Cloning and transcriptional activity analysis of the bovine CDH11 gene promoter: transcription factors Sp1 and GR regulate bovine CDH11 expression

Cloning and transcriptional activity analysis of the bovine CDH11 gene promoter: transcription factors Sp1 and GR regulate bovine CDH11 expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cloning and transcriptional activity analysis of the bovine CDH11 gene promoter: transcription factors Sp1 and GR regulate bovine CDH11 expression Zhanxin Liu, Yanbin Bai, Zongchang Chen, Yanmei Niu, Xue Jia, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5023144/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Growth rate of bovine skeletal muscle has a major impact on beef yield. Cadherin-11 ( CDH11 ) was found to be a potential candidate gene for growth and development in beef cattle. This study confirmed the high expression of CDH11 in bovine longissimus dorsi muscle by tissue expression analysis. To understand the transcriptional regulation mechanism of CDH11 gene, we constructed a double luciferase vector for the promoter region of CDH11 gene and determined that the core transcriptional regulatory region was located at -129/+55 bp relative to the transcription start site (TSS). In addition, we confirmed that skeletal muscle growth and development-related transcription factor-specific protein 1 (SP1) and glucocorticoid receptor (GR) bind to the CDH11 gene promoter region at -36/-27 bp and − 20/-11 bp, respectively, to regulate CDH11 expression. These interactions provide valuable information for understanding the mechanism of transcriptional regulation of the bovine CDH11 gene during muscle growth and development. bovine CDH11 gene transcriptional regulation skeletal muscle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1 Introduction Muscle development plays a pivotal role in determining the growth rate of beef cattle. Skeletal muscle, which constitutes the major component of muscular tissue in animals, exerts a direct influence on the economic aspects of intensive beef cattle production [ 1 , 2 ]. The process of skeletal myogenesis is intricate and involves coordinated actions among multiple cell types. It is noteworthy that this process is finely regulated by different signaling pathways and transcription factors at various stages, encompassing prenatal myoblast proliferation, fusion for differentiation into myofibers, and postnatal myofiber hypertrophy [ 3 – 6 ]. Although Genome-Wide Association analyses (GWAS) have identified numerous genes associated with growth traits in beef cattle, limited investigation has been conducted regarding the epigenetic mechanisms underlying these specific genes and their interactions with specific binding transcription factors [ 6 , 7 ]. Cadherin-11 ( CDH11 ) is a transmembrane protein gene that encodes type II classical calreticulin, playing a crucial role in organismal growth and development primarily through the trans dimerization of extracellular calreticulin structural domain 1 to generate calreticulin complexes [ 8 , 9 ]. GWAS have demonstrated associations between CDH11 gene polymorphisms and growth and development traits in Quichuan cattle, as well as economic traits such as height, body size, growth and development of meat rabbits, and weaning weight of calves in Charolais beef cattle [ 10 – 13 ]. The proliferation and maturation of myofibroblasts are pivotal processes in myogenesis. Previous studies have reported the involvement of CDH11 gene in the growth and development of myofibroblasts. In chicken cells, CDH11 forms a complex with miR-205a that participates in the TGFβII pathway. This complex regulates the TGF-β1 pathway at both transcriptional and post-transcriptional levels, thereby enhancing the proliferation and differentiation of QM7 cells along with primary myofibroblasts [ 14 ]. Moreover, CDH11 plays a crucial role in myogenic differentiation by acting as a mesenchymal calcineurin mediating adhesion between homologous cells. When high-density myoblasts come into contact with each other, intercellular adhesion occurs viacal cineurins which promote tight junction formation leading to fusion of mononuclear myoblasts into multinucleated myotubes. This process induces myoblast differentiation resulting in muscle fibers formation ultimately contributing to increased meat production [ 15 – 17 ]. Additionally, apart from nutritional and environmental factors influencing animal growth and development, genetic regulation involving transcription factors also plays an important role specifically for skeletal muscle growth and development [ 18 – 20 ]. However, research on the regulatory mechanisms associated with CDH11 has primarily focused on human diseases. A case in point that FOXF1 regulates TGF-β1-induced damage in BEAS-2B cells by modulating CDH11 gene-mediated Wnt/β connexin signaling [ 21 ]. Furthermore, it has been discovered that the transcription factor homology box C8 (HOXC8) specifically binds to the promoter region of the CDH11 gene and maintains high levels of CDH11 gene expression in breast cancer [ 22 ]. Given the the critical role of the CDH11 gene in beef cattle growth and development, coupled with limited knowledge regarding its involvement in the transcriptional regulation of skeletal muscle growth and development, this study was aimed to comprehensively investigate the structural composition of the proximal promoter with the 5'UTR region of the CDH11 gene. We successfully identified both the core promoter region and as two transcription factors closely associated with skeletal muscle growth and development. Furthermore, we conducted comprehensive analyses to elucidate these two transcription factors intricate roles in controlling CDH11 gene transcription. These discoveries provide a theoretical foundation for the future research on the functional significance of the CDH11 gene in beef cattle's muscular growth and development as well as genetic improvement. 2 Results 2.1 Sequence structure, homology analysis, and phylogenetic tree construction of the bovine CDH11 gene The CDH11 gene sequences of seven different species were selected and compared with those of Bos taurus (XP_005218806.1). The results revealed that the similarity was 100% for Bos mutus (XP_005905649.1), while the similarity with Capra hircus (XP_ 017917568.1) and Ovis aries (XP_014955948.1) were both with 99.87%. Additionally the similarity with Sus scrofa (XP_020949024.1), Homo sapiens (XP_054235326.1), Mus musculus (XP_ 030099132.1), and Gallus gallus (XP_046781520.1) were found to be 99.25%, 98.42%, 97.49% and 90.58% respectively (Fig. 1B). It is evident from these findings that the CDH11 gene exhibits a higher degree of conservation in ruminants compared to non-ruminants. In comprehensive understand the evolution of the CDH11 gene, we selected Bos taurus (NM_001081624), Bos mutus (XM_005905587.1), Bubalus bubalis (NM_001081624.2), Capra hircus (NM_009866), Ovis aries (CM_001595.2), Camelus dromedarius (NM_011515430), Homo sapiens (NM_001308392.2), Mus musculus (XM_006530624.3), Sus scrofa (NM_001244482.1) and Gallus gallus (NC_006098.5) using the EMGA11 software in conjunction with the amino acid sequences published by UCSC, with their CDH11 amino acid sequences used for phylogenetic tree construction. The results showed that Bos taurus formed a cluster with ruminants including Bubalus bubalis, Bos mutus, Capra hircus, Ovis aries, and Camelus dromedarius followed by a cluster comprising non-ruminants comprising Sus scrofa, Homo sapiens, and Mus musculus in another cluster while Gallus gallus was the most distantly related group (Fig. 1C). Figure1 Structural homology and phylogenetic tree of the CDH11 gene. (A) This section mainly includes the CDH11 gene promoter, 5'UTR, 3'UTR, exons, introns, coding length of CDS region, and number of coding amino acids. (B) A comparison of the degree of amino acid clustering in the CDH11 protein among seven different species. (C) An analysis of the protein-amino acid phylogenetic tree for the CDH11 gene among 10 different species. 2.2 Expression assay of bovine CDH11 gene in various tissues To investigate the mRNA expression pattern of the CDH11 in various tissues, total RNA was extracted from the heart, liver, spleen, lung, kidney, longissimus dorsi muscle, and subcutaneous fat. Subsequently, the isolated RNA was reverse-transcribed into cDNA. Quantitate real-time polymerase chain reaction (qRT-PCR) analysis revealed that the CDH11 gene exhibited differential expression across different tissues with the lung display the highest expression. Moreover, moderate to high levels of CDH11 gene expression were observed in liver, kidney, longissimus dorsi muscle, and subcutaneous fat which were significantly compared to spleen and heart tissues. Notably, spleen exhibited the lowest level of the CDH11 gene expression (Fig. 2 ). 2.3 Identification of the core transcriptional regulatory region of the bovine CDH11 gene To identify the core transcriptional regulatory region of the promoter region of bovine CDH11 gene. We amplified seven segment-by-segment fragments from the 5'UTR proximal promoter region and successfully ligated NheI and XhoI restriction enzyme fragments into the pGL3-Basic vector to construct a recombinant double-luciferase reporter plasmid (Figure S1 ). Subsequently, the corresponding recombinant luciferase reporter plasmids were transfected into mouse C2C12 cells to assess luciferase activity. The results showed no significant change in luciferase activity compared to -1855/-429. Notably, luciferase activity was significantly higher in the − 129/+55 region compared to the − 429/+55 region, indicating elevated promoter activity compared to the pGL3-Basic vector plasmid (Fig. 3A). In conclusion, our results suggest that the core transcriptional regulatory region of the bovine CDH11 gene is located at -129/+55 bp relative to the TSS. Additionally, in order to investigate the potential impact of methylation on CDH11 gene expression within its promoter region, Methprimer, an online software tool, was utilized to predict the presence of CpG islands. Notably, analysis revealed a lack of CpG islands within the promoter region of the bovine CDH11 gene (Fig. 3B). Figure 3 Identification of the core transcriptional regulatory region of bovine CDH11 gene and prediction of CPG islands. (A) The recombinant plasmid was co-transfected with pGL3-TK into mouse C2C12 cells, and firefly and kidney luciferase activities were measured after 48 h. The results were expressed as the ratio of firefly luciferase activity to kidney luciferase activity. (B) A schematic diagram of the proximal promoter region (-1855/+55) of the 5'UTR of the bovine CDH11 gene is shown. y-axis indicates the percentage of GC and x-axis indicates the position of non-coding segments. TSS (+ 1) indicates the transcription start site. ** denotes P < 0.01. Error lines represent SD. 2.4 The validation of transcription factors in core regions of transcriptional regulatory To elucidate the role of potential trans-acting elements within the core promoter region of the bovine CDH11 gene, this study aimed to predict its transcription factors using Gene Regulation and Jaspar online platforms. As a result, SP1 and GR, two transcription factor binding sites associated with muscle growth and development, were found to be present at the − 36/-27 bp and − 20/-11 bp sites in the core transcriptional regulatory region of the bovine CDH11 gene (Fig. 4 A). Furthermore, conservation analysis demonstrated that SP1 and GR exhibited remarkable conservation across multiple species (Fig. 4 B). To further investigate their functional significance, this experiment constructed vectors containing mutated core transcriptional regulatory regions of these two binding sites. Interestingly, deletion of the SP1 binding site significantly increased luciferase activity while deletion of the GR binding site led toa significant decrease in luciferase activity (Fig. 4 C). Based on these findings, in the present study, siRNA-GR and siRNA-SP1 (Fig. 4 D, E ), which have better interference efficiency, were co-transfected with the pGL3-129/+55 plasmid, respectively, in bovine adult myoblasts, and their luciferase activities were determined. The results of the dual-luciferase reporter system showed that the luciferase activity of siRNA-GR + pGL3-129/+55 was significantly reduced, whereas that of siRNA-SP1 + pGL3-129/+55 was significantly elevated, compared with that of the control (Fig. 4 F, G). It was also observed that knockdown of GR significantly inhibited the expression level of CDH11 , whereas knockdown of SP1 significantly promoted the expression of CDH11 (Fig. 4 H, I). 2.5 Electrophoretic Mobility Shift Assays (EMSA)to validate the interaction of GR and SP1 with the promoter To confirm the binding of transcription factors GR and SP1 to the core transcriptional regulatory region of the bovine CDH11 gene, we performed in vitro EMSA experiments. Biotin-labelled oligonucleotide probes GR and SP1 were synthesized and incubated with nucleoproteins from C2C12 cells. The biotin-labelled probes formed a DNA-protein complex band with nuclear proteins (Fig. 5 A B, lane 2). Addition of the noncompetitive probe had minimal impact on the DNA protein complex bands (Fig. 5 A B, lane 3), while the addition of a competitive probe resulted in attenuation and disappearance of the DNA protein complex bands (Fig. 5 A B, lane 4). Furthermore, inclusion of GR antibody and SP1 antibody individually attenuated the DNA protein complex band and lead to formation of a super shifted band above it (Fig. 5 A B, lane 5). These findings indicate that both transcription factors GR and SP1 bind to the core transcriptional regulatory region of the CDH11 gene. 3 Materials and methods 3.1 Sample collection Samples utilized for the experiment were heart, liver, spleen, lung, kidney, longissimus dorsi muscle and subcutaneous fat samples from three healthy 1-year-old bulls were obtained from the livestock farm of Gansu Agricultural University (Lanzhou, China). Subsequently, these samples were rapidly immersed in liquid nitrogen and subsequently stored at -80°C. 3.2 Isolation of DNA and the detection of CDH11 gene mRNA expression This genomic DNA from the longissimus dorsi muscle was isolated using the TRlzol method described by Ye et al [ 23 ]. Total RNA was extracted from tissues and cells using AG RNAex Pro Reagent (Accurate, Biotechnology, China). Integrity of the RNA samples was assessed through agarose gel electrophoresis (JUNYI, Beijing, China), while concentration and purity were determined using a Nano Dro8000 spectrophotometer (ND8000-GL, NanoDrop Technologies, Wilmington, NC, USA). Subsequently, cDNA synthesis was performed according to the TransScript One-Step gDNA Removal and cDNA Synthesis Super Mix (AT311, Transgen, Beijing, China). Fluorescent quantitative primers were designed based on the bovine CDH11 gene family published by UCSC (accession number: NM_001081624.2) using Clone Manage software (Table S1 ). Quantification of mRNA expression levels was carried out with the Perfect Start Green qPCR Super Mix kit (AQ601, Transgen, Beijing, China) The reaction mixture consisted of 20 µL total volume containing 0.4 µL each of forward and reverse primers, 10 µL of 2× PerfectStart Green qPCR Super Mix, 0.4 µL of Passive Reference Dye (50 x), 2 µL Template and 6.8 µL of Nuclease-free Water. qRT-PCR reaction conditions included pre-denaturation at 94°C for 30 secs; denaturation at 94°C for 5 secs; annealing at 60°C for 30 secs, with a total of 42 cycles. Gene expression level were normalized to Glyceraldehyde-3 phosphate dehydrogenase ( GAPDH ) expression and calculated using the − 2 ΔΔCt method. 3.3 Proximal initiation sub-bioinformatics analysis and prediction The 5'UTR proximal − 1855/+55 bp DNA sequence of the bovine CDH11 gene (GenBank: NM_001081624.2) was obtained from from the UCSC Genome Brower Home database ( https:/genome.ucsc.edu ). A neighbor-joining method through Mega 11 (Philadelphia, USA) was emplaoyed to constructed a phylogenetic tree. Uniport ( https://www.uniprot.org ) was utilized toanalyze protein homology of the CDH11 gene across Bos mutus, Capra hircus, Ovis aries, Sus scrofa, Homo sapiens, Mus musculus and Gallus gullus. CpG sites were predicted using Methprimer ( http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi ), while the transcription start sites were predicted using BDGP ( https://www.fruitfly.org/seq-tools/promoter.html ). Furthermore, Gene Regulation ( http://gene-regulation.com ) combined with Jaspar ( https://jaspar.elixir.no ) predictions were used predict transcription factor binding sites within the core transcriptional regulatory region of the promoter. 3.4 Construction of luciferase deletion vector plasmid in the 5 ' UTR proximal promoter region of bovine CDH11 gene. In this experiment, the purified fragments were digested with NheI and XhoI (JN301, JX201, Transgen, Beijing, China) to generate sticky ends. Subsequently, gel extraction was performed using the Easy Pure Quick Gel Extraction Kit (EG101, Transgen, Beijing, China). The resulting fragments were then ligated to the pGL3-Basic plasmid using T4 DNA Ligase (FL101, Transgen, Beijing, China). Finally, transformation into bacteriophage was carried out using Trans1-T1 Phage Resistant Chemically Competent Cells (CD501, Transgen, Beijing, China), followed by plasmid extractions using Endofree MINI Plasmid kit II (DP118, TINGEN, Beijing, China). The resulting plasmids were named pGL-1855/+55 (P1), pGL-1629/+55 (P2), pGL-1329/+55 (P3), pGL-1029/+55 (P4), pGL-729/+55 (P5), pGL-429/+55 (P6), and pGL-129/+55 (P7). 3.5 Cell culture, transfection, and luciferase activity analysis The medium consisted of 10% fetal bovine serum (A6904, Invitrogen, Carlsbad, CA, USA) and 4% dual antibiotics (G4003-100ML, Servicebio, Wuhan, China), supplemented with 86% DMEM high glucose medium (C11995500BT, GIBCO, Grand Island, NY, USA). Mouse C2C12 cells (TCM-C720, Starfish Bio, Suzhou, China) were cultured in the above configured medium. Cells were seeded into 24-well plates at a density of 1×10 5 cells per well prior to transfection and then cultured at 37°C in a CO 2 incubator with a concentration of 5% until reaching a cell density of 75%-80%. For detection purposes following co-transfection according to the manufacturer protocol from Invigentech (Invitrogen, Carlsbad, CA, USA), using the TransDetect Dual Luciferase Reporter Gene Assay Kit (FR201, Transgen, Beijing, China), cells were transfected with the target plasmid along with pGL3-TK at an ng ratio of 800:20 for a duration of 48 h. Subsequently, collected cells were passed through through an enzyme labeler (VL0000D0, Thermo Scientific, Waltham, MAUSA). Firefly luciferase and Renilla luciferase activities were measured and promoter activity was determined as firefly luciferase activity/Renilla luciferase activity in three replicate wells per transfection. In accordance with this, bovine adult myoblasts were obtained from cells isolated and identified as having better activity by the previous group [ 24 ]. Their medium consisted of 13% FBS, 4% antibiotics and 83% DMEM/F12 medium (C11330500BT, Hyclone, New York, NY, USA). Prior to transfection, cells were seeded into 12-well plates at a density of 1x10 6 cells per well and then cultured in a CO 2 incubator at 37°C at a concentration of 5% until cell densities reached 75%-80% for later RNA interference experiments. 3.6 Site-directed mutagenesis The PCR amplification kit was utilized for the amplification, employing custom-designed glucocorticoid receptor (GR) and specificity protein 1 (SP1) sentinel mutation primers based on the Fast MultiSite Mutagenesis System (FM201, Transgen, Beijing, China) (Table S1 ). The reaction system comprised of 25 µL of 2× TransStart FastPfu Fly PCR SuperMix, 1 µL each of the upstream and downstream primers, 9 µL of Plasmid, and finally 14 µL of Nuclease-free Water. The amplified target sequences were subsequently purified and assembled. The reaction was carried out at 50°C for 15 mins followed by rapid cooling on ice for a few secs. Subsequently, the receptor cells underwent transformation and single clones were selected for sequencing. 3.7 RNA interference assay The interfering sequences targeting transcription factor GR and Sp1 (siRNA-GR, siRNA-SP1) were custom-synthesized by Suzhou Hongxun Biotechnology Co. (Suzhou, China). The specific details of these interfering sequences are provided (Table S1 ). 3.8 Electrophoretic mobility shift assay (EMSA) To obtain nuclear protein extracts from mouse C2C12 cells, logarithmic growth phase cells were cultured into T25 culture flasks. The Nuclear Protein Extraction Kit (Active, Motif Corp, Carlsbad, CA, USA) was used for treating the C2C12 cells. Biotin-labelled DNA probes for the 5'UTR proximal core transcriptional regulatory region were designed and synthesized (Table S1 ). In brief, a binding reaction mixture consisting of 2 µL of 5× binding buffer, 10 µg of nuclear extract, and 1 µL of poly (dI-dC) was prepared in a volume of 20 µL and incubated on ice for 15 mins. Subsequently, biotin-labeled DNA (200 fmol) was added to the reaction mixture and incubated at room temperature for an additional 20 mins. For competition assays, unlabelled or mutant probes were added to the reaction mixture 15 mins prior to adding the labelled probe. For the super-shift assay, antibodies (10 µg). including anti-GR (TA7647A, Abmart, Shanghai, China) and anti-SP1 (PS02143, Abmart, Shanghai, China) were included in the reaction mixture after pre-incubation at low temperature for 30 mins. Following this step, DNA-protein complexes were separated by non-denaturing polyacrylamide gel electrophoresis using polyacrylamide and a borate -EDTA buffer for one hour duration. 3.9 Statistical analysis. In this study, the Duncan method was employed for multiple comparisons. Comparisons between the two groups were conducted using a two-tailed test, and significance was assessed using IBM SPSS 26 statistical software. Graphs were generated using GraphPad Prism 9.0 software. The values in this study are presented as mean ± standard deviation (SD). * denotes p < 0.05, ** denotes p < 0.01, in organizational expressions, different letters represent significant differences, same letters represent non-significant differences, n = 3 denotes sample size. 4 Discussion The rate of growth in skeletal muscle plays a crucial role in determining the carcass weight of livestock. Elucidating the transcriptional regulation of genes related to skeletal muscle can serve as a theoretical foundation for breeding meat-producing livestock and poultry. GWAS conducted in livestock and poultry have facilitated the identification of numerous genes associated with their growth and development. For instance, the CDH11 gene has been identified as a potential regulator influencing beef cattle growth and development [ 25 ]. In this study, we observed that CDH11 gene mRNA is highly expressed in the longissimus dorsi muscle compared to cardiac expression, suggesting its potential role in regulating bovine skeletal muscle production. To further elucidate the epigenetic regulatory role of the cattle CDH11 gene in muscle growth and development, this study conducted an analysis on the promoter sequence of the bovine CDH11 gene obtained from the UCSC database. Our analysis revealed that the promoter region of bovine CDH11 lacks a traditional TATA box and CpG island structure, which is with previous investigations demonstrating limited presence of TATA boxes in mammalian gene promoters and 50% occurrence of CpG islands either inter or intra-genic ally [ 26 , 27 ].Therefore, it can be speculated that methylation of promoter region may not affected transcriptional regulation of the bovine CDH11 gene. Furthermore, employing a segment-by-segment deletion approach, we identified − 129/+55 bp as the core transcriptional regulatory region of the CDH11 gene expression. In our study, we predicted two transcription factor binding sites (SP1 and GR) in the core transcriptional regulatory regions − 36/-27 bp and − 20/-11 bp regions that are highly conserved across multiple species and associated with muscle development. To investigate their role further, mutated vectors containing mutated core transcriptional regulatory regions were constructed and luciferase activity was measured. Deletion of SP1 binding site significantly increased luciferase activity while deletion of GR binding site significantly decreased it. Hence, these results suggest that SP1 and GR play a crucial role in mediating the transcriptional active of bovine CDH11 gene. The GR is ligand-dependent transcription factor belong to the nuclear receptor superfamily, which plays an active role in all stages of muscle strength and development. Previous studies have demonstrated its essentiality in maintaining and establishing a quiescent state during the growth and development phase of satellite cells. Knocking down GR in myosatellite cells has been shown to increase the amount of circulating cells in the proliferative phase, ultimately leading to muscular atrophy [ 28 ]. During myofibrilla genesis, GR synergistically binds with MyoD at the GC-rich DNA corresponding element region of the promoter of the target gene, resulting in programmed expression of muscle fiber-related genes [ 29 – 31 ]. Although MyoD has been identified as a specific transcription factor for skeletal muscle proliferation and differentiation, It may also act through alternative pathways or transcription factors on muscle precursors [ 32 , 33 ]. In this study, interference with the GR led to a significant decrease in luciferase activity within the core transcriptional regulatory region of the CDH11 gene as well as a reduction in CDH11 gene expression. Additionally, results from the EMSA assay demonstrated that the GR transcription factor could bind to the promoter region sequence of the CDH11 gene. These findings suggest that GR transcription factors positively regulate the expression of the cattle CDH11 gene in bovine muscle growth and development. Transcription factor SP1, a member of the Sp/Kruppel superfamily, is an eukaryotic transcription factor that has been identified to directionally regulate mammalian muscle growth and development [ 34 , 35 ]. Previous studies have indicated that the SP1 recruits MyoD, MyoG, and MEF-2C elements to specifically bind to GC-box and GT-box progenitors in gene promoters for the regulation of myosatellite cell proliferation and differentiation [ 36 , 37 ]. In this study, mutation of the SP1 binding site significantly enhanced the transcriptional activity of the core transcriptional regulatory region of the CDH11 gene. Overexpression of the CDH11 gene has been shown to inhibit MyoD and MyoG expression [ 38 ]. Therefore, we hypothesize that the SP1 specifically binds to the promoter region of CDH11 gene and plays a role in regulating bovine myofibroblasts proliferation and differentiation by recruiting MyoD, MyoG, MEF-2C, or itself. Ultimately, this positively impacts bovine myogenesis [ 39 , 40 ]. Interference with the SP1 transcription factor led to increased luciferase activity in the core regulatory region of CDH11 gene resulting in reduced levels of CDH11 gene expression. EMSA results confirmed that the SP1 can bind to the core regulatory region of the CDH11 gene. In conclusion, our study demonstrates that the transcription factor SP1 plays a crucial role in regulating CDH11 gene expression in bovine skeletal muscle. However, further investigation is required to understand the epigenetic modifications with its promoter as well as its specific transcriptional regulatory mechanisms related to the CDH11 gene function. These findings provide insights into understanding how bovine CDH11 gene is regulated at a transcriptional level and its biological function. 5 Conclusion In conclusion, this study establishes that the CDH11 gene is highly expressed in bovine longissimus dorsi muscle and identifies its core promoter region within − 129/+55 bp. Our results suggest that CDH11 is positively regulated by GR and negatively regulated by SP1 in the regulation of muscle growth and development. These findings enhance our understanding of the transcriptional regulation mechanisms governing CDH11 gene and suggest potential molecular breeding strategies for improving beef cattle yield and quality. Declarations Author Contributions: Conceptualization, ZXL and ZDZ; Methodology, YBB, ZCC, YMN, XJ and LL, JQW, XL, JH; Validation, ZXL, YBB, ZCC; Formal analysis, ZXL, ZCC, YMN. and YBB; Investigation, ZXL, YBB, ZCC, YMN, XJ, LL; Resources, ZDZ and JH; Writing—original manuscript preparation, ZXL; Writing—review and editing, ZXL, ZDZ and YBB, Supervision, JQW, BGS, FFZ, and ZDZ; Project Management, XLZ, SBL, XL, BGS, and ZDZ; Funding Acquisition, JH and ZDZ. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the integration and demonstration of key technologies for the healthy and efficient breeding of beef cattle in Linxia（KJJC-LX-2023-3-01）; the discipline Team Project of Gansu Agricultural University (GAU-XKTD-2022-22); the Efficient Production Technology of Yak in Qilian Mountains Pastoral Areas and Livestock Recycling Development and Demonstration (2022CYZC-43). Institutional Review Board Statement: The animal study was reviewed and approved by the Ethical Commission of Gansu Agricultural University, as well as the Ministry of Science and Technology of the People’s Republic of China (ethical permit number: GSAU-ETH-AST-2021-025). Informed Consent Statement: Not applicable. Data Availability Statement: The dataset supporting the conclusions of this article is available via email to the corresponding author. Acknowledgments: We thank all the members of the Gansu Key Laboratory of Herbivorous Animal Biotechnology of the College of Animal Science and Technology, Gansu Agricultural University, who contributed their efforts to these experiments. Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest References Hu Z et al. 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Myod1 and GR coordinate myofiber-specific transcriptional enhancers. Nucleic Acids Res, 2021. 49(8). Bablok M et al. Spatiotemporal expression pattern of the chicken glucocorticoid receptor during early embryonic development. Ann Anat, 2023. 247. Rovito D et al. Myod1 and GR coordinate myofiber-specific transcriptional enhancers. Nucleic Acids Res, 2021. 49(8). Hayashi S et al. Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice. Elife, 2016. 5. Zammit PS. Function of the myogenic regulatory factors Myf5, MyoD, Myogenin and MRF4 in skeletal muscle, satellite cells and regenerative myogenesis. Semin Cell Dev Biol, 2017. 72. O'Connor L, Gilmour J, Bonifer C. The Role of the Ubiquitously Expressed Transcription Factor Sp1 in Tissue-specific Transcriptional Regulation and in Disease. Yale J Biol Med, 2016. 89(4). Beishline K, Azizkhan-Clifford J. Sp1 and the 'hallmarks of cancer'. FEBS J, 2015. 282(2). Philipsen S, Suske G. A tale of three fingers: the family of mammalian Sp/XKLF transcription factors. Nucleic Acids Res, 1999. 27(15). Zhang R et al. Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene. Int J Mol Sci, 2016. 17(1). Wang Z et al. Gga-miR-205a Affecting Myoblast Proliferation and Differentiation by Targeting CDH11. Front Genet, 2018. 9. Wang Y et al. miR-130b inhibits proliferation and promotes differentiation in myocytes via targeting Sp1. J Mol Cell Biol, 2021. 13(6). Dai Y et al. MicroRNA-128 regulates the proliferation and differentiation of bovine skeletal muscle satellite cells by repressing Sp1. Mol Cell Biochem, 2016. 414(1–2). Additional Declarations No competing interests reported. Supplementary Files TableS1.docx FigureS1..docx FigureS2.OriginalEMSAfigures.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5023144","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":350128071,"identity":"b8302ff1-f791-4a53-b991-7817a2462a34","order_by":0,"name":"Zhanxin Liu","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhanxin","middleName":"","lastName":"Liu","suffix":""},{"id":350128072,"identity":"85ce19b8-4b28-49ee-8471-c9fbe6e618ea","order_by":1,"name":"Yanbin Bai","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanbin","middleName":"","lastName":"Bai","suffix":""},{"id":350128073,"identity":"8285c53b-ca76-40e6-b844-8d739c4dd7b3","order_by":2,"name":"Zongchang Chen","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zongchang","middleName":"","lastName":"Chen","suffix":""},{"id":350128074,"identity":"a92fd9df-bd2d-4902-9155-aa17d161fa5a","order_by":3,"name":"Yanmei Niu","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanmei","middleName":"","lastName":"Niu","suffix":""},{"id":350128075,"identity":"05510f98-50a4-432a-94d4-846225953a1b","order_by":4,"name":"Xue Jia","email":"","orcid":"","institution":"Gansu Agricultural 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University","correspondingAuthor":false,"prefix":"","firstName":"Jiang","middleName":"","lastName":"Hu","suffix":""},{"id":350128081,"identity":"7003c8b0-c283-4b65-8b03-72f7bccae1db","order_by":10,"name":"Jiqing Wang","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jiqing","middleName":"","lastName":"Wang","suffix":""},{"id":350128082,"identity":"9e299983-b727-45d6-8125-f50888ad0a51","order_by":11,"name":"Xiu Liu","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiu","middleName":"","lastName":"Liu","suffix":""},{"id":350128083,"identity":"36eecdb6-cc0f-4ab1-8cf0-eb0e99e24266","order_by":12,"name":"Shaobin Li","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shaobin","middleName":"","lastName":"Li","suffix":""},{"id":350128084,"identity":"a9118b6f-9b19-4325-afe6-25a1882dcfb4","order_by":13,"name":"Fangfang Zhao","email":"","orcid":"","institution":"Gansu Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Fangfang","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2024-09-03 08:16:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5023144/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5023144/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66188198,"identity":"c9f3479e-d916-4a55-bc9b-ff93b985f385","added_by":"auto","created_at":"2024-10-08 13:49:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":78541,"visible":true,"origin":"","legend":"\u003cp\u003eStructural homology and phylogenetic tree of the \u003cem\u003eCDH11\u003c/em\u003e gene. (A) This section mainly includes the \u003cem\u003eCDH11\u003c/em\u003e gene promoter, 5'UTR, 3'UTR, exons, introns, coding length of CDS region, and number of coding amino acids. (B) A comparison of the degree of amino acid clustering in the \u003cem\u003eCDH11\u003c/em\u003eprotein among seven different species. (C) An analysis of the protein-amino acid phylogenetic tree for the\u003cem\u003e CDH11\u003c/em\u003e gene among 10 different species.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5023144/v1/30383e139cc6507085f7a644.png"},{"id":66187890,"identity":"c1b681b0-7e3f-4e36-ac3e-d0d58fbcae36","added_by":"auto","created_at":"2024-10-08 13:41:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24408,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of the \u003cem\u003eCDH11\u003c/em\u003e gene in various bovine tissues was analyzed using qRT-PCR with the heart as a reference. The results are presented as the mean ± standard deviation (SD) of three experimental groups. Different letters indicate significant differences between groups, while the same letter indicates no significance. Error bars represent SD.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5023144/v1/be969319e8d8ac365158bce1.png"},{"id":66187892,"identity":"58217d73-e181-404e-bf85-893d4c0e154b","added_by":"auto","created_at":"2024-10-08 13:41:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44513,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the core transcriptional regulatory region of bovine \u003cem\u003eCDH11\u003c/em\u003egene and prediction of CPG islands. (A) The recombinant plasmid was co-transfected with pGL3-TK into mouse C2C12 cells, and firefly and kidney luciferase activities were measured after 48 h. The results were expressed as the ratio of firefly luciferase activity to kidney luciferase activity. (B) A schematic diagram of the proximal promoter region (-1855/+55) of the 5'UTR of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene is shown. y-axis indicates the percentage of GC and x-axis indicates the position of non-coding segments. TSS (+1) indicates the transcription start site. ** denotes \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01. Error lines represent SD.\u0026nbsp;\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5023144/v1/cfa406d838c13adbfb98b22e.png"},{"id":66187894,"identity":"ae356238-96eb-4a2e-b700-b760c816bcc5","added_by":"auto","created_at":"2024-10-08 13:41:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":206566,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of transcription factors SP1 and GR on the transcriptional activity and expression of the \u003cem\u003eCDH11\u003c/em\u003e gene. (A) The promoter region of the \u003cem\u003eCDH11\u003c/em\u003e gene (-1855/+55) is schematically represented, with the arrow TSS (+1) indicating the transcriptional start site. The GR binding site to the SP1 transcription factor is shown in the box, and predicted transcriptional regulatory elements are marked with black markers. (B) The -129/+55 vector sequence containing a mutant GR and SP1 binding site was constructed, and luciferase activity was measured by transfecting mouse C2C12 cells for 48 h using the -129/+55 vector plasmid as a negative control. The black and white filled graphs represent wild type and mutant. (C) Conservation analysis of GR and SP1 in multiple species. (D E) Detection of siRNA-GR and siRNA-SP1 interference efficiency in bovine adult myocytes using siRNA-NC as a negative control. (F G) Luciferase activity was measured after co-transfection of bovine adult myoblasts with pGL3-129/+55+siRNA-NC for 48 h using pGL3-129/+55+siRNA-GR and pGL3-129/+55+siRNA-SP1 as negative controls. (H I) Bovine adult myoblasts were transfected with siRNA-GR, siRNA-SP1 using siRNA-NC as a negative control, and\u003cem\u003e CDH11\u003c/em\u003e gene expression was detected by qRT-PCR. * indicatesP\u0026lt;0.05, while ** indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 compared to the control group.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5023144/v1/bdd1214f1d8ba82072e32cc2.png"},{"id":66188199,"identity":"86817a22-6709-4637-82f0-d25f1a298a08","added_by":"auto","created_at":"2024-10-08 13:49:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":124861,"visible":true,"origin":"","legend":"\u003cp\u003eEMSA confirmed the in vitro binding of transcription factors GR and SP1 to the core transcriptional regulatory region of the \u003cem\u003eCDH11\u003c/em\u003e gene. (A) shows EMSA validation of transcription factor GR binding, while figure. (B) demonstrates EMSA validation of transcription factor SP1 binding to the core transcriptional regulatory region of the \u003cem\u003eCDH11\u003c/em\u003e gene. A biotin-labelled probe containing two transcription factors from the core transcriptional regulatory region of the \u003cem\u003eCDH11\u003c/em\u003e gene was incubated with C2C12 cytosolic protein (lane 2), mutated probe (lane 3), and unmutated probe (lane 4), Sup remigration assays were performed using anti-GR and anti-SP1 antibodies (lane 5).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5023144/v1/a9c63c106d59a4d3a1bdb67b.png"},{"id":78536506,"identity":"ea646beb-d143-49aa-ba04-929fd496a906","added_by":"auto","created_at":"2025-03-14 15:01:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1408370,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5023144/v1/21643057-4c3c-4c21-9862-eb836815be94.pdf"},{"id":66187896,"identity":"19608bf5-6b2f-43de-9f36-6c53cac09bbe","added_by":"auto","created_at":"2024-10-08 13:41:52","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":20658,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5023144/v1/d9c71dbb5fbd94b724c4a0c9.docx"},{"id":66187895,"identity":"0925e04b-06cf-4ce9-9dd7-c4d3d4611e82","added_by":"auto","created_at":"2024-10-08 13:41:51","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":278067,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS1..docx","url":"https://assets-eu.researchsquare.com/files/rs-5023144/v1/ce721652e19e00c5d7d7b740.docx"},{"id":66187905,"identity":"dfc1de12-a485-43e9-89c7-43346bb41818","added_by":"auto","created_at":"2024-10-08 13:41:52","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4720961,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.OriginalEMSAfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5023144/v1/1814f0b30a4132b87f91eb47.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cloning and transcriptional activity analysis of the bovine CDH11 gene promoter: transcription factors Sp1 and GR regulate bovine CDH11 expression","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eMuscle development plays a pivotal role in determining the growth rate of beef cattle. Skeletal muscle, which constitutes the major component of muscular tissue in animals, exerts a direct influence on the economic aspects of intensive beef cattle production [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The process of skeletal myogenesis is intricate and involves coordinated actions among multiple cell types. It is noteworthy that this process is finely regulated by different signaling pathways and transcription factors at various stages, encompassing prenatal myoblast proliferation, fusion for differentiation into myofibers, and postnatal myofiber hypertrophy [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although Genome-Wide Association analyses (GWAS) have identified numerous genes associated with growth traits in beef cattle, limited investigation has been conducted regarding the epigenetic mechanisms underlying these specific genes and their interactions with specific binding transcription factors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCadherin-11 (\u003cem\u003eCDH11\u003c/em\u003e) is a transmembrane protein gene that encodes type II classical calreticulin, playing a crucial role in organismal growth and development primarily through the trans dimerization of extracellular calreticulin structural domain 1 to generate calreticulin complexes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. GWAS have demonstrated associations between \u003cem\u003eCDH11\u003c/em\u003e gene polymorphisms and growth and development traits in Quichuan cattle, as well as economic traits such as height, body size, growth and development of meat rabbits, and weaning weight of calves in Charolais beef cattle [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The proliferation and maturation of myofibroblasts are pivotal processes in myogenesis. Previous studies have reported the involvement of \u003cem\u003eCDH11\u003c/em\u003e gene in the growth and development of myofibroblasts. In chicken cells, \u003cem\u003eCDH11\u003c/em\u003e forms a complex with miR-205a that participates in the TGFβII pathway. This complex regulates the TGF-β1 pathway at both transcriptional and post-transcriptional levels, thereby enhancing the proliferation and differentiation of QM7 cells along with primary myofibroblasts [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Moreover, \u003cem\u003eCDH11\u003c/em\u003e plays a crucial role in myogenic differentiation by acting as a mesenchymal calcineurin mediating adhesion between homologous cells. When high-density myoblasts come into contact with each other, intercellular adhesion occurs viacal cineurins which promote tight junction formation leading to fusion of mononuclear myoblasts into multinucleated myotubes. This process induces myoblast differentiation resulting in muscle fibers formation ultimately contributing to increased meat production [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, apart from nutritional and environmental factors influencing animal growth and development, genetic regulation involving transcription factors also plays an important role specifically for skeletal muscle growth and development [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, research on the regulatory mechanisms associated with \u003cem\u003eCDH11\u003c/em\u003e has primarily focused on human diseases. A case in point that FOXF1 regulates TGF-β1-induced damage in BEAS-2B cells by modulating \u003cem\u003eCDH11\u003c/em\u003e gene-mediated Wnt/β connexin signaling [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Furthermore, it has been discovered that the transcription factor homology box C8 (HOXC8) specifically binds to the promoter region of the \u003cem\u003eCDH11\u003c/em\u003e gene and maintains high levels of \u003cem\u003eCDH11\u003c/em\u003e gene expression in breast cancer [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven the the critical role of the \u003cem\u003eCDH11\u003c/em\u003e gene in beef cattle growth and development, coupled with limited knowledge regarding its involvement in the transcriptional regulation of skeletal muscle growth and development, this study was aimed to comprehensively investigate the structural composition of the proximal promoter with the 5'UTR region of the \u003cem\u003eCDH11\u003c/em\u003e gene. We successfully identified both the core promoter region and as two transcription factors closely associated with skeletal muscle growth and development. Furthermore, we conducted comprehensive analyses to elucidate these two transcription factors intricate roles in controlling \u003cem\u003eCDH11\u003c/em\u003e gene transcription. These discoveries provide a theoretical foundation for the future research on the functional significance of the \u003cem\u003eCDH11\u003c/em\u003e gene in beef cattle's muscular growth and development as well as genetic improvement.\u003c/p\u003e"},{"header":"2 Results","content":"\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003e2.1 Sequence structure, homology analysis, and phylogenetic tree construction of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene\u003c/h2\u003e\n \u003cp\u003eThe \u003cem\u003eCDH11\u003c/em\u003e gene sequences of seven different species were selected and compared with those of Bos taurus (XP_005218806.1). The results revealed that the similarity was 100% for Bos mutus (XP_005905649.1), while the similarity with Capra hircus (XP_ 017917568.1) and Ovis aries (XP_014955948.1) were both with 99.87%. Additionally the similarity with Sus scrofa (XP_020949024.1), Homo sapiens (XP_054235326.1), Mus musculus (XP_ 030099132.1), and Gallus gallus (XP_046781520.1) were found to be 99.25%, 98.42%, 97.49% and 90.58% respectively (Fig. 1B). It is evident from these findings that the \u003cem\u003eCDH11\u003c/em\u003e gene exhibits a higher degree of conservation in ruminants compared to non-ruminants.\u003c/p\u003e\n \u003cp\u003eIn comprehensive understand the evolution of the \u003cem\u003eCDH11\u003c/em\u003e gene, we selected Bos taurus (NM_001081624), Bos mutus (XM_005905587.1), Bubalus bubalis (NM_001081624.2), Capra hircus (NM_009866), Ovis aries (CM_001595.2), Camelus dromedarius (NM_011515430), Homo sapiens (NM_001308392.2), Mus musculus (XM_006530624.3), Sus scrofa (NM_001244482.1) and Gallus gallus (NC_006098.5) using the EMGA11 software in conjunction with the amino acid sequences published by UCSC, with their \u003cem\u003eCDH11\u003c/em\u003e amino acid sequences used for phylogenetic tree construction. The results showed that Bos taurus formed a cluster with ruminants including Bubalus bubalis, Bos mutus, Capra hircus, Ovis aries, and Camelus dromedarius followed by a cluster comprising non-ruminants comprising Sus scrofa, Homo sapiens, and Mus musculus in another cluster while Gallus gallus was the most distantly related group (Fig. 1C).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure1\u003c/strong\u003e Structural homology and phylogenetic tree of the \u003cem\u003eCDH11\u003c/em\u003e gene. (A) This section mainly includes the \u003cem\u003eCDH11\u003c/em\u003e gene promoter, 5\u0026apos;UTR, 3\u0026apos;UTR, exons, introns, coding length of CDS region, and number of coding amino acids. (B) A comparison of the degree of amino acid clustering in the \u003cem\u003eCDH11\u003c/em\u003e protein among seven different species. (C) An analysis of the protein-amino acid phylogenetic tree for the \u003cem\u003eCDH11\u003c/em\u003e gene among 10 different species.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003e2.2 Expression assay of bovine \u003cem\u003eCDH11\u003c/em\u003e gene in various tissues\u003c/h2\u003e\n \u003cp\u003eTo investigate the mRNA expression pattern of the \u003cem\u003eCDH11\u003c/em\u003e in various tissues, total RNA was extracted from the heart, liver, spleen, lung, kidney, longissimus dorsi muscle, and subcutaneous fat. Subsequently, the isolated RNA was reverse-transcribed into cDNA. Quantitate real-time polymerase chain reaction (qRT-PCR) analysis revealed that the \u003cem\u003eCDH11\u003c/em\u003e gene exhibited differential expression across different tissues with the lung display the highest expression. Moreover, moderate to high levels of \u003cem\u003eCDH11\u003c/em\u003e gene expression were observed in liver, kidney, longissimus dorsi muscle, and subcutaneous fat which were significantly compared to spleen and heart tissues. Notably, spleen exhibited the lowest level of the \u003cem\u003eCDH11\u003c/em\u003e gene expression (Fig. \u003cspan\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\"\u003e\n \u003ch2\u003e\u003cstrong\u003e2.3 Identification of the core transcriptional regulatory region of the bovine\u003c/strong\u003e \u003cstrong\u003eCDH11\u003c/strong\u003e \u003cstrong\u003egene\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eTo identify the core transcriptional regulatory region of the promoter region of bovine \u003cem\u003eCDH11\u003c/em\u003e gene. We amplified seven segment-by-segment fragments from the 5\u0026apos;UTR proximal promoter region and successfully ligated \u003cem\u003eNheI\u003c/em\u003e and \u003cem\u003eXhoI\u003c/em\u003e restriction enzyme fragments into the pGL3-Basic vector to construct a recombinant double-luciferase reporter plasmid (Figure \u003cspan\u003eS1\u003c/span\u003e). Subsequently, the corresponding recombinant luciferase reporter plasmids were transfected into mouse C2C12 cells to assess luciferase activity. The results showed no significant change in luciferase activity compared to -1855/-429. Notably, luciferase activity was significantly higher in the \u0026minus;\u0026thinsp;129/+55 region compared to the \u0026minus;\u0026thinsp;429/+55 region, indicating elevated promoter activity compared to the pGL3-Basic vector plasmid (Fig. 3A). In conclusion, our results suggest that the core transcriptional regulatory region of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene is located at -129/+55 bp relative to the TSS.\u003c/p\u003e\n \u003cp\u003eAdditionally, in order to investigate the potential impact of methylation on \u003cem\u003eCDH11\u003c/em\u003e gene expression within its promoter region, Methprimer, an online software tool, was utilized to predict the presence of CpG islands. Notably, analysis revealed a lack of CpG islands within the promoter region of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene (Fig. 3B).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eFigure 3\u003c/strong\u003e Identification of the core transcriptional regulatory region of bovine \u003cem\u003eCDH11\u003c/em\u003e gene and prediction of CPG islands. (A) The recombinant plasmid was co-transfected with pGL3-TK into mouse C2C12 cells, and firefly and kidney luciferase activities were measured after 48 h. The results were expressed as the ratio of firefly luciferase activity to kidney luciferase activity. (B) A schematic diagram of the proximal promoter region (-1855/+55) of the 5\u0026apos;UTR of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene is shown. y-axis indicates the percentage of GC and x-axis indicates the position of non-coding segments. TSS (+\u0026thinsp;1) indicates the transcription start site. ** denotes \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01. Error lines represent SD.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\"\u003e\n \u003ch2\u003e2.4 The validation of transcription factors in core regions of transcriptional regulatory\u003c/h2\u003e\n \u003cp\u003eTo elucidate the role of potential trans-acting elements within the core promoter region of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene, this study aimed to predict its transcription factors using Gene Regulation and Jaspar online platforms. As a result, SP1 and GR, two transcription factor binding sites associated with muscle growth and development, were found to be present at the \u0026minus;\u0026thinsp;36/-27 bp and \u0026minus;\u0026thinsp;20/-11 bp sites in the core transcriptional regulatory region of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene (Fig. \u003cspan\u003e4\u003c/span\u003eA). Furthermore, conservation analysis demonstrated that SP1 and GR exhibited remarkable conservation across multiple species (Fig. \u003cspan\u003e4\u003c/span\u003eB). To further investigate their functional significance, this experiment constructed vectors containing mutated core transcriptional regulatory regions of these two binding sites. Interestingly, deletion of the SP1 binding site significantly increased luciferase activity while deletion of the GR binding site led toa significant decrease in luciferase activity (Fig. \u003cspan\u003e4\u003c/span\u003eC).\u003c/p\u003e\n \u003cp\u003eBased on these findings, in the present study, siRNA-GR and siRNA-SP1 (Fig. \u003cspan\u003e4\u003c/span\u003eD, E ), which have better interference efficiency, were co-transfected with the pGL3-129/+55 plasmid, respectively, in bovine adult myoblasts, and their luciferase activities were determined. The results of the dual-luciferase reporter system showed that the luciferase activity of siRNA-GR\u0026thinsp;+\u0026thinsp;pGL3-129/+55 was significantly reduced, whereas that of siRNA-SP1\u0026thinsp;+\u0026thinsp;pGL3-129/+55 was significantly elevated, compared with that of the control (Fig. \u003cspan\u003e4\u003c/span\u003eF, G). It was also observed that knockdown of GR significantly inhibited the expression level of \u003cem\u003eCDH11\u003c/em\u003e, whereas knockdown of SP1 significantly promoted the expression of \u003cem\u003eCDH11\u003c/em\u003e (Fig. \u003cspan\u003e4\u003c/span\u003eH, I).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\"\u003e\n \u003ch2\u003e\u003cstrong\u003e2.5 Electrophoretic Mobility Shift Assays (EMSA)to validate the interaction of GR and SP1 with the promoter\u003c/strong\u003e\u003c/h2\u003e\n \u003cp\u003eTo confirm the binding of transcription factors GR and SP1 to the core transcriptional regulatory region of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene, we performed in vitro EMSA experiments. Biotin-labelled oligonucleotide probes GR and SP1 were synthesized and incubated with nucleoproteins from C2C12 cells. The biotin-labelled probes formed a DNA-protein complex band with nuclear proteins (Fig. \u003cspan\u003e5\u003c/span\u003eA B, lane 2). Addition of the noncompetitive probe had minimal impact on the DNA protein complex bands (Fig. \u003cspan\u003e5\u003c/span\u003eA B, lane 3), while the addition of a competitive probe resulted in attenuation and disappearance of the DNA protein complex bands (Fig. \u003cspan\u003e5\u003c/span\u003eA B, lane 4). Furthermore, inclusion of GR antibody and SP1 antibody individually attenuated the DNA protein complex band and lead to formation of a super shifted band above it (Fig. \u003cspan\u003e5\u003c/span\u003eA B, lane 5). These findings indicate that both transcription factors GR and SP1 bind to the core transcriptional regulatory region of the \u003cem\u003eCDH11\u003c/em\u003e gene.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3 Materials and methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Sample collection\u003c/h2\u003e \u003cp\u003eSamples utilized for the experiment were heart, liver, spleen, lung, kidney, longissimus dorsi muscle and subcutaneous fat samples from three healthy 1-year-old bulls were obtained from the livestock farm of Gansu Agricultural University (Lanzhou, China). Subsequently, these samples were rapidly immersed in liquid nitrogen and subsequently stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Isolation of DNA and the detection of \u003cem\u003eCDH11\u003c/em\u003e gene mRNA expression\u003c/h2\u003e \u003cp\u003eThis genomic DNA from the longissimus dorsi muscle was isolated using the TRlzol method described by Ye et al [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Total RNA was extracted from tissues and cells using AG RNAex Pro Reagent (Accurate, Biotechnology, China). Integrity of the RNA samples was assessed through agarose gel electrophoresis (JUNYI, Beijing, China), while concentration and purity were determined using a Nano Dro8000 spectrophotometer (ND8000-GL, NanoDrop Technologies, Wilmington, NC, USA). Subsequently, cDNA synthesis was performed according to the TransScript One-Step gDNA Removal and cDNA Synthesis Super Mix (AT311, Transgen, Beijing, China).\u003c/p\u003e \u003cp\u003eFluorescent quantitative primers were designed based on the bovine \u003cem\u003eCDH11\u003c/em\u003e gene family published by UCSC (accession number: NM_001081624.2) using Clone Manage software (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Quantification of mRNA expression levels was carried out with the Perfect Start Green qPCR Super Mix kit (AQ601, Transgen, Beijing, China) The reaction mixture consisted of 20 \u0026micro;L total volume containing 0.4 \u0026micro;L each of forward and reverse primers, 10 \u0026micro;L of 2\u0026times; PerfectStart Green qPCR Super Mix, 0.4 \u0026micro;L of Passive Reference Dye (50 x), 2 \u0026micro;L Template and 6.8 \u0026micro;L of Nuclease-free Water. qRT-PCR reaction conditions included pre-denaturation at 94\u0026deg;C for 30 secs; denaturation at 94\u0026deg;C for 5 secs; annealing at 60\u0026deg;C for 30 secs, with a total of 42 cycles. Gene expression level were normalized to Glyceraldehyde-3 phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) expression and calculated using the \u0026minus;\u0026thinsp;2\u003csup\u003eΔΔCt\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.3 Proximal initiation sub-bioinformatics analysis and prediction\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe 5'UTR proximal \u0026minus;\u0026thinsp;1855/+55 bp DNA sequence of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene (GenBank: NM_001081624.2) was obtained from from the UCSC Genome Brower Home database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps:/genome.ucsc.edu\u003c/span\u003e\u003cspan address=\"https://genome.ucsc.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). A neighbor-joining method through Mega 11 (Philadelphia, USA) was emplaoyed to constructed a phylogenetic tree. Uniport (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized toanalyze protein homology of the \u003cem\u003eCDH11\u003c/em\u003e gene across Bos mutus, Capra hircus, Ovis aries, Sus scrofa, Homo sapiens, Mus musculus and Gallus gullus. CpG sites were predicted using Methprimer (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.urogene.org/cgi-bin/methprimer/methprimer.cgi\u003c/span\u003e\u003cspan address=\"http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), while the transcription start sites were predicted using BDGP (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.fruitfly.org/seq-tools/promoter.html\u003c/span\u003e\u003cspan address=\"https://www.fruitfly.org/seq-tools/promoter.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Furthermore, Gene Regulation (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gene-regulation.com\u003c/span\u003e\u003cspan address=\"http://gene-regulation.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) combined with Jaspar (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://jaspar.elixir.no\u003c/span\u003e\u003cspan address=\"https://jaspar.elixir.no\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) predictions were used predict transcription factor binding sites within the core transcriptional regulatory region of the promoter.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.4 Construction of luciferase deletion vector plasmid in the 5\u003c/b\u003e' \u003cb\u003eUTR proximal promoter region of bovine\u003c/b\u003e \u003cb\u003eCDH11\u003c/b\u003e \u003cb\u003egene.\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn this experiment, the purified fragments were digested with \u003cem\u003eNheI\u003c/em\u003e and \u003cem\u003eXhoI\u003c/em\u003e (JN301, JX201, Transgen, Beijing, China) to generate sticky ends. Subsequently, gel extraction was performed using the Easy Pure Quick Gel Extraction Kit (EG101, Transgen, Beijing, China). The resulting fragments were then ligated to the pGL3-Basic plasmid using T4 DNA Ligase (FL101, Transgen, Beijing, China). Finally, transformation into bacteriophage was carried out using Trans1-T1 Phage Resistant Chemically Competent Cells (CD501, Transgen, Beijing, China), followed by plasmid extractions using Endofree MINI Plasmid kit II (DP118, TINGEN, Beijing, China). The resulting plasmids were named pGL-1855/+55 (P1), pGL-1629/+55 (P2), pGL-1329/+55 (P3), pGL-1029/+55 (P4), pGL-729/+55 (P5), pGL-429/+55 (P6), and pGL-129/+55 (P7).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Cell culture, transfection, and luciferase activity analysis\u003c/h2\u003e \u003cp\u003eThe medium consisted of 10% fetal bovine serum (A6904, Invitrogen, Carlsbad, CA, USA) and 4% dual antibiotics (G4003-100ML, Servicebio, Wuhan, China), supplemented with 86% DMEM high glucose medium (C11995500BT, GIBCO, Grand Island, NY, USA). Mouse C2C12 cells (TCM-C720, Starfish Bio, Suzhou, China) were cultured in the above configured medium. Cells were seeded into 24-well plates at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well prior to transfection and then cultured at 37\u0026deg;C in a CO\u003csub\u003e2\u003c/sub\u003e incubator with a concentration of 5% until reaching a cell density of 75%-80%. For detection purposes following co-transfection according to the manufacturer protocol from Invigentech (Invitrogen, Carlsbad, CA, USA), using the TransDetect Dual Luciferase Reporter Gene Assay Kit (FR201, Transgen, Beijing, China), cells were transfected with the target plasmid along with pGL3-TK at an ng ratio of 800:20 for a duration of 48 h. Subsequently, collected cells were passed through through an enzyme labeler (VL0000D0, Thermo Scientific, Waltham, MAUSA). Firefly luciferase and Renilla luciferase activities were measured and promoter activity was determined as firefly luciferase activity/Renilla luciferase activity in three replicate wells per transfection.\u003c/p\u003e \u003cp\u003eIn accordance with this, bovine adult myoblasts were obtained from cells isolated and identified as having better activity by the previous group [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Their medium consisted of 13% FBS, 4% antibiotics and 83% DMEM/F12 medium (C11330500BT, Hyclone, New York, NY, USA). Prior to transfection, cells were seeded into 12-well plates at a density of 1x10\u003csup\u003e6\u003c/sup\u003e cells per well and then cultured in a CO\u003csub\u003e2\u003c/sub\u003e incubator at 37\u0026deg;C at a concentration of 5% until cell densities reached 75%-80% for later RNA interference experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Site-directed mutagenesis\u003c/h2\u003e \u003cp\u003eThe PCR amplification kit was utilized for the amplification, employing custom-designed glucocorticoid receptor (GR) and specificity protein 1 (SP1) sentinel mutation primers based on the Fast MultiSite Mutagenesis System (FM201, Transgen, Beijing, China) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The reaction system comprised of 25 \u0026micro;L of 2\u0026times; TransStart FastPfu Fly PCR SuperMix, 1 \u0026micro;L each of the upstream and downstream primers, 9 \u0026micro;L of Plasmid, and finally 14 \u0026micro;L of Nuclease-free Water. The amplified target sequences were subsequently purified and assembled. The reaction was carried out at 50\u0026deg;C for 15 mins followed by rapid cooling on ice for a few secs. Subsequently, the receptor cells underwent transformation and single clones were selected for sequencing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.7 RNA interference assay\u003c/h2\u003e \u003cp\u003eThe interfering sequences targeting transcription factor GR and Sp1 (siRNA-GR, siRNA-SP1) were custom-synthesized by Suzhou Hongxun Biotechnology Co. (Suzhou, China). The specific details of these interfering sequences are provided (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Electrophoretic mobility shift assay (EMSA)\u003c/h2\u003e \u003cp\u003eTo obtain nuclear protein extracts from mouse C2C12 cells, logarithmic growth phase cells were cultured into T25 culture flasks. The Nuclear Protein Extraction Kit (Active, Motif Corp, Carlsbad, CA, USA) was used for treating the C2C12 cells. Biotin-labelled DNA probes for the 5'UTR proximal core transcriptional regulatory region were designed and synthesized (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In brief, a binding reaction mixture consisting of 2 \u0026micro;L of 5\u0026times; binding buffer, 10 \u0026micro;g of nuclear extract, and 1 \u0026micro;L of poly (dI-dC) was prepared in a volume of 20 \u0026micro;L and incubated on ice for 15 mins. Subsequently, biotin-labeled DNA (200 fmol) was added to the reaction mixture and incubated at room temperature for an additional 20 mins. For competition assays, unlabelled or mutant probes were added to the reaction mixture 15 mins prior to adding the labelled probe. For the super-shift assay, antibodies (10 \u0026micro;g). including anti-GR (TA7647A, Abmart, Shanghai, China) and anti-SP1 (PS02143, Abmart, Shanghai, China) were included in the reaction mixture after pre-incubation at low temperature for 30 mins. Following this step, DNA-protein complexes were separated by non-denaturing polyacrylamide gel electrophoresis using polyacrylamide and a borate -EDTA buffer for one hour duration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Statistical analysis.\u003c/h2\u003e \u003cp\u003eIn this study, the Duncan method was employed for multiple comparisons. Comparisons between the two groups were conducted using a two-tailed test, and significance was assessed using IBM SPSS 26 statistical software. Graphs were generated using GraphPad Prism 9.0 software. The values in this study are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). * denotes \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** denotes \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, in organizational expressions, different letters represent significant differences, same letters represent non-significant differences, n\u0026thinsp;=\u0026thinsp;3 denotes sample size.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe rate of growth in skeletal muscle plays a crucial role in determining the carcass weight of livestock. Elucidating the transcriptional regulation of genes related to skeletal muscle can serve as a theoretical foundation for breeding meat-producing livestock and poultry. GWAS conducted in livestock and poultry have facilitated the identification of numerous genes associated with their growth and development. For instance, the \u003cem\u003eCDH11\u003c/em\u003e gene has been identified as a potential regulator influencing beef cattle growth and development [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study, we observed that \u003cem\u003eCDH11\u003c/em\u003e gene mRNA is highly expressed in the longissimus dorsi muscle compared to cardiac expression, suggesting its potential role in regulating bovine skeletal muscle production.\u003c/p\u003e \u003cp\u003eTo further elucidate the epigenetic regulatory role of the cattle \u003cem\u003eCDH11\u003c/em\u003e gene in muscle growth and development, this study conducted an analysis on the promoter sequence of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene obtained from the UCSC database. Our analysis revealed that the promoter region of bovine \u003cem\u003eCDH11\u003c/em\u003e lacks a traditional TATA box and CpG island structure, which is with previous investigations demonstrating limited presence of TATA boxes in mammalian gene promoters and 50% occurrence of CpG islands either inter or intra-genic ally [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].Therefore, it can be speculated that methylation of promoter region may not affected transcriptional regulation of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene. Furthermore, employing a segment-by-segment deletion approach, we identified \u0026minus;\u0026thinsp;129/+55 bp as the core transcriptional regulatory region of the \u003cem\u003eCDH11\u003c/em\u003e gene expression. In our study, we predicted two transcription factor binding sites (SP1 and GR) in the core transcriptional regulatory regions \u0026minus;\u0026thinsp;36/-27 bp and \u0026minus;\u0026thinsp;20/-11 bp regions that are highly conserved across multiple species and associated with muscle development. To investigate their role further, mutated vectors containing mutated core transcriptional regulatory regions were constructed and luciferase activity was measured. Deletion of SP1 binding site significantly increased luciferase activity while deletion of GR binding site significantly decreased it. Hence, these results suggest that SP1 and GR play a crucial role in mediating the transcriptional active of bovine \u003cem\u003eCDH11\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003eThe GR is ligand-dependent transcription factor belong to the nuclear receptor superfamily, which plays an active role in all stages of muscle strength and development. Previous studies have demonstrated its essentiality in maintaining and establishing a quiescent state during the growth and development phase of satellite cells. Knocking down GR in myosatellite cells has been shown to increase the amount of circulating cells in the proliferative phase, ultimately leading to muscular atrophy [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. During myofibrilla genesis, GR synergistically binds with MyoD at the GC-rich DNA corresponding element region of the promoter of the target gene, resulting in programmed expression of muscle fiber-related genes [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Although MyoD has been identified as a specific transcription factor for skeletal muscle proliferation and differentiation, It may also act through alternative pathways or transcription factors on muscle precursors [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, interference with the GR led to a significant decrease in luciferase activity within the core transcriptional regulatory region of the \u003cem\u003eCDH11\u003c/em\u003e gene as well as a reduction in \u003cem\u003eCDH11\u003c/em\u003e gene expression. Additionally, results from the EMSA assay demonstrated that the GR transcription factor could bind to the promoter region sequence of the \u003cem\u003eCDH11\u003c/em\u003e gene. These findings suggest that GR transcription factors positively regulate the expression of the cattle \u003cem\u003eCDH11\u003c/em\u003e gene in bovine muscle growth and development.\u003c/p\u003e \u003cp\u003eTranscription factor SP1, a member of the Sp/Kruppel superfamily, is an eukaryotic transcription factor that has been identified to directionally regulate mammalian muscle growth and development [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Previous studies have indicated that the SP1 recruits MyoD, MyoG, and MEF-2C elements to specifically bind to GC-box and GT-box progenitors in gene promoters for the regulation of myosatellite cell proliferation and differentiation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, mutation of the SP1 binding site significantly enhanced the transcriptional activity of the core transcriptional regulatory region of the \u003cem\u003eCDH11\u003c/em\u003e gene. Overexpression of the \u003cem\u003eCDH11\u003c/em\u003e gene has been shown to inhibit MyoD and MyoG expression [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Therefore, we hypothesize that the SP1 specifically binds to the promoter region of \u003cem\u003eCDH11\u003c/em\u003e gene and plays a role in regulating bovine myofibroblasts proliferation and differentiation by recruiting MyoD, MyoG, MEF-2C, or itself. Ultimately, this positively impacts bovine myogenesis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Interference with the SP1 transcription factor led to increased luciferase activity in the core regulatory region of \u003cem\u003eCDH11\u003c/em\u003e gene resulting in reduced levels of \u003cem\u003eCDH11\u003c/em\u003e gene expression. EMSA results confirmed that the SP1 can bind to the core regulatory region of the \u003cem\u003eCDH11\u003c/em\u003e gene. In conclusion, our study demonstrates that the transcription factor SP1 plays a crucial role in regulating \u003cem\u003eCDH11\u003c/em\u003e gene expression in bovine skeletal muscle. However, further investigation is required to understand the epigenetic modifications with its promoter as well as its specific transcriptional regulatory mechanisms related to the \u003cem\u003eCDH11\u003c/em\u003e gene function. These findings provide insights into understanding how bovine \u003cem\u003eCDH11\u003c/em\u003e gene is regulated at a transcriptional level and its biological function.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eIn conclusion, this study establishes that the \u003cem\u003eCDH11\u003c/em\u003e gene is highly expressed in bovine longissimus dorsi muscle and identifies its core promoter region within \u0026minus;\u0026thinsp;129/+55 bp. Our results suggest that \u003cem\u003eCDH11\u003c/em\u003e is positively regulated by GR and negatively regulated by SP1 in the regulation of muscle growth and development. These findings enhance our understanding of the transcriptional regulation mechanisms governing \u003cem\u003eCDH11\u003c/em\u003e gene and suggest potential molecular breeding strategies for improving beef cattle yield and quality.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, ZXL and ZDZ; Methodology, YBB, ZCC, YMN, XJ and LL, JQW, XL, JH; Validation, ZXL, YBB, ZCC; Formal analysis, ZXL, ZCC, YMN. and YBB; Investigation, ZXL, YBB, ZCC, YMN, XJ, LL; Resources, ZDZ and JH; Writing\u0026mdash;original manuscript preparation, ZXL; Writing\u0026mdash;review and editing, ZXL, ZDZ and YBB, Supervision, JQW, BGS, FFZ, and ZDZ; Project Management, XLZ, SBL, XL, BGS, and ZDZ; Funding Acquisition, JH and ZDZ. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was supported by the integration and demonstration of key technologies for the healthy and efficient breeding of beef cattle in Linxia（KJJC-LX-2023-3-01）; the discipline Team Project of Gansu Agricultural University (GAU-XKTD-2022-22); the Efficient Production Technology of Yak in Qilian Mountains Pastoral Areas and Livestock Recycling Development and Demonstration (2022CYZC-43).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u0026nbsp;\u003c/strong\u003eThe animal study was reviewed and approved by the Ethical Commission of Gansu Agricultural University, as well as the Ministry of Science and Technology of the People\u0026rsquo;s Republic of China (ethical permit number: GSAU-ETH-AST-2021-025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The dataset supporting the conclusions of this article is available via email to the corresponding author.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank all the members of the Gansu Key Laboratory of Herbivorous Animal Biotechnology of the College of Animal Science and Technology, Gansu Agricultural University, who contributed their efforts to these experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHu Z et al. 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Mol Cell Biochem, 2016. 414(1\u0026ndash;2).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"bovine, CDH11 gene, transcriptional regulation, skeletal muscle","lastPublishedDoi":"10.21203/rs.3.rs-5023144/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5023144/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGrowth rate of bovine skeletal muscle has a major impact on beef yield. Cadherin-11 (\u003cem\u003eCDH11\u003c/em\u003e) was found to be a potential candidate gene for growth and development in beef cattle. This study confirmed the high expression of \u003cem\u003eCDH11\u003c/em\u003e in bovine longissimus dorsi muscle by tissue expression analysis. To understand the transcriptional regulation mechanism of \u003cem\u003eCDH11\u003c/em\u003e gene, we constructed a double luciferase vector for the promoter region of \u003cem\u003eCDH11\u003c/em\u003e gene and determined that the core transcriptional regulatory region was located at -129/+55 bp relative to the transcription start site (TSS). In addition, we confirmed that skeletal muscle growth and development-related transcription factor-specific protein 1 (SP1) and glucocorticoid receptor (GR) bind to the \u003cem\u003eCDH11\u003c/em\u003e gene promoter region at -36/-27 bp and \u0026minus;\u0026thinsp;20/-11 bp, respectively, to regulate \u003cem\u003eCDH11\u003c/em\u003e expression. These interactions provide valuable information for understanding the mechanism of transcriptional regulation of the bovine \u003cem\u003eCDH11\u003c/em\u003e gene during muscle growth and development.\u003c/p\u003e","manuscriptTitle":"Cloning and transcriptional activity analysis of the bovine CDH11 gene promoter: transcription factors Sp1 and GR regulate bovine CDH11 expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-08 13:41:47","doi":"10.21203/rs.3.rs-5023144/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7a472c97-0767-4710-8392-3f72134eeb7c","owner":[],"postedDate":"October 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-14T14:53:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-08 13:41:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5023144","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5023144","identity":"rs-5023144","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}