Salmonella Typhimurium genome. In parallel, we have monitored changes in transcription start site use resulting from expression of the regulators. Together, these data allow direct and indirect gene regulatory effects to be disentangled. Promoter architecture across the regulon can also be deduced. At a phylogenetic scale, around one third of regulatory targets are conserved in most organisms encoding MarA, SoxS, Rob or RamA. We focused our attention on the control of csgD, which encodes a transcriptional activator responsible for stimulating production of curli fibres during biofilm formation. We show that expression of csgD is particularly sensitive to SoxS that binds upstream to repress transcription. This differs to the situation in Escherichia coli, where MarA regulates csgD indirectly.The closely related transcription factors MarA, SoxS, Rob and RamA control overlapping stress responses in many enteric bacteria. Furthermore, constitutive expression of such regulators is linked to clinical antibiotic resistance. In this work we have mapped the binding of MarA, SoxS, Rob and RamA across the  Escherichia coli, the multiple antibiotic resistance (mar) locus was identified in a screen for mutations conferring resistance to tetracycline  with 150 mM NaCl and either 2 or 4 mg ml−1 lysozyme. Following incubation at 37 °C for 30 min, the suspension was briefly chilled on ice before sonication with a Bioruptor Plus (Diagenode) for 30 cycles of 30 s on 30 s off at 4 °C. Cell debris was removed by centrifugation at 21 000 g for 5 mins and the supernatant divided into four microfuge tubes and diluted with 800 µl of IP buffer with 150 mM NaCl. One aliquot of the lysate was used per immunoprecipitation. Protein A sepharose beads were washed, then resuspended as a 50 % (v/v) slurry, using 1×TBS. Blunt pipette tips were used in these and all subsequent steps to avoid damaging the beads. For precipitations, 25 µl of Protein A beads, and 2 µl of the appropriate antibody, were added to the aliquot of cell lysate. The cocktails were incubated at room temperature, with constant mixing by inversion, for 90 min.All ChIP-seq experiments follow the procedure described previously and were done in duplicate . An overg for 1 min before resuspending in 700 µl fresh IP buffer with 150 mM NaCl. The mixtures were transferred to Spin-X columns and mixed for 3 min at room temperature. The buffer was removed by centrifuged at 1600 g for 1 min and discarded. After equivalent wash steps with IP buffer with 150 NaCl, and then with 10 mM Tris-HCl pH 7.5 (the latter done twice) DNA fragments were blunted using a quick blunting kit (NEB). Note that these reactions were set up in the Spin-X columns and mixed at room temperature for 30 min without inversion. The beads were then washed twice each with IP buffer containing 150 mM NaCl and 10 mM Tris-HCl pH 8. The next enzymatic step added an ‘A tail’ to each DNA fragment using the Klenow fragment (3′>5′ exo-). The reactions were incubated at 37 °C for 30 min with rotation but not inversion. Further washes (twice each) were done using IP buffer having 150 mM NaCl and 10 mM Tris-HCl pH 7.5. The final enzymatic step used ligation to add NEXTflex barcoded adaptors (BioOscientific) to the DNA fragments. Beads were then washed twice in IP buffer with 150 mM NaCl. Single wash steps were then done with IP buffer containing 500 mM NaCl, ChIP wash buffer  and TE. DNA was eluted by transferring the Spin-X column basket to a fresh dolphin-nosed tube and incubated at 65 °C for 10 min in 100 µl ChIP elution buffer . Following incubation, reactions were transferred to a centrifuge and the eluate collected. Samples were then de-crosslinked by boiling for 10 min.After immunoprecipitation, Protein A beads were collected by centrifugation at 1600 Prior to library amplification, DNA fragments were subjected to a 1.1×volume Agencourt AMPure XP bead clean up and eluted in 13 µl ddH2O. Next, 2 µl of the library was used in a qPCR reaction to determine the number of amplification cycles needed to maximized library amplification but minimised both NEXTflex barcode adapter and PCR primer dimers. Amplified libraries were diluted to 200 µl with ddH2O and subjected to a 0.7×AMPure XP bead clean up before imaging on an Agilent TapeStation 2200 (Agilent). Library concentration was quantified using an NEBNext Library Quant Kit (NEB) then adjusted to between 0.5 and 2 nM. Samples were then pooled and sequenced using an Illumina MiSeq. Sequence reads are accessible in ArrayExpress (accession number E-MTAB-12627).S. Typhimurium SL1344 chromosome (NC_016810.1) or plasmids pCol1B9 (NC_017718.1), pRSF1010 (NC_017719.1) and pSLT (NC_017720.1) using Bowtie 2 for Illumina [blastn and used to search the genomes of the strains shown in Fig. 2(a) as described previously [Bioinformatic analysis of ChIP-seq data was done as described previously . Raw FASIllumina . The resIllumina . To seleS. Typhimurium SL1344 carrying pAMNF or pAMNM derivatives encoding epitope tagged derivatives of MarA, SoxS or RamA. Cells were grown to mid-log phase in LB medium. RNA extraction was done using the SV Total RNA Isolation System (Promega). Sequence reads are accessible in ArrayExpress (accession number E-MTAB-12628). As a control, we used cappable-seq data for S. Typhimurium SL1344 carrying empty pAMNF (E-MTAB-12506) [This method exploits the ability of vaccinia capping enzyme to specifically modify triphosphorylated RNA 5′ ends with biotinylated GTP. This allows primary unprocessed transcripts  to be isolated using streptavidin beads . CappablB-12506) .S. Typhimurium SL1344 reference genome  using Bowtie2 and SAMtools (version 1.3.1). Transcription start sites were identified using the software of Ettwiller et al. [Sequencing reads were mapped to the r et al. . Brieflyr et al. . DNA seqr et al. . When caS. Typhimurium MarA, SoxS, Rob or RamA were cloned in pET28a and His6 tagged variants overexpressed in E. coli T7 Express cells. Purification was as described by Kettles et al. [−1 using vivaspin columns and stored at −20 °C.Genes encoding s et al. . Purifie−1 of Congo red. The agar plates were then incubated at 37 °C overnight. The morphology and colour of colonies were recorded by digital photography. The experiments were done at least three times to check that colony phenotypes were reproducible, and images shown are representative. The crystal violet assay described by Baugh et al. [600 of 0.1. A 200 µl aliquot was added to a flat-bottomed 96-well microtitre plate, with four replicate wells per culture. The plate was incubated at 30 °C for 48 h. Wells were washed with water to remove unattached cells and 200 µl of 0.1 % (w/v) crystal violet was added for 15 min. Wells were then washed with water again to remove unbound crystal violet and 200 µl of 70 % ethanol was added to solubilise the retained crystal violet. The A600 was then measured using a CLARIOstar plate reader (BMG Labtech) to give a quantitative measure of biofilm formation.For Congo red binding assays, strains were cultured overnight in LB lacking salt. The next day, 5 µl of the culture was spotted onto LB agar lacking salt and supplemented with 40 µg mlh et al.  was usedS. Typhimurium SL1344 genome consists of a 4 878 012 bp chromosome and three plasmids  [E. coli genome [acrZ gene regulatory region was a target for all four factors, whilst ypeC and yhcC each had binding peaks for only one factor.The nd pSLT) . We usednd pSLT) . The ovex-axis) [y-axis lists different binding targets with gene names coloured according to their function. Overall, genes adjacent to binding targets for MarA, SoxS, Rob and RamA, are most likely to encode factors involved in cell envelope biology (purple), gene regulation  or metabolism (grey). Genes with other functions are labelled with cyan text. Where gene and species names intersect, the heatmap cell is coloured according to conservation of the identified binding site. Around one third of the binding targets are conserved in almost all genomes examined . The most common reason for a binding target not being detected in another species is that a sequence aligning with the equivalent S. Typhimurium regulatory DNA is not present (white squares). For instance, the dppA gene is found in many enteric bacteria but the upstream regulatory DNA often has a drastically different sequence. Comparatively, it was rare for an equivalent DNA region to be found but the binding site absent (grey squares). S. Typhimurium specific genes, are least likely to be conserved. We also compared binding site conservation data for essential and non-essential genes [We next turned our attention to understanding the conservation of binding targets and the function of adjacent genes. x-axis) . The y-aal genes  Fig. 2cx-axis [yfpr, gshB and acrAB, respectively.Whilst ChIP-seq captures DNA binding events, consequences for transcription from nearby promoters are not determined. Hence, we used cappable-seq to better understand regulatory outcomes associated with expression of the MarA, SoxS or RamA fusions . Note that, whilst standard RNA-seq maps overall transcript abundance, cappable-seq targets the RNA 5′ end. Consequently, as well as providing a measure of transcriptional activity, cappable-seq identifies transcription start sites (TSSs) . Fig. 3. Whilst promoters dependent on σ70 can often also be used by σ38, during periods of starvation, we did not identify any motifs associated with binding of alternative σ factors.We next focused our attention on understanding the properties of promoters targeted by SoxS, MarA or RamA. As a starting point, we made an inventory of regulatory regions where the binding site identified by ChIP-seq could be unambiguously assigned to a TSS identified by cappable-seq. Hence, we excluded regulatory regions with multiple TSSs unless the TSS subject to regulation was clear . Similarly, to minimize erroneous designations, we excluded regions with either no, or multiple, potential binding sites for SoxS, MarA or RamA. Taking this cautious approach, we allocated TSSs to 36 of the binding sites identified by ChIP-seq. The regulatory region sequences are shown in Fig. S1, with TSSs (green) and binding motifs for SoxS, MarA or RamA (blue) highlighted. We searched DNA sequences upstream of the TSSs for motifs indicative of RNA polymerase σ factor specificity. In all cases, we identified appropriately positioned DNA elements upstream of transcription start sites. Universally, these matched the sequences recognized by the house keeping σE. coli promoters activated by MarA have previously been divided into two classes [70 binding. Targets for SoxS and RamA conform to the same organizational rules [yadG promoter is bound, and activated, by MarA . Wild-type cells carrying empty pAMNF, form red colonies with a nonuniform rough/wrinkled surface Ectopic expression of SoxS from plasmid pAMNF resulted in pale colonies with a uniformly smooth surface, consistent with repression of csgD by SoxS. We also monitored biofilm formation directly by staining with crystal violet. Briefly, in these assays, liquid cultures are incubated overnight in a polystyrene microtitre plate. The next day, after removing planktonic microbes, cells attached to the solid surface can be detected by crystal violet staining. The results are shown in CsgD is required for the production of curli fibres and for biofilm formation. Production of curli can be monitored using Congo red dye that binds the fibres. S. Typhimurium MarA, SoxS, Rob and RamA targets identified here, 34 map to promoters where the binding site sequence is conserved in at least 75 % of organisms encoding a MarA-like protein  and RNA polymerase, revealed displacement of σ70 from the promoter −35 element [E. coli, the marbox at the ycgZ-ymgABC promoter is in a class I position but the reverse orientation [zwf promoter does not match the rules for class I or class II organization [Overall, half of the MarA, SoxS and RamA targets to which we could assign a TSS could be designated as class I or class II promoters . With re element . By cont element . For insentation . Similarnization .S. Typhimurium csgDEFG regulatory region cannot be classified according to conventional class I or class II rules; SoxS binds 294 bp upstream of the csgD P1 TSS to repress transcription  upon expression of MarA or SoxS, rather than de novo biofilm formation.The cription . Given tormation . Previouoduction . BrieflyClick here for additional data file.


Mytilus coruscus, and the findings demonstrated that McNrf2 effectively protected the mussels against oxidative stress induced by benzopyrene (Bap). In order to delve deeper into the underlying mechanism, we utilized Chromatin Immunoprecipitation followed by sequencing (ChIP-seq) technology to systematically identify potential novel target genes of McNrf2. A total of 3,465 potential target genes were screened, of which 219 owned binding sites located within the promoter region. During subsequent experimental verification, it was found that McSLC35E2, a candidate target gene of McNrf2, exhibited negative regulation by McNrf2, as confirmed through dual luciferase and qRT-PCR detection. Further, the enzyme activity tests demonstrated that McNrf2 could counteract Bap induced oxidative stress by inhibiting McSLC35E2. The current study provides valuable insights into the application of ChIP-seq technology in the research of marine mollusks, advancing our understanding of the key role of Nrf2 in antioxidant defense mechanisms, and highlighting the significance of SLC35E2 in the highly sophisticated regulation of oxidative stress response in marine invertebrates.NF-E2-related factor 2 (Nrf2) plays a crucial role in the oxidative regulatory process, which could trigger hundreds of antioxidant elements to confront xenobiotics. In the previous study, we identified Nrf2 from the marine mussel  Mytilus coruscus has gradually developed into a model organism for studying marine invertebrates responses to environmental changes, including natural influences such as temperature rise and acidification, as well as environmental pollution from organic and inorganic substances (M. coruscus to polycyclic aromatic hydrocarbons (PAHs) pollution, particularly on its member benzo(a)pyrene (Bap). Bap has been proven to cause severe harm to marine organisms, including immune system disruption, metabolic inhibition, induction of mutagenic reactions, and tissue damage  genes bind to HSF1. This research unveiling the application of ChIP-seq technology in marine mollusks.Understanding transcriptional regulation is essential to comprehending the gene regulatory networks behind various cellular pathways and processes. Accurate mapping of transcription factor binding sites (TFBS) on a genome-wide scale can provide invaluable insights into gene regulation. Protein-DNA interactions are key to this mapping process and an extensive genome-wide map of interaction data is necessary to build meaningful models of TFBS . ChromatMcNrf2, followed by subsequent experimental validation. We revealed for the first time that solute carrier family 35 member E2 (SLC35E2) functions as a target gene for McNrf2, which is demonstrated by the binding of McNrf2 to the promoter region of McSLC35E2. Subsequent dual-luciferase and qRT-PCR assays further confirmed this fact. Further, the enzyme activity tests determined that McNrf2 could target McSLC35E2 to antagonize Bap induced oxidative stress. The current study provides valuable insights into the application of ChIP-seq technology in the research of marine mollusks. Moreover, the research findings have advanced our understanding of the key role of Nrf2 in antioxidant defense mechanisms and highlights the significance of SLC35E2 in the highly sophisticated regulation of oxidative stress response in marine invertebrates.In the present study, we employed ChIP-seq assay to comprehensively screen for potential novel target genes of M. coruscus mussels were obtained from Donghe Market, Zhoushan City, Zhejiang Province. These mussels were acclimated in a tank at a temperature of 20°C for 1 week. The seawater used had a salinity of 30% ± 1% and a pH of 8.0 ± 0.3. The seawater was renewed every 2 days, and the mussels were fed with Spirulina powder on a daily basis.A total of 200 healthy g for 10 min, the cells were added 10 mL of glycine termination solution with a 5 min centrifugation at 300 × g, 4°C. The cells were then washed twice with phosphate buffer containing 1 mM PMSF to remove any remaining formaldehyde. After washing, 1 mL of lysis buffer was added, lysed on ice for 30 min, followed by a cells collection by centrifugation at 5,000 × g, 4°C for 10 min. Next, 350 μL of pre-warmed digestion buffer was added, and the mixture was incubated at 37°C for 5 min. The cut chromatin was separated, and 10 μL of input DNA was labeled and kept as a control for ChIP samples. The ChIP reaction system was prepared and then incubated overnight at 4°C on a rotating shaker. Subsequently, the magnetic beads were washed, and the chromatin was eluted. Uncross linking and proteinase K treatment were performed afterward. Finally, the resulting DNA was purified, and the detailed steps were described in the Magnetic Chromatin Immunoprecipitation Kit .The digestive gland cells of mussels were extracted and 20 mL formaldehyde fixative was added to make the final concentration 1%. After incubation at room temperature on a 100 × ChIP-seq libraries were generated following the Illumina ChIP-seq library construction protocol. The chip DNA was fragmented into fragments of approximately 200 bp in length. These DNA fragments then underwent end repair and A-tailing processes. Subsequently, adaptor ligation was performed to attach sequencing adaptors to the DNA fragments. To ensure high-quality libraries, the quality assessment of DNA library products was conducted using the Agilent 2200 TapeStation  and Qubit . Subsequently, the libraries were subjected to pair-end 150 bp sequencing on the Illumina platform  at Ribobio Co., Ltd. .M. coruscus genome (unpublished) (p < 0.05 were considered to be significant.The raw fastq sequences were processed using Trimmomatic tools (v0.36) with the following options: TRAILING: 20, SLIDINGWINDOW: 4:15, MINLEN: 52. This process was performed to eliminate trailing sequences with a phred quality score below 20 and to obtain uniform sequence lengths for subsequent clustering procedures . The genblished) . Subsequblished) . Then usblished) . Motif cblished) . Kyoto EMcNrf2 with a candidate target gene McSLC35E2, dual luciferase assays were performed using the Dual-Glo® Luciferase Assay System . The experimental procedure followed the instructions provided by the manufacturer. The ChIP-seq data revealed the enrichment of the potential binding site, i.e., the region of the SLC35E2 promoter where Nrf2 is capable of binding. The region approximately 1 kb upstream of the SLC35E2 gene was cloned into the pGL3-control luciferase reporter plasmid. Additionally, the Nrf2 fragment was cloned for insertion into the pcDNA3.1 plasmid. After cloning, the recombinant plasmids were transfected into the recipient cells, and subsequent extraction was carried out for sequencing verification. Plasmids that underwent successful sequencing were co-transfected into cells. Then, the activity of the reporter gene was assessed using the Dual-Luciferase Reporter Assay System. The fluorescence signals for both Firefly and Renilla luciferase were captured using the Varioskan Flash Multimode Reader from Thermo Fisher Scientific . The recorded fluorescence values for each experimental group were then utilized to evaluate the regulatory effect of Nrf2.To validate the relationship between M. coruscus were collected at 24 h. Three individuals were selected from each group for sampling. Total RNA was extracted using the RNA extraction kit from Solarbio , followed by a reverse transcription using cDNA synthesis kit . The housekeeping gene β-actin was employed as a control gene in our study. The qRT-PCR was conducted utilizing the SYBR Green Real-Time PCR Mix  on a ABI 7500 Fast Real-Time PCR System  and. Data analysis was carried out using the 2−ΔΔCT method , ML385 -1H-indol-5-yl]-5-methyl-2-thiazolyl]-1,3-benzodioxole-5-acetamide), and PBS were administered via injection. Digestive gland tissues of T method . The priMcNrf2 and McSLC35E2, which were prepared in our laboratory, were collected and their final concentration was diluted to 300 ng/μL. M. coruscus individuals (Net weight: 19.8 ± 0.3 g) were randomly divided into 5 groups, each consisting of 6 mussels. The adductor muscle of each mussel was injected with either 100 µL McNrf2, McSLC35E2, or 200 µL of both McNrf2 and McSLC35E2. After injection, the individuals were exposed to Bap separately. Subsequently, the reactive oxygen species (ROS) production and total antioxidant capacity (T-AOC) were detected by using kits .The eukaryotic expression recombinant plasmids of t-test was utilized. Data with more than two sets were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s multiple range test for post hoc comparisons. Statistical significance was considered for probabilities of p < 0.05.All data were analyzed using SPSS 27.0 software . The results were presented as mean ± SD. Before conducting statistical analysis, normality tests and tests for homogeneity of variances were performed. For comparing two sets of data, the M. coruscus genome. The statistical analysis revealed a total of 3,465 peaks, with an average peak length of 383.88 bp and a median peak length of 311 bp. The majority of peak lengths clustered around 200–300 bp (The raw data has been uploaded to the GEO database with the accession number GSE242277. The raw data from the sample (Nrf2 chip) and control (Nrf2 input) groups were 5.96 billion bp and 5.75 billion bp, respectively. After removing low-quality bases or filtering for valid data . The qua0–300 bp . Annotat0–300 bp . Among a0–300 bp . The maj0–300 bp .M. coruscus. In total, 3,465 peaks were identified as potential binding sites, and among them, 89.24% of the peaks were successfully annotated to the nearest gene. Out of all the peaks, 219 were localized within the promoter zone. Gene functions were established through the utilization of information sourced from diverse databases, notably Swiss-Prot, Interpro, TrEMBL, and KEGG databases. A KEGG enrichment analysis was conducted on a total of 3,465 screened peaks, revealing significant signaling pathways linked to Nrf2 target genes, which included Phosphatidylinositol signaling system (map04070), cAMP signaling pathway (map04024), and Peroxisome (map04146)  . The anaap04146) . Bindingut group .Transcription factors’ DNA binding sites often exist as conserved short sequence fragments. Therefore, motif analysis of the ChIP-seq results aids in analyzing the recognition pattern of transcription factors on DNA sequences. Predictions were screened to assess the potential binding of Nrf2 to the identified motif .p < 0.05)    . This in < 0.05) . In cont < 0.05) .McNrf2 and McSLC35E2 plasmids showed differences in ROS levels after injection and Bap exposure. As depicted in p < 0.05). However, when Bap was exposed and Nrf2 was overexpressed, the level of ROS was reduced (p < 0.05). On the other hand, elevating the level of SLC35E2 was associated with increased ROS levels (p < 0.05). Nevertheless, when both Nrf2 and SLC35E2 were overexpressed, the level of ROS was lower than when only SLC35E2 was overexpressed (p < 0.05). After injection, there was a notable difference in T-AOC levels in the digestive gland, as depicted in p < 0.05). Moreover, T-AOC was decreased in the SLC35E2 overexpression group compared to the Nrf2 overexpression group (p < 0.05). However, when both Nrf2 and SLC35E2 were overexpressed, T-AOC levels were elevated compared to SLC35E2 overexpression (p < 0.05).Crassostrea gigas. To our knowledge, this is the first application of this technology in marine mollusks. In this study, the researchers employed ChIP-seq technique to scan genes regulated by HSF1. The sequencing yielded a set of unique reads, with a 34.2% match rate to the C. gigas genome. Ultimately, a total of 916 peaks corresponding to HSF1 binding sites were identified, of which 6% were located in the TSS region, and a subset of HSP genes displayed a direct binding to HSF1. In the present study, unique reads showed a higher genome matching degree (79.44%), indicating high sequencing quality. Statistical analysis revealed a total of 3,465 peaks corresponding to Nrf2 binding sites, 7.24% of which were located at TSS region, and most of the peaks were located at intergenic regions and introns. Our results aligned with the results of a prior investigation, that Nrf2-ChIP-seq data from A549 cells also revealed an approximately 7% gene binding sites on the TSS promoter , NAD(P)H, quinone oxidoreductase 1 (NQO-1), UDP-glucuronosyltransferase (UGT), and hemeoxygenase-1 (HO-1), in hepatic pathophysiology. They found that these genes play complex and multifaceted roles in liver inflammation, fibrosis, and hepatocarcinogenesis. Nrf2 plays a positive role in the equilibrium state, however, the imbalance of Nrf2 and its target gene expression will inflict severe damage upon the organism. NQO1 and B-cell lymphoma-2 (BCL-2), induced dysplasia of cell proliferation and apoptosis, and were associated with malignant transformation of human bronchial epithelial cells induced by arsenite. Indeed, the most important role of Nrf2 target genes lies in their contribution to the antioxidant defense system and their ability to mitigate oxidative damage. Nrf2 target genes ensure cell integrity and overall health in the face of oxidative challenges by upregulating antioxidant enzymes and detoxifying proteins (proteins . UnfortuMcNrf2 with binding sites located within the promoter region, and the enriched peaks corresponding to the putative binding sites of SLC35E2 and Nrf2 were identified by comparing the sequence reads with and without Nrf2 antibody treatment. Laboratory experiments including the dual luciferase and qRT-PCR assays were employed to verify the in silico prediction. Dual luciferase assay showed that compared with McSLC35E2 alone, the luciferase activity in the Nrf2 supplemental group was lower, indicating that McNrf2 could target McSLC35E2 and was negatively correlated. The qRT-PCR further confirmed this fact, that the transcriptional expression of McSLC35E2 was activated by Nrf2 inhibitor ML385 while inhibited by Nrf2 agonist SFN. These results suggested that McNrf2 may be involved in the regulation of physiological processes in M. coruscus mussels by inhibiting McSLC35E2.In the present study, ChIP-seq scanned a total of 219 candidate target genes of in vivo using a mouse tumor model (McNrf2 in Bap-induced antioxidant effects through its targeting of McSLC35E2, enzyme activities were determined in the digestive glands injected with the recombinant plasmids of McNrf2 and McSLC35E2 under or non-Bap.Members of the SLC family play a crucial role in human physiology as transporters that facilitate the transportation of hydrophilic compounds into and out of cells and subcellular organelles. For instance, SLC30 and SLC39 transport zinc, SLC11 and SLC40 transport iron, and SLC19 transport folic acid and thiamine . In addior model . In addior model . In summM. coruscus exposed to Bap were significantly increased in the present study compared with the NC group, indicating that the Bap burst caused severe oxidative stress to the mussels. Overexpression of McNrf2 leads to a significant reduction in ROS production, on the contrary, a significant increase in T-AOC levels is observed. Similar results were found in zebrafish studies, where McSLC35E2 is overexpressed, ROS production is significantly increased and T-AOC is significantly decreased. This result demonstrated that McSLC35E2 may exacerbate oxidative damage, leading to increased oxidative stress in mussels. However, when McNrf2 and McSLC35E2 were both overexpressed, the situation is exactly the opposite, suggesting that this two had antagonistic effects on the oxidative stress induced by Bap in mussels.Bap, being one of the most toxic types of PAHs, has been extensively characterized toxicologically . The detMcNrf2 in M. coruscus. After comprehensive genome-wide survey, 3,465 candidate target genes of McNrf2 were scanned, of which 219 owned binding sites located within the promoter region. Following, a typical target gene termed McSLC35E2 was selected to perform the experimental verification. Specifically, the targeting of McNrf2 to McSLC35E2 was successfully verified using both dual luciferase and qRT-PCR assay. In order to investigate the involvement of McNrf2 in Bap-induced antioxidant effects through its targeting of McSLC35E2, enzyme activities were determined in the digestive glands injected with the recombinant plasmids of McNrf2 and McSLC35E2 under or non-Bap. The results revealed that McNrf2 could participate in the anti-Bap oxidative stress process by inhibiting McSLC35E2. Overall, these findings lay the groundwork for applying ChIP-seq technology in mollusks, opening up new avenues for understanding the function of Nrf2 in the antioxidant defense system of marine mollusks. The study contributes valuable knowledge that may have implications for future research on environmental responses and stress adaptation in mollusks.In this study, ChIP-seq technique was employed to identify new target genes of


Three-dimensional (3D) genomics is an emerging field of research that investigates the relationship between gene regulatory function and the spatial structure of chromatin. Chromatin folding can be studied using chromosome conformation capture (3C) technology and 3C-based derivative sequencing technologies, including chromosome conformation capture-on-chip (4C), chromosome conformation capture carbon copy (5C), and high-throughput chromosome conformation capture (Hi-C), which allow scientists to capture 3D conformations from a single site to the entire genome. A comprehensive analysis of the relationships between various regulatory components and gene function also requires the integration of multi-omics data such as genomics, transcriptomics, and epigenomics. 3D genome folding is involved in immune cell differentiation, activation, and dysfunction and participates in a wide range of diseases, including autoimmune diseases. We describe hierarchical 3D chromatin organization in this review and conclude with characteristics of C-techniques and multi-omics applications of the 3D genome. In addition, we describe the relationship between 3D genome structure and the differentiation and maturation of immune cells and address how changes in chromosome folding contribute to autoimmune diseases. As we khromatin . In 2002hromatin  to studyhromatin , chromoshromatin , high-thhromatin , and chrhromatin . The effhromatin . For exahromatin . Chen ethromatin  investigIRF8 expression by spatially interacting with the IRF8 promoter and influencing methylation levels. DNA methylation, histone post-translational modification, and non-coding RNAs have been extensively explored at the epigenetic level in many autoimmune diseases  have made significant advancements in identifying variations associated with complicated genetic disorders, including autoimmune diseases . Single-diseases , 22. DesThis review summarized current concepts in 3D genome organization and multi-omics research techniques and applications of the 3D genome. We focus on how the 3D organization of genomes has an impact on the feature of immune cells of innate and adaptive immunity. Furthermore, we discuss how variants in non-coding regions, as well as epigenetic changes, might disrupt spatial genomic folding and generate an abnormal state of autoimmune diseases, as well as the functional consequences of autoimmune diseaseassociated variants and chromosomal rearrangements. Furthermore, following changes in gene expression and cellular function regulation throughout disease progression may give clues for developing new biomarkers and designing focused therapy regions.Chromosomes are not randomly distributed within the nucleus but tend to occupy distinct, non-overlapping regions known as chromosome territory (CT), representing the highest level of chromatin organization . Chromosin situ hybridization (FISH), which makes it possible to visualize the spatial length between two or more loci by hybridizing fluorescently labeled probes to DNA after fixation and moderate denaturation  is a novel technique that constructs a sequencing library by Tn5 transposase with reduced starting material  technique, a 3C or Hi-C library is subjected to capture by hybridization with pools of DNA or RNA oligos to concentrate ligation products that fit specific sites , 58. Witmaterial Table 1.cis-regulatory elements and trans-acting factors can physically interact with chromatinized DNA is referred to as chromatin accessibility   , DNase Hory DNA) , HiCoP (ory DNA) , and Oceory DNA) , have beTo accurately assess the interrelationships between different regulatory components and gene function during cell development and differentiation, the integration of multi-omics data to define the biological effects of associated genes, transcriptional regulatory elements, and other biomolecules involved in the transcriptional regulation of specific genes is required. Numerous loci have been identified as statistically associated with various diseases and traits based on GWASs. However, because more than 80% of disease-associated variants are found in non-coding areas, it is difficult to ascertain the biological mechanism behind a genetic risk factor . GeneralDifferent types of cells make up the immune system with specialized functions, concluding rapidly responding innate immune cells and highly specialized lymphocytes . It is nB-cell receptors (BCRs), consisting of pairs of immunoglobulin heavy (IgH) and immunoglobulin light (IgL) chains, and T-cell receptors (TCRs), composed of either an α and β chain or a γ and δ chain, confer the ability to detect foreign antigens . The antEbf1) is sequestered in the nuclear lamina of progenitor cells switching between an inactive B compartment and an active A compartment. By developing new intradomain and interdomain interactions at the pro-B- cell stage, B-lineage transcriptional signatures are developed . ThymoD transcription stimulates CpG residue demethylation and enhances the recruitment of cohesin, bringing the Bcl11b promoter and enhancer together into a loop domain , resulting in naïve CD4 T cells . BCL11B  T cells . Similarp domain . Recentlp domain . Taken t+ T cells are at the center of the adaptive immune system, which is phenotypically specialized in various ways by cytokine stimulation, for example, Th1, Th2, and Th17 cells. T-bet TF regulates the differentiation of naïve CD4+ T helper lymphocytes by increasing transcription of Th1-specific genes (such as IFN-γ) and suppressing transcription of Th2-specific genes (such as IL-4)  TFs across the genome. STAT4-deficient cells are unable to produce Th1-selective Ifng contact due to the prevented 3D genome reorganization  . 3C expene locus . Th1 andnization . The basnization . Naïve Bnization . It appenization .In summary, the activation of lymphocytes is followed by comprehensive, multistep 3D genome organization dynamics that enable cell proliferation and the formation of an inflammatory transcription program.TNFAIP3) hindered TF binding and disrupted 3D chromatin folding in human CD4+ T cells  and T- cell receptor locus  binds NF-κB with a greater affinity and is more accessible that the risk allele (T), leading to an increase in miR-146a production in monocytes through a special chromosomal 3D structure proved by 4C-seq. It has been determined how miR-146a controls the inflammatory response in SLE . Differevia a chromatin loop not only with TNFAIP3 but also with the more distant IL20RA instead of OLIG3 in B and T cells. Furthermore, there are more interactions between enhancer and IL20RA in patients with the risk G allele of the SNP rs6927172 compared to those with the non-risk allele  are crucial for the development of RA, there are few functional genetics databases for these cells. FLS samples from patients with RA were studied to characterize 3D chromatin interactions, DNA accessibility, and gene expression . EpigeneIL22RA2, IFNGR1, and TNFAIP3, suggesting that these variants could play a role in regulating numerous immune pathways , which is characterized by persistent demyelination, oligodendrocyte mortality, and loss of axons and neurons, is an excellent illustration of an autoimmune disorder driven by abnormal activation of tissue-derived immune cells . Some chpathways . SNPs at T cells . The fun T cells . SOCS1 p T cells . Neverthammation .Even though diabetes is regarded as a metabolic disorder, exogenous insulin dependency is typically caused by the autoimmune destruction of insulin-producing cells of the pancreas in type 1 diabetes (T1D). GWASs have found over 60 loci associated with T1D across the human genome . To iden+ T cells are one of the principal drivers of pathogenesis in systemic sclerosis (SSc), a highly complex autoimmune disease regulated by the interaction of hereditary and epigenetic factors , GSDMB (rs9303277), IL12RB1 (rs2305743), and CSK (rs1378942), which potentially interact with DMP-DEG  pairs cg17239269–ANXA6, cg19458020–CCR7, cg10808810–JUND, and cg11062629–ULK3, respectively (+ cells from SSc patients, which may be a result of abnormal overexpression of the CTCF gene (IRF5. ZBTB3, a putative structural regulator, mediates the formation of site-specific chromatin loops in the chromosomal rs4728142 locus. The presence of the rs4728142-A risk allele facilitated the tight binding of ZBTB3 and regulated the upregulation of IRF5-induced genes related to autoimmunity. This process leads to the polarization of M1 macrophages, contributing to the occurrence and development of autoimmune diseases by improving the inflammatory and immune response (CD4 factors . DNA metectively . MoreoveTCF gene . CTCF reTCF gene . TakayasSK rs13782, which TCF gene . Based oTCF gene . FollowiTCF gene . In CD pTCF gene . By perfresponse  (Table 2As previously mentioned, the application of 3D genomics has significantly advanced our knowledge of the genetics of lymphocyte development and the etiology of autoimmune diseases. The 3D genome can help to determine the functional effect of disease genetics on the associated target genes, involving the gene expression and regulatory networks or pathways, which provides a great grasp of the mechanisms of spatial conformational changes in chromatin, transcriptional regulation, and the generation of biological features. Our previous research identified the affecting gene regulatory elements, such as active enhancers and promoters within the genome loops, revealing the new mechanisms of SLE from the 3D genome landscape .GWASs are based on the assumption that common diseases are often influenced by a large number of genetic variants. After analyzing information from Hi-C sequencing, histone markers, and chromatin accessibility, some risk-associated SNPs were found to be located in the promoter or enhancer region. Risk variants can affect gene expression by interacting with distal genomic regions through 3D chromatin folding. These interactions can bring the regulatory elements closer to the target gene and increase or decrease its expression levels, depending on the nature of the interaction and the specific regulatory elements involved. However, it is especially challenging to demonstrate the functions of enhancers through experiments. Through targeting genomic deletions and mutations, CRISPR/Cas9 and CRISPR-based approaches, such as CRISPRi and CRISPRa, were used to identify the function of potential regulatory regions coupled with transcriptional quantification or RNA-seq. The integration of GWASs and omics data allows the elucidation of the function of individual risk variants and the identification of disease and tissue specificity. This helps to elucidate the detailed pathogenesis process of autoimmune diseases and thus leads to precise diagnosis and treatment.Although 3C and its derivatives are unquestionably powerful tools for structural and functional genomics research, those technologies still face challenges on several fronts, such as difficulties in detecting weak interactions between chromatin, long experimental cycles that do not allow for a rapid and realistic response to interactions and a low ratio of signal to noise. Moreover, current visualization tools are incapable of integrating genomic information across the interaction intensity, spatial conformation, and gene synergy, as 3D genome data indicate the spatial composition and functional interconnections in multiple dimensions. The spatial conformation of the genome in the nucleus varies with time in order to sustain proper cell growth and development. As the 4D nucleosome project proceeds, it will be feasible to map the spatial and temporal organization and dynamics of the genomic structure of humans . There aYQ, QL, and MZ made substantial contributions to the study’s conception and design. All authors drafted the article or revised it critically for important intellectual content. All authors gave their final approval of the version to be submitted and any revised versions.


Epstein–Barr Virus (EBV) is a human gamma-herpesvirus that is widespread worldwide. To this day, about 200,000 cancer cases per year are attributed to EBV infection. EBV is capable of infecting both B cells and epithelial cells. Upon entry, viral DNA reaches the nucleus and undergoes a process of circularization and chromatinization and establishes a latent lifelong infection in host cells. There are different types of latency all characterized by different expressions of latent viral genes correlated with a different three-dimensional architecture of the viral genome. There are multiple factors involved in the regulation and maintenance of this three-dimensional organization, such as CTCF, PARP1, MYC and Nuclear Lamina, emphasizing its central role in latency maintenance. Epstein–Barr Virus, or EBV, is a human gammaherpesvirus that infects 95% of the population worldwide ,2,3. EBVIn latently infected cells, EBV expresses a limited set of viral proteins: six Epstein–Barr Nuclear Antigens (EBNAs); three integral membrane proteins called Latency Membrane Protein (LMP1 and LMP2a and b); two small non-polyadenylated RNAs (EBERs); and several miRNAs generated from the BamHI-A Rightwards Transcripts (BARTs) . EBV expEBV can adopt at least three different latency types in latently infected cells . In TypeEpigenetic regulation of EBV latency programs. Changes in the viral promoter that the virus uses determine which latency state the EBV-infected B cells adopt ,40. EBNAFurther studies determined that the EBV genome was highly methylated, restricting viral genes expression in both B and epithelial cells . SubsequThe notion that each latency type is characterized by a specific epigenetic landscape across the EBV genome is further supported by early studies demonstrating that the deposition of different patterns of histone modifications correlates with different EBV latency types ,56,57. TSystemic mapping of chromatin has shown that the epigenome is organized into distinct domains of transcriptionally active and inactive regions . These dIn recent years, several studies showed the role of CTCF in the context of EBV infection. CTCF has been shown to bind to the EBV genome during latency ,55,69,70Furthermore, several studies have revealed that CTCF can regulate viral gene expression in other double-stranded DNA viruses. For instance, in Kaposi’s sarcoma herpesvirus (KSHV) ,80,81,82CTCF also plays a critical role in regulating gene expression by influencing the three-dimensional structure of chromatin and promoting or inhibiting interactions between enhancer and promoter gene regions . RecentlThe role of CTCF and Cohesin in regulating the chromatin architecture of the genome in higher eukaryotes prompted similar studies to determine the 3D structure of the EBV genome in latently infected cells. Earlier studies focusing on the 3D structure of the Cp and Qp regions of the EBV genome demonstrated that these regions adopt alternative 3D chromatin structures between latency types  Figure . For exaMost recently, studies employing EBV-specific Capture-HiC assay revealed the chromatin architecture of the EBV genome in Type I and Type III EBV+ B cells  24]. Th. Th24]. Between latency types, the EBV genome can therefore assume alternative 3D structures, indicating that the chromatin architecture provides an additional layer of epigenetic regulation for EBV gene expression during latency. Genetic studies show that CTCF is essential for forming chromatin loops, and EBV chromatin loops occur between viral regions occupied by CTCF. Yet, the CTCF binding profile across the EBV genome is similar between Type I and Type III EBV+ cells . Most reIn eukaryotes, an additional level of epigenetic regulation that can influence the chromatin structure is the interaction of genomic regions with the Nuclear Lamina (NL). The Nuclear Lamina is formed by proteins named lamins. They are grouped into two different families (type A and B lamins) that are located at the nuclear peripheral space. The nuclear lamina–genome interactions occur at chromatin regions called Lamin-Associated Domains (LADs) that when located at the nuclear periphery are associated with different repressive histone marks, such as H3K9me2/3 and H3K27me3 ,114,115,In the context of herpesvirus biology, the nuclear lamina has been reported to play many different roles in regulating different stages of viral infection. To be specific, the nuclear lamina can control viral replication by acting as a physical barrier that prevents the viral capsid egress by inducing the expression of lytic genes that target the nuclear lamina itself. For example, EBV lytic gene BGLF4 encodes a viral kinase that phosphorylates lamina A causing its degradation . AlternaThe nuclear lamina has been also reported to play a role in EBV latency . LADs weLamin B1 binding is conserved in both latency types at both the origins of lytic replication OriLyt Left and RMPS1-OriLyt, which are regions inactive during latency , indicatThe expression of Lamin A/C was also involved in the modulation of expression of both latent and lytic genes, suggesting a role of lamin A/C in fine tuning EBV gene expression during latency .Furthermore, the nuclear lamina–genome interactions regulate gene expression by modifying the chromatin composition without affecting its 3D structure. In Type III latency, the lamin A/C depletion resulted in reduced H3K9me2 deposition at viral loci bound by both lamin A/C and lamin B1 . The H3KBesides the central role of CTCF/Cohesin complex in regulating the 3D structure of EBV, another transcription factor involved in this process is Myc. The Myc protooncogenes are upregulated in a wide variety of cancers and their expression is induced upon EBV infection . The worDepletion of Myc promotes the reactivation of the lytic cycle through the formation of loops between the promoter of the early lytic gene BZLF1 and the OriLyt enhancer and Terminal Repeats (TR) regions.As discussed above, Cohesin and CTCF are essential to the regulation of gene expression, placing distal enhancers and promoters in close proximity. The enhancers are enriched in two histone modifications, H3K4me1 and H3K27ac, which, respectively, characterize poised and active enhancers. Extensive mapping of the H3K27ac histone mark revealed that the EBV genome is enriched for this histone mark at key latent promoters such as Ori P, Cp, Qp, LMP and some newly discovered regions such as the BILF2 promoter . In accoWith newly developed techniques such as HiChIP that combine chromatin immunoprecipitation with HiC, it has been possible to map the intragenomic interactions that are enriched for H3K27ac in B cells harboring all three latency types . In thisThe three-dimensional structure of EBV is closely associated with the latency program and, therefore, with the regulation of viral gene expression. The central role of viral chromatin architecture is underscored by the fact that multiple factors are exploited by the virus to maintain it, including CTCF, Cohesin, PARP1, MYC, and Lamins. However, our present understanding of the role of chromatin structure in EBV gene expression is still incomplete due to technological limitations. For example, multiple copies of EBV episomes exist in each infected cell, yet HiC technologies only provide the viral 3D genome structure obtained from a cell population. Therefore, it is unclear whether all EBV episomes within an infected cell adopt the same chromatin structure or whether sub-populations of episomes exist, each adopting a specific 3D chromatin structure consisting of just a sub-set of loops. Overall, shedding light on these regulatory layers can be of considerable importance in the treatment of EBV-associated malignances.In recent years, numerous drugs targeting epigenetic mechanisms have been developed and utilized to improve the efficacy of chemotherapy. Considering the significant role of epigenetics in regulating both EBV infection and the development of EBV-associated malignancies, leveraging epigenetic drugs may offer a promising and innovative therapeutic strategy for treating EBV-positive malignancies.


Η) and sigma E (σE) subunits of Corynebacterium glutamicum RNA polymerase belong to Group 4 of sigma factors, also called extracytoplasmic function (ECF) sigma factors. Genes of the C. glutamicum σΗ regulon that are involved in heat and oxidative stress response have already been defined, whereas the genes of the σE regulon, which is involved in cell surface stress response, have not been explored until now. Using the C. glutamicum RES167 strain and its derivative C. glutamicum ΔcseE with a deletion in the anti-σΕ gene, differential gene expression was analyzed by RNA sequencing. We found 296 upregulated and 398 downregulated genes in C. glutamicum ΔcseE compared to C. glutamicum RES167. To confirm the functional link between σΕ and the corresponding promoters, we tested selected promoters using the in vivo two-plasmid system with gfpuv as a reporter gene and by in vitro transcription. Analyses with RNAP+σΗ and RNAP+σΕ, which were previously shown to recognize similar promoters, proved that the σΗ and σE regulons significantly overlap. The σE-controlled genes were found to be involved for example in protein quality control , the regulation of Clp proteases (clgR), and membrane integrity maintenance. The single-promoter analyses with σΗ and σΕ revealed that there are two groups of promoters: those which are exclusively σΗ-specific, and the other group of promoters, which are σΗ/σE-dependent. No exclusively σE-dependent promoter was detected. We defined the consensus sequences of exclusively σΗ-regulated promotors to be −35 GGAAt and − 10 GTT and σΗ/σE-regulated promoters to be −35 GGAAC and − 10 cGTT. Fifteen genes were found to belong to the σΗ/σΕ regulon. Homology modeling showed that there is a specific interaction between Met170 in σΗ and the nucleotides −31 and − 30 within the non-coding strand (AT or CT) of the σΗ-dependent promoters. In σE, Arg185 was found to interact with the nucleotides GA at the same positions in the σE-dependent promoters.The sigma H (σ Transcription of the genes involved in starvation, stationary phase, and general stress response largely depends on the presence of the primary-like σ factor usually named σΒ in Gram-positive bacteria. The stress-responsive genes, which are active during various adverse environmental conditions, are in many cases transcribed by RNAP with an ECF (extracytoplasmic function) sigma factor. This group of σ factors is the most numerous and variable. The ECF sigma factors σH and σE are responsible for the transcription of large gene groups in Actinobacteria. These σ factors share some significant features in streptomycetes, mycobacteria, and corynebacteria, and probably also in rhodococci  holoenzyme that includes a primary sigma subunit , encoded by an ortholog of M. tuberculosis SigH, is responsible for many stress response functions, e.g., thiol homeostasis (redox control), antibiotic stress response, sulfur metabolism, ribosome modulation, energy metabolism, DNA repair, and protein turnover  and σR (in S. coelicolor) regulatory networks and the variety of their functions, these σ factors can be considered to be global regulators.σno acids . Moreoveno acids . In Streturnover . σΗ in CA repair . Genes rol of σΗ . Ten tra/cGTTgaa . The settor RshA . We prov derived . ConsideM. tuberculosis ECF sigma factor, σE, forms a regulatory network that has a role in heat, cell surface, and oxidative stress response. It is also essential for various virulence functions  were found to be most likely transcribed by either RNAP+σE or σH.Recently, a basic regulatory network operated by σ factors was described in utamicum . The genE-deficient C. glutamicum strain was shown to be sensitive to heat, SDS, EDTA, and lysozyme , dnaJ2 (chaperone), and clgR  are under the control of σE  were found to be closely similar to the consensus of C. glutamicum σΗ-dependent promoters  were cultivated under the same conditions, but at 30°C. The defined chromosomal deletion ΔcseE in the C. glutamicum chromosome was constructed using the sacB gene as a conditionally lethal marker and a double-crossover event as described previously (oli DH5α  was cultoli DH5α  at 37°C.m RES167  and C. geviously . The plaE. coli were done using the standard techniques (sigH and sigE were constructed with a Q5 Site-Directed Mutagenesis kit (New England BioLabs Inc.).DNA isolation, PCR, cutting with restriction enzymes, ligation, and transformation of chniques . MutatioC. glutamicum cells were cultivated in 2xYT medium at 30°C and disrupted with glass beads and a FastPrep FP120 (BIO101) disintegrator. The cell debris was removed by centrifugation, and total RNA was isolated from the extract using a High Pure RNA Isolation Kit (Roche Diagnostics). The primer extension analysis was essentially done using SuperScript III transcriptase  with 30 μg RNA and 5 pmol Cy-5-labeled primer CM4 complementary to the vector pET2. The synthesized cDNA was run on PAA gel simultaneously with the DNA sequencing products generated with the same labeled primer in an A.L.F. Sequencer .Non-radioactive primer extension (PEX) analysis was carried out as described previously . C. glutCorynebacterium glutamicum RES167 or C. glutamicum ∆cseE was cultivated in minimal medium CGXII (The whole procedure was done in principle according to the protocols used previously  with a fum CGXII  supplemeC. glutamicum cells by a Quick-RNA Miniprep Plus kit (Zymo Research). The samples were treated with DNase (Roche Diagnostics) and RNA was purified with an RNA Clean&Concentrator-5 kit (Zymo Research). Ribosomal rRNA was removed with a Ribo-Zero rRNA Removal Kit for bacteria (Illumina). The purity of RNA was then tested with an Agilent RNA Pico 6,000 kit and an Agilent 2,100 Bioanalyzer (Agilent Technologies). TruSeq Stranded mRNA Sample Preparation guide (Illumina) was then used to construct the cDNA library. The constructed cDNA library was then sequenced with Illumina HiSeq 1,500 using a read length of 70 bases.Total RNA was isolated from 3 biological replicates of A primary 5′-end-specific cDNA library was constructed for sequencing with the aim of defining TSSs using the protocol described previously  Briefly,The primary 5′-triphosphate ends of the primary transcripts were treated with RNA 5′-polyphosphatase to convert them into 5′-mono phosphate ends, and the 5′-adapter was ligated to the produced 5′-ends. Then, reverse transcription with a stem-loop DNA adapter was carried out and the library was amplified. The primary 5′-end cDNA library was then purified and size-selected for fragments approximately 100–1,000 nt in size by gel electrophoresis and quantified. The fragments were finally sequenced with an Illumina MiSeq.C. glutamicum reference genome sequence accession number BX927147 as described previously  was calculated by the log2 mean of normalized read counts, and the signal intensity ratio  by log2 fold change. The evaluation of the differential RNA-seq data was done with an adjusted value of p cutoff of p ≤ 0.01 and a signal intensity ratio  cutoff of ≥1 or ≤ −1.Differential gene expression analysis of rer v2.2 , includirer v2.2 . The sigC. glutamicum ∆cseE were included in the further analysis .Genes with upregulated or downregulated expression in in vitro transcription assay was carried out essentially as described previously  in a molar ratio of 1:15 (σA) or 1:30  The holo-RNAP was assembled for 10 min at 37°C. The transcription mixture was incubated for 10 min at 37°C. The transcripts labeled with [α-32P]UTP were separated in 5.5% (w/v) polyacrylamide gel. In vitro transcription assays were done 2 or 3 times for each promoter, with essentially the same results.The eviously . The proeviously  were useeviously . The holeviously . The RNAin vivo using the recently described two-plasmid system for C. glutamicum , stress  SDS and 4% (v/v) ethanol, respectively) was applied. Promoter activity was measured as the fluorescence intensity of the reporter protein GFPuv.Sigma factors were assigned to individual promoters utamicum . In prinor pEPR1 , 2019. Tor pEPR1 , 2019. TH and σE domains which recognize the −10 and − 35 sequences of the respective promoters were produced by using the Swiss-Model server  forms an operon with the downstream genes cseE  and tatB . The sub-operon cseE-tatB was also detected (sigE TSSs. DNA fragments (256 bp upstream of sigE and 227 bp upstream of cseE) were cloned in the vector pET2 and the constructs were used for PEX assay. Total RNA for PEX was isolated from C. glutamicum (pET2 carrying the sigE promoter region) and C. glutamicum (pET2 with-cseE promoter) cells, which were cultivated to the exponential growth phase. Three TSSs were found within the upstream region of sigE (data not shown). Two of them, TSS1 at A and TSS3 at G , TAATCT (P2sigE), and TATAGT  genes . The signal of TSS2 corresponding to the C. glutamicum cells cultivated under standard conditions was weak, whereas the respective peak obtained with the cells cultivated with 0.01% (w/v) SDS was very large  genes . NeverthTTGA/CCA  or TTGNNTTGA/CCA , but thiromoters . Upstreasition A ,C, whichsition A . In addisition A ,C. The ssition A . The −10romoters . The P2c–N18–GTT , howeverromoters . The strry large . This isS stress . This susigE and P2cseE) were further examined using a transcriptional fusion of these promoters with the gfpuv reporter gene in the promoter-test vector pEPR1  ethanol. The effects of these stresses on P2cseE activity were only weak and thus not completely convincing.  showed that in total 83 genes in 61 transcriptional units were directly or indirectly σΗ-dependent.To view the transcription initiated by σus study  using thC. glutamicum ΔcseE with that of C. glutamicum RES167 is shown in C. glutamicum ΔcseE compared to C. glutamicum RES167. These genes may be considered as genes whose expression is directly or indirectly modulated by σE, i.e., σE modulon . We decided therefore to search for the σE-specific promoters upstream of the differentially transcribed genes.The ratio/intensity (M/A) plot deduced from RNA-seq results comparing the gene expression of  modulon , S3. OurC. glutamicum σA-and σD-controlled genes, respectively (Ε stimulon), 157 genes were in operons and without TSSs (and therefore without closely located promoters). Upstream of the remaining 139 upregulated genes, 168 TSSs were detected. Some of the genes were transcribed from 2 or 3 or even as much as 4 TSSs. Closely upstream of the detected TSSs, we recognized 150 sequences similar to the consensus sequence of vegetative promoters . Using the software Improbizer (A-dependent genes in C. glutamicum. The sequence GGAAC–N18-19–GTT (with a single exception GGAAA–N19–GTT in amtR), which was identical to the sequences of a few already found σΕ-dependent promoters  were used for the in vivo two-plasmid assay. The promoter activity was measured as a fluorescence intensity of the GFPuv reporter protein. The chosen promoters were further tested with the in vitro transcription system for C. glutamicum  gene, which was also found to be σΗ-dependent  thus constitute a gene group which is entirely contained in the σΗ regulon. All σE-dependent promoters (with a single exception of PamtR) contained the consensus sequence with the −35 and −10 sequences GGAAC–N18-19–GTT.In addition to the promoters, which were mapped by RNA-seq, we selected the promoter Ped by σΗ  and possependent , and whichniques ,D,K,L, w in vivo ,H. As fovo assay , whereascription . All in o assays . AccordiΗ-dependent promoters Pmca, PuvrD3, PmshC, and PsufR with the −35 region GGAAT  was successful. The results confirmed that the cseE gene located closely downstream of the sigE gene encodes the specific anti-σE as suggested previously  at position −31 from the core consensus GGAAt–N18-19–GTT of the σΗ-controlled genes .The sequences of the 16 σromoters  and 44 eromoters  were sepromoters . The coned genes . HoweverE- and σH-dependent promoters in similar way as was done previously for the σD and σH-dependent promoters  was taken as a typical strong exclusively σΗ-specific promoter.The 2H27 structure for σ E. coli  was usedE) or methionine M170 (in σH) interact with nucleotide bases (within the non-coding strand) at positions −31 and − 30 of the promoters  or methionine M170  were proposed. The wild-type σH, σE, and the modified sigma factors σHmut and σEmut can form with Pcg3309, PtrxB1, and PcseE in total four unique combinations of key interactions between amino acids R or M and dinucleotides AT and GA at positions −31 and − 30 in the template strand, i.e., R-GA, M-AT, R-AT, M-GA , σE (R185), σHmut (R170), and σEmut (M185) and constructs of pEPR1 with the selected promoters were used for creating combinations of the two plasmids (pEC-XT99A + sig and pEPR1 + promoter) in C. glutamicum clones. In addition to the predominantly σE-controlled PcseE and Pcg3309 promoters and exclusively σH-dependent PtrxB1 promoter, we decided to also test PrshA. This promoter is one of a few exceptions among the strong σH-dependent promoters with G at position −31 (instead of T) and it can be thus supposed that M170 in σH would interact with −31 and − 30 CT (instead of AT) in the template strand. The promoter activities in the resulting clones were measured by the standard in vivo procedure as the intensity of fluorescence produced by GFPuv  also impaired their activity: to a large extent for PtrxB1 and completely for PrshA . Most of these genes  which confer bacteria resistance to various compounds, e.g., antibiotics. The tatB gene (twin-arginine translocation pathway protein) which is in the operon with sigE and cseE is most likely also connected to the stress response functions in C. glutamicum. The Tat proteins generally have a role in translocating fully folded proteins from the cytoplasm across the cytoplasmic membrane. Among them, redox proteins may be connected to the oxidative stress response , an important role in controlling nitrogen metabolism is played by alternative sigma factor σ54, which is structurally and functionally different from the σ70-type sigma factors , although it was not detected as upregulated according to the RNA-seq results. Moreover, many genes of the AmtR regulon  were induced in C. glutamicum ΔcseE, but they were not found to be directly under an σE-dependent promoter. We can speculate that σE and σH together with AmtR  form a functional module which is responsible for regulating the uptake and assimilation of nitrogen sources.In some bacteria  was found mainly in the −35 region of the consensus sequence: σE could only recognize promoters with the −35 GGAAC sequence, whereas σΗ recognized −35 GGAAN, although −35 GGAAT was the most frequent.One of the main aims of this study was to find differences between the σM. tuberculosis, similar features were found : C at position −31 was essential for recognition by σΕ, whereas any base at −31 allowed recognition by σΗ, although T prevailed  and the corresponding σ regulons to various extent overlap. These similarities in structure and activity are most probably based on similar sequences of the key amino acids in σ factors.In revailed . Howeverhropolis  in our r, and σD  in C. glMycobacterium  of 82 Corynebacterium species. A stress promoter with the consensus motifs (GGAAC–N18-20–GTT) was only found upstream of the sigE gene of 5 Corynebacterium species. The autoregulation of sigE is thus apparently rare in corynebacteria. Further, a stress promoter with the same structure was found upstream of cseE in 11 Corynebacterium species. In conclusion, both the sigE and cseE genes are mostly transcribed from vegetative promoters in the majority of the corynebacteria. In four species  the cseE gene is clearly missing. The strategy for the control of sigE expression thus developed divergently in corynebacteria.The genes encoding stress σ factors in various Actinobacteria, such as acterium , Rhodocodococcus , and S. elicolor , are mosC. glutamicum cseE is transcribed from two promoters, σΑ-dependent P1cseE and σΗ/σΕ-dependent P2cseE  in C. glutamicum (A) to another close promoter.t P2cseE . Primer t P2cseE  showed t factors . Some stutamicum . AnothersigH-rshA operon showed that the transcription of sigH is also rarely autoregulated (in only 7 Corynebacterium species). In contrast to cseE, upstream of the rshA gene, conserved stress promoters (GGAAT–N18–GTT) were recognized in 63 Corynebacterium species. We can infer from these sequence analyses that the transcription of sigE and sigH in response to stress conditions should therefore be regulated mostly by DNA-binding transcriptional factors. Moreover, a certain level of σ factors could be permanently present in the cell in an inactive form bound by the cognate anti-σ factor. The additional σΗ- or σΕ-dependent promoter upstream of rshA or cseE, respectively, probably developed as a different strategy to ensure sufficient production of the anti-σ that can inhibit the σ factor when the stress is over , which might be a binding site for a transcriptional regulator. We tested the activity of the promoter fragment −42 to +5 and various shortened versions, including those in which the inverted repeats were missing, but did not find any significant effect of these deletions on the P1sigE activity (data not shown). Measurements in the C. glutamicum strain deficient in the two-component system CgtRS2 . This double deletion is probably lethal due to the fact that these two σ factors secure too many functions in stress responses and therefore cannot be replaced by any of the three remaining C. glutamicum ECF sigma factors  which provide a limited number of activities. Moreover, σΗ and σΕ drive the transcription of sigB that also controls a wide range of functions. The σΒ-σΕ-σΗ “sigma triangle” or “Big Three” thus forms the essential basis for maintaining C. glutamicum cell viability during transition and stationary growth phases and diverse types of stress conditions. The integration of σΒ, σΕ and σΗ in an intricate regulatory network was also observed in M. tuberculosis  under accession numbers E-MTAB-12457 and E-MTAB-12458.The transcriptome sequencing raw data files are available in the ArrayExpress database , and Deutscher Akademischer Austauschdienst.The authors declare that this work was carried out in the absence of any personal, professional, or financial relationships that could potentially be construed as a conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


A chromatin immunoprecipitation (ChIP) assay evaluated promoter-associated modifications. Nuclear association between SIRT1, p300, and NFκB-p65 and acetylation changes of p65 were determined using immunoprecipitation and Western blot analyses. A Student’s t-test and one-way ANOVA determined the significance. Results: Ethanol significantly increased promoter-associated histone-H3-lysine-9 acetylation (H3K9Ac), reflecting a transcriptionally permissive state with a resultant increase in hepatic CCL2 mRNA and protein expression. Moreover, increased lysine-310-acetylation of nuclear RelA/p65 decreased its association with SIRT1, a class III HDAC, but concomitantly increased with p300, a histone acetyltransferase. This further led to enhanced recruitment of NF-κB/p65 and RNA polymerase-II to the CCL2 promoter. Oral TB administration prevented ethanol-associated acetylation changes, thus downregulating CCL2 expression, hepatic neutrophil infiltration, and inflammation/ injury. Conclusion: The modulation of a protein acetylation state via ethanol or TB mechanistically regulates hepatic CCL2 upregulation in ALD.Purpose: Chemokine-driven leukocyte infiltration and sustained inflammation contribute to alcohol-associated liver disease (ALD). Elevated hepatic CCL2 expression, seen in ALD, is associated with disease severity. However, mechanisms of CCL2 regulation are not completely elucidated. Post-translational modifications (PTMs) of proteins, particularly acetylation, modulate gene expression. This study examined the acetylation changes of promoter-associated histone-H3 and key transcription factor-NFκB in regulating hepatic CCL2 expression and subsequent inflammation and injury. Further, the effect of therapeutic modulation of the acetylation state by tributyrin (TB), a butyrate prodrug, was assessed. Methods: Hepatic CCL2 expression was assessed in mice fed control (PF) or an ethanol-containing Lieber–DeCarli (5%  Sustained hepatic inflammation is an important factor in alcohol-associated liver disease (ALD) development and progression. Neutrophil infiltration and monocyte recruitment in the liver are associated with chronic alcohol-induced liver inflammation and injury ,2. ChemoEmerging studies have documented the role of the gut–liver axis in ALD in the context of alcohol-induced alterations in gut microbiota, barrier dysfunction, and endotoxemia leading to sustained hepatic inflammation. Alcohol consumption is known to affect short-chain fatty acids (SCFAs) levels, particularly butyrate . ButyratEpigenetic regulation is critical for gene expression. In particular, a post-translational acetylation modification is known to dictate the transcriptional activation of a given gene, as this modification regulates functions of both histones and non-histone proteins, like transcription factors . HistoneIn the present study, we demonstrated that ethanol-induced pathogenic alterations in acetylation states of promoter-associated histone H3 and essential transcription factor NFκB contribute to the upregulation of hepatic CCL2 expression. Further, tributyrin, a dietary butyrate prodrug, prevents ethanol-mediated modulation of SIRT-1-NFκB-p300 interactions and resultant acetylation changes. v/v)) ad libitum for 7 weeks (EF). The control-pair-fed (PF) mice were given the isocaloric maltose-dextrin-containing liquid diet. For tributyrin treatment groups, both pair-fed (TB) and ethanol-fed (EF + TB) mice received tributyrin  by oral gavage  for 7 weeks as a preventive strategy (EF + TB (7 + 7)) or for the last 3 weeks as an interventional strategy (EF + TB (7 + 3)). The tributyrin-alone group (TB) received oral TB administration  for 7 weeks.Animal Model: Eight-week-old male C57BL/6N mice were obtained from Harlan . All mice were housed in a pathogen-free, temperature-controlled animal facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care with 12 h light/12 h dark cycles. All experiments were carried out according to the criteria outlined in the Guide for Care and Use of Laboratory Animals and with the approval from the University of Louisville Animal Care and Use Committee (IACUC# 20754). Mice were fed a modified Lieber–DeCarli liquid diet enriched in unsaturated fat (corn oil), which provided 40% of energy from fat, 43% from carbohydrate, and 17% from protein . Mice were pair-fed a Lieber–DeCarli liquid diet containing either ethanol (EF) or isocaloric maltose dextrin (PF) for 7 weeks. Ethanol was gradually increased for one week, and then, mice were fed the ethanol diet , F4/80+, and CAE staining and examined under light microscopy with 20× magnification. Assays were performed according to the manufacturer’s protocols. Assessment of Liver Injury: The liver injury was assessed by measuring alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using commercially available kits and manufacturer’s instructions .Blood Endotoxin Assay: Serum endotoxin levels were measured using a Limulus Amebocyte Lysate kit  according to the manufacturer’s instructions.g for 15 min. The MPO levels were measured according to the manufacturer’s instructions  at 450 nm using a plate reader. Results are expressed as mg of MPO levels/gram of protein, as determined via a Lowry protein assay MPO Assay: For hepatic myeloperoxidase activity (MPO), liver samples were homogenized (10 μg/200 μL) in 200 mM NaCl, 5 mM EDTA, 10 mM tris, and 10% glycerin and centrifuged at 1500× Hepatic CCL2 Protein Measurements: Liver lysates were homogenized in a lysis buffer , and analysis was performed using a U-Plex kit from Mesoscale Discovery (MSD) . Over 200 μg of protein was loaded into each well. The plate was read using a MESO QuickPlex SQ 120 imager and analyzed using Discovery Workbench v4.0 software. The assay was performed according to the manufacturer’s instructions.−ΔΔCt method by normalizing with TBP (TATA-binding protein) gene expression in all the experiments and is presented as a fold change over untreated/pair-fed, which was set at 1.RNA Isolation and Real-Time PCR Analysis: Total RNA was isolated using TRIzol , and cDNA was made using Quanta qScript . The real-time PCR was performed with Quanta Perfecta SYBR green fast mix and an ABI Prism 7500 sequence detection system . The relative gene expression was analyzed using the 2Mouse CCL2 mRNA primersForward: GGCTCAGCCAGATGCAGTReverse: TGAGCTTGGTGACAAAAACTACAGMouse TBP mRNA primersForward: CCTAAAGACCATTGCACTTCGTReverse: GCTCCTGTGCACACCATTTTChromatin Immunoprecipitation (ChIP) and qChIP PCR Analysis: The ChIP assay was conducted using the ChIP assay protocol established in the lab . ChIP an(Region I) mmCcl2_F1CAAGCACCCTGCCTGACT(Region I) mmCcl2_R1CTCCCGTCTGGCTCTCTG(Region II) mmCcl2_F2TCCCAGGAGTGGCTAGAAAA(Region II) mmCcl2_R2TCCGCTGAGTAAGTGCAGAG(Region III) mmCcl2_F3CATCTGGAGCTCACATTCCA(Region III) mmCcl2_R3GGCAGGTCAGAGGCAGAGTAThree ChIP-PCR primers were designed for each region of the mouse-CCL2 promoter. The primer sequences are as follows:2VO3, and 10 mM NaF). All IP and WB analyses were performed as described earlier [Immunoprecipitation (IP) and Western Blot (WB) Analysis: Total nuclear extracts were prepared by lysing liver tissue in a RIPA lysis buffer  had significantly higher hepatic steatosis, as shown by H&E staining A and neuChemokines act as a critical mediator for the recruitment of neutrophils and macrophages, and elevated expression of hepatic CCL2 has been well documented to correlate with PMN infiltration and disease severity in ALD . Hence, Epigenetic modifications are known to orchestrate the interplay between the chromatin state and gene expression. To understand whether the post-translational histone modifications occurring at the CCL2 promoter contribute to its gene expression in response to alcohol and tributyrin, a Chromatin immunoprecipitation (ChIP) assay was performed. Promoter-associated histone modifications were investigated via ChIP qPCR at three transcriptionally relevant sites known for NFκB occupancy on the CCL2 promoter ,28,29. RThe status of histone H3 lysine 9 acetylation (H3K9Ac), which plays a key role in the transcriptional activation of gene expression, was examined. ChIP analysis demonstrated that correspondent to CCL2 gene expression, ethanol feeding significantly increased transcriptionally permissive histone H3 lysine 9 acetylation (H3K9Ac) at all three regions of the CCL2 promoter in murine livers B. AdditiHistone acetylation is regulated by histone acetyltransferases (HATs), and p300 is a known HAT that has been previously reported to increase histone acetylation at the CCL2 promoter . Hence, NFκB is one of the major transcription factors for CCL2 expression. Additionally, it is known that NFκB interacts with the co-activator protein p300 as part of the transcription initiation complex and recruits the complex to the target gene promoters . Hence, Acetylation of lysine 310 (K310) of the p65 subunit of NFκB is required for its transcriptional activation and DNA-binding affinity ,34. SincImportantly, it has been reported that the nuclear acetylation status of K310 is maintained by the interaction of NFκB with a class III histone deacetylase—SIRT1—and histone acetyltransferase p300 ,36,37. HIn correlation with promoter histone hyper-acetylation and increased NFκB binding, ethanol enhanced the transcription initiation process by recruiting RNA Pol II at the TSS region I of the CCL2 promoter . ImportaTaken together, our study indicates that chronic ethanol-induced hyperacetylation of histone H3(K9) and the transcription factor NFκB (K310) drive hepatic CCL2 expression and, in turn, contribute to liver inflammation. Importantly, transcriptional repression of CCL2 via tributyrin administration, under both preventive and interventional treatment strategies, was associated with hypoacetylation and diminished binding of chromatin-associated proteins, including p300, NFκB, and RNA polymerase II.Alcohol-associated liver disease (ALD) is a major cause of liver-related morbidity and mortality ,38,39. DUnder both normal and disease states, the epigenetic machinery is important for regulating gene expression. Post-translational modifications (PTMs) of proteins are considered the main components of epigenetic regulatory machinery for gene expression and are also broadly accepted as a therapeutic target . VariousIn addition to histones, acetylation of several non-histone proteins, including transcription factors, also plays an important role in regulating their function and, in turn, target gene transcription ,47. NFκBThus, these data suggest that ethanol exposure enhanced the transcription potential of NFκB, increasing its binding to the CCL2 promoter and consequent CCL2 upregulation. Additionally, it is known that the interaction of NFκB with co-activator proteins, such as p300, allows their recruitment to target gene promoters ,51. The In contrast to ethanol, both preventive and interventional treatment strategies of tributyrin administration showed that TB treatment inhibited the ethanol-inducible CCL2 expression. Tributyrin is a triglyceride containing three butyrate moieties that gets rapidly absorbed and hydrolyzed to butyrate ,52. We hTB treatment also interferes with p65 binding to p300, which occurs in response to ethanol exposure. This resultant decrease in p300-p65 association likely contributed to further deacetylation of NFκB and reduced recruitment of p300 to CCL2 promoter, decreasing the H3K9 acetylation levels. Thus, oral administration of tributyrin can induce close chromatin structure and transcriptional suppression of CCL2 expression. It has been reported that SIRT1 can physically interact with p300, inactivating its acetyltransferase capacity ,59. HencOverall, our data support the idea that not only acetylation of histone H3 at the CCL2 promoter but also acetylation and transactivation of the non-histone protein, NFκB-p65, contribute to alcohol-induced upregulation of CCL2 in the liver.