 M, the transcript levels of proVWX increase by up to ~8-fold  increases ~2-fold, the P2 distal amplicon of proW (amplicon proW2) is ~7-fold higher, and proX transcripts increase by a factor of ~6.5. During exponential growth at 0.3 M NaCl, in comparison to 0.08 M NaCl, the transcript levels of proV, proW1, proW2, and proX increase by factors of ~1.8, ~1.3, ~2.2, and ~1.5, respectively  promoter positioned 60 bp upstream of the proV ORF. The proVWX σS-dependent (P1) promoter located at −250 bp of proV is cryptic and is activated by rho and hns mutations, and by cold stress47. To record the contribution, if any, of P1 to the expression of proVWX in a wild-type NT331 background, the relative transcript level of amplicon proU1 positioned between the P1 and P2 promoters was determined. At 0.08 M NaCl, transcript level of proU1 was ~6-fold lower than that of amplicon proV1 positioned 345 bp downstream of P2. Upon a hyperosmotic shock, the transcript level of proU1 increased ~2-fold, but was ~25-fold lower than that of proV1. During exponential growth at 0.3 M NaCl, the transcript level of proU1 was ~10-fold lower than proV1  and both ygaY amplicons (amplicons ygaY1 and ygaY2) were detectably higher after a hyperosmotic shock. This may occur due to an increased local density of RNAP at this osmotic condition or the decompaction of the local chromatin following the shock providing a more conducive environment for transcription.To detect cross-talk between the expression of ock Fig. , indicatproU repression was alleviated by introducing a series of point mutations in the AT-rich H-NS-binding regions that were experimentally detected in the proU DRE with an in vitro DNase-I protection assay55. This approach was chosen over hns deletion to prevent the pleiotropic effects associated with the latter. In this design, the −35 and −10 promoter elements of P2, the P2 TSS, and the Shine-Dalgarno sequence of proVWX were not edited. However, four mutations were introduced in the high-affinity H-NS binding site extending between −7 and +15 of the P2 TSS. AA > GC and TA > GC mutations were carried out at −3 to −2 and at +7 to +8 positions, respectively. AT-rich codons in H-NS binding sites in the proV ORF were switched out for GC-rich variants with the most similar codon usage ratio in the E. coli chromosome. Identical mutations decided upon with the aforementioned criteria, were incorporated into both high-affinity H-NS binding sites occurring in the DRE56. Following mutation, the AT-content of the DRE was lowered from 66.4% to 51.6%  is comparable to the relative transcript level in the wild-type background (NT331) after a hyper-osmotic shock (from 0.08 M to 0.3 M NaCl)  is similar to the specific activity of proV-β-galactosidase in a wild-type background at high osmolarity . The differences between these two sets of results may be accounted for by the post-translational evaluation of osmoresponse in ref. 49. β-galactosidase is a stable protein. Hence, expression analyses using lacZ reporter fusions49 mask down-shifts of expression upon adaptation to high osmolarity.The relative transcript level of ound NT33 after a ound NT33 after a proVWX by ~2-fold  and NT331 (wild-type) have similar transcript levels of proU at 0.3 M NaCl .A hyper-osmotic shock to NT644, increases the transcript levels of old Fig. , and upoaCl Fig. . Our reshns background50. The mutations at the proVWX regulatory element in NT644 are designed to disrupt H-NS binding to the DRE . Indeed, chromosome contact maps of NT331 generated with Hi-C show chromosome rearrangements in the vicinity of the proVWX operon  for corresponding biological replicates (R1-R4) overlapping with each other within error. We tested four interactions that form in the rpoD and hcaT genes that have been used as internal controls in the RT-qPCR experiments reported in this article, and two additional interactions in rpoB that was not a reliable RT-qPCR internal control for our experimental conditions, but has been used previously. Each of these resulted in the misalignment of one or more of the biological replicates of a set of 3C libraries. This may arise since several factors contribute to regulating gene expression, and while the transcription output of rpoD and hcaT, or rpoB in other cases, may remain unchanged, chromatin architecture may differ. The Escherichia coli chromosome is organised into four structured, and two unstructured macrodomains. Since the ligation efficiency between two fragments is not only influenced by their proximity to each other, but also by their proximity to other loci, we inferred that chromatin interactions in one macrodomain may not be a reliable internal control for chromatin structure studies in another macrodomain, or perhaps even another sub-domain in the same macrodomain. Thus, we checked if a test interaction within our region of observation could serve as an internal control. The proU3-proU6 interaction best-fulfilled the criteria we set for a reliable internal control.To account for differences in the restriction digestion and ligation efficiencies of 3C libraries, and the differences in the starting quantity of 3C libraries in a qPCR reaction, interaction frequencies calculated from 3C-qPCR experiments are normalised to an Internal controlproVWX in NT331 differs in cultures growing exponentially in M9 medium with 0.08 M NaCl , cultures subjected to a hyperosmotic shock , and cultures growing exponentially in a medium with 0.3 M NaCl   Fig. . 3C-qPCRproVWX in NT331 by ~2-8-fold  and proU11_NlaIII (ygaY)  and the URE (proU2_NlaIII)  and the nrdEF genes   and partially-bound (bend angle 115o ± 30o) conformations105. In the partially-bound state, the DNA on one side of the IHF-mediated bend is not folded onto the body of the protein105. In vitro fluorescence lifetime measurements of the conformations sampled by IHF bound to its high affinity H’ site show that the formation of a partially bent IHF-H’ complex increases from 22% to 32% upon an increase in the concentration of KCl from 100 mM to 200 mM105. Thus, the intracellular influx of K+ upon a hyperosmotic shock may shift the equilibrium of IHF binding to favour the formation of a partially-bent IHF-P2 conformation, promoting the disassembly of the DRE—H-NS—URE bridge that is already weakened by K+.The 3C-qPCR profiles of proVWX at low osmolarity. This contradicts an earlier report that shows the expression of proV-GFP cloned on a plasmid downstream of the P2 promoter is activated by four-fold in the presence of IHF at an osmolarity of 0.17 M NaCl in LB due to the distortion to the structure of the promoter upon IHF binding69. We propose that the impact of IHF on the expression of proVWX may be a consolidation of two opposing effects: repression mediated by facilitating the formation of the DRE—H-NS—URE bridge, and activation of P2 mediated by direct binding of IHF to the promoter, facilitating the formation of the open promoter complex.The model above suggests that IHF favours repression of proVWX in Escherichia coli  for RNAP binding and by exerting a weaker roadblocking effect. Active transcription from P2 then remodels the local chromatin structure of the proVWX template causing the disassembly of the weakened bridge between the DRE and ygaY. The expression of proVWX and the subsequent synthesis of the ProU transporter allows E. coli to import osmoprotectants such as glycine-betaine, proline-betaine, and proline into the cell45. These maintain a low osmotic potential in the cytoplasm without adverse effects on cellular physiology. Hence, K+ is exported from the cell. The decrease in cytoplasmic K+ stabilises the open conformation of H-NS favouring the formation of bridges and re-establishing a repressed operon.Upon a hyperosmotic shock, proVWX osmotic regulation has not been sampled in our report, for instance, there is at least an order of magnitude of repression of proVWX between 0.08 M NaCl and 0.01 M NaCl54. Other regulatory mechanisms may function in these ranges, adding to the complexity of proVWX osmoregulation.Notably, the full range of proVWX operon of Escherichia coli as a model system, we provide the evidence that, in vivo, the nucleoid-associated protein H-NS regulates gene expression in response to environmental stimuli by local chromatin remodelling. The tuning of the architectural properties of H-NS by changes in osmolarity either directly remodels the chromosome to regulate gene expression, or renders the chromosome susceptible to remodelling by, for instance, the transcriptional machinery.Bacterial chromosomes are organised by active transcription and by architectural nucleoid-associated proteins (NAPs) that also function as transcription factors. NAPs are sensitive to physicochemical signals. Hence, the proteins organise the chromosome as a dynamic structure that is remodelled in response to physicochemical environmental stimuli, and they coordinate gene transcription with the extracellular environment. These characteristics have been studied separately in the past and direct evidence of the environmentally-regulated interplay between local three-dimensional chromatin organisation and gene expression in prokaryotes has been lacking. Using ensemble RT-qPCR and 3C-qPCR approaches, and the H-NS-regulated osmoresponsive Escherichia sp., Shigella sp., and Salmonella sp. The model of transcription regulation by chromatin reorganisation presented here provides clues of how H-NS may operate at other genes and operons within its regulon that encompasses pathogenicity-related loci. The model may also be extrapolated to explain how H-NS-like proteins such as MvaT in Pseudomonas sp., and Lsr2 in Mycobacterium sp., may function in response to physicochemical environmental signals that can be ‘sensed’ by the protein112, and, how the regulons of atypical H-NS-like proteins such as Rok in Bacillus sp. may be regulated by environment-sensing protein partners like sRok113.H-NS is a global gene regulator in, among others, proU operon encodes an ABC transporter – a member of the largest group of paralogous protein complexes114. The components of ABC transporters are contained within operons, the earliest of which is predicted to have arisen before the divergence of bacteria and archaea116. The organisation of ABC operons is evolutionarily conserved and is specific for each orthologous group118. This ubiquity permits a systematic study of the evolution of the environmentally-regulated interplay between local chromatin structure and gene expression.The Escherichia coli MG1655 using a two-step genome editing method based on the λ-red recombinase system119 as described in120. Chemically competent bacteria were transformed with pKD46119, a temperature-sensitive plasmid encoding the λ-red proteins under an arabinose-inducible promoter. Electrocompetent pKD46+ cells were prepared from a culture grown at 30 °C in LB medium (1.0% bactotryptone (BD), 0.5% yeast extract (Alfa Aesar), 170 mM NaCl, pH 7.5) supplemented with 10 mM arabinose, for plasmid maintenance and to induce the expression of the λ-red proteins, respectively. The cells were transformed with a kanR-ccdB cassette120 encoding kanamycin resistance and the ccdB toxin under a rhamnose-inducible promoter119. The cassette was designed to carry ~1.5 kb extensions on either side homologous to the flanks of the genomic region to be edited. KanR-ccdB was amplified from plasmid pKD45119 and the homology regions from genomic DNA. The complete construct was assembled using overlap extension PCR (oePCR). Recombinants were selected for on LB agar (1.0% bactotryptone (BD), 0.5% yeast extract (Alfa Aesar), 170 mM NaCl, 1.5% bacteriological agar (Oxoid), pH 7.5) supplemented with 40 μg/mL kanamycin at 37 °C and verified using colony PCR and Sanger sequencing (BaseClear B.V.). The mutant allele construct with ~1.5 kb homology regions was assembled into a plasmid using Gibson assembly121. The allele was amplified and used to replace the genomic kanR-ccdB cassette via the λ-red recombinase system. Recombinants were selected for on M9 agar plates ) supplemented with 1% rhamnose and verified with colony PCR and Sanger sequencing (BaseClear B.V.). All bacterial strains and intermediates were stored at -80 °C as glycerol stocks. Strains and plasmids used in this study are listed in Tables Bacterial strains used in this study were derived from 2HPO4, 22 mM KH2PO4, 19 mM NH4Cl, 2.0 mM MgSO4, 0.1 mM CaCl2, 80 mM NaCl, 1X trace elements, 1% Bacto™ casamino acids (BD), 10 μg/mL thiamine (Sigma-Aldrich), 0.4% glycerol (PanReac Applichem)). The overnight culture was used to inoculate fresh low salt LB or M9 medium to a starting OD600 of 0.05 for up to four biological replicates and grown at 37 °C to an OD600 of ~1.0. The culture was split into two aliquots. For hyper-osmotic shock, 5.0 M NaCl was added to one of the aliquots at a ratio of 46 μL per mL of culture. An equivalent volume of milliQ water was added to the second aliquot for the low salt condition. The cultures were grown for 10 minutes at 37 °C, and then immediately harvested for RNA isolation, and cell fixation for 3C-based experiments. For high salt studies, a single bacterial colony was grown overnight in high salt LB medium (LS-LB: 1.0% bactotryptone (BD), 0.5% yeast extract (Alfa Aesar), 300 mM NaCl, pH 7.5) or high salt M9 medium , 10 μg/mL thiamine (Sigma-Aldrich), 0.4% glycerol (PanReac Applichem)). The overnight culture was used to inoculate fresh high salt LB or M9 medium to an OD600 of 0.05 for up to four biological replicates. The cells were cultured at 37 °C to an OD600 of ~1.0, and for an additional 10 minutes. The cells were immediately harvested for RNA isolation, and cell fixation. For experiments requiring rifampicin treatment, cultures at an OD600 of ~1.0 in were incubated with 100 μg/mL rifampicin for two hours at 37 °C. The addition of NaCl for a hyperosmotic shock and the respective treatments for low and high osmolarity conditions were performed after incubation with rifampicin.For low salt and hyper-osmotic shock conditions, a single bacterial colony from a freshly streaked plate was grown overnight at 37 °C in low salt LB medium (LS-LB: 1.0% bactotryptone (BD), 0.5% yeast extract (Alfa Aesar), 80 mM NaCl, pH 7.5) or low salt M9 medium  pre-heated to 95 °C. The lysate was incubated at 95 °C for 5 minutes. The preparation was treated with 1 mL of TRIzol® reagent  and incubated at room temperature for 5 minutes. RNA isolation was paused at this step to accommodate cell fixation for 3C and Hi-C library preparation by flash-freezing the TRIzol®-lysate mix in liquid nitrogen and storing at -80 °C for up to 2 weeks. The TRIzol®-lysate mix was thawed at room temperature and RNA isolation was continued using the TRIzol® Max™ Bacterial RNA Isolation Kit  according to the manufacturer’s instructions. The concentration and purity (A260/A280) of RNA was measured with a NanoDrop™ 2000 spectrophotometer  and NanoDrop 2000/2000c Software Version 1.6. Accordingly, RNA samples were diluted to a final concentration of 20 ng/μL with UltraPure™ DNase/RNase-Free Distilled Water . DNA contamination in the RNA samples was checked with RNase A  treatment and agarose gel electrophoresis. 50 μL of the RNA samples were transferred into wells of a 96-well RNase/DNase-free plate in triplicate to facilitate multi-channel pipetting for RT-qPCR experiments. RNA samples in the multi-well plate were stored at 4 °C and placed on ice during reaction set-up to avoid freeze-thaw cycles. Stock RNA samples were stored at -20 °C.Bacterial cells in 1 mL of culture were collected by centrifugation at 13,000 x 122, the details of RNA yield and purity (A260/A280 measurements), and the results of RNA inhibition testing are provided in Supplementary Data In compliance with MIQE guidelinesEscherichia coli K-12 MG1655 sequence (Accession number: NC_000913.3 – https://www.ncbi.nlm.nih.gov/nuccore/556503834). In silico specificity, screening was performed on SnapGene® Viewer 5.2. RT-qPCR primers were ordered as dried pellets from Sigma-Aldrich. The oligonucleotides were dissolved in 1X TE pH 8.0 to a final concentration of 100 μM and stored at 4 °C. The list of primers used for the RT-qPCR assay and corresponding amplicon details are provided in Supplementary Data proVWX operon are provided in Supplementary Data Primers for RT-qPCR experiments were designed using the RT-qPCR experiments were performed with the Luna® Universal One-Step RT-qPCR Kit  with a final reaction volume of 10 μL per well. The reactions were prepared according to the manufacturer’s instructions with modifications. Reaction composition is provided in Supplementary Table rpoD, hcaT, and rrsA, were selected for this study. rpoD encoding the RNA polymerase σ70 factor, is a house-keeping gene that has been validated as a stably-expressed mRNA suitable as an internal control in a diversity of bacterial species including Klebsiella pneumoniae123, Pseudomonas aeruginosa124, Pseudomonas brassicacearum GS20125, and Gluconacetobacter diazotrophicus126. hcaT encodes a predicted 3-phenylpropionic transporter and has been validated as a suitable internal control in Escherichia coli128. rrsA codes for 16 S rRNA and occurs in six copies in the E. coli MG1655 chromosome. It is accepted as a stably expressed house-keeping gene and was accepted as a validated internal control for E. coli RT-qPCR experiments128. rrsA has been used to normalise relative transcript levels in RT-qPCR studies of the proU operon54. We tested the amplicon as a potential internal control, but, the low Cycle of quantification (Cq) values of rrsA in comparison to the Cq values of amplicons of the proU operon  and incubated at 4 °C for 10 minutes. The cells were collected by centrifugation at 3000 x g for 5 minutes at 4 °C and washed with 20 mL of LS/HS-M9 medium. The pellet of washed, methanol-treated cells was resuspended in 3% formaldehyde  in LS/HS-M9 medium. The reaction was incubated at 4 °C for 1 hour and subsequently quenched with a final concentration of 0.375 M glycine for 15 minutes at 4 °C. The fixed cells were collected by centrifugation and washed twice with 1X TE pH 8.0 . The cell suspension was split into three aliquots and pelleted at 10000 x g at room temperature for 2 minutes. The supernatant was removed, and the cell pellet was flash-frozen in liquid nitrogen for storage at -80 °C until use (up to 1 month).2HPO4, 1.8 mM KH2PO4), 1X HS-PBS , or 1X PBS  as required.Bacterial cultures grown in LB were fixed in the same manner, except that all wash steps and 3% formaldehyde preparation were performed with 1X LS-PBS . The cells were lysed and solubilised with 2000 U of Ready-Lyse Lysozyme  and 0.5% SDS . The extracted chromatin was digested for 3 hours at 37 °C in a volume of 250.0 μL with 100 U of NlaIII  for 3C library preparation, or 100 U of PsuI  for preparing a Hi-C library. The reaction was performed in the presence of 0.5% Triton X-100  to sequester SDS in the cell lysate. 3C libraries were treated with 0.5% SDS  for 20 minutes at 37 °C to terminate restriction digestion. For Hi-C libraries, the overhangs in the restriction digested chromatin were filled in with biotin-14-dATP  using 30 U of DNA Polymerase I, Large (Klenow) fragment  in a volume of 300.0 μL. The reaction was performed for 45 minutes at 25 °C, and later terminated with 0.5% SDS  for 20 minutes at 25 °C.Hi-C and 3C libraries were prepared as described previouslyThe chromatin for both 3C and Hi-C libraries was centrifuged at 25000 x g for 1 hour at 4 °C. The supernatant was discarded, and the gel-like pellet was resuspended in 200.0 μL of nuclease-free water . The DNA concentration of the preparation was measured with the Qubit® dsDNA HS Assay Kit . 1.0-3.0 μg of DNA was used for a ligation reaction in a volume of 1 mL using 1.0 μL of 2000 U/μL T4 DNA Ligase  for 3C experiments, and 2.0 μL of the enzyme for Hi-C experiments. The reaction was incubated at 16 °C for 16 hours, and then at 25 °C for 1 hour. The reaction was terminated with 20.5 μL of 0.5 M EDTA .The libraries were purified with a 30-minute incubation at 37 °C in the presence of 160 μg/mL RNase A , a treatment with 200 μg/mL Proteinase K  for 16 hours at 65 °C in the presence of 500 mM NaCl, two extractions with 25:24:1 phenol:chloroform:isoamyl alcohol , and one extraction with chloroform . The DNA was precipitated at -20 °C overnight with 0.025 V of 5 mg/mL glycogen , 0.1 V of 1.0 M NaOAc  pH 8.0, and 2.5 V of 100% ethanol. Precipitated DNA was pelleted by centrifugation, washed twice with 70% ethanol, and air-dried. The pellet was dissolved in 30.0 μL nuclease-free water . At this step, 3C libraries were stored at -20 °C until use.129.Hi-C libraries were treated with 3 U of T4 DNA polymerase  in the presence of 0.1 mM dGTP  for 3 hours at 16 °C to remove biotin from unligated restriction fragment ends. The library was then extracted once with 25:24:1 phenol:chloroform:isoamyl alcohol , and once with chloroform . The DNA was precipitated overnight at -20 °C with 0.025 V of 5 mg/mL glycogen , 0.1 V of 3.0 M NaOAc  pH 5.6, and 2.5 V of 100% ethanol. Precipitated DNA was collected by centrifugation. The DNA pellet was washed once with 70% ethanol, air-dried, and dissolved in 20.0 μL nuclease-free water . Hi-C next-generation sequencing (NGS) libraries were prepared using the KAPA HyperPlus Kit  according to instructions260/A280 ratio was not determined since the presence of glycogen in the library preparation interfered with absorbance at these wavelengths. The quality of the 3C libraries, and effectively, their reliability for 3C-qPCR, was determined with an inhibition testing experiment akin to the Purity Assessment98. Details of the yield of 3C libraries and inhibition testing experiments are provided in Supplementary Data The concentration of the 3C libraries was measured with the Qubit™ dsDNA HS Assay Kit  using the Qubit 2.0 fluorometer . The AThe 3C libraries were diluted to a final concentration of 0.2 ng/μL with 10 mM Tris, pH 8.0. The libraries were individually pipetted into a 96-well plate for 3C-qPCR. 3C libraries were stored at 4 °C and placed at room temperature during reaction set-up to avoid freeze-thaw cycles. Library stocks were stored at -20 °C.proVWX operon were designed using the Escherichia coli K-12 MG1655 sequence (Accession number: NC_000913.3 – https://www.ncbi.nlm.nih.gov/nuccore/556503834) on SnapGene® Viewer 5.2. The program was also used for in silico specificity screening. For the fragments of interest for 3C-qPCR test reactions and cross-linking controls, primers and TaqMan probes were designed on opposite DNA strands within 100 bp of the downstream NlaIII site, with TaqMan probes positioned closer to the restriction site than the primers. All primers were designed to anneal to the genomic DNA with the same directionality to ensure that the signal observed during 3C-qPCR assays arises only from re-ligation between the pair of fragments being tested and not due to incomplete restriction digestion. Furthermore, the hybridisation of the TaqMan probe to the non-primed strand of a fragment ensures that the hydrolysis of the probe, and hence, the detection of a fluorescence signal in qPCR only occurs if amplification crosses the ligation junction being tested.Primers and TaqMan probes for 3C-qPCR experiments of the proU operon and their annealing sites on the chromosome are provided in Supplementary Data HPLC-purified, double-quenched TaqMan probes with a 5’ 6-FAM fluorophore, 3’ Iowa Black™ Fluorescence Quencher, and an internal ZEN quencher positioned 9 bases from the fluorophore were ordered as dried pellets from Integrated DNA Technologies, Inc. Primers  were ordered from Integrated DNA Technologies, Inc. or Sigma-Aldrich. The oligonucleotides were dissolved in 1X TE pH 8.0  to a final concentration of 100 μM and stored at 4 °C. The lists of primers and probes used for 3C-qPCR, their positions within the Three sets of control libraries were prepared for 3C-qPCR: digested and randomly re-ligated NT331 genomic DNA, digested and randomly re-ligated NT644 gDNA, and a synthetic control template where chimeric fragments of interest were separately prepared by PCR and pooled in equimolar ratios. The control libraries were serially diluted to prepare the standard samples for 3C-qPCR.99 was performed using the PrimeTime™ Gene Expression Master Mix Kit  using 1 ng of the 3C libraries, corresponding to ~2 × 105 genome equivalents, as the template. A detailed reaction composition is provided in Supplementary Table 3C-qPCRRT- and 3C-qPCR data was processed using the Bio-Rad CFX Manager™ Version 3.1 program (Bio-Rad) and analysed with Microsoft Excel (Microsoft 365). All the raw data files, and the exported.csv files are available as Supplementary material (Supplementary Data 2 > 0.95) and that the range of Cq values spanned by the standard curve encompassed the Cq values of the test reactions as much as possible. The slope, y-intercept, PCR efficiency, and R2 value were extracted from the standard plot.The Cq values of the standard samples were used to plot a standard curve of Cq against the logarithm of the starting quantity of the template. Standard samples with pipetting errors were eliminated from the plot. To verify that quantification was performed in the linear dynamic range, it was ensured that the standard curve was linear (Ravg) and standard deviation of Cq (σCq) for each target. Cqavg was used to determine the relative transcript level of the amplicons in RT-qPCR experiments and the relative interaction frequency in 3C-qPCR. σCq was used to determine the negative and positive errors for the biological samples. The calculations were done using the formula: 10^((Cq-Intercept)/Slope). This processing was applied for all Test amplicons and Internal control candidates . The cell suspension was incubated with 1.0% Sarkosyl, 100 μg/mL RNase A , and 100 μg/mL Proteinase K  at 65 °C until the solution cleared. 400 μL of 4 M NH4OAc was added to the lysate, and the solution was extracted twice with 25:24:1 phenol:chloroform:isoamyl alcohol  and once with chloroform . Genomic DNA was precipitated with an equal volume of isopropanol  for 10 minutes at room temperature and pelleted by centrifuged for 20 minutes at 25000 x g at 4 °C. The pellet was dissolved in 400 μL of 0.1 M NaOAc  pH 6.0, and re-precipitated with 800 μL of cold 100% ethanol for 15 minutes at room temperature. The precipitated DNA was collected by centrifugation at 25000 x g for 15 minutes at 4 °C. The pellet was washed with cold 70% ethanol and air dried. Genomic DNA was dissolved in 1X TE pH 8.0 .An isolated E. coli MG1655 was grown in LB medium at 37 °C with shaking until exponential phase (OD600 = ~0.5). Cells were harvested by centrifugation and flash frozen in liquid nitrogen. The cells were lysed using the RNAsnap protocol130 and total RNA was purified using the Qiagen RNeasy Plus Mini kit . Term-seq library preparation and sequencing was performed by Vertis Biotechnologie AG. Briefly, the 5’ TruSeq Illumina adapter was ligated to 3’ hydroxyl ends of rRNA-depleted RNA followed by first-strand cDNA synthesis using M-MLV reverse transcriptase. First strand cDNA was fragmented and the 3’ TruSeq Illumina adapter was ligated to the 3’ ends of single-strand cDNA. cDNA was amplified with a high-fidelity DNA polymerase and purified with Agencourt AMPure XP beads . Libraries were analysed by capillary electrophoresis, and sequencing was performed on an Illumina NextSeq 500 platform using 1 × 75 bp read length. Term-seq experiments were performed in duplicate.E. coli reference genome U00096.3 (https://www.ebi.ac.uk/ena/browser/view/U00096) using Bowtie2 to give binary alignment map (BAM) files131. To obtain read-depth data for aggregate plots, BAM files were converted to wiggle plots with bam2wig.py133.Due to the method of library preparation, raw Term-seq reads are in the anti-sense orientation. The reads were reverse complemented and aligned to the Escherichia coli BL21 Δhns pLysE pRD18 was cultured in LB medium (1.0% bactotryptone (BD), 0.5% yeast extract (Alfa Aesar), 170 mM NaCl, pH 7.5) at 37 °C to an OD600 of 0.4. Expression of H-NS from pRD18 was induced with a final concentration of 120 μg/mL IPTG for 3 hours. The cells were collected by centrifugation at 7000 x g for 15 minutes at 25 °C. Cell pellets were resuspended in 20 mL low salt H-NS buffer  containing 100 mM NH4Cl with 1 μg/mL DNase, 100 μg/mL lysozyme, and 1 mM PMSF. The cells were lysed by sonication and the soluble fraction was loaded onto a P11 column. H-NS was eluted in low salt H-NS buffer with NH4Cl using a gradient of 100 mM to 1.0 M NH4Cl in 100 mL at a rate of 1 mL/min. Fractions with high concentrations of H-NS were pooled and dialysed overnight at 4 °C into low salt H-NS buffer. The dialysed sample was loaded onto a 1 mL HisTrapTM HP column  and eluted in H-NS buffer  using a gradient of 130 mM to 1.0 M NaCl in 20 mL at a rate of 1 mL/min. The fractions with high H-NS concentrations were pooled and dialysed at 4 °C overnight into low salt H-NS buffer. The H-NS was loaded onto a 1 mL Resource-Q column  and eluted into a highly concentrated and pure peak with a block elution in H-NS buffer from 200 mM to 500 mM NaCl. The purified H-NS was dialysed at 4 °C overnight into H-NS buffer with 300 mM KCl. H-NS was separated into 50.0 μL aliquots, flash-frozen in liquid nitrogen, and stored at -80 °C until use.mut or DREwt . The dilutions were mixed with an equivalent volume of 400 nM DREFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Reporting SummarySource Data