Micromelalopha troglodyta (Graeser) (Lepidoptera: Notodontidae) is a notorious pest of poplar. Coevolution with poplars rich in plant secondary metabolites prompts M. troglodyta to expand effective detoxification mechanisms against toxic plant secondary metabolites. Although glutathione S-transferases (GSTs) play an important role in xenobiotic detoxification in M. troglodyta, it is unclear how GSTs act in response to toxic secondary metabolites in poplar. In this study, five GST gene core promoters were accurately identified by a 5’ loss luciferase reporter assay, and the core promoters were significantly induced by two plant secondary metabolites in vitro. Two transcription factors, cap ‘n’ collar C (CncC) and aryl hydrocarbon receptor nuclear translocator (ARNT), were cloned in M. troglodyta. MtCncC and MtARNT clustered well with other insect CncCs and ARNTs, respectively. In addition, MtCncC and MtARNT could bind the MtGSTt1 promoter and strongly improve transcriptional activity, respectively. However, MtCncC and MtARNT had no regulatory function on the MtGSTz1 promoter. Our findings revealed the molecular mechanisms of the transcription factors MtCncC and MtARNT in regulating the GST genes of M. troglodyta. These results provide useful information for the control of M. troglodyta. Plants are the main food source for animals, while insects serve as important consumers of plants. They constantly fight against each other. From a plant perspective, many plants produce defensive toxins or inhibitors to repel insects, including secondary metabolites such as isoflavones, furanocoumarins, terpenoids, alkaloids and cyanogenic glycosides . From anMicromelalopha troglodyta (Graeser), which is mainly found in China, is an important leaf-feeding pest of poplar trees and can be widely spread causing heavy losses to the forestry industry [M. troglodyta [M. troglodyta that are involved in the metabolism of plant secondary metabolites [Myzus persicae could be induced by different host plants of Brassica species, suggesting that GSTs might play roles in host plant adaptation in M. persicase [GST genes in Lymantria dispar, and the adaptation of L. dispar to plant secondary metabolites was reduced after interference with GST genes [GST genes [industry ,5. Poplaoglodyta ,7. It isoglodyta . There aoglodyta . GSTs arabolites ,8. Mountabolites ,11. GST ersicase . MultiplST genes . Red palST genes . Hence, cis- and trans-acting factors, that can enhance or repress the initiation of gene transcription by binding to specific promoter sequences [Spodoptera litura Nrf2 acted as a cis-regulatory element that activated the excretion and degradation of indole-3-methanol (I3C) and chlorpyrifos by upregulating GSTe1, which could improve the response of S. litura to phytochemicals and insecticides [Leptinotarsa decemlineata, several imidacloprid-resistant GST genes were regulated by the transcription factors CncC and Maf [Spodoptera exigua transcription factors AhR and ARNT coordinately regulated the expression of multiple GSTs conferring insecticide resistance [LmGSTd7 [GST genes, and they play an important role in governing the expression of GSTs. However, the mode of regulation of GST genes by the two transcription factors in M. troglodyta is not clear.The detoxification genes are transcriptionally activated by a common mechanism of transcription factors . Transcrequences . Cap ‘n’equences ,16,17,18equences ,20. CncCequences . Spodoptcticides . In Lept and Maf . The ary and Maf ,25. Enha and Maf . The Sposistance . In locu[LmGSTd7 . CombiniGST promoters of M. troglodyta and demonstrated that tannic acid could induce the activity of these five MtGST promoters [GST in M. troglodyta is regulated to adapt to plant secondary metabolite stress. In this study, we focused on the following three questions: (1) Where is the core region of the MtGST promoter located? (2) Will the core region of the MtGST promoter be responsive to plant secondary metabolites? and (3) Do MtCncC and MtARNT interact with the promoters of GST? This study elucidated the role of MtCncC and MtARNT in regulating GST metabolism of plant secondary metabolites, which would be helpful to find new target genes to control M. troglodyta.We cloned five romoters . To dateMtGST promoters. To determine the regions that are essential for GST expression, we constructed several deletion structures containing 5’ loss fragments and inserted them into the pGL4.10-Basic vector , which showed that sequences were essential for the transcription of MtGSTd2 located in the 240 bp region between −642 and −402 bp (MtGSTo1 promoter exhibited relatively high luciferase expression compared with that of pGL4.10-(−360/+414) and pGL4.10-(−127/+414), which suggested that −596 to −360 region was critical for MtGSTo1 transcription (MtGSTs1 promoter displayed relatively high luciferase expression than that of the pGL4.10-(−361/+385), and pGL4.10-(−70/+385) of MtGSTs1 had low luciferase expression compared to pGL4.10-(−361/+385), which suggested that sequences were essential for the transcription of MtGSTs1 located in the 215 bp region between −576 and −361 bp (MtGSTt1 promoter had relatively high luciferase expression compared with that of pGL4.10-(−334/+340), and the pGL4.10-(−334/+340) vector of MtGSTt1 had high luciferase expression levels compared to pGL4.10-(−1052/+340) and pGL4.10-(−1555/+340), which showed that the 240 bp transcription region between −574 and −334 was critical for MtGSTt1 expression (MtGSTz1 promoter had relatively high luciferase expression compared with that of pGL4.10-(−500/+521) and pGL4.10-(−220/+521), which suggested that sequences between −350 and −220 were critical for MtGSTz1 expression (MtGST promoters were shown in The pGL4.10-(−642/+290) vector of  −402 bp A. The pGcription B. The pG −361 bp C. The pGpression D. The pGpression E. The maMtGSTt1 (−574/+340) and MtGSTz1 (−350/+521) promoters, we used them as examples to explore the promoter response to tannic acid and quercetin (MtGSTt1 (−574/+340) was significantly induced by tannic acid at a low concentration (0.56 mg/L), while it was inhibited by tannic acid at 2.8, 14 and 70 mg/L (MtGSTt1 (−574/+340) promoter showed an increasing and then decreasing trend under quercetin stress, and the highest promoter activity was observed when the quercetin concentration was 7 mg/L (MtGSTt1 (−574/+340) could be induced by tannic acid and quercetin. For MtGSTz1 (−350/+521) promoter, the promoter activity was strongly induced by 2.8 mg/L tannic acid (MtGSTz1 (−350/+521) was notably increased when Sf9 cells were treated with quercetin at 0.28, 1.4 and 7 mg/L and sharply repressed at 35 mg/L  sequence and encodes 556 AA residues. The amino acid sequence of MtCncC is listed in To explore the regulatory mode of promoters by transcription factors. The ARNT was also identified from the transcriptome database and confirmed by cloning and resequencing. MtARNT is a 1440 bp ORF sequence and encodes 479 AA residues. The amino acid sequence of MtARNT is listed in Another transcription factor MtGST promoters, we selected the MtGSTt1 (−574/+340) and MtGSTz1 (−350/+521) promoters with high transcription activity to verify the regulatory role of ARNT and CncC. pGL4.10-MtGSTt1 (−574/+340) and pGL4.10-MtGSTz1 (−350/+521) promoters were transfected into Sf9 cells. MtCncC and MtARNT sequences were constructed into pAC-V5 basic expression vector and named pAC-CncC and pAC-ARNT, respectively. The pAC-V5 basic expression vector was used as a negative control. The transcription factors MtCncC/MtARNT and the promoter constructs were cotransfected into Sf9 cells. As shown in MtGSTt1 (−574/+340) promoter strongly increased when cotransfected with pAC-CncC or pAC-ARNT. These results showed that the expression of MtCncC/MtARNT in Sf9 cells facilitates MtGSTt1 (−574/+340) promoter transcription, but there was no significant change when cotransfected with pAC-CncC and pAC-ARNT. When pAC-CncC/pAC-ARNT was cotransfected with pGL4.10-MtGSTz1 (−350/+521) promoter, the transcriptional activity of the MtGSTz1 (−350/+521) promoter was not increased  binding to endogenous and exogenous compounds and excrete them outside cells, thus reducing their damage to the cells ,30. In iand zeta . In a prnic acid .Drosophila heat shock protein (Hsp70) could enhance the expression of Hsp70 more than 200-fold after heat stimulation treatment [DREB1A gene in plants could enhance the drought, high salt and low temperature resistance of transgenic plants [Bombyx mori nucleopolyhedrovirus (BmNPV)-inducible promoters were applied for gene therapy [GST have been reported in a few insects. In S. litura, the GST promoter acted as an important element for upregulating the expression of GST and improved S. litura tolerance to insecticides [S. exigua GSTs were coregulated by two transcription factors, which enhanced the resistance of insects to xenobiotic stress [MtGST promoters, their regulatory mechanisms for MtGST genes are not clear. In the present study, we further identified the core regions of the five MtGST promoters in vitro by a 5’ loss fragment assay. The activity of the core regions of MtGSTt1 (−574/+340) and MtGSTz1 (−350/+521) promoters were also well induced by low concentrations of tannic acid and quercetin, which suggested that the core sequences of MtGST promoters have significant activity in response to plant secondary metabolites. However, we also found that higher concentrations of tannic acid and quercetin would decrease the activity of luciferase despite that they were not toxic to Sf9 cells. Based on a previous study, when human cells were exposed to xenobiotic stress, low concentrations of xenobiotics induced the promoter activity of pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 1 (PAC1-R) while high concentrations of xenobiotics inhibited PAC1-R promoter activity [GST promoter activity and plant secondary metabolites (tannic acid and quercetin) also presented a dose-dependent way. Once the concentrations of plant secondary metabolites exceeded the range causing induction, they might inhibit the activity of promoters. These findings provided useful information for understanding the mechanism of GST transcriptional regulation in M. troglodyta.The promoter is a very important regulatory element in gene transcription that determines the pattern and intensity of gene expression . Many inreatment . Adding c plants . Bombyx  therapy . Currentcticides . It was c stress . Althougactivity . Thus, wNrf2 in mice makes it more sensitive to xenobiotic stress [CncC and detoxification genes (including GSTs and P450s) regulated by CncC were upregulated after phoxim treatment [Drosophila, and it was found that CncC could upregulate GSTd expression to enhance the ability to resist oxidative stress [CYP6A2 and increase resistance to DTT in Drosophila [Tribolium castaneum, the transcription factors CncC and Maf could regulate the expression of the CYP6BQ gene and increase resistance to deltamethrin [MtCncC is relatively conserved and has similar characteristics to other CncCs. In our study, by cotransfecting constructs containing MtCncC sequences and MtGST promoter sequences, we observed a significant induction of the MtGSTt1 (−574/+340) promoter by MtCncC, which suggests that MtCncC acts as a transcription factor responsible for the activity of the MtGSTt1 (−574/+340) promoter.The transcription factor Nrf2, which is a member of the basic leucine-zipper family, is an oxygen-sensitive transcription factor and a vital physiological stress response mechanism in organisms . Under oc stress . Nrf2 cac stress . CncC inreatment . The Cncosophila . In Tribamethrin . Based oGSTs were regulated by AhR/ARNT [Aphis gossypii Glover CYP450 to improve its tolerance to spirotetramat [M. persicae, AhR/ARNT could upregulate the expression levels of CYP450 to confer resistance to pesticides [CarE7 promoter and strongly induce transcriptional activity to enhance resistance to xenogenic stress in Nilaparvata lugens [MtARNT sequences and MtGST promoter sequences, the MtGSTt1 (−574/+340) promoter was significantly induced by MtARNT, which suggests that MtARNT acts as an important cis-regulatory element responsible for the transcriptional activity of MtGSTt1 (−574/+340). CncC and ARNT coordinately regulated the expression of GST in S. exigua [Nrf2 as a target gene of AhR, indirect activation of Nrf2 via CYP1A1-generated reactive oxygen species, and direct cross-interaction of AhR/XRE and Nrf2/ARE signaling [MtGST promoters and even appeared to reduce the transcriptional activity of the promoters. Thus, we speculated that MtCncC and MtARNT regulate the GST genes of M. troglodyta in a complex process.ARNT is also a regulatory element of xenobiotic stress response genes and a member of the bHLH-PAS transcription factor superfamily . AhR is AhR/ARNT ,52. In itetramat . In M. psticides . NlARNT a lugens . In this. exigua . In mammignaling . AccordiMtGST promoters and demonstrated their involvement in the response to tannic acid and quercetin stress. Furthermore, we identified two important transcription factors MtCncC and MtARNT involved in the regulation of the GST gene promoter. These results suggested that transcription factors regulate the expression of GSTs conferring resistance to plant secondary metabolites in M. troglodyta, and provided useful information for a better understanding of the regulatory mechanism between transcription factors and GSTs in M. troglodyta. Future studies will need to examine the mechanism of posttranscriptional regulation of GSTs in M. troglodyta.In summary, this study identified the core regions of the five M. troglodyta larvae were gathered from poplar (Populus × euramericana ‘Nanlin 895’) trees in Nanjing, Jiangsu Province, China. The larvae were fed fresh poplar leaves with a photoperiod of 16 h:8 h (light: dark), a temperature of 26 ± 1 °C and a relative humidity of 70–80%. Third-instar larvae were used for subsequent experiments. Sf9 cells were routinely maintained with SF-900 II serum-free medium  with 10% fetal bovine serum , 50 mg/mL streptomycin and 50 mg/mL penicillin (Invitrogen) at 28 °C. Sf9 cells were cultured for 3 days and then used for transfection experiments.MtGST promoter sequences were predicted on the website http://alggen.lsi.upc.es (accessed on 10 November 2022) to avoid disrupting the integrity of the binding sites when the promoters of different fragments were cloned. The 5’ loss fragments of each MtGST promoter were amplified from the full-length sequences of the MtGST promoters using TaKaRa Ex Premier™ DNA Polymerase . All primers were designed by Primer 5 software and were listed in MtGST promoter was ligated to a TA clone vector pMD-19T , and the correct clone product was obtained by sequencing. The PGL4.10-Basic vector and the pMD-19T with the 5’ loss fragment were digested with Nhe I and Xho I. Then, the 5’ loss fragment of MtGST promoter was ligated to the PGL4.10-Basic vector using T4 DNA ligase , and the ligation product was transformed into E. coli cells. The plasmid DNA was purified from E. coli cells for subsequent cell transfection experiments.First, the transcription factor-binding sites for all full-length 6 /well) were cultured in a 24-well culture plate, and each 5’ loss fragment promoter plasmid (700 ng/well) and pRL-TK  (70 ng/well) were cotransfected using 2 µL/well Cellfectin II reagent (Invitrogen) in accordance with our previous method . After 48 h, Sf9 cells were harvested and lysed in 1 × passive lysis buffer (Promega), and the renilla and firefly luciferase activities were measured using the Dual-Luciferase® Reporter Assay System kit (Promega) on an FLx800TM fluorescence microplate reader . The promoter activity was calculated by normalizing the relative activity of firefly luciferase with that of renilla luciferase. Three replicates were performed for each treatment independently.Sf9 cells (2.0 × 106 /well) were cultured in a 24-well culture plate, and then each 5’ loss fragment promoter plasmid (700 ng/well) and pRL-TK (interference plasmid) (70 ng/well) were cotransfected using 2 µL/well Cellfectin II reagent (Invitrogen). At 5 h posttransfection, we changed the transfection solution to cell culture medium containing 10 µL of tannic acid or quercetin with serum and double antibiotics. After 48 h, we measured luciferase activity using a Dual-Luciferase® Reporter Assay System kit on a microplate reader. The luciferase activity was calculated according to the above method.Tannic acid was initially solubilized in a small volume of acetone and then diluted in sterilized water to 70, 14, 2.8 and 0.56 mg/L, and sterilized water was used as a control. Quercetin was serially diluted in acetone to 35, 7, 1.4 and 0.28 mg/L, and acetone was used as a control. The concentrations of tannic acid and quercetin were determined according to previous study . Sf9 celM. troglodyta RNA was reverse transcribed using the PrimeScriptTM 1st Strand cDNA Synthesis Kit , and the cDNA was used for cloning MtCncC and MtARNT. The primers for cloning MtCncC and MtARNT were designed according to the transcriptome database and were listed in Ex Taq™ . The PCR program was as follows: 98 °C for 3 min; 35 cycles of 98 °C for 10 s, approximately 60 °C for 30 s and 72 °C for 90 s; an extension cycle of 72 °C for 5 min. The MtCncC and MtARNT DNA were ligated to the pMD-19T clone vector. The constructs were transformed into E. coli cells and sequenced by Sangon Biotech (Shanghai) Co., Ltd. The amino acid (AA) sequences of MtCncC and MtARNT were deduced from the NCBI Open Reading Frame (ORF) finder .Total RNA was extracted from third-instar larvae using TRIzol Reagent  according to the protocol. The quality and integrity of RNA were examined by a NanoDrop spectrophotometer and agarose gel electrophoresis, respectively. https://www.ncbi.nlm.nih.gov/ (accessed on 10 November 2022)). Multiple AA sequence alignment analysis was carried out using MEGA X (version 10.1) and Clustal X software (version 2.1). The phylogenetic tree was inferred by the maximum likelihood (ML) method in MEGA X with 1000 bootstrap replicates. The phylogenetic tree of MtARNT was inferred using the same methods.A phylogenetic tree was constructed to investigate the relationship between MtCncC and other insect CncC, and we picked Cap ‘n’ Collar as a keyword to query the nonredundant database . Sf9 cells (2.0 × 106/well) were cultured in a 24-well culture plate, and 350 ng of the promoter plasmid and 350 ng of the pAC-V5, pAC-V5-MtARNT, pAC-V5-MtCncC or pAC-V5-MtARNT and pAC-V5-MtCncC expression plasmids were cotransfected using 2 µL/well Cellfectin II reagent (Invitrogen). After 48 h induction, Sf9 cells were harvested and lysed to measure the renilla and firefly luciferase activities.Using two primer pairs pAC-V5--V5-ARNT , the MtCt test (two-tailed unpaired t test). The statistical significance of multisample comparisons was assessed with one-way ANOVA followed by Tukey’s multiple comparisons. A value of p < 0.05 was considered significantly different.ANOVA of the data collected from these experiments was performed using InStat software . The significant differences of all two samples were evaluated using Student’s


The fundamental cause of transcription dysregulation in hepatocellular carcinoma (HCC) remains elusive. To investigate the underlying mechanisms, comprehensive 3D‐epigenomic analyses are performed in cellular models of THLE2  and HepG2 (a hepatocellular carcinoma cell line) using integrative approaches for chromatin topology, genomic and epigenomic variation, and transcriptional output. Comparing the 3D‐epigenomes in THLE2 and HepG2 reveal that most HCC‐associated genes are organized in complex chromatin interactions mediated by RNA polymerase II (RNAPII). Incorporation of genome‐wide association studies (GWAS) data enables the identification of non‐coding genetic variants that are enriched in distal enhancers connecting to the promoters of HCC‐associated genes via long‐range chromatin interactions, highlighting their functional roles. Interestingly, CTCF binding and looping proximal to HCC‐associated genes appear to form chromatin architectures that overarch RNAPII‐mediated chromatin interactions. It is further demonstrated that epigenetic variants by DNA hypomethylation at a subset of CTCF motifs proximal to HCC‐associated genes can modify chromatin topological configuration, which in turn alter RNAPII‐mediated chromatin interactions and lead to dysregulation of transcription. Together, the 3D‐epigenomic analyses provide novel insights of multifaceted interplays involving genetics, epigenetics, and chromatin topology in HCC cells. THLE2 cells of normal liver hepatocytes and HepG2 cells of hepatocellular carcinoma (HCC) are comprehensively analyzed via integrated approaches including ChIA‐PET for 3D‐genome mapping and RNA‐seq, ChIP‐seq, and whole genome bisulfite sequencing for epigenome mapping, thus the 3D‐epigenome. The comparative analyses of the 3D‐epigenomes for normal hepatocytes and HCC cells reveal HCC‐associated genes for suppressing normal functions (HCC‐suppressed genes) in hepatocytes and activating new properties (HCC‐specific genes) in HCC. Thus, there is a pressing need for the development of novel therapies for HCC, which depends on a better mechanistic understanding of its pathogenesis.Hepatocellular carcinoma (HCC) is the sixth most common cancer in the world and the second leading cause of cancer‐related mortality in men.1 In s However, the underlying mechanism of transcription regulation remains elusive. In the past decade, cis‐regulatory elements  and trans‐regulatory factors  have been recognized as essential for the establishment of specific patterns of gene expression. In addition, it is becoming increasingly evident that distinct transcriptional profiles responsible for cell‐specific identity not only rely on the linear genome landscape, but also the 3D genome architecture that orchestrates the interaction of distal cis‐regulatory element mediated by trans‐acting TFs. Disorganization of 3D genome architecture has been implicated in the development of gastrointestinal stromal tumors (GIST), T cell acute lymphoblastic leukemia (T‐ALL), glioma, prostate cancer, and multiple myeloma. Nevertheless, the characteristics of 3D genome configuration with related epigenomic features, the extent and function of interactome, and their roles in establishing and maintaining HCC‐specific transcriptional programs have not been fully explored.Recently, large‐scale RNA sequencing efforts from the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) have revealed that gene expression is pervasively dysregulated in HCC.2, 3In this study, we report our integrated 3D‐epigenomic analyses to interrogate the underlying topological and epigenomic mechanisms that control gene transcription specific in HCC cells. Our results provide novel insights into the interplays of genetic and epigenetic elements that alter 3D chromatin architecture and lead to HCC‐associated transcription regulation.22.1 and RNA polymerase II (RNAPII) involved in most gene transcription, we specifically performed RNAPII ChIA‐PET experiments to map chromatin interactions between promoters and enhancers, and CTCF ChIA‐PET experiments to map chromatin folding architectures. Furthermore, to examine the impact in HCC by genetic and epigenetic variants, we incorporated GWAS and TCGA data along with epigenomic profiling data generated in this study for comparative analyses , epigenome , and 3D genome mapping . Considering CCCTC‐binding zinc finger protein (CTCF) as a major chromatin architectural protein18 and2.2AFP, GPC3, CCL15, HULC, Table SAA1, SAA2, NNMT, CXCL2, Table To identify and characterize HCC‐associated genes, we generated high quality RNA‐seq and RNAPII ChIA‐PET data for HepG2 and THLE2 cells , we further characterize HCC‐associated genes according to their participation in RAIDs. We found that most of HCC‐specific genes (386/488 = 79%) and HCC‐suppressed genes (924/1053 = 88%) are located within RAIDs in HepG2 and THLE2 cells, respectively  as a topological mechanism for co‐transcription‐regulation20 in y Figure . For exan Figure . Taken t2.3cis‐acting elements such as enhancers through long‐range chromatin interactions mediated by trans‐acting TFs. We first characterized all potential cis‐regulatory enhancers that are associated with RNAPII‐mediated chromatin interactions in HepG2 and THLE2 cells, showing that most of the enhancers with RNAPII are in active (H3K4me1+H3K27ac) or intermediate (H3K4me1 only) states, with only a small portion are in poised state (H3K4me1+H3K27me3)  or multiple genes (MG)  on HCC‐specific genes in MG domains and assigned the promoter as enhancer‐like (log2 ratio of H3K4me3/H3K4me1 < 0) and strong promoter TSS (log2 ratio of H3K4me3/H3K4me1 >0). In this analysis, we found that 48 (19.6%) out of 245 promoters in MG domains harboring HCC‐specific genes possess enhancer‐like property  analysis by Genomic Regions Enrichment of Annotations Tool (GREAT)24 fors Figure . The GO s Figure . This en on 421 enhancers of HCC‐specific genes  is the most significantly enriched. To further validate the specific binding by these TFs in HepG2 at specific enhancers with E‐P interaction, we analyzed ENCODE ChIP‐seq data for 124 TFs in HepG2 cells (Table CCL16 enhancer and promoter (enhancer‐like), which interact with the CCL15 promoter (a strong promoter) as shown by RNAPII ChIA‐PET data, and CCL15 is actively transcribing as indicated by RNA‐seq data in HepG2 cells. In contrast, there is no HNF4G/HNF4A binding and also no RNAPII present at the CCL16/CCL15 loci, as well as CCL16/CCL15 are inactive in THLE2 cells.To explore potential transcription factors that might be participated in specific E‐P interactions with HCC‐associated genes, we performed motif scan analysis using MEME25 on s Figure , and ides Figure , includils Table . Other t However, we have yet to distinguish DNA variants that directly influence risk for liver cancer  from those that are merely in linkage disequilibrium (LD) with them. In particular, it has not been systematically addressed if non‐coding variants in distal regulatory elements may have a role in HCC pathogenesis from a 3D genome perspective. In this direction, we integrated GWAS SNPs with liver/liver disease‐related traits and LD SNPs from the 1000 Genomes Project with our E‐P interaction data  interactions showed significant high‐correlation in transcription as measured by RNA‐seq, as in contrast with the randomly rewired the gene pairs , and found that 61% (16/26) of gene pairs were transcriptionally correlated in the TCGA data (Pearson's Correlation Coefficient > 0.25)  are connected by strong RNAPII‐associated chromatin interactions for possible co‐transcription  also showed co‐transcriptional property  Figure . Similarn Figure . However2.5STXBP6 is expressed in HepG2 but not in THLE2 cells, where this gene in THLE2 is located in a B compartment (inactive); whereas in HepG2 it is in an A compartment and associated with extensive histone marks of active transcription as well as RNAPII mediated chromatin loops  in HepG2 and THLE2 cells  is significantly associated with gene expression level, suggesting that enhancer activity quantitatively contributes to transcription.In this study, we leveraged multi‐omics analysis to shed light on the 3D epigenomic features and their impacts on transcriptional regulation in HCC cells. First, we identified a significant number of HCC‐associated genes (HCC‐specific and HCC‐suppressed), whose promoters and distal enhancers were in connection in long‐range distance in forms of interaction clusters often involving multiple genes and regulatory elements, in line with previous reports.32 Thi together suggesting HNF4A a central TF in the pathogenesis of NASH. Although the potential function of HNF4G in HCC is unknown, a recent study revealed that HNF4G is a pioneer factor that reprograms the enhancer landscape at gastrointestinal genes and mediates androgen‐receptor therapy resistance in prostate cancer. We thus propose that HNF4G/HNF4A may activate the expression of NASH‐associated genes via E‐P interaction, potentially leading to liver fibrosis/cirrhosis and HCC.As TFs are key determinants of enhancer activity, we hypothesized that the TFs enriched in HepG2‐specific and connected enhancers would be implicated in HCC transformation. As a proof of principle, we performed TF motif scan on enhancers with E–P interactions to HCC‐associated genes and identified HNF4G/HNF4A as the most enriched TF motif. Roles for these two transcription factors in HCC have not been previously described, but supported by a recent study showing that HNF4A is upregulated in livers of nonalcoholic steatohepatitis (NASH) patients and positively correlated with nonalcoholic fatty liver disease (NAFLD) fibrosis score,36 tog and maintained in vivo. A recent report described an improved dCas9 method to precisely modulate mCG states at TF binding motifs at single base resolution to clarify the roles of mCG in shaping TF occupancy. Future investigations in applying those tools will be valuable to test whether perturbation of DNA methylation on the CTCF motif in the promoter of HCC‐specific genes in HepG2 and HCC‐suppressed genes in THLE2 can reprogram the 3D genome architecture and subsequent enhancer–promoter interaction and transcription via reconfiguring CTCF binding and loop formation. Collectively, as DNA hypomethylation is a known hallmark of HCC, methylation‐dependent changes to CTCF binding may represent a previously unrecognized mechanism during HCC pathogenesis through its genome‐wide impact on 3D chromatin structure. Thus, modulation of CTCF binding by therapeutic epigenome editing may evolve as a novel strategy for future HCC treatment.In addition, we found that the majority of RNAPII‐associated interaction domains (RAIDs) are within, or overlapping with, CTCF‐mediated chromatin chromatin domains (CCDs), suggesting that CTCF‐mediated chromatin architecture may provide the topological framework that may constrain transcription regulation. We also found that the overall CCD distribution is conserved between HepG2 and THLE2 cells. However, unexpectedly, we discovered that the majority of HCC‐specific genes in HepG2 (HCC) cells have concomitant increase of CTCF binding comparing to THLE2 cells, whereas the HCC‐suppressed genes showed higher CTCF binding and looping in THLE2 cells than in HepG2 cells. To gain mechanistic insight into the differential CTCF binding on the promoter, we first examined the DNA sequence in CTCF binding sites for potential course of genetic variants, but found only a tiny fraction of DNA sequence variation. Remarkably, our analysis of whole genome bisulfite DNA sequencing revealed that the differential CTCF binding and extended looping associated with HCC‐specific genes in normal hepatocytes and HCC cells are highly correlated with epigenetyic states of 5mC methylation or demethylation, suggesting a robust imprinting and reprogramming mechanism involved in regulating the dynamics of DNA methylation/demethylation that in turn alter chromatin folding architecture and control transcription of HCC‐associated genes during the transformation from normal hepatocytes to HCC cells. Recently developed tools for epigenome editing provided evidence that the DNA methylation status can be modulated by dCas9/TALE fused with engineered DNA (de)methylation 39 and4−1 penicillin/streptomycin. THLE2 cells were cultured by using BEGM Bullet Kit . The culture dish used for THLE2 were precoated with a mixture of 0.01 mg mL−1 fibronectin, 0.03 mg mL−1 bovine collagen type I and 0.01 mg mL−1 bovine serum albumin dissolved in BEBM medium.HepG2 (ATCC HB‐8065) and THLE2 (ATCC CRL‐2706) cell lines were purchased from ATCC. HepG2 cells were cultured in DMEM supplemented with 10% FBS and 100 U mL Briefly, when the cells were grown to 80% confluency, 40 mL of ethylene glycol bis (EGS) /1 X PBS solution was added to each culture dish followed by shaking on orbital shaker for 45 min at room temperature (RT). After that, 1.1 mL of 37% formaldehyde   was added to each dish followed by shaking on orbital shaker for 20 min at RT. Subsequently, 3.57 mL of 2.5 m glycine   was added to each dish followed by shaking on orbital shaker for 10 min at RT. The cells were scraped off the dish by cell scraper and transferred into 50 mL corning tube and then centrifuged at 2000 rpm for 10min, 4 °C. Media was discarded by pipetting and the cells were washed for twice by adding 20 mL chilled PBS and then centrifuged at 2000 rpm for 5 min, 4 °C. After removing the supernatant, the cells pellets were stored at −80 °C.Long Reads ChIA‐PET libraries with antibody against RNAPII and CTCF were generated with HepG2 and THLE2 cells by following the ChIA‐PET protocol reported previously.44 Brim; NEB, cat.#N0440S). On‐bead proximity ligation was performed using in‐house bridge linker  and T4 DNA ligase . After that, proximity ligated chromatin complexes were eluted from beads and proteinase K  was added to reverse cross‐linked chromatin complexes for overnight. After Phenol:chloroform:IAA  extraction and isopropanol precipitation, tagmentation was performed on the proximity‐ligated DNA by Tn5 transposase from Nextera DNA Sample Prep Kit . Then tagmented DNA was immobilized on M280 streptavidin dynabeads . PCR amplification was performed on beads using Nextera DNA Sample Prep Kit and the products were purified by AMPure XP beads  and subjected to size selection (300–600 bp) on BluePippin instrument (Sage Science) using Blue Pippin Cassette Kit . DNA library was sequenced on Illumina Nextseq 500 by paired‐end 150 bp.300 millions of fixed HepG2/THLE2 cells were used to generate one ChIA‐PET library. Briefly, after cell lysis and nuclear lysis, the nucleus was sonicated into ≈1 kb size. Then chromatin was precleared using protein G beads and immunoprecipitation was performed using anti‐RNAPII /anti‐CTCF antibody  coated protein G beads . Then on‐beads A‐tailing was performed using Klenow Fragment (3′>5′ exo‐)  and dATP  . Chromatin was isolated by the addition of lysis buffer and lysates were sonicated and the DNA sheared to an average length of 200–300 bp by Bioruptor (Diagenode). Genomic DNA (Input) was prepared by treating aliquots of chromatin with proteinase K  at 65 °C for de‐crosslinking overnight, followed by purification using MinElute PCR Purification Kit . Sonicated chromatin was precipitated by H3K4me1 , H3K4me3 , H3K27Ac , H3K27me3  antibodies. Precipitated chromatin complexes were washed, eluted from the beads with SDS buffer, and subjected to proteinase K treatment. De‐crosslinking was performed by incubation overnight at 65 °C. ChIPed DNA was purified by Qiagen MinElute kit. Library preparation was performed using the KAPA Hyper Prep kit  and sequenced on Illumina Nextseq 500 by single‐end 50 bp.Ten million of HepG2 and THLE2 cells were fixed by adding 1%  formaldehyde . Fixation was stopped by the addition of 0.125 Total RNA was extracted from HepG2 and THLE2 using PureLink RNA Mini kit  and subjected to on‐column DNase I  treatment. 1 µg RNA was performed ribosomal RNA depletion using the riboZero rRNA removal kit . RNA was fragmented into 200 bp in 5X NEBNext First Strand Synthesis Reaction Buffer . First strand cDNA was synthesized using random hexamer primer and M‐MuLV Reverse Transcriptase . Second strand cDNA synthesis was subsequently performed using DNA Polymerase I  and RNase H . Library preparation was performed using the KAPA Hyper Prep kit . DNA library was sequenced on Hiseq 2500 by paired‐ end 100 bp.A total amount of 2 µg DNA per sample (HepG2/THLE2) was used as input material for the DNA library preparations. Sequencing library was generated using Truseq Nano DNA HT Sample Prep Kit (Illumina) as per manufacturer's instruction and index codes were added to each sample. Briefly, genomic DNA sample was fragmented by sonication to a size of 300–500 bp by Covaris M220. Then DNA fragments were end‐polished, A‐tailed, and ligated with the full‐length adapter for Illumina sequencing, followed by further PCR amplification. After PCR products were purified using AMPure beads, libraries were analyzed for size distribution by Agilent 2100 Bioanalyzer and quantified using KAPA Hyper Prep kit  and sequenced on Illumina HiSeqXten by pair‐end 150 bp.2 µg of HepG2/THLE2 genomic DNA was sonicated using a Covaris M220 into a size of 300–500 bp. Sodium bisulfite conversion of all DNA samples was performed using the EZ DNA Methylation kit . All libraries were subjected to quality control by Agilent Bioanalyzer examination and quantified using the KAPA Hyper Prep kit  and sequenced on Illumina HiSeqXten by pair‐end 150 bp.See Supporting Information.Genome‐wide sequencing reads were deposited at GEO. The accession number for the ChIA‐PET, ChIP‐Seq, whole genome sequencing, and RNA‐Seq datasets for HepG2 and THLE2 cells reported in this paper is GEO: GSE144893. Software used in this study is listed in the Software and Algorithms Table.The authors declare no conflict of interest.Y.F., P.W., L.C., and M.Z. contributed equally to this work. X.C., and L.L. conceptualized and supervised this study. Y.F., P.W., and M.Z. designed the experiments and generated all genomic data. L.C. analyzed the data. E.G. cultured cells and treated cells. J.W. and S.L. processed the whole genome bisulfate sequencing data and provided valuable suggestions. F.H., Y.L., W.Z., W.Z., Y.X., X.H. provided expertise in liver cancer biology and contributed to manuscript preparation. Y.F., P.W, L.C., and Y.R. interpreted the results and wrote the manuscript.Supporting InformationClick here for additional data file.


Styela clava, a wildly distributed ascidian with strong vitality. Cross-species comparison assures the function of the endostyle as a thyroid and parathyroid gland and the key composition of the digestive tract. Meanwhile, the high correlation with blood-producing organs, such as the head kidney and bone marrow in advanced vertebrates, implies a potential function of the endostyle.Formation of the complex organ has long been an arresting topic in evolution. The prototype of the complex organ is more likely to exist in creatures of ancient evolutionary hierarchy. In sister groups to vertebrates, including cephalochordate and urochordate, and the basal vertebrate, cyclostomate, a specialized pharyngeal organ is uniquely possessed by animals in these groups, named the endostyle. The endostyle serves as a filter-feeding and thyroid hormone-synthesizing function and is recognized as a prototype of the thyroid gland. Here, we comprehensively uncovered the expression profile of the endostyle in Styela clava was conducted and distinct markers were selected by multileveled comparative analysis. Transcriptional data assay and qRT-PCR-verified results showed the regional expression patterns of Hox genes in the longitudinal axis. Organ-specific markers of the endostyle was proposed by comparing expression with the main organs of the ascidian. A cross-species transcriptional profile projection between the endostyle and organs from Danio rerio and Homo sapiens indicates a robust homogenous relationship to the thyroid and digestive system of the endostyle. The high similarity between the endostyle and the head kidney in zebrafish/the bone marrow in human implies uniquely profound functions of the pharyngeal organ in proto-vertebrates. Our result revealed that the transcriptional profile of the human parathyroid gland was similar to the ascidian endostyle, indicating the evolutionary origin of vertebrate hormone secretion organs.The endostyle is a pharyngeal organ with an opening groove and cilia in invertebrate chordates (amphioxus and ascidian) and cyclostomate (lamprey), serving as a filter-feeding tract and thyroid-secreting location. Emerging evidence implies its complex cellular composition and potentially versatile functions. Multiple cell types in the endostyle have been thought to be progenitors of complex organs in advanced vertebrates. To describe the expression profile and the potential functions, bulk RNA sequencing on the endostyle in ascidian  The endostyle is a specialized pharyngeal organ existing in evolutionary transitional groups between invertebrates and vertebrates, including urochordates, cephalochordates, and ammocoete in Cyclostomata. The endostyle is a grooved organ partially encircled by ciliated epithelia tissue and longitudinally extended through the ventral wall of the pharynx . In asciBesides the mucus secretion for food particle packaging and digestion, research has shown certain epithelial cell clusters are capable of concentrating iodine ,5,6,7,8.During the process of individual development, the endostyle is generally believed to be originated from the endoderm . DiffereAs the particularity and novelty of the endostyle are possessed only by non-vertebrate chordates and ammocoete in Cyclostomata, the evolutionary status of the endostyle has long been discussed along with the origin of the thyroid organ. In lamprey larvae, the transformation to thyroid follicles during endostyle metamorphosis gives a recapitulation of thyroid organ emergence in vertebrate evolution, which makes it acknowledged that the evolutionary origin of the thyroid gland lies in endostyle epithelial compartments . HoweverImmune activity is also a key function in the endostyle as the direct exposure to seawater. Kinds of immune effector cells with certain markers were defined in the endostyle and subendostylar region bathed with circulatory cells. In solitary ascidian, Toll-like receptor 2 and vasoactive intestinal peptide and lectin histochemistry were used in defining immune zone cells and proved zone 8 with a multilabeled immune function . AdditioBotryllus schlosseri has proved the anterior ventral region of the endostyle and adhering blood cells from the subendostylar sinus as an adult somatic stem cell niche that supports the organ regeneration and development of new zooids [B. schlosseri was believed to be homologous to the hematopoietic bone marrow of advanced species as the discovery of hematopoietic stem cells, blood cells, and immune effector cells in the endostyle niche [The colonial ascidians have a strong capacity for asexual reproduction and stem-cell mediated development process. Research on ascidian w zooids . Along ww zooids . The endle niche . The resle niche , the resSlit was specifically expressed in the endostyle [By comparing multi-organ within-species and cross-species organ transcriptional profiles, the endostyle was defined as having a high similarity to other vertebrate tissue . Previoundostyle . All theS. clava, whose genome has been sequenced and whose transcription factors have been deeply investigated [Ciona robusta was conducted in defining the OSGs in the endostyle by comparing the expression profile of the endostyle with other organs. Based on the OSG, similarities between the endostyle and the organs of D. rerio and H. sapiens were calculated and interpreted.Here, we utilized the bulk RNA sequencing data on the endostyle in ascidian stigated ,26. The S. clava was collected from the coastal areas of Weihai City, Shandong province . The ascidian was temporarily persevered in the aquarium system in the laboratory at a temperature of 18 °C with a consistent oxygen supply. The healthy animal was dissected manually with disinfected instruments initiated by the removal of the stem tunic part and scissored from the ventral body mid-line. The endostyle was gently dissected from the dorsal pharyngeal wall and segmented into three longitudinal parts of the same length for the convenience of the following experiments. Three individual ascidians were sacrificed for the endostyle collection. The guidelines for animal experiments were approved by the Ocean University of China Institutional Animal Care and Use Committee (OUC-IACUC) with approval number 2021-0032-0027.The adult ascidian ® O.C.T. Compound) in the model of a proper shape. Liquid nitrogen was used to snap-freeze the block. The embedded block after freezing was fully covered by aluminum foil and preserved in sealed containers at −80 °C.To visualize morphological features of the endostyle cross-section, a cryosection preparation and staining technique was carried out on the endostyle sample. Fresh endostyle tissue was carefully dissected from the ascidian and immediately immersed into the precooled 4% paraformaldehyde (PFA) and persevered on a gentle shaker table for 16 h at 4 °C. The tissue after fixation was washed with cooled PBS to wash off PFA and the tissue was transferred into 30% sucrose solution (made with PBS solution) in the flat-bottomed container. All processes of fixation and sucrose sinkage were conducted at the temperature of 4 °C. After full sinkage, the solution on the surface of the tissue was dried with Kimtech wiper (FIS: 06666) and the tissue was merged into the optimal cutting temperature compound  Stain Kit from Solarbio (G1121).® Ultra™ RNA Library Prep Kit for Illumina®. The library was sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. Clean reads were obtained from Novogene.The total RNA of the endostyle samples was extracted with RNAiso Plus . The integrity and quality of the total RNA was evaluated by agarose gel electrophoresis and Nanodrop spectrophotometry (Eppendorf). Before library construction, the quality of RNA was finally analyzed by Agilent 2100 bioanalysis. The library construction and sequencing experiment were accomplished by the Novogene experimental department. mRNA was enriched and the library was constructed with NEBNexthttp://ccb.jhu.edu/software/stringtie/index.shtml?t=manual#deseq, accessed on 31 May 2022). The FPKM and TPM results were transferred based on the count file. Differential expressing genes were analyzed with R package DEseq2 [https://CRAN.R-project.org/package=pheatmap, accessed on 31 May 2022). Gene enrichment analysis was performed with clusterProfiler [Clean reads were aligned against the reference genome with hisat2 . Resultse DEseq2 . All thrProfiler . Top funhttps://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 2 November 2022)  on Light Cycler 480 (Roche). All RT-qPCR primers were listed in −ΔΔCt method. Visualization of qRT-PCR results was carried out with R package ggplot2 and ggsignif .qRT-PCR was used in verifying the expression of DEGs and the transcriptome results. The cDNA of the endostyle was synthesized by reverse transcription reagent, HiScript II Q RT SuperMix for qPCR . The primers for qRT-PCR were designed utilizing the NCBI primer design tool (er 2022) . The uniC. robusta organs were obtained from a published dataset [Styela was aligned against the transcript dataset of C. robusta and the max_target_seqs was set to 1. The expression matrix of C. robusta in reads per kilobase per million mapped reads (RPKM) format was merged with Styela endostyle expression profile in fragments per kilobase per million mapped reads (FPKM) version. Homolog gene pairs with an expression value larger than 0.5 in Ciona organs (except endostyle) were eliminated. Genes with expression values larger than 1 in Styela endostyle were defined as OSGs of S. clava endostyle. A total of 104 OSGs were obtained  genome (Version GRCz10) with BLASTP. One hundred unique pairs of homolog genes between zebrafish and Styela endostyle OSGs were obtained. To obtain as many organs expressional data as possible, multiple transcriptomic datasets were involved in the analysis. The raw FASTQ files of organs such as tail, spleen, skin, muscle, liver, heart, and brain were obtained from GSE171906 [D. rerio, genes with TPM larger than 1 in D. rerio organs were defined as identical expressed genes. The ratio of highly expressed genes in each organ of zebrafish was defined as a similarity ratio.OSGs in the endostyle were aligned against the zebrafish . Shared and organ-only expressed gene lists were utilized for GO enrichment analysis with the R package clusterProfiler [The transcriptional matrix of the endostyle and pharynx, gill, head kidney, and thymus were compared manually with the Venny online interactive tool . RNA consensus tissue gene data were obtained from the website, which included the consensus transcript expression levels summarized per gene in more than 50 tissues of the human. Twenty-seven main tissues were taken into account. The tissue-enriched gene was defined with an expression level of at least a four-fold higher mRNA level in a particular tissue compared with any other tissue . The protein sequence of all tissue-enriched genes (in UNIPROT gene id) was aligned against all proteins of Styela. Based on the homolog correlation, all paired homolog tissue-enriched genes were evaluated with the expression level in the endostyle. Homolog pairs with FPKM larger than 5 were defined as positive-expressed human-tissue-enriched genes in the endostyle. The similarity index was the ratio of the positively expressed genes in each list of tissue-enriched genes for human organs. The human anatomical graph was plotted with gganatogram [Human tissue-enriched genes were obtained from the protein atlas  was conducted C. CategoMeanwhile, the top 300 expressed genes of the endostyle were filtered and selected manually based on the functional annotation of the genes. The functions of four main categories were selected and the relative expression level was visualized by clustered heatmap D. For exThe immune function of the ascidian exclusively relies on innate immunity . As a phAs an organ-facilitated filter-feeding , the endDifferential expression analysis was conducted on the longitudinal levels of the endostyle. The expression profile of each two segments was compared to obtain DEGs. The volcano plot shows DEGs of each two comparing groups . The filA few genes with expressional longitudinal change gradients can be seen in the heatmap A. For exC. robusta juvenile [Meanwhile, some genes with expression patterns enriched in the posterior part of the endostyle were also defined B. Here, juvenile , which iStyela, to decipher the function of endostyle in the ascidian we choose to use Bulk RNA sequencing data of 11 organs or tissues from adult C. robusta as a reference dataset to identify OSGs of the endostyle [C. robusta includes the oral siphon, atrial siphon, neural complex, heart, ovary, pharynx, stomach, proximal intestine, middle intestine, distal intestine, and endostyle, which are the main organs of an ascidian adult individual. Unique transcripts with the longest length of S. clava were aligned against the total protein sequence of C. robusta with BLASTP for obtaining comparable homologous gene pairs. A total of 18,203 homolog transcript pairs were obtained as the cornerstone of two ascidian cross-species’ comparison. All gene or transcript functional annotations were adopted from the version of C. robusta  has a significantly high expression in the endostyle of both species. The expression of B-cell CLL/lymphoma 6 member B protein (BCL6B) was also detected in both species, which was reported as a functional factor related to early B-cell development events in the mammal model [slit homolog, was also detected.As the selected result  showed A, some gal model . The higD. rerio was conducted , 5528 shared-expressed homolog gene pairs were detected, while 1254 and 1725 genes were uniquely expressed by either the pharynx or the endostyle , which was reported as a functional factor related to early B-cell development events in mammal models [By comparing with the expression profile of  markers A. For ex mammals . The disl models .Fibropellin, a family of extracellular matrix proteins, has the function of assisting epidermal growth and the differentiation of mesenchymal and epithelial cells. The high expression of fibropellin caters to the structure feature of the endostyle. The transcription factor homolog of aristaless, which plays a vital role in both the neuron system and pancreas development , is alsoThe Origin of Species. Nowadays, we assure that complex eyes could be evolved from very simple ones with the driving force of natural selection. From this example, the origin or evolutionary node pattern of the complex organ is necessary for drawing a realistic evolving path in forming a complex organ in advanced vertebrates. The endostyle is only preserved by cephalochordates, urochordate, and larva lamprey (which is thought to be a homolog organ to the thyroid gland); more specifically, only zone 7 is capable of concentrating iodine. However, as a specialized pharynx organ shares the same body location as the pharynx of advanced animals, the endostyle is very likely to preserve another prototype of pharynx organs.The formation of complex organs in advanced vertebrates has long been an attractive topic in evolution. For example, the evolution of the eye was thought to be “absurd to the highest degree” by Charles Darwin in S. clava. Based on these OSGs, a cross-species transcriptional profile comparison was conducted in probing the possible correlation of the endostyle to more advanced organs. From the perspective of the shared expression of the endostyle OSG homolog, the expressional similarity to organs in zebrafish was obtained  [Ciona pharynx and the Styela endostyle to conduct a similar comparison and the enrichment functional terms are basically consistent with results of the pharynx–endostyle of Ciona  , the phaof Ciona , while rIn an OSG-based similarity ratio analysis, the head kidney is the second highest in organ similarity ranking. The head kidney in fish is believed to be analogous to the mammalian bone marrow and the primary site of hematopoiesis. The high similarity between the endostyle and the head kidney hints at potential functional similarity. In a transcriptional expression profile comparison analysis C,D, sharMeanwhile, the level of shared expression of the organ/tissue-enriched gene homolog of human was used as a similarity between human tissues and the endostyle. Interestingly, in this result, except for the generally acknowledged thyroid and digestive-related tissue , the parathyroid was reported for the first time as a highly similar organ, which needs further research and evidence in supporting the possible evolutionary lineage. Similar to the result of zebrafish, the blood system, and hematopoiesis corresponding organs, the choroid and bone marrow both have a high similarity to the endostyle. The choroid is a middle layer of tissue of the wall of the eye, which is composed almost entirely of blood vessels functioning as the blood supplier of the retina. The high similarity of the choroid and the endostyle showed potential similarity between these two organs, which is consistent with the research on the colonial ascidian endostyle . These rCell types are the basic building blocks for multicellular organisms, which are also fundamental evolution units such as genes and species ,67. The S. clava provided a detailed profile of the endostyle function and the multileveled comparison selected certain markers in representing its distinct features. Basic functions of the endostyle, including thyroid hormone synthesis, immune function, digestion, and neuron function, were supported by distinct markers. The Hox A3 and Hox A4 are mainly longitudinal divergent genes in the endostyle. A total of 104 OSGs were defined as organ markers of the endostyle in S. clava. The expression similarity between the endostyle and the organs of D. rerio, including the gill and the head kidney, was proved in the OSGs-based cross-species alignment and organ transcriptomic profile comparison. The similarity between the endostyle and human thyroid/the stomach was proved to be of the highest rank in human tissue-enriched marker-based similarity calculations. Adapting to the structure consisting mainly of epithelial cells, similarities to the fallopian structure and skin were also seen. Interestingly, high similarities were also obtained in the comparison of endostyle against parathyroid, choroid, bone marrow, and retina. These similarities of the endostyle against organs from D. rerio and H. sapiens imply the potential function homolog of the pharynx organs and hematopoietic system of the endostyle.Bulk RNA sequencing on the endostyle in


YY2 overexpression and knockdown increased and decreased osteoblast differentiation, respectively, in BMP4-induced C2C12 cells. Mechanistically, YY2 overexpression increased the mRNA and protein levels of Osterix (Osx), whereas YY2 knockdown had the opposite effect. To investigate whether YY2 regulates Osx transcription, the effect of YY2 overexpression and knockdown on Osx promoter activity was evaluated. YY2 overexpression significantly increased Osx promoter activity in a dose-dependent manner, whereas YY2 knockdown had the opposite effect. Furthermore, vectors containing deletion and point mutations were constructed to specify the regulation site. Both the Y1 and Y2 sites were responsible for YY2-mediated Osx promoter activation. These results indicate that YY2 is a positive regulator of osteoblast differentiation that functions by upregulating the promoter activity of Osx, a representative osteogenic transcription factor in C2C12 cells.Yin Yang 2 (YY2) is a paralog of YY1, a well-known multifunctional transcription factor containing a C-terminal zinc finger domain. Although the role of YY1 in various biological processes, such as the cell cycle, cell differentiation and tissue development, is well established, the function of YY2 has not been fully determined. In this study, we investigated the functional role of YY2 during osteoblast differentiation.  Bone development is maintained by the formation and resorption of osteoblasts. Disruptions in the dynamic balance of formation and resorption, such as excessive resorption, can cause loss of bone mass, density, and strength, resulting in systemic diseases such as osteoporosis ,2. ThereOsx clearly exhibit an osteoblastic phenotype, suggesting that Osx plays an important role in osteoblast differentiation. The mechanism of Osx function in osteoblast differentiation occurs via the binding of a GC-rich domain in the osteoblast marker gene promoter region [Osx transcription is absent in Runx2-null mice. In contrast, the expression levels of Runx2 in Osx-null mice are similar to those in wild-type mice, suggesting that Runx2 is an important upstream regulator of Osx in the differentiation pathway [Osteoblasts are the main cell type responsible for bone formation. They are derived from mesenchymal stem cells (MSCs), which differentiate into myoblasts, chondrocytes, adipocytes, or osteoblasts, depending on the conditions . MSC difr region . Osx tra pathway .YY1, YY2 is not ubiquitously expressed; therefore, the biological function of YY2 has not been identified. Recently, YY2 has been shown to be involved in tumorigenesis as a novel tumor suppressor, with a role in cell cycle regulation and cell proliferation [IL-4) by exerting an antagonistic effect that opposes the effect of YY1 [Runx2 transcriptional activity and inhibits osteoblast differentiation [Yin Yang 2 (YY2) is a member of the Yin Yang (YY) family and has a highly conserved DNA-binding zinc finger structural domain at the C-terminus. YY2 is highly homologous to another member of the YY family, Yin Yang 1 (YY1). YY2 may function as a transcriptional regulator, similar to YY1 ,13. Howeferation . YY2 is t of YY1 . During ntiation . YY2 is Osx and increasing Osx expression.Here, we present evidence of the role of YY2 in osteoblast differentiation. YY2 promoted BMP4-induced osteoblast differentiation by regulating the promoter activity of YY2 significantly enhanced BMP4-induced ALP activity in a dose-dependent manner  after 2 days of treatment with BMP4  expression in C2C12 cells. ALP is an enzyme that is involved in bone mineralization so it is commonly used as an early marker for osteoblast differentiaion . Under tt manner A,B. We aith BMP4 C,D. ThesYY2 knockdown on osteoblast differentiation. The efficiency of the inhibition of endogenous YY2 was described previously [YY2 knockdown dramatically reduced BMP4-induced ALP activity  upstream regulatory sequence of the human Osx gene to analyze Osx promoter activity  and ctgctCCATttgctgagct (Y2 site) using genematix software. To determine whether the effect of YY2 on Osx occurs at the YY1 consensus motif, we used a reporter assay to analyze the effects of deletions in the 3.0 kb Osx promoter (Osx occurs at the YY1 consensus site.activity . To inveactivity A. Overext manner B. It waspromoter C. The prpromoter D. This sYY2 exerted a significantly weaker effect on the mutant Osx promoters than on the wild-type promoters (Osx expression stimulates Osx promoter activity at the YY1 consensus motif. The above results indicated that YY2 stimulates the Osx promoter at the YY1 consensus motif. To investigate whether YY2 could bind to the proposed binding site (Y2), we performed a ChIP assay using Myc antibody and a primer set flanking the binding site  A. Overexromoters B. These ing site C. We foupromoter D. These Bone formation is divided into endochondral ossification and intramembranous ossification. These two different patterns of bone development involve a variety of transcription factors and secreted molecules, indicating the complexity of the bone formation process and the diversity of the regulatory processes . Bone coOsx expression in Runx2-null mice. Recently, BMP was found to induce Osx expression in a Runx2-dependent or non-Runx2-dependent manner [Osx expression is also regulated by IGF [Osx expression by various signal transduction proteins and transcription factors is important for the regulation of osteoblast differentiation [Sp7/Osx is an osteoblast-specific transcription factor that regulates bone formation and osteoblast differentiation in vivo and in vitro. Osx was concluded to be a downstream factor of Runx2 due to the absence of t manner ,24. Osx d by IGF , the infd by IGF , and pard by IGF . This evntiation .YY2 is similar to that of YY1, the N-terminal region is very different, suggesting that the YY2 protein functions differently from the YY1 protein [IL)-4, share the DNA binding sequence of YY1 and YY2 but have opposite biological functions [YY2 is a paralog of YY1 with a highly similar structure. Therefore, YY2 exhibits either transcriptional activation or transcriptional repression. Although the C-terminal region of  protein ,29,30. Y protein . For exaunctions . To dateYY2 overexpression dramatically increased BMP4-induced osteoblast differentiation and stimulated mRNA expression of the osteoblast differentiation markers ALP, OC, and the transcription factors Runx2 and Osx. YY2 also significantly upregulated the promoter activity of ALP, BSP, and OC. Therefore, YY2 and YY1 exhibited opposite phenotypes during osteoblast differentiation. To clarify the molecular mechanisms of YY2-mediated promotion of osteoblast differentiation, we further investigated the relationship between YY2 and osteoblast transcription factors. YY2 specifically stimulated the expression of the transcription factor Osx during osteoblast differentiation. While YY2 did not affect both mRNA and protein levels of Runx2.In the present study, our results emphasized the essential role of YY2 in osteoblast differentiation. Osx promoter. In agreement with our hypothesis, YY2 upregulated Osx promoter activity in a dose-dependent manner. Using Genomatix software, we identified two YY1 consensus sequences located in the Osx promoter region. Two YY1 consensus sequences were identified as essential for YY2-mediated regulation of the Osx promoter. More importantly, YY2 regulated Osx promoter activity in a YY1-independent manner. Subsequent deletions in the Osx promoter revealed that the reduction in promoter activity at the Y1 site in response to YY2 overexpression was significantly smaller than that in the wild-type promoter. Nevertheless, the presence of the Y2 site allowed the activity of the deleted promoter to be maintained. This suggests that the two YY1 consensus sequences are essential for YY2-mediated regulation of the Osx promoter.Based on our hypothesis that YY2-mediated gene regulation is promoter-dependent, we constructed a luciferase reporter plasmid containing the human Osx promoter, and we determined that YY2 regulated Osx promoter activity and expression by binding to the YY1 consensus motif, especially the Y2 site. Moreover, YY2 knockdown decreased ALP activity and osteogenic gene expression, resulting in the suppression of osteoblast differentiation.To further demonstrate the importance of the consensus sequences, mutations were introduced into the Y1 and Y2 sites, separately and simultaneously. Consistent with our hypothesis, the promoter activity associated with mutations in the Y1 or Y2 sites was significantly reduced. Furthermore, we found that YY2 binds to the Y2 site in the promoter region of Osx, but not the Y1 site. The mutated Y1 site loses the induction of YY2 and we retain the possibility that there are other factors on the Y1 site involved in the regulation of the Osx promoter by YY2 that require further elucidation. These findings highlighted the importance of the YY1 consensus motif for YY2-mediated regulation of the Osx is a major target of YY2. YY2 increased osteoblast differentiation by binding with the Osx promoter to regulate promoter activity and gene expression.In summary, we provide novel evidence of the essential role of YY2 in osteoblast differentiation. Our findings also indicate that 2 at 37 °C and maintained in Dulbecco’s modified Eagle medium  containing 10% FBS  and 1% antibiotic-antimycotic solution (Gibco). Transient transfections were performed using the polyethyleneimine (PEI)-mediated method , and transfection controls were established using empty vectors.Mouse pre-myogenic C2C12 and human embryonic kidney 293 (HEK293) cells were cultured in 5% COC2C12 cells were cultured for 72 h in 2% FBS medium containing BMP4 to induce differentiation. The cells were washed twice with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde at room temperature (RT) for 15 min, washed twice again with PBS, and then incubated in BCIP/NBT substrate  for 15 min at RT. Quantification of ALP staining was performed at an absorbance of 480 nm.4 cells/well. The cells were transfected with a CMV promoter-driven β-galactosidase reporter gene  (0.05 μg) and luciferase reporter gene (0.3 μg), and the cell lysates were analyzed for luciferase activity 36 h after differentiation. A luciferase reporter gene assay kit  was used to measure luciferase activity on the TriStar² Multimode Reader platform. All experiments were performed in triplicate.C2C12 cells were seeded in 24-well plates at a density of 2 × 10Total mRNA was extracted from cultured C2C12 cells using RNAiso Plus . cDNA was synthesized from 1 μg of total RNA using Oligo dT primers and reverse transcriptase . cDNA was synthesized using SYBR Premix Ex Taq kit (TaKaRa) on a CFX96 real-time PCR system. The following PCR conditions were used: initial denaturation at 94 °C for 3 min, 40 cycles of denaturation at 94 °C for 30 s, and annealing at the optimal temperature for each primer pair for 30 s. The primer sequences used for PCR were as follows: mALP forward 5′-ATC TTT GGT CTG GCT CCC ATG-3′ and reverse 5′-TTT CCC GTT CAC CGT CCA C-3′; mOC forward 5′-GCA ATA AGG TAG TGA ACA GAC TCC-3′ and reverse 5′-GTT TGT AGG CGG TCT TCA AGC-3′; mRunx2 forward 5′-CCT GAA CTC TGC ACC AAG TCC T-3′ and reverse 5′-TCA TCT GGC TCA GAT AGG AGG G-3′; mOsx forward 5′-TCG CAT CTG AAA GCC CAC TT-3′ and reverse 5′-CTC AAG TGG TCG CTT CTG GT-3′; GAPDH forward 5′-AGG TCG GTG TGA ACG GAT TTG-3′ and reverse 5′-GGG GTC GTT GAT GGC AAC A-3′.3VO4, 250 μM PMSF, 10 μg/mL leupeptin, 10 μg/mL peptidase, and 10 μg/mL aprotinin) for 15 min, followed by centrifugation at 13,000 rpm and 4 °C. The supernatant was then removed. Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes . The membranes were blocked with 5% skim milk and incubated with the appropriate antibodies against Runx2 , Osx , Myc  (Santa Cruz Biotechnology), HA , and α-tubulin . After washing and incubation with the appropriate horseradish peroxidase (HRP)-conjugated mouse and rabbit secondary antibodies, visualization was performed using Immobilon Western Chemiluminescent HRP Substrate  on the AmershamTM ImageQuantTM 800 biomolecular imager .Cells were washed twice with PBS and lysed in ice-cold lysis buffer  and a standard antisense primer, 5′-CCG CTC GAG GGG GGG TAG AGA GAG AAA AGG-3′. To mutate the putative YY1 consensus sites in the 3.0 kb promoter, mutant primers for aac ctC CAT att cct tgt c  and ctg ctC CAT ttg ctg agct  were applied in combination with the primers used to amplify the normal 3.0 kb promoter. The overlap-extension PCR method was used for mutagenesis. Amplicons were cut with KpnI and XhoI restriction endonucleases and incorporated into a pGL3-basic vector cut with the same enzymes, resulting in several promoter-firefly luciferase constructs.ChIP tests were carried out according to the manufacturer’s instructions using a commercial kit . Chromatin samples were prepared from HEK 293 cells at the indicated time after transfection of Myc-empty or Myc-YY2. Briefly, HEK 293 cells were cross-linked with formaldehyde and incubated for 15 min at 37 °C, and the cell lysates were treated with SDS lysis buffer prior to sonication to shear DNA into 200–1000 bp fragments. Protein cross-linked with DNA was then immunoprecipitated with Myc antibody or anti-IgG antibody  and add 60 μL of protein A Agarose/Salmon Sperm DNA. After wash and elution, chromatin was then reverse-crosslinked for 4 hr at 65 °C, and extracted DNA was used as a template for PCR. The following ChIP PCR primers were used: YY2 binding site in the Osx promoter (Y2-site), forward 5′-CTC CCT GAT CCC TTC TTT GG-3′ and reverse 5′-AAG GCT CCA GAT CCA ATG AG-3′YY2 gene. Sense and antisense oligonucleotides were annealed and ligated into a pSuper-retro vector .Small interfering RNA oligonucleotides were generated by targeting a 22 base sequence, GTA GTA GAG ATC ATG ATA A, of the mouse t-test was used to analyze the data, with p < 0.05 indicating significance.All experiments were repeated at least twice with three independent, generating qualitatively identical results. The results are given as the mean ± standard error of the mean. The student’s


Enterotoxigenic E. coli F18 induces post-weaning diarrhea in piglets, which results in severe economic damage for pig farmers. However, the mechanism of resistance to E. coli F18 infection in IPEC-J2 cells is still unclear. Here, we determined the regulatory mechanism of CXCL11 expression in IPEC-J2 against infection by E. coli F18. Briefly, CXCL11 expression was dramatically downregulated in IPEC-J2 cells infected with E. coli F18. In addition, CXCL11 overexpression significantly reduced the interaction ability of E. coli F18 with IPEC-J2 cells. The methylation level of the mC-7 site of the CpG island in the promoter region of the CXCL11 gene and mRNA expression were inversely linked. In addition, the mC-7 site is located in the transcription factor OSR1-binding domain, which is linked to the susceptibility of E. coli F18. In this study, the epigenetic regulation mechanism of CXCL11 on resistance to E. coli F18 was revealed from the level of DNA methylation, and the resistance function genes were further identified, laying a foundation for the prevention and control strategies of resisting bacterial diarrhea in pigs. Escherichia coli F18 (E. coli F18), hence it is essential to investigate the mechanism governing E. coli F18 resistance in native pig breeds. Based on the previous RNA-seq results of the duodenum from E. coli F18-resistant and -susceptible Meishan piglets, CXCL11, an important functional gene, was preliminarily screened. In this investigation, in order to further examine the expression regulation mechanism of E. coli F18 in intestinal porcine epithelial cells (IPEC-J2) against E. coli F18 infection, CXCL11 gene expression on IPEC-J2 cells infected by E. coli F18 was detected, which was significantly downregulated (p < 0.01). Secondly, the overexpression on the IPEC-J2 cell line was successfully structured, and a relative quantification method of the PILIN, bacteria enumeration, and immunofluorescence assay indicated that the CXCL11 overexpression significantly reduced the ability of E. coli F18 to interact with IPEC-J2 in vitro. The promoter region of the CXCL11 gene was predicted to contain a CpG island (−619 ~ −380 bp) of which 13 CpG sites in the sequencing region were methylated to varying degrees, and the methylation level of one CPG site (mC-7) positively linked negatively with the expression of the CXCL11 gene (p < 0.05). Meanwhile, a dual luciferase assay detected the mutation of the mC-7 site that significantly inhibited the luciferase activity of the CXCL11 gene promoter (p < 0.01). Transcription factor prediction and expression verification indicated that mC-7 is located in the OSR1-binding domain, and that its expression level is related to E. coli F18 susceptibility. We speculated that methylation modification of the mC-7 site of the CpG island in the promoter region of the CXCL11 gene might inhibit the binding of transcription factor OSR1 with the mC-7 site, and then affect its expression level to regulate the susceptibility to E. coli F18.The primary pathogen causing post-weaning diarrhea in piglets is  Escherichia coli F18 is the primary causative pathogen of post-weaning diarrhea in piglets [E. coli can increase the protein expression level of inflammatory factors when it infects intestinal epithelial cells [E. coli F18 infection in weaned piglets and its regulatory mechanism. In previous studies, our research team confirmed E. coli F18-resistant and -susceptible Meishan weaned piglets [CXCL11—was screened out. CXCL11, known as interferon-γ-inducible T-cell a-chemoattractant (I-TAC), is an important immunomodulator in the CXC chemokine family [CXCL11 is the ligand with the highest affinity for receptor CXCR3, which can specifically activate Th1 cells expressing chemokine receptor CXCR3. Th1 mainly secretes IFN-γ and TNF-β, which are capable of mediating cellular immunity [CXCL11 is mainly focused on human tumor diseases [E. coli F18 resistance in the swine intestinal immune system are still unclear.Enterotoxigenic  piglets ,3. Numer piglets . The int piglets . Previoual cells ,7,8 and al cells ,10, affe piglets . Combinee family . Chemokie family , which iimmunity ,15. Currdiseases ,17. HoweCXCL11 gene expression and methylation modification on susceptibility to E. coli F18, the mRNA and protein expression changes of the CXCL11 gene on the IPEC-J2 cells infected by E. coli F18 and those not infected by E. coli F18, and the IPEC-J2 cell line overexpressed CXCL11 gene were constructed. The effects of CXCL11 expression on E. coli F18 adhesion were verified by a relative quantification method, bacteria enumeration, and immunofluorescence assay. Next, bisulfite sequencing PCR (BSP) was used to determine the methylation degree of the CpG island in the promoter region of the CXCL11 gene in both E. coli F18-infected IPEC-J2 cells and untreated IPEC-J2 cells, and we performed a correlation analysis to investigate the effect of the methylation modification of different CpG sites in the CXCL11 gene promoter region on mRNA expression. Finally, point mutations, a dual luciferase assay, and overexpression were used to validate the effect of key CpG sites and transcription factors (TFs) on transcriptional activity in the CXCL11 gene promoter region. Based on the new candidate molecule CXCL11 identified previously, this study validated and explored the effect of the regulatory mechanism of its expression on resistance to porcine E. coli F18 and further deepened understanding of the regulatory mechanism underlying resistance to weaned piglet E. coli F18 from the level of DNA methylation modification. The purpose of this study was to investigate the epigenetic mechanism of porcine CXCL11 gene expression and methylation modification on the susceptibility of E. coli F18 from the perspective of epigenetics, and to establish a foundation for the breeding of pigs with disease resistance.In eukaryotes, DNA methylation is a significant form of epigenetic alteration that occurs and occupies an extremely crucial position in regulating gene expression . The priE. coli F18ab {107/86 (O139:K12:H1)} and E. coli F18ac {2134P (O157:H19)} were donated by the laboratory of livestock and poultry pathogenic microorganisms, College of Veterinary Medicine, Yangzhou University. Human embryonic kidney cells (293T) were purchased from Guangzhou Jennio Biotech Co., Ltd., Guangzhou, China.The University of Pennsylvania in the USA contributed Intestinal porcine epithelial cells (IPEC-J2). 5 cells per well, and cells were cultured under the culture conditions of 5% CO2 in a 37 °C constant temperature incubator with DMEM containing 10% fetal bovine serum  until the cell density achieved about 80%. E. coli F18 cells were added to LB and incubated for 12 h on a shaking table while being a rotated at a velocity of 200 rev/min. The thalli of E. coli F18 were centrifuged for 10 min and gathered at a velocity of 4000 rev/min. The bacterium precipitate was resuspended in PBS, then washed three times and centrifuged. Each culture hole received 1.0 mL of bacteria that had been diluted with DMEM to 1.0 × 109 CFU/mL. The cells were then harvested after being cultured for 4 h at 37 °C and 5% CO2.IPEC-J2 cells were plated onto 12-well plates at a density of 5.0 × 10E. coli F18-infected and -uninfected IPEC-J2 cells was extracted. Using a NanoDrop 2000 spectrophotometer , agarose gel electrophoresis in formaldehyde (1%) was used to measure the whole RNA’s thickness and purity. The synthesis of cDNA was divided into two steps. The first step reaction occurred via 4 × gDNA wiper Mix 4 µL, RNA 1000 ng, and RNase free ddH2O up to 16 µL. Reaction conditions: 42 °C for 2 min. The second reaction system: the first reaction system was added to 5 × HiScript III qRT SuperMix 4 µL, and the total system was 20 µL. The reaction qualification was set at 85 °C for 15 min, 37 °C for 30 s, and 4 °C for preservation. The 10 µL reaction mixtures used for the qPCR assay contain 1 µL cDNA, 0.2 µL of each forward and reverse primer (10 µmol L−1), 5 µL 2 × AceQ Universal SYBR qPCR Master Mix, and RNase free ddH2O up to 10 µL. To guarantee the reproducibility of the results, each sample was tested in duplicate in three separate trials. An internal reference was GAPDH gene. All primers were synthesized by Sangon Biotech Co., Ltd., Shanghai, China , the total RNA of both the i, China .CXCL11 (1:1000) (Affinity Biosciences LTD) and GAPDH (1:5000)  overnight at 4 °C on a shaking table, followed by the isolated protein being incubated for 2 h with secondary goat anti-rabbit or goat anti-mouse antibodies (1:500 dilution) . Finally, the proteins were made visible with an enhanced chemiluminescence (ECL) detection technique . The GAPDH protein was used as a reference.Proteins were extracted on the basis of instructions after IPEC-J2 cells were collected, and then they were quantified and isolated through 10% SDS-PAGE . PVDF membranes  were used to receive the separated protein bands. The 5% skim milk powder was used to block the membrane, and then incubated with primary antibodies against CXCL11 gene sequences of CDS were confirmed by the NCBI. The complete CDS sequence of the porcine CXCL11 gene was synthesized by Wuhan GeneCreate Biological Engineering Co., Ltd. , and connected to the PdoubleEX-EGFP-1170593 empty vector (CXCL11-OE); the PdoubleEX-EGFP-1170593 empty vector was set as a negative control group (NC-OE). JetPRIME Transfection Reagent was used to transfect CXCL11-OE and NC-OE vectors into IPEC-J2 cells cultured once cells density reached 80%. The normal cells without transfection were used as the control group. Cells were cultured in an incubator for 4 h before replacing the original medium with fresh medium. Cells were cultured overnight to monitor the expression of green fluorescent protein (GFP). G418 100 µg/mL was used to apply the drug sieve. After two weeks of G418 selection, when all living cells were G418 resistant, RNA and protein were extracted, and GAPDH was used as reference to verify CXCL11 overexpression efficiency by qPCR and Western blot.Porcine CXCL11 overexpression and control cells were added into cell culture plates at a density of 5.0 × 104 cells per well, and cultured in a thermostatic incubator with DMEM containing 10% FBS until the cell density reached roughly 80%. A relative quantification method, bacteria enumeration, and immunofluorescence assay were employed to evaluate E. coli F18 adhesion in vitro, investigating the interaction ability of E. coli F18 with IPEC-J2 cells. Bacteria enumeration: The supernatant was collected after E. coli F18 infection, and diluted 2000 times with PBS that was coated on the plate, at 37 °C incubator for 12 h. The bacteria colonies on the plate were counted by Image J software. A relative quantification method of PILIN has been published [E. coli, 1:20) and 200 μL red fluorescence-labeled secondary antibody (1:200) were successively incubated. Finally, the cells were investigated and recorded under an inverted fluorescent microscope in a dark environment.ublished . IndirecCXCL11 5′ end upstream promoter region was downloaded from the NCBI database. Using MethPrimer software, we predicted the CXCL11 gene promoter region CpG island. According to the kit instructions , the genomic DNA was identified using bisulfite sequencing PCR. The MethPrimer software was employed to create the PCR primers  based on the sequence of the sulfite transformation. The ingredients for the 50 μL PCR amplification reactions of bisulfite-treated DNA were 4 μL of DNA template, 25 μL of ZYMO Taq Premix, 4 μL each of forward primer (10 μ M/L) and reverse primer (10 μ M/L), and 13 μL of distilled water. The purified products (230 bp) were connected to the pMD19-T vector , and overnight connection at 16 °C, then transformation to DH-5α competent cells for cultivation. The transformation system contained 4.3 μL of the PCR purified product, 0.7 μL pMD-19T, and 5.0 μL solution I. All samples were sequenced using 20 positive clones in total , and the methylation degree of sequence data was assessed using QUMA (http://quma.cdb.riken.jp/ (accessed on 4 January 2022)) software.The 2000 bp sequence of the pig CXCL11 promoter region CpG island was connected to the pGL3-Basic vector to construct a mutant-type vector (CXCL11-MUT). The original sequence without mutation at the mC-7 site was linked to the pGL3-Basic vector to construct a wild-type vector (CXCL11-WT). The pGL3-Basic empty vector was set as a negative control group (Mock) at the same time, and three equal replicate experiments replicates were employed for each group. The 293T cells were cultured in 24-well plates and the culture conditions were 5% CO2 in a 37 °C constant temperature incubator. After growing to 80% density, 293T cells were co-transfected using JeTPRIM Transfection Reagent (Polyplus) with Mock, CXCL11-WT vector, CXCL11-MUT vector, respectively, and pRL Renilla luciferase reporter vector . After 48 h, the firefly luciferase activity was determined and standardized to Renilla. The operation procedure was implemented in rigorous accordance with the kit instructions. A segment of the mC-7 site mutation of the CXCL11-WT: GAACATTTCTCTATCGATAGGTACCCACCACAGAGGGAAAGATTTTATGAGTTTGGTTTTGGACATGTTGAGTTTGGGATTAAAAGGCACTCAAATGGGGATATTGAGTAGACAGTTTATATACAGGTCTAGAACTCAGAGAAGAAGTCTAGATTAGAAATATAATTTGGGGAGTTCCCGTCGTGGCGCAGTGGTTAACGAATCCGACTAGGAACCATGAGGTTGCAGGTTCGCTCCCTGCCCTTGCTCAGTGGGTTAAAGGATCCGGCGTTGCCGTGAGCTGTGGTGTAGGTTGCAGACGCGGCTTGGATCCCGCGTTACTGTGGCTCTGGCGTAGGCCAGTGGCTACAGCTCCGATTCGACCCCTAGCCTGGGAACCTCCATATGCCGCGGGAGCGGCCCAAAGAAATCTCGAGATCTGCGATCTAAGTAAGCXCL11-MUT: GAACATTTCTCTATCGATAGGTACCATGCACCACAGAGGGAAAGATTTTATGAGTTTGGTTTTGGACATGTTGAGTTTGGGATTAAAAGGCACTCAAATGGGGATATTGAGTAGACAGTTTATATACAGGTCTAGAACTCAGAGAAGAAGTCTAGATTAGAAATATAATTTGGGGAGTTCCCGTCGTGGCGCAGTGGTTAACGAATCCGACTAGGAACCATGAGGTTGCAGGTTCGCTCCCTGCCCTTGCTCAGTGGGTTAAAGGATCCGGCGTTGCCGTGAGCTGTGGTGTAGGTTGCAGACGCGGCTTGGATCCCCTTCCTTAACTTACCTGGCGTAGGCCAGTGGCTACAGCTCCGATTCGACCCCTAGCCTGGGAACCTCCATATGCCGCGGGAGCGGCCCAAAGAAATCTCGAGATCTGCGATCTAAGTAAGhttp://jaspar.genereg.net/search?q=&collection=CORE&tax_group=vertebrates (accessed on 20 February 2022)) and Alibaba2 ) were adopted to estimate transcription factor-binding sites (TFBSs) on the CXCL11 promoter region’s sequence. Based on the CpG sites of methylation level significantly altering mRNA expression, we further screened out the key TFs, and we carried out validation investigations using a dual luciferase activity assay.JASPAR  was performed to run statistical analyses. In three separate experiments, each sample was examined in triplicate, and the relative quantitative results were processed using the 2t method . Data weCXCL11 expression and E. coli F18 susceptibility in IPEC-J2, we investigated the mRNA and protein level of CXCL11 in the E. coli F18-infected IPEC-J2 cells and the normal IPEC-J2 cells without E. coli F18 infection. As shown in CXCL11 in the normal group was found to be substantially greater (p < 0.01) than that in the infection group by qPCR assays. Immunoblotting was used to further demonstrate the results, which showed that the protein levels of CXCL11 was considerably elevated in the normal group, as shown in In order to examine the connection between CXCL11 expression impacted E. coli F18 invasion, this study constructed a CXCL11 overexpression cell line (CXCL11-OE) and a negative control cell line (NC-OE). A relative quantification method, fimbria protein gene PILIN of E. coli, bacteria enumeration, and immunofluorescence assay were used to measure the interaction ability of E. coli F18 with IPEC-J2 cells in vitro by CXCL11 overexpression. As indicated in CXCL11 RNA showed signs of GFP expression. The overexpression potential of CXCL11 in overexpression CXCL11-treated IPEC-J2 cells was 143,151.12% ; bacteria enumeration (PILIN (p < 0.05). The immunofluorescence assay revealed that the presence of E. coli F18 was noticeably decreased in the CXCL11-OE group compared to the control group  (CXCL11 promoter region CpG island in E. coli F18-infectedand -uninfected IPEC-J2 cells via bisulfite amplicon sequencing (BSAS), and the length of the BSP-PCR amplification product was 230 bp (CXCL11 promoter region were methylated to varying degrees (MethPrimer online software was used to predict the CpG island of the −380 bp) A. We invs 230 bp B. As sho degrees C, and on degrees D.CXCL11 promoter region on gene expression by luciferase assays. As shown in CXCL11 promoter (p < 0.01). Secondly, the Pearson method was adopted to evaluate the connection between the methylation level of the CpG island in the CXCL11 gene promoter region and mRNA expression. The results indicated that the correlation coefficient showed significantly negative associations between mC-7 site methylation levels and CXCL11 mRNA expression   , suggestCXCL11 expression, according to the JASPAR and Alibaba databases, we found 14 putative TFBSs in the CpG island of the pig CXCL11 genes’ promoter region   B.OSR1 on CXCL11 mRNA expression, the impact of OSR1 overexpression on CXCL11 gene transcriptional activity was detected by double luciferase assay in this study. The results indicated (OSR1 significantly increased luciferin activity (p < 0.05).In order to further prove the impact of ndicated  that oveE. coli F18 is the most common adhesin pili in post-weaning bacterial diarrhea (PWD) [E. coli F18′s pathogenicity [E. coli F18 infection of the intestine relies on adhesins binding to the host’s intestinal epithelial cells, and the intestinal mucosal immune response is an important line of defense against pathogen invasion of the body [CXCL11, which affects the immune response of the intestinal mucosa in weaning piglets against invasion by E. coli F18. Based on our research, we obtained E. coli F18-resistant and -susceptible Meishan weaned piglets, and transcriptome analysis was performed on the duodenal tissue combined with quantitative tissue verification that screened out an important functional gene, CXCL11. CXCL11, a chemokine belonging to the CXC subfamily, is induced by IFN-c and IFN-b. The study demonstrated that chemokines play an important role in immune cell migration-mediated immune response [CXCL11, a chemokine affecting intestinal mucosal immune response, and then to effectively resist E. coli F18 invasion in weaned piglets. In this investigation, qPCR and Western blot were used to detect the CXCL11 gene expression level on the IPEC-J2 cells stimulated by different strains of E. coli F18 and the normal IPEC-J2 cells. The findings indicated that CXCL11 gene expression was dramatically downregulated on IPEC-J2 cells infected by E. coli F18ab and E. coli F18ac (p < 0.01). In conclusion, the high expression of the CXCL11 gene may benefit weaned piglets in their resistance to E. coli F18 infection. Thus, the overexpression IPEC-J2 cell line of the porcine CXCL11 gene was constructed to explore the biological function of CXCL11. The detection included a relative quantification of the PILIN, bacteria enumeration, and immunofluorescence assay, which indicated that the CXCL11 overexpression significantly reduced the E. coli F18 ability to interact with IPEC-J2 in vitro, which suggested that CXCL11 plays a vital role in the immune regulation of E. coli F18 infection, and the upregulation of its expression may enhance the resistance of piglets to E. coli F18.ea (PWD) ,26. Adhegenicity . The firthe body ,4. Thereresponse . TherefoE. coli F18 infection at the cellular level, MethPrimer was used in this study to predict the presence of a CpG island in the CXCL11 gene’s promoter region. Methylation sequencing revealed that the 13 CpG sites in the sequencing region were methylated to varying degrees, and one of the CpG sites (mC-7) had a significant difference in methylation level between the E. coli F18-infected and -uninfected IPEC-J2 cells. Studies have confirmed that the DNA methylation of FUT1 and FUT2 promoters controls their expression, which in turn controls weaned piglets’ resistance to the ETEC F18 pathogen [E. coli F18 may trigger significant levels of methylation at the mC-7 site in the CXCL11 gene’s promoter region, and then show susceptibility to E. coli F18 in weaned piglets. In addition, the impact of the mC-7 site mutation on CXCL11 gene promoter activity was detected by dual luciferase assay. The findings determined that the mutation of the mC-7 site significantly inhibited the luciferase activity of the CXCL11 gene promoter (p < 0.01). The two above conclusions suggest that the mC-7 site may be a key regulatory site in the CXCL11 gene’s promoter region for resistance to E. coli F18 infection in piglets. In our investigation, based on the fact that promoter methylation is directly related to gene transcription [CXCL11 gene and mRNA expression. The results revealed that the methylation degree of the mC-7 site was inversely linked with the mRNA expression of the CXCL11 gene (p < 0.05), and the mC-7 site was located in the OSR1-binding domain. OSR1 is a protein composed of 266 amino acids, encoding a zinc finger transcription factor. Recently, studies have shown that OSR1, a novel tumor suppressor gene, is preferentially methylated in gastric cancer cell lines and its expression is reduced or silenced [CXCL11 gene may block the binding of OSR1, further inhibiting the expression of the CXCL11 gene. The study determined that most TFs also take part in the regulation of gene expression during pathogenic infection. In order to more accurately determine whether OSR1 was involved in the regulatory mechanism of E. coli F18 resistance, qPCR was adopted to detect the mRNA expression of OSR1 on IPEC-J2 cells infected by E. coli F18 and IPEC-J2 cells not infected by E. coli F18. The findings showed that the mRNA expression level of OSR1 in the infected group was dramatically lower than in the normal group (p < 0.05), which implied a connection between the degree of OSR1 expression and the susceptibility to E. coli F18. Meanwhile, OSR1 may have a role in the mechanism governing E. coli F18 resistance. In order to further verify the influence of OSR1 on the mRNA expression of the CXCL11 gene, the double luciferase assay was employed to investigate the influence of OSR1 overexpression on CXCL11 gene transcriptional activity. We found that luciferase activity was significantly increased after overexpression of OSR1 (p < 0.05). The results suggest that OSR1 may be an important transcription factor in the transcription regulation of the CXCL11 gene. In conclusion, this study theorized that methylation modification of the mC-7 site of the CpG island in the promoter region of the CXCL11 gene might inhibit the binding of OSR1 with the mC-7 site and then affect its expression level to regulate the susceptibility to E. coli F18 (CXCL11 promoter at the cellular level using an electrophoresis mobility shift assay (EMSA) method, etc., in order to offer a more complete experimental underpinning for the epigenetic regulation mechanism of the CXCL11 gene.At present, DNA methylation is a prominent topic in epigenetics study because it regulates gene expression and has a crucial role in the genesis of illness. ,29,30. Apathogen ,36. Addipathogen . Therefocription , the Peasilenced , thus itsilenced ,40. In msilenced ,42. Basecoli F18 . HoweverCXCL11 expression is closely related to the resistance of IPEC-J2 to E. coli F18 infection. In addition, the CXCL11 gene CpG island was identified through bioinformatics analysis. Methylation and double luciferase analysis determined that the key CpG site (mC-7) had an important effect on the activity of the CXCL11 gene promoter region, which indicated that the high methylation of the mC-7 site may block the binding of OSR1 and then affect its expression level to regulate the susceptibility to E. coli F18. This research further clarified the resistance functional genes, identified the epigenetic regulatory mechanism of CXCL11 resistance to E. coli F18 from the level of DNA methylation, deepened our understanding of the regulatory mechanism of E. coli F18 resistance in weaned piglets from the standpoint of DNA methylation modification, and established a foundation for the future development of prevention and control measures as well as breeding for disease resistance of porcine bacterial diarrhea.This study verified at the cellular level that