Method for improving beef quality, action mechanism and experimental method

Abstract

The invention discloses a method for improving beef quality, an action mechanism and an experimental method, in a cattle body, vitamin A can activate the expression of EBF2 through an active metabolite RA of the vitamin A, and the EBF2 can inhibit the transcription process of CYP26B1 in a targeted manner to maintain the activity of a retinol signal channel, so that PPAR gamma and downstream lipid metabolism related genes thereof are activated, and the activity of the retinol signal channel is improved. Fatty acid transport and lipid accumulation in fat cells in the cattle muscle are promoted, fat deposition in the cattle muscle is promoted, and the beef quality is improved. By constructing the molecular network for regulating and controlling the formation of the fat in the cattle muscle, a complex regulation and control mechanism for controlling the fat deposition in the cattle muscle can be understood more deeply, a method for improving the beef quality based on regulating and controlling the vitamin A mediated key factor EBF2 is provided, and a new way is provided for improving the beef quality in a targeted manner.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for improving beef quality, an action mechanism and an experimental method. Background Art

[0002] Intramuscular fat content is a core indicator of beef quality, directly affecting its juiciness, tenderness, and flavor. The proliferation, differentiation, and lipid metabolism of intramuscular preadipocytes play a crucial role in regulating the production and deposition of intramuscular fat. Specifically, proliferation ensures an adequate supply of intramuscular preadipocytes, while differentiation transforms these cells into mature intramuscular adipocytes capable of storing lipids. Lipid metabolism controls the uptake, synthesis, and storage of fatty acids. Improving the quality of beef is a key issue in beef cattle farming.

[0003] Vitamin A and its derivatives play important roles in various physiological processes, including lipogenesis and lipid metabolism. In biological systems, vitamin A primarily exerts its physiological effects through its active metabolite, retinoic acid (RA). Cutting-edge research has revealed that RA has a multidimensional and complex mechanism of action in regulating lipid metabolism or lipogenesis. RA can mobilize triglycerides for hydrolysis into free fatty acids, providing cellular energy or participating in other physiological processes. Furthermore, RA is involved in the regulation of hepatic lipid metabolism and has shown potential for disease treatment. Studies have shown that RA can reduce the expression of the lipid metabolism marker SREBP-1c through a SIRT1-mediated mechanism, enhance antioxidant capacity, and thus ameliorate high-fat diet-induced hepatic steatosis. Importantly, the effects of vitamin A on intramuscular fat deposition in cattle may be developmental stage-dependent: vitamin A supplementation in calves promotes the proliferation of adipocyte progenitor cells and increases intramuscular fat deposition; whereas in adult cattle, vitamin A, through its active metabolite RA, inhibits adipocyte differentiation and reduces intramuscular fat deposits. Current research mainly focuses on macroscopic effects, and there is insufficient understanding of the molecular network of RA regulating fat production. Analyzing this mechanism can provide new strategies for targeted improvement of beef quality at various stages of cattle growth and development.

[0004] Studies have shown that RA promotes the proliferation, differentiation, and lipid metabolism of bovine intramuscular adipocytes, and that EBF2 may play a key role in this process. EBF2, a member of the EBF family, has been reported to be a specific marker for brown and beige preadipocytes. This discovery provides a new avenue for in-depth analysis of the early development and differentiation trajectory of adipocytes. EBF2-expressing adipocytes exhibit unique differentiation potential in adult animal adipose tissue. These cells have the ability to differentiate into brown (or beige) adipocytes but do not express myoblast or dermal cell marker proteins. This characteristic suggests that EBF2 may function as a key "molecular switch" during adipocyte lineage differentiation, precisely directing cells to differentiate into specific adipocyte subtypes while preventing them from transitioning to other cell types. Furthermore, EBF2 enhances the activity and expression of the cold-inducible transcription factors ERRα and PGC1α, stimulating the transcription of UCP1, thereby controlling adipocyte development and the genetic programming of adaptive thermogenesis. ZFP423 can synergize with EBF2 to regulate UCP1 expression, thereby finely controlling the thermogenic plasticity of white adipocytes. Based on these studies, EBF2 is a pleiotropic regulator, including maintaining functional coordination during adipocyte differentiation and maturation. However, the specific mechanism of EBF2 in bovine IMF formation and the role of RA in this process remain unclear. Summary of the Invention

[0005] In response to the above problems, the present invention proposes a method for improving beef quality, a mechanism of action and an experimental method, which effectively solves the problems in the prior art.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for improving beef quality by regulating the expression of EBF2, a key downstream target of vitamin A, to promote fat deposition in cattle muscles.

[0008] One mechanism of action is that vitamin A is first converted into retinal through a dehydrogenation reaction in the body. Retinal is catalyzed into RA by the Raldh1-Raldh3 enzyme. RA binds to its receptor RAR / RXR, activating the expression of the transcription factor EBF2. EBF2 inhibits the transcriptional activity of the retinol metabolizing enzyme CYP26B1, thereby activating RA signal transduction and regulating the activity of the retinol metabolic pathway. This cascade reaction further activates the lipogenesis marker PPARγ and its downstream lipid metabolism markers, promoting fatty acid transport and lipid accumulation in bovine intramuscular adipocytes.

[0009] Alternatively, silencing of EBF2 activates the CDK2 / CCNE2 axis, promoting the G1 / S phase transition of bovine intramuscular preadipocytes and promoting their proliferation.

[0010] Alternatively, EBF2 can directly target CYP26B1 and inhibit its transcriptional activity, binding sites: -112 to -120.

[0011] An experimental method, which is the experimental method for the mechanism of improving beef quality, comprises:

[0012] Step S1: Incubating bovine intramuscular adipocytes with RA, an active metabolite of vitamin A, and conducting integrated transcriptomic and metabolomic analyses to identify EBF2, a key regulatory factor in the regulation of bovine intramuscular adipogenesis by vitamin A;

[0013] Step S2, siRNA screening to determine si-EBF2-907 as the most effective silencing siRNA;

[0014] Step S3, setting up an EBF2 control group, a si-EBF2 interference group, and a siEBF2+RA group, and detecting the regulatory effects of EBF2 interference on the proliferation, differentiation, and lipid metabolism of bovine intramuscular preadipocytes and the regulatory effects of RA on these processes, respectively;

[0015] Step SS4: Transcriptome and non-target metabolomics sequencing of EFB2-silenced bovine intramuscular adipocytes to screen for CYP26B1, a potential downstream target of EBF2;

[0016] Step S5: Multiple verification of the targeting relationship between EBF2 and CYP26B1 based on site-directed mutagenesis, dual-luciferase reporter gene assay, and ChIP-qPCR assay.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] By constructing the molecular mechanism of the "vitamin A-EBF2-CYP26B1-retinol metabolic signaling pathway" axis regulating intramuscular fat formation, we can gain a deeper understanding of the complex regulatory mechanism that controls intramuscular fat deposition in cattle, and propose a method to improve beef quality based on regulating EBF2, a key factor in the vitamin A metabolic pathway, providing a new way to improve beef quality in a targeted manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the mechanism of action of an embodiment of the present invention;

[0020] Figure 2 This is a data diagram related to the screening of the downstream key factor EBF2 mediated by vitamin A in regulating bovine intramuscular fat production according to an embodiment of the present invention;

[0021] Figure 3 This is a graph showing experimental data on the regulatory effects of EBF2 interference on bovine intramuscular adipocyte differentiation and lipid metabolism and the effects of RA in an embodiment of the present invention;

[0022] Figure 4 This is a graph showing experimental data on the regulatory effect of EBF2 interference on the proliferation of bovine intramuscular preadipocytes and the effect of RA in an embodiment of the present invention;

[0023] Figure 5 This is a graph showing the experimental data of integrated analysis of transcriptome and metabolome after interference with EBF2 according to an embodiment of the present invention;

[0024] Figure 6 This is a data diagram of the screening and identification experiment of EBF2 downstream target genes in an embodiment of the present invention. DETAILED DESCRIPTION

[0025] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] The present invention discloses a method for improving beef quality, which promotes fat deposition in cattle muscles by regulating the expression of EBF2, a key downstream target of vitamin A. The binding site of EBF2 in the promoter region of CYP26B1 in cattle fibroblasts is edited to increase its binding activity. Based on the transcriptional inhibitory effect of EBF2 on CYP26B1, somatic cell nuclear transfer technology is used to improve the quality of beef.

[0027] Among some feasible approaches, EBF2 expression can be targeted and enhanced through vitamin A feeding or RA drug delivery, thereby improving beef quality.

[0028] To facilitate understanding of the method for improving beef quality disclosed herein, the present application also discloses a mechanism of action, in which vitamin A is first converted into retinal through a dehydrogenation reaction in the body. Retinal is catalyzed into RA by the Raldh1-Raldh3 enzyme. RA binds to its receptor RAR / RXR, activating the expression of the transcription factor EBF2. EBF2 inhibits the transcriptional activity of the retinol metabolizing enzyme CYP26B1, thereby activating RA signal transduction and regulating the activity of the retinol metabolic pathway. This cascade reaction further activates the lipogenesis marker PPARγ and its downstream lipid metabolism markers, promoting fatty acid transport and lipid accumulation in bovine intramuscular adipocytes, thereby improving beef quality.

[0029] Silencing of EBF2 activates the CDK2 / CCNE2 axis, promotes the G1 / S phase transition of bovine intramuscular preadipocytes, and promotes their proliferation.

[0030] EBF2 can directly target CYP26B1 and inhibit its transcriptional activity, binding sites: -112 to -120.

[0031] In this way, by establishing the regulatory network of EBF2, a key factor in vitamin A regulating the production of bovine IMF, on the lipid accumulation and proliferation of bovine IMF, it is beneficial to have a deeper understanding of the complex regulatory mechanism that controls bovine intramuscular fat deposition. Specifically, by constructing the molecular mechanism of regulating intramuscular fat formation through the "vitamin A-EBF2-CYP26B1-retinol metabolic signaling pathway" axis, a method of improving beef quality by regulating the expression of EBF2, a key downstream target of vitamin A, is proposed, providing a new strategy for targeted improvement of beef quality.

[0032] In order to study the mechanism of action disclosed in this application, the present invention also discloses an experimental method:

[0033] Step S1: Incubating bovine intramuscular adipocytes with RA, an active metabolite of vitamin A, and conducting integrated transcriptomic and metabolomic analyses to identify EBF2, a key regulatory factor in the regulation of bovine intramuscular adipogenesis by vitamin A;

[0034] Step S2, siRNA screening to determine si-EBF2-907 as the most effective silencing siRNA;

[0035] Step S3, setting up an EBF2 control group, a si-EBF2 interference group, and a siEBF2+RA group, and detecting the regulatory effects of EBF2 interference on the proliferation, differentiation, and lipid metabolism of bovine intramuscular preadipocytes and the regulatory effects of RA on these processes, respectively;

[0036] Step S4: transcriptome and non-target metabolomics sequencing of EFB2-silenced bovine intramuscular adipocytes to screen for CYP26B1, a potential downstream target of EBF2;

[0037] Step S5: Multiple verification of the targeting relationship between EBF2 and CYP26B1 based on site-directed mutagenesis, dual-luciferase reporter gene assay, and ChIP-qPCR assay.

[0038] See also Figure 2 , showing the correlation analysis diagram of differentially expressed genes related to adipogenesis and differentially expressed metabolites based on the O2PLS model; the correlation analysis diagram of differentially expressed genes related to adipogenesis and differentially expressed metabolites based on the correlation model; the protein interaction analysis diagram of candidate genes and markers related to adipogenesis and lipid accumulation; the change diagram of EBF2 expression level at different stages of bovine intramuscular adipocyte differentiation and the optimal siRNA screening diagram for bovine EBF2.

[0039] Bovine intramuscular preadipocytes were treated with RA, the active metabolite of vitamin A, and transcriptome and metabolome analysis was performed on day 6 of differentiation. Based on O2PLS modeling, strong correlations were found between 10 differentially expressed genes associated with lipogenesis or lipid accumulation (e.g., EBF2, EGR2, GPD1L, CAMK2A, and ELOVL2) and differentially expressed metabolites (e.g., behenic acid, estriol, proline-tryptophan dipeptide, and choline). Correlation modeling further confirmed a significant negative correlation between EBF2 and behenic acid (r = -0.930, P = 0.007).

[0040] Protein interaction analysis of candidate genes (EBF2, EGR2, GPD1L, CAMK2A, ELOVL2, CXCL2, PANK2, BDH2, PECR, and CAV1) and adipogenic markers (PPARγ, CEBPB, CEBPA, FABP4, LPL, and CD36) revealed interactions between EBF2 and PPARγ, CEBPB, and FABP4. Integrated analysis of multi-omics data and protein interactome profiles identified EBF2 as a key regulatory target.

[0041] The expression pattern of EBF2 during bovine intramuscular adipocyte differentiation was also analyzed. The results showed that EBF2 expression levels were significantly different between days 2 and 10 of differentiation (P < 0.01). Subsequently, siRNA screening identified si-EBF2-907 as the most effective silencing siRNA, and it was therefore selected for subsequent experiments.

[0042] After the key transcriptional regulatory factor EBF2 and its silencing siRNA were identified, an EBF2 control group (NC), a si-EBF2 interference group, and a si-EBF2+RA group were set up, and the regulatory effects of EBF2 interference on the proliferation, differentiation, and lipid metabolism of bovine intramuscular preadipocytes and the effects of RA on this process were detected respectively.

[0043] The cells were cultured in a cell culture incubator at 37°C. When the confluence of bovine intramuscular preadipocytes reached about 70%, Lipofectamine TM NC and si-EBF2 were transfected into cells at 3000. Meanwhile, RA (10 -5 In the cell differentiation experiment, when the cell confluence reached 100%, the cells were induced to differentiate with differentiation medium for 2 days, and then the maintenance medium was replaced every two days.

[0044] The treated cells were subjected to Oil Red O staining analysis, BODIPY lipid droplet staining analysis, Nile red lipid droplet staining analysis, real-time fluorescence quantitative PCR detection, EdU staining analysis, cell cycle determination and protein content determination.

[0045] See also Figure 3 , showing the results of Oil Red O staining of bovine intramuscular adipocytes in the NC group, si-EBF2 group and RA-added (si-EBF2+RA group); the quantification results of Oil Red O staining; the results of Bodipy and Nile Red staining of bovine intramuscular adipocytes in the NC group, si-EBF2 group and si-EBF2+RA group; the changes in mRNA expression of EBF2, PPARγ, C / EBPβ, FASN, FABP4 and LPL in bovine intramuscular adipocytes after EBF2 interference, and the results of the effect of adding RA on the expression of these genes; the changes in protein expression of EBF2, PPARγ and LPL in bovine intramuscular adipocytes after EBF2 interference, and the results of the effect of adding RA on the expression of these genes; the quantification results of proteins.

[0046] Oil Red O staining revealed a significant decrease in lipid droplet numbers in bovine intramuscular adipocytes following EBF2 silencing compared to controls. Quantitative analysis revealed that lipid content decreased by approximately 30.34% (P < 0.05) after EBF2 inhibition, a phenotype reversed by co-incubation with RA. Fluorescence-based lipid quantification confirmed these observations: BODIPY staining revealed a decrease in lipid-associated fluorescence intensity following EBF2 silencing, while Nile Red staining analysis demonstrated similar inhibition and RA-mediated restoration of the phenotype.

[0047] Silencing EFB2 suppressed the expression of adipogenic markers (PPARγ and C / EBPβ) and lipid accumulation markers (FABP4, LPL, and FASN) in bovine intramuscular adipocytes (P<0.01). RA treatment reversed this transcriptional repression, restoring marker expression to baseline levels. Western blot analysis revealed that EBF2 knockdown resulted in a corresponding decrease in protein expression levels of PPARγ (51.74%) and LPL (46.95%) in bovine intramuscular adipocytes, a phenomenon reversed by RA supplementation. This finding further confirms that EBF2 knockdown leads to a decrease in lipid content in bovine intramuscular adipocytes, while RA partially restores lipid accumulation.

[0048] See also Figure 4, showing the EdU staining results of bovine intramuscular preadipocytes in the NC group, si-EBF2 group, and si-EBF2+RA group; the cell cycle results of the NC group, si-EBF2 group, and si-EBF2+si-EBF2 group; the EdU staining quantification graph; the cell cycle quantification result graph; the interference efficiency of EBF2 in the proliferation period of bovine intramuscular preadipocytes and the effect of RA on it; the changes in the mRNA expression levels of CDK2 and CCNE2 in the si-EBF2 group compared with the NC group, and the effect of adding RA on the expression of these genes; the changes in the protein expression levels of EBF2, CDK2, and CCNE2 in bovine intramuscular preadipocytes after interfering with EBF2, and the effect of adding RA on them; the quantitative analysis results of protein content.

[0049] EdU staining revealed a significant increase in the proliferation rate of cells treated with si-EBF2 compared to the NC control group. Quantitative analysis revealed a 13.61% increase in cell proliferation (P < 0.01). Flow cytometry analysis of cell cycle distribution revealed a significant decrease in the number of G1-phase positive cells and a significant increase in the number of S-phase positive cells in bovine intramuscular preadipocytes following EBF2 interference, indicating that EFB2 interference promotes the G1 / S phase transition.

[0050] EBF2 knockdown increased the expression of CDK2 and CCNE2, consistent with the observed proliferative phenotype. Protein level analysis confirmed the upregulation of CDK2 and CCNE2 expression in EBF2-knockdown cells. Overall, EBF2 silencing promotes the proliferation of bovine intramuscular preadipocytes by modulating cell cycle progression and enhancing the expression of key proliferation regulators.

[0051] To further clarify the downstream target genes of EBF2, a key factor in vitamin A regulating bovine intramuscular fat deposition, transcriptome and non-target metabolomics sequencing were performed on bovine intramuscular adipocytes with EFB2 interference.

[0052] See also Figure 5 , showing the volcano plot of differentially expressed genes; the volcano plot of differentially expressed metabolites in POS mode; the volcano plot of differentially expressed metabolites in NEG mode; correlation heat map analysis of the top 30 adipogenesis-related differentially expressed gene-metabolite relationship pairs with Pearson correlation coefficients; correlation network analysis of the adipogenesis-related differentially expressed gene-metabolite relationship pairs in POS mode; and correlation network analysis of the adipogenesis-related differentially expressed gene-metabolite relationship pairs in NEG mode.

[0053] Transcriptome sequencing identified 509 differentially expressed genes, of which 220 were upregulated and 289 were downregulated. Non-targeted metabolomics sequencing in POS mode identified 51 differentially expressed metabolites, of which 14 were upregulated and 37 were downregulated. In NEG mode, 11 differentially expressed metabolites were identified, of which 6 were upregulated and 5 were downregulated.

[0054] Correlation analysis of differentially expressed genes and metabolites related to lipogenesis revealed that EBF2 was significantly associated with five key metabolites, M100T378 _ POS (cyclohexylamine), M184T350 _ POS (1-homocysteine), M183T395 _ Significant associations were found between POS (unknown), M159T435NEG (3-methyldiacid), and M432T372NEG. Correlation network analysis revealed that differentially expressed metabolites in the POS pattern were more closely associated with 23 differentially expressed genes, such as INHBB, APOD, and CYP26B1. In contrast, differentially expressed metabolites in the NEG pattern were primarily associated with 24 differentially expressed genes, such as PLTP, GSTM, and PDSS1. These results collectively depict a metabolic-gene interaction network in a specific pattern and establish EBF2 as a core regulator connecting transcriptional regulation with the dynamic changes of lipid-related metabolites.

[0055] Based on the correlation analysis between differentially expressed genes and metabolites in lipogenesis, the core downstream target genes of EBF2-mediated intramuscular fat deposition were further screened, and the targeting relationship between EBF2 and its target genes was multiplexed and verified based on site-directed mutagenesis, dual-luciferase reporter gene experiments and ChIP-qPCR experiments.

[0056] See also Figure 6 , showing the Venn diagram analysis of differentially expressed genes that are strongly correlated with lipogenesis-related differential metabolites in both POS and NEG modes; PPI network analysis of candidate key genes; changes in CYP26B1 expression after interfering with EBF2; dual luciferase reporter assay showing changes in enzyme activity after mutation of the EBF2 binding site in the CYP26B1 promoter compared with the wild type; ChIP-qPCR result graph; KEGG enrichment analysis bar chart and differentially expressed gene heat map showing genes enriched in the retinol metabolism pathway.

[0057] Venn diagram analysis revealed that 10 differentially expressed genes were significantly correlated with lipogenesis-related metabolites in both the POS and NEG modes. PPI network analysis revealed that CYP26B1 may be a core downstream target of EFB2 and has potential interactions with PDSS1, HTR2A, CXCL8, and IMPA2. These results suggest that CYP26B1 may play an important role in bovine intramuscular fat deposition as a downstream target of EBF2.

[0058] Analysis of the CYP26B1 promoter region revealed an EBF2 binding site at positions -112 to -120 of the promoter. RNA interference-mediated inhibition of EBF2 resulted in significant upregulation of CYP26B1, consistent with RNA-seq data. Dual-luciferase reporter assays revealed that EFB2 inhibited CYP26B1 transcriptional activity, and mutation of the EBF2 binding site (CYP26B1-MT) abolished this regulatory effect. ChIP-qPCR analysis revealed that the EBF2 transcription factor specifically bound to the CYP26B1 promoter, with a 3.21-fold enrichment compared to the IgG control. Furthermore, CYP26B1 was enriched in the retinol metabolism pathway (ko00830), a key enzyme in this biochemical cascade. These results suggest that binding of the EBF2 transcription factor to the CYP26B1 promoter region inhibits its activity. This negative regulatory relationship between EBF2 and CYP26B1 provides a key theoretical basis for elucidating the molecular mechanisms of bovine intramuscular fat deposition.

[0059] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for improving beef quality, characterized by: Promotes intramuscular fat deposition in cattle by regulating the expression of EBF2, a key downstream target of vitamin A.

2. A mechanism of action characterized by: Vitamin A is converted into retinal through dehydrogenation reaction in the body. Retinal is catalyzed into RA by Raldh1-Raldh3 enzymes. RA binds to its receptor RAR / RXR and activates the expression of EBF2. EBF2 inhibits the transcriptional activity of the retinol metabolizing enzyme CYP26B1, thereby activating RA signaling and regulating the activity of the retinol metabolic pathway. This cascade reaction further activates the lipogenesis marker PPARγ and its downstream lipid metabolism markers, promoting fatty acid transport and lipid accumulation in bovine intramuscular adipocytes.

3. A mechanism of action according to claim 2, characterized in that: Silencing of EBF2 activates the CDK2 / CCNE2 axis, promotes the G1 / S phase transition of bovine intramuscular preadipocytes, and promotes their proliferation.

4. A mechanism of action according to claim 2, characterized in that: EBF2 can directly target CYP26B1 and inhibit its transcriptional activity, binding sites: -112 to -120.

5. An experimental method for studying the mechanism of action of improving beef quality as claimed in any one of claims 2 to 4, characterized in that: include, Step S1: Incubating bovine intramuscular adipocytes with RA, an active metabolite of vitamin A, and conducting integrated transcriptomic and metabolomic analyses to identify EBF2, a key regulatory factor in the regulation of bovine intramuscular adipogenesis by vitamin A; Step S2, siRNA screening to determine si-EBF2-907 as the most effective silencing siRNA; Step S3, setting up an EBF2 control group, a si-EBF2 interference group, and a siEBF2+RA group, and detecting the regulatory effects of EBF2 interference on the proliferation, differentiation, and lipid metabolism of bovine intramuscular preadipocytes and the regulatory effects of RA on these processes, respectively; Step S4: transcriptome and non-target metabolomics sequencing of EFB2-silenced bovine intramuscular adipocytes to screen for CYP26B1, a potential downstream target of EBF2; Step S5: Multiple verification of the targeting relationship between EBF2 and CYP26B1 based on site-directed mutagenesis, dual-luciferase reporter gene assay, and ChIP-qPCR assay.

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