Histone Methyltransferase MLL1 Mediates Oxidative Stress and Apoptosis upon Deoxynivalenol Exposure in the Intestinal Porcine Epithelial Cells

Deoxynivalenol (DON), as a secondary metabolite of fungi, is continually detected in livestock feed and has a high risk to animals and humans. Moreover, pigs are very sensitive to DON. Recently, the role of histone modification has drawn people’s attention; however, few studies have elucidated how histone modification participates in the cytotoxicity or genotoxicity induced by mycotoxins. In this study, we used intestinal porcine epithelial cells (IPEC-J2 cells) as a model to DON exposure in vitro. Mixed lineage leukemia 1 (MLL1) regulates gene expression by exerting the role of methyltransferase. Our studies demonstrated that H3K4me3 enrichment was enhanced and MLL1 was highly upregulated upon 1 μg/mL DON exposure in IPEC-J2 cells. We found that the silencing of MLL1 resulted in increasing the apoptosis rate, arresting the cell cycle, and activating the mitogen-activated protein kinases (MAPKs) pathway. An RNA-sequencing analysis proved that differentially expressed genes (DEGs) were enriched in the cell cycle, apoptosis, and tumor necrosis factor (TNF) signaling pathway between the knockdown of MLL1 and negative control groups, which were associated with cytotoxicity induced by DON. In summary, these current results might provide new insight into how MLL1 regulates cytotoxic effects induced by DON via an epigenetic mechanism.


Introduction
Deoxynivalenol (DON), also known as vomitoxin, belongs to the Trichothecene group, which is mainly Fusarium graminearum, F. culmorum and F. crookwellense, and has one of the highest contamination ratio and detection rates among mycotoxins [1]. DON contaminates wheat, barley, maize, and oats, etc. Moreover, DON is hard to be degraded even in the condition of high temperature and strong acid due to its stable chemical structure [2]. DON can be "modified" to produce more derivatives, such as 3-acetyl-DON, 15-acetyl-DON, deoxynivalenol-3-beta-D-glucopyranoside (D3G), de-epoxy DON (DOM-1), 3-epi-DON, and 3-keto-DON [3]. Pigs are very susceptible to DON, especially the intestine of pigs, which is the primary target of DON attacks [4]. In China, the National Standard GB 2761-2017 has stipulated that the standard limit of DON in cereals and grain products (including corn, barley, wheat, cereal, and wheat flour) should be 1000 µg/kg. In vivo, chronic DON exposure not only led to decreased feed intake and body weight in animals, but also affected their susceptibility to diseases and vaccination efficiency. For instance, a previous study suggested that low-dose DON exposure influenced the replication of porcine epidemic diarrhea virus (PEDV), increased diarrhea rates, and exacerbated the gut barrier injury [5]. Moreover, the exposure of pigs to DON for 4 weeks reduced the efficiency of vaccination against porcine reproductive and respiratory virus (PRRSV) [6]. In vitro or in vivo, acute or chronic DON both alters the expression of tight junction proteins thereby disrupting the intestinal barrier [7,8]. Mechanically, DON inhibits protein and nuclei acid synthesis by binding to the 60 S ribosome [9]. Previous studies have shown that DON caused accumulation levels of reactive oxygen species (ROS) in the mitochondria which could active the oxidative stress response and induce apoptosis and immune dysfunction [10].
Epigenetics aims to explain the regulation of gene expression by altering transcriptional activity and chromosomal structure [11]. Recently many studies have shown that epigenetic regulation can be involved in a variety of biological processes, including cytotoxicity induced by mycotoxin. For instance, our previous study indicated that the genomewide methylation levels significantly changed and found 3030 differentially methylated regions (DMRs) after the IPEC-J2 cells were treated with 1 µg/mL DON for 48 h, then combined with RNA-sequencing analysis; the promotor region methylation of 29 genes was negatively correlated with gene expression [12]. DON exposure may play an important role in DNA methylation, but the underlying mechanism remains to be clarified. Histone methylation is a highly dynamic modification regulated by histone methyltransferases and demethylases in vitro and in vivo, which can result in the activation or suppression of gene expression, depending on the specific methylation site [13]. Lysine 4 (K4) methylation on histone 3 (H3) is a wide spectrum of modification and conservatively plays a crucial role in gene expression. The trimethylation of Lysine 4 on histone 3 (H3K4me3) generally marks the transcriptional activating of genes, whilst the trimethylation of histone 3 lysine 27 is usually related to silence gene expression [14].
Mixed lineage leukemia 1 (MLL1), also known as KMT2A, was initially uncovered in the research of cancer and the translocation and rearrangement of the MLL1 gene sequence results in the leukemia [15]. Subsequently, other studies discovered that MLL1 could specifically catalyze the H3K4 to undergo methylation modification [16]. MLL1 plays an important role in stem cell proliferation and differentiation, and regulates the expression of homeobox (HOX) gene clusters which are the major regulators during early embryonic development [17].
In the past several years, MLL1 was deeply investigated in the regulation of leukemogenesis, but few studies were performed regarding its own role under mycotoxins [18]. In the present study, the intestinal porcine epithelial cells (IPEC-J2 cells) were used as an in vitro model to explore the potential mechanism regulation of MLL1 upon DON stimulation. We uncovered that H3K4me3 modification enrichment was enhanced and the levels of MLL1 mRNA and MLL1 protein were highly expressed, upon IPEC-J2 cells exposure to 1 µg/mL DON. Silencing the MLL1 increased the rate of apoptosis and the level of ROS, the cell cycle was also arrested, and the MAPKs pathway was activated. Combined with the RNA-seq, TNF receptor superfamily member 1A (TNFRSF1A) is probably the potential target of MLL1 to participate in the regulation of DON.

Cell Viability
To determine whether the cell growth was affected by DON, the cell viability assay was conducted using a Cell Counting Kit-8 (Yeasen Biotechnology (Shanghai) Co., Ltd., Shanghai, China) according to the protocols provided from the manufacturer. Briefly, Antioxidants 2022, 11,2006 3 of 17 IPEC-J2 cells (4000 cells/well) were put into 96-well culture plates. After cell confluence of approximately 50%, DON was added to the medium at a final concentration of 1 µg/mL. The cells were incubated with no FBS medium containing 10% reagent for 2 h at 37 • C. The absorbance was monitored using the Tecan Infinite 200 microplate reader (Sunrise, Tecan, Switzerland) at a wavelength of 450 nm. All experiments were performed in triplicate.

Immunofluorescence
The IPEC-J2 cells were seeded on the glass slide in a 12-well plate and exposed with 1 µg/mL DON for 48 h or not. The cells were fixed with 4% paraformaldehyde for 30 min, then permeabilized with 0.5% Triton X-100 for 15 min and blocked with 5% BSA for 2 h at 37 • C incubator. Afterwards, anti-alpha-tubulin (ab7291, Abcam, Cambridge, UK) was incubated with cells overnight at 4 • C, followed by the cells being washed in PBST thrice and stained with secondary antibodies for 1h at 37 • C incubator. DAPI (Invitrogen) was used to counterstain the nucleus. The cells were photographed with an inverted microscope (Olympus, Japan).

RNA Extraction and Real Time Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted from cells after treatment using RNAiso (Takara, Dalian, China) following the instructions from the manufacturer. Next, 1 µg RNA was reversely transcribed into complementary DNA (cDNA) using a cDNA synthesis kit (Vazyme Biotech Co., Ltd., Nanjing, China). Briefly, the reactions were performed in an ABI StepONEPlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with a 10 µL mixture system following the instructions. All the primers used are shown in Table S2. GAPDH and β-ACTIN were chosen for normalizing the other gene levels. 2 −∆∆Ct method was used to calculate the relative gene expression [19].

Analysis Cell Cycle and Apoptosis using Flow Cytometry
Cell apoptosis was evaluated using Annexin V-fluorescein isothiocyanate (FITC)/ propidium iodide (PI) kit (Solarbio, Beijing, China). IPEC-J2 cells were transfected with si-MLL1-NC or si-MLL1, si-TNFRSF1A-NC or si-TNFRSF1A, and then treated with 1 µg/mL DON for 48 h in a 6-well plate when the cell fusion reached 50%. There are several different groups including si-MLL1-NC + DON, si-MLL1 + DON, si-TNFRSF1A-NC and si-TNFRSF1A. After treatments, the samples were performed as described previously [20]. Cell apoptosis and cell cycle were detected using flow cytometry (Beckman Coulter, Brea, CA, USA), and then analyzed by CytExpert 2.3 and FlowJo 7.6. Each measurement was performed three times using a total of 10,000 events per sample.

Western Blot
After treatments, cells were collected and lysed with radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor and phosphatase inhibitor on ice. Total protein was extracted and the concentration was measured by BCA protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). The protein samples were diluted in 5X SDS-PAGE buffer and denatured by heating at 95 • C for 10 min. Each lane was loaded equal mass sample (20 µg). Samples were separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon, Darmstadt, Germany). Next, the membranes were blocked for 2 h at room temperature with TBST buffer containing 5% skim milk, followed by being incubated overnight at 4 • C with specific primary antibodies and then incubated for 1 h at RT with secondary antibodies (goat anti-mouse IgG HRP, goat anti-rabbit IgG HRP; CWBIO, Beijing, China). Finally, the membrane was detected by ECL and the relative intensities were analyzed by ImageJ software.

Small Interfering RNA (siRNA) Transfection
Three pig MLL1 and TNFRSF1A siRNAs and a negative control siRNA sequence were synthesized from GenenPharma (Suzhou, China). The siRNA MLL1 sequences were listed in Table S1, and the siRNAs of TNFRSF1A sequences are listed in Table S1, of which si-MLL1-3 and si-TNFRSF1A-3 were efficient and used in the study for analysis. Cells (1 × 10 6 cells/well) were seeded in 6-or 12-well plates, then the transfection was performed using jet PRIME (PolyPlus, France) according to the manufacturer's instructions when the cells had reached approximately 60-70% confluence. After transfection, the medium was changed for 24 h, and cells were harvested for another 24 h.

Reactive Oxygen Species Assessment
Reactive oxygen species (ROS) levels were determined with 2 ,7 -dichlorofluorescein diacetate (DCFH-DA) probe using a reactive oxygen species assay kit (Solarbio, Beijing, China). After treatments, cells were washed twice in cooled-PBS, then incubated with serum-free DMEM containing DCFH-DA (10 µM) for 30 min at 37 • C incubator. Following, fluorescence signals of DCF were measured at 488 nm excitation and 525 nm emission using the confocal microscope (Leica, Heidelberg, Germany) within 1 h.

RNA-Sequencing and Library Construction
Total RNA of samples transfected with si-MLL1 and si-MLL1-NC was isolated using RNAiso (Takara, Dalian, China) for RNA-seq library construction. The quality of RNA was inspected with a 1% agarose gel, and the RNA integrity and concentration were monitored using a Qubit RNA Assay Kit at the Qubit ® 2.0 Fluorometer (Life Technologies, Camarillo, CA, USA) and a NanoDrop spectrophotometer (IMPLEN, Westlake Village, CA, USA), respectively. Afterwards, the NEBNext@ UltraTM II Directional RNA Library Prep Kit (New England Biolabs (NEB), Beijing, China) was conducted to prepare the RNA-seq library of each sample following the provided guidelines, and the libraries were sequenced on Illumina HiSeq2000 platform. The quality control of sequences raw data was filtered to discard low-quality reads using fastQC, the clean reads were aligned to the pig reference genome (Ensembl, Sscrofa11.1) using Bowtie2, the read counts mapped to each gene were calculated using HTSeq program, the FPKM (fragments per kilobase of transcript sequence per million of mapped fragments) was determined using Gfold, the differentially expressed genes (DEGs) between the samples were verified with DESeq2, and the threshold p-values < 0.05 and |log2 (fold change)| > 0 were considered to be statistically significant [21][22][23][24]. GO (Gene Ontology) functional annotation of DEGs was conducted with GOseq, which divided biological function of DEGs into three aspects including cellular component, molecular function, and biological process. KOBAS software was employed to check the enrichment of DEGs in the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways. The GO terms and KEGG pathways analysis were performed using a correction with cutoff of 0.05.

Determination of Porcine TNFRSF1A Core Promotor Region with Reporter Assay
The 2kb upstream sequence of pig TNFRSF1A gene was derived from UCSC database, and then the core promotor region was predicted using the online website of Berkeley Drosophila Genome Project (BDGP) and Promotor 2.0. According to the predicted loci, the sequence was cut into four different lengths (-500-(-1) bp, -750-(-1) bp, -1010-(-1) bp and -1610-(-1) bp) to amplify corresponding products. The primers are listed in Table S3. The pGL3-basic vector was digested with XhoI and HindIII for 3 h, and then the four amplification fragments of the promotor were ligated in vector for 15 min at 50°C using a ClonExpress Ultra One Step Cloning Kit (Vazyme biotech Co., Ltd., Nanjing, China). Next, the ligations were transformed into Trelief TM 5α Chemically competent cells (TSINGKE, Nanjing, China) and then cultured on a solid LB-medium containing ampicillin at 37 • C overnight. Monoclonal colonies were taken and replicated in the liquid LB-medium, consequently, plasmids were extracted, and sequenced by company (Songon, Shanghai, China).
HEK293T cells were co-transfected into 500 ng Renilla plasmid and four recombinant plasmids and an empty vector as negative control, and afterwards cultured for 48 h at 37 • C incubator. The Substrate fluorescence intensity was detected on a Tecan Infinite 200 microplate reader (Tecan, Switzerland) using a Dual Luciferase Reporter Assay Kit (Vazyme biotech Co., Ltd., Nanjing, China). The promotor activities were calculated by the ratio Firefly Luciferase/Renilla Luciferase.

Chromatin Immunoprecipitation (ChIP) Assay
IPEC-J2 cells treated with 1 µg/mL DON (60% density) or not were digested (1 × 10 7 ) and washed twice with chilled PBS. The cell precipitation was resuspended with medium and cells and were cross-linked with 1% formaldehyde for 10 min on a shaker at room temperature, then crosslinking was ended with 50 mM glycine for 5 min [25]. The chromatin fragments of 200-700 bp were obtained through lysing cell by lysed buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 1% Nonidet P-40, 1 × protease inhibitor cocktail) and sonicated by Bioruptor (Covaris) for 21 min below 7 • C. ChIP-grade antibodies (anti-tri-methylhistone H3 (Lys 4), ab8580, Abcam) and rabbit immunoglobulin G (IgG) were incubated with 400 µL TBST containing 50 µL Protein A/G Magnetic Beads (MedChemExpress, Monmouth Junction, NJ, USA) for 4 h on a rotator at 4 • C, and then the chromatin and the beads connected antibody were co-incubated overnight with rotation at 4 • C. Next, the ChIP DNA was de-crosslinked by Protease K digested at 65 • C overnight and enriched and purified using a FastPure Gel DNA Extraction Mini Kit (Vazyme biotech Co., Ltd., Nanjing, China). At last, purification of DNA was performed and detected via qPCR using TNFRSF1A primers listed in Table S3.

Statistical Analysis
The results were analyzed using Excel and GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). The data were represented as means ± SD, Student's t-test was applied to compare significance between two groups. p < 0.05 was regarded as statistically significant.

The Toxic Impacts of DON Exposure on IPEC-J2 Cells
To examine the effect of DON on IPEC-J2 cells, we treated cells with a concentration of 1 µg/mL for 24 h and 48 h. The results indicated that 1 µg/mL DON significantly reduced the cell viability; specifically, the cell viability declined to about 50% at 48 h (p < 0.001) ( Figure 1A). We further monitored the cell morphological changes of IPEC-J2 cells exposed to 1 µg/mL DON; the cells exhibited an irregular and shrinkage morphology ( Figure 1B). Furthermore, apoptosis analysis found that the DON-treated group obviously increased the ratio of apoptosis compared with the control group (p < 0.001) ( Figure 1C). qPCR assay revealed that the expression of antioxidative gene SOD was decreased ( Figure 1D), the pro-apoptotic factors (caspase 3 and BAK) were elevated and antiapoptotic factor (Bcl-2) was reduced ( Figure 1E). We also revealed that the expression of cytokines (IL-6, IL-8, IL-18 and TNF-α) and NF-κB were increased upon 1 µg/mL DON exposure (p < 0.001) ( Figure 1F,G). These results suggest that DON causes toxicity in IPEC-J2 cells. ure 1B). Furthermore, apoptosis analysis found that the DON-treated group obviously increased the ratio of apoptosis compared with the control group (p < 0.001) ( Figure 1C). qPCR assay revealed that the expression of antioxidative gene SOD was decreased ( Figure  1D), the pro-apoptotic factors (caspase 3 and BAK) were elevated and antiapoptotic factor (Bcl-2) was reduced ( Figure 1E). We also revealed that the expression of cytokines (IL-6, IL-8, IL-18 and TNF-α) and NF-κB were increased upon 1 μg/mL DON exposure (p < 0.001) ( Figure 1F, G). These results suggest that DON causes toxicity in IPEC-J2 cells.

Elevation of H3K4me3 and MLL1 Expression Induced by DON
The H3K4me3 modification was significantly enriched by 1 μg/mL DON treatment (p < 0.01) (Figure 2A). H3K4me3 is a dynamic process that is regulated by histone methyltransferases and demethylases. We further investigated a variety of genes that participate in the H3K4me3, the methyltransferase genes (MLL1, MLL2, MLL3, MLL4, MLL5, SETD1A and SETD1B) were increased with 1 μg/mL DON treatment ( Figure 2B); especially, MLL1 was markedly elevated. As shown in Figure 2C

Elevation of H3K4me3 and MLL1 Expression Induced by DON
The H3K4me3 modification was significantly enriched by 1 µg/mL DON treatment (p < 0.01) (Figure 2A). H3K4me3 is a dynamic process that is regulated by histone methyltransferases and demethylases. We further investigated a variety of genes that participate in the H3K4me3, the methyltransferase genes (MLL1, MLL2, MLL3, MLL4, MLL5, SETD1A and SETD1B) were increased with 1 µg/mL DON treatment ( Figure 2B); especially, MLL1 was markedly elevated. As shown in Figure 2C, we found that DON increased the MLL1 protein. The IPEC-J2 cells transfected with si-MLL1-3 possessed the highest knockdown efficiency (p < 0.01) ( Figure 2D). The protein level of MLL1 was obviously reduced by treatment with si-MLL1 ( Figure 2E), and H3K4me3 was also subsequently declined ( Figure 2F). These above-mentioned results suggest that MLL1 may participate in the regulation of DON induced IPEC-J2 cells.
Antioxidants 2022, 11, x FOR PEER REVIEW 7 of 17 efficiency (p < 0.01) ( Figure 2D). The protein level of MLL1 was obviously reduced by treatment with si-MLL1 ( Figure 2E), and H3K4me3 was also subsequently declined (Figure 2F). These above-mentioned results suggest that MLL1 may participate in the regulation of DON induced IPEC-J2 cells.

MLL1 Knockdown Resulted in Cell Cycle Arrest and Exacerbated the Oxidative Stress
Ample reports suggest that DON can arrest the cell cycle and induce ROS in many cell lines [26,27]. The flow cytometry and fluorescent probe were exerted to investigate whether MLL1 participates in the cell cycle and ROS biological process and plays potential functions. The cell cycle assay showed that the rate of the S phase increased and G2/M phase declined after knockdown of MLL1 ( Figure 3A). The mRNA expression levels of cell cycle related genes (CDK2, CDK4, Cyclin A2 and p21) were all upregulated ( Figure 3B). The ROS levels were elevated after treatment with MLL1 siRNA ( Figure 3C). Furthermore, the expression of antioxidative genes, SOD and CAT, decreased by si-MLL1 ( Figure 3D). Taken together, the findings indicated that the knockdown of MLL1 caused cell cycle arrest at the S phase and intensified the ROS. Ample reports suggest that DON can arrest the cell cycle and induce ROS in many cell lines [26,27]. The flow cytometry and fluorescent probe were exerted to investigate whether MLL1 participates in the cell cycle and ROS biological process and plays potential functions. The cell cycle assay showed that the rate of the S phase increased and G2/M phase declined after knockdown of MLL1 ( Figure 3A). The mRNA expression levels of cell cycle related genes (CDK2, CDK4, Cyclin A2 and p21) were all upregulated ( Figure 3B). The ROS levels were elevated after treatment with MLL1 siRNA ( Figure 3C). Furthermore, the expression of antioxidative genes, SOD and CAT, decreased by si-MLL1 ( Figure 3D). Taken together, the findings indicated that the knockdown of MLL1 caused cell cycle arrest at the S phase and intensified the ROS.

MLL1 Knockdown Aggravated Cell Apoptosis
The increasing ROS level was closely associated with apoptosis and was proved by a variety of studies [28,29]. The IPEC-J2 cells were transfected with si-MLL1-NC and si-MLL1 to further test the influence of MLL1 on apoptosis upon DON exposure. As shown in Figure 4A, the rate of apoptotic cells was elevated by MLL1 knockdown (p < 0.001). Furthermore, the proteins of cleaved-caspase 3/caspase 3 and Bax were upregulated (Figure 4B). Collectively, the results suggested that the loss of MLL1 indeed aggravated apoptosis induced by DON.

MLL1 Knockdown Aggravated Cell Apoptosis
The increasing ROS level was closely associated with apoptosis and was proved by a variety of studies [28,29]. The IPEC-J2 cells were transfected with si-MLL1-NC and si-MLL1 to further test the influence of MLL1 on apoptosis upon DON exposure. As shown in Figure 4A, the rate of apoptotic cells was elevated by MLL1 knockdown (p < 0.001). Furthermore, the proteins of cleaved-caspase 3/caspase 3 and Bax were upregulated ( Figure 4B). Collectively, the results suggested that the loss of MLL1 indeed aggravated apoptosis induced by DON.

RNA-Sequencing Analysis between si-NC and si-MLL1 Transfected into IPEC-J2 Cells
To identify and figure out the underlying mechanism induced by MLL1, we performed RNA sequencing. First, principal component analysis (PCA) indicated an obvious separation of the si-MLL1-NC group from si-MLL1 group ( Figure 5A), and the heatmap analysis of sample-to-sample correlation also validated this ( Figure 5B). Adjusted p < 0.05 and |log2 fold change| > 0 were utilized as criteria to filter differentially expressed genes. As shown in Figure 5C, there are 2591 and 2760 mRNAs upregulated and downregulated, respectively ( Figure S1). As well, the differentially expressed genes between two groups were provided in Tables S4 and S5. Gene Ontology (GO) enrichment analysis was divided into three terms, including biological process, cellular component, and molecular function (Figures 5D and S1C, D; Table S6). For all DEGs, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that MLL1 mediates the cell cycle, apoptosis and TNF signaling pathway ( Figure 5E; Tables S7 and S8). To validate the reliability of RNA-sequencing analysis, nine genes were randomly selected and their expression levels were examined by qPCR; we then found the result is consistent with the expression changes detected by RNA-seq ( Figure 5F).

RNA-Sequencing Analysis between si-NC and si-MLL1 Transfected into IPEC-J2 Cells
To identify and figure out the underlying mechanism induced by MLL1, we performed RNA sequencing. First, principal component analysis (PCA) indicated an obvious separation of the si-MLL1-NC group from si-MLL1 group ( Figure 5A), and the heatmap analysis of sample-to-sample correlation also validated this ( Figure 5B). Adjusted p < 0.05 and |log2 fold change| > 0 were utilized as criteria to filter differentially expressed genes. As shown in Figure 5C, there are 2591 and 2760 mRNAs upregulated and downregulated, respectively ( Figure S1). As well, the differentially expressed genes between two groups were provided in Tables S4 and S5. Gene Ontology (GO) enrichment analysis was divided into three terms, including biological process, cellular component, and molecular function ( Figures 5D and S1C,D; Table S6). For all DEGs, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that MLL1 mediates the cell cycle, apoptosis and TNF signaling pathway ( Figure 5E; Tables S7 and S8). To validate the reliability of RNAsequencing analysis, nine genes were randomly selected and their expression levels were examined by qPCR; we then found the result is consistent with the expression changes detected by RNA-seq ( Figure 5F).

Loss Function of MLL1 Exacerbated MAPKs Activation
Accumulating reports have validated that ROS could trigger the MAPKs signaling pathway and lead to the secretion of pro-inflammatory cytokines which promotes inflammation response [30,31]. Western blot analysis represented that the phosphorylated protein levels of p38, ERK, and JNK markedly increased after the IPEC-J2 cells were transfected with si-MLL1 compared with si-MLL1-NC upon 1 µg/mL DON exposure ( Figure 6).

Loss Function of MLL1 Exacerbated MAPKs Activation
Accumulating reports have validated that ROS could trigger the MAPKs signaling pathway and lead to the secretion of pro-inflammatory cytokines which promotes inflammation response [30,31]. Western blot analysis represented that the phosphorylated protein levels of p38, ERK, and JNK markedly increased after the IPEC-J2 cells were transfected with si-MLL1 compared with si-MLL1-NC upon 1 μg/mL DON exposure ( Figure  6).

Identification of TNFRSF1A Core Promoter Region and Function of TNFRSF1A
According to the RNA-seq, we found that TNFRSF1A may be the potential target of MLL1. Previous studies indicated that tumor necrosis factor receptor 1 (TNFR1), which is encoded by TNFRSF1A, is a hub to mediate the apoptosis and inflammation response pathway [32]. The qPCR that detected the relative mRNA expression of TNFRSF1A was elevated when induced by 1 μg/mL DON, and knockdown of MLL1 also obviously increased its expression ( Figure 7A). According to the prediction of the TNFRSF1A core promoter region from the website, we amplified the truncated products and recombined it with the pGL3-basic vector to construct four insertional vectors (Figures 7B and S2). Then we performed a dual-luciferase reporter and identified that the luciferase activity of pGL3-p1 was remarkedly higher than the pGL3-basic (p < 0.01) ( Figure 7C), which suggested that the core promoter region of TNFRSF1A was located at −441~−391. Furthermore, we also examined that H3K4me3 modification was significantly enriched in the TNFRSF1A promoter region through a ChIP-qPCR (p < 0.001) (Figures 7D and S3). Afterwards, we constructed a small interfering RNA of TNFRSF1A and validated the si-TNFRSF1A-3 possessing the best interference efficiency ( Figure 7E). Silencing of TNFRSF1A resulted in the rate of later apoptosis being increased when a flow cytometry assay was used ( Figure 7F). Moreover, knockdown of TNFRSF1A led to the relative expression of TNF-α and IL-8 being declined upon 1 μg/mL DON exposure ( Figure 7G, H).

Identification of TNFRSF1A Core Promoter Region and Function of TNFRSF1A
According to the RNA-seq, we found that TNFRSF1A may be the potential target of MLL1. Previous studies indicated that tumor necrosis factor receptor 1 (TNFR1), which is encoded by TNFRSF1A, is a hub to mediate the apoptosis and inflammation response pathway [32]. The qPCR that detected the relative mRNA expression of TNFRSF1A was elevated when induced by 1 µg/mL DON, and knockdown of MLL1 also obviously increased its expression ( Figure 7A). According to the prediction of the TNFRSF1A core promoter region from the website, we amplified the truncated products and recombined it with the pGL3-basic vector to construct four insertional vectors (Figures 7B and S2). Then we performed a dual-luciferase reporter and identified that the luciferase activity of pGL3-p1 was remarkedly higher than the pGL3-basic (p < 0.01) ( Figure 7C), which suggested that the core promoter region of TNFRSF1A was located at −441~−391. Furthermore, we also examined that H3K4me3 modification was significantly enriched in the TNFRSF1A promoter region through a ChIP-qPCR (p < 0.001) (Figures 7D and S3). Afterwards, we constructed a small interfering RNA of TNFRSF1A and validated the si-TNFRSF1A-3 possessing the best interference efficiency ( Figure 7E). Silencing of TNFRSF1A resulted in the rate of later apoptosis being increased when a flow cytometry assay was used ( Figure 7F). Moreover, knockdown of TNFRSF1A led to the relative expression of TNF-α and IL-8 being declined upon 1 µg/mL DON exposure ( Figure 7G,H). Antioxidants 2022, 11, x FOR PEER REVIEW 12 of 17

Discussion
DON is one of the most prevalent environmental toxins that is produced by fungi, which attracts cytotoxicity and causes immune system dysregulation and is severely harmful to animals and human health [33]. Furthermore, recently accumulating studies uncovered that mycotoxins could elicit different types of cancer [34]. In 2016, the International Agency for Research on Cancer (IARC) and World Health Organization (WHO) urged for action to be taken against widespread mycotoxin contamination in developing countries. DON is regarded by the IARC as a Group 3 carcinogen that is suspected of carcinogenicity in humans and there are no adequate human and animal data. Increasing studies explored the effects of DON exposure to the body; however, the potential regulatory mechanism remains to be clarified.
Recently, epigenetic modification was reported to be related to the regulation of intracellular oxidative stress response caused by mycotoxin. The protein post-translational modifications have drawn much attention to their ability to regulate gene expression and and TNF-α after silenced TNFRSF1A. All data are presented as the mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, ns > 0.05 (Student t-test).

Discussion
DON is one of the most prevalent environmental toxins that is produced by fungi, which attracts cytotoxicity and causes immune system dysregulation and is severely harmful to animals and human health [33]. Furthermore, recently accumulating studies uncovered that mycotoxins could elicit different types of cancer [34]. In 2016, the International Agency for Research on Cancer (IARC) and World Health Organization (WHO) urged for action to be taken against widespread mycotoxin contamination in developing countries. DON is regarded by the IARC as a Group 3 carcinogen that is suspected of carcinogenicity in humans and there are no adequate human and animal data. Increasing studies explored the effects of DON exposure to the body; however, the potential regulatory mechanism remains to be clarified.
Recently, epigenetic modification was reported to be related to the regulation of intracellular oxidative stress response caused by mycotoxin. The protein post-translational modifications have drawn much attention to their ability to regulate gene expression and impact chromatin structure while not directly affecting the sequence of genes [35]. Czakai et al. found that histone acetyltransferases participating in Ochratoxin A (OTA) led to toxicity and carcinogenicity [36]. They reported that OTA caused core histones to be phosphorylated and acetylated, after which the mitotic chromosomes were condensed and the sister chromatid was separated. The histone modification alterations, including the levels of H4K20me3 and H3K9me3 that increased and levels of H4K20me2 and H3K27me3 that decreased, were monitored in the mouse oocytes upon Aflatoxin B1 (AFB1) exposure [37]. In this study, we proved that after IPEC-J2 cells' exposure to 1 µg/mL DON, H3K4me3 modification was enriched and the gene expression of histone methyltransferases were elevated; in particular, the levels of MLL1 mRNA expression were dramatically increased. The results suggest that MLL1 may take part in the regulation of DON-induced cytotoxicity. Consistent with the previous study conducted by Ansari et al., they cultured human H358 cells and treated them with different concentrations of DON (up to 33 µM) for 7.5 h, then observed that the levels of MLL1 and Set1 mRNA induced a two-to five-fold upregulation in a concentration-dependent manner, and the protein level of MLL1 was also obviously increased (8.3-fold) [38]. Moreover, they reported that the transcription factor Sp1 plays a crucial role in the regulation of MLL1 under stress. As shown in Figure 8, we found that MLL1 plays a crucial role during DON exposure to IPEC-J2 cells. Previous investigations indicated that MLL1 could promote cell proliferation, self-renewal and skeletal muscle regeneration, and also participate in the biological process, including hematopoiesis and development [39,40]. MLL1 attracted people's attention in the study of mixed lineage leukemia. However, the study of its own function in other fields was limited. Besides, MLL1 was regarded as one of the histone methyltransferases and specifically regulated the methylation on histone 3 lysine 4 [41]. In the present study, a small interfering RNA was further exerted to explore the function of MLL1. The silencing of MLL1 resulted in an attenuation of MLL1 mRNA and MLL1 protein, and H3K4me3 modification also decreased. Furthermore, cell cycle was arrested after the loss of MLL1. A previous study indicated that the knockdown of MLL1 induced cell cycle arrest at G2/M phase in HeLa cells [42]. Histone methylation modification could regulate gene expression through changing the state and structure of chromatin [43]. In the myogenesis process, Myf5 is considered an important transcription factor. The si-MLL1 decreased H3K4me3 modification. Therefore, MLL1 probably directly regulates Myf5 and affects the expression of Cyclin D1, resulting in cell cycle arrests in the G1/S phase [44]. Herein, we also illustrated that the loss function of MLL1 increased cell apoptosis and ROS levels. A knockdown of MLL1 declined the expression of p16 INK4A and induced cellular senescence after cells were treated with ambient air particulate PM with a diameter < 2.5 (PM 2 . 5 ) [45]. Besides, N-acetylcysteine (NAC), known as an antioxidant, attenuated the ROS increase upon PM 2 . 5 exposure in NHEK and HaCaT cells. The cells were pretreated with NAC followed by PM 2 . 5 treatment; the ROS levels and epigenetic enzymes' (DNMT, DNMT3B, TET1, EZH2 and MLL1) expression levels returned to normal. In this study, we demonstrated that si-MLL1 aggravated the apoptosis rate and increased the expression of cleaved-caspase 3 and Bax upon IPEC-J2 cell exposure to 1 µg/mL DON.
We performed an RNA-sequencing profile between si-MLL1 and negative control groups to investigate the function of MLL1. Adjusted p < 0.05 and |log2 fold change| > 0 were used as criteria to screen differentially expressed genes. As shown in Figure 5C, 2591 mRNAs were upregulated and 2760 mRNAs were downregulated. The KEGG enrichment analysis proved that MLL1 participates in the cell cycle, apoptosis, TNF signaling pathway, and so on.
The MAPKs include three subfamilies, ERK 1/2, p38 and JNK; its phosphorylated proteins performed the function of regulating inflammation, cell growth, development, and apoptosis [46,47]. The p38/MAPK and ERK 1/2/MAPK are correlated with the inflammation response via mediated proinflammatory cytokines secretion, such as interleukin families and TNF-α [48]. Our study revealed that the silencing of MLL1 significantly ele-vated the phosphorylated levels of ERK, p38, and JNK after IPEC-J2 cells were induced by 1 µg/mL DON. These results suggested that oxidative stress probably regulates the MAPKs (ERK, p38 and JNK) signaling pathway to trigger cell apoptosis and cell cycle arrest.
Antioxidants 2022, 11, x FOR PEER REVIEW Figure 8. The diagram of regulation mechanism of MLL1 upon IPEC-J2 cell exposure DO According to our RNA-seq, TNFRSF1A may be the target to regulate the cy and genotoxicity induced by DON. TNFRSF1A is regarded as a transmembran for TNF-α, and TNFRSF1A encodes a protein called TNFR1 which can assist signal into cells [49]. TNFR1 possesses a death domain and is involved in a cellular processes, such as cell apoptosis [50]. Consistent with the previous stud pression of TNF-α and TNFRSF1A mRNA was significantly increased when c oxidative stress. In addition, the modification of H3K4me3 was increased in the region of TNFRSF1A after IPEC-J2 cells were treated with 1 μg/mL DON. Be silencing of MLL1 induced an elevation of the mRNA expression of TNFRSF1 theless, we discovered that a knockdown of TNFRSF1A attenuated apoptosis a ited the expression of IL-8 and TNF-α mRNA. These evidences suggested that the of TNFR1 attenuated the inflammatory response induced by DON through t signaling pathway.

Conclusions
In summary, we confirmed that a histone methyltransferase MLL1 and mo of H3K4me3 significantly increased upon DON exposure, and the knockdown could aggravate ROS levels, cell apoptosis, induce cell cycle arrest and ac MAPKs pathway. Moreover, we also performed an RNA-seq profile to identify potential target gene. We provide new insight that MLL1 probably mediates the modification of the TNFRSF1A promoter region to regulate gene expression and in DON-induced cytotoxicity.
Supplementary Materials: The following supporting information can be down www.mdpi.com/xxx/s1, Figure S1: Differentially expressed genes and its function enrich ysis between si-MLL1-NC and si-MLL1; Figure S2: Construct the TNFRSF1A gene prom truncated vector; Figure S3: Evaluation of the condition of Chromatin sonication by a electrophoresis; Table S1: The siRNA sequences of MLL1 and TNFRSF1A; Table S2: The RT-qPCR; Table S3: Amplification primers of different fragments of pig TNFRSF1A gen According to our RNA-seq, TNFRSF1A may be the target to regulate the cytotoxicity and genotoxicity induced by DON. TNFRSF1A is regarded as a transmembrane receptor for TNF-α, and TNFRSF1A encodes a protein called TNFR1 which can assist TNF-α to signal into cells [49]. TNFR1 possesses a death domain and is involved in a variety of cellular processes, such as cell apoptosis [50]. Consistent with the previous study, the expression of TNF-α and TNFRSF1A mRNA was significantly increased when cells upon oxidative stress. In addition, the modification of H3K4me3 was increased in the promoter region of TN-FRSF1A after IPEC-J2 cells were treated with 1 µg/mL DON. Besides, the silencing of MLL1 induced an elevation of the mRNA expression of TNFRSF1A. Nevertheless, we discovered that a knockdown of TNFRSF1A attenuated apoptosis and inhibited the expression of IL-8 and TNF-α mRNA. These evidences suggested that the blocking of TNFR1 attenuated the inflammatory response induced by DON through the TNF-α signaling pathway.

Conclusions
In summary, we confirmed that a histone methyltransferase MLL1 and modification of H3K4me3 significantly increased upon DON exposure, and the knockdown of MLL1 could aggravate ROS levels, cell apoptosis, induce cell cycle arrest and activate the MAPKs pathway. Moreover, we also performed an RNA-seq profile to identify the MLL1 potential target gene. We provide new insight that MLL1 probably mediates the H3K4me3 modification of the TNFRSF1A promoter region to regulate gene expression and take part in DON-induced cytotoxicity.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.