肌肉重量和质量对养猪业经济效益至关重要。骨骼肌是猪肉的主要来源，鉴定骨骼肌生长发育的关键调控因子与调控机理能够为猪产肉性状的精准遗传改良提供理论依据与潜在靶点。哺乳动物骨骼肌来源于骨骼肌中胚层间充质干细胞 (Mesenchymal Stem Cells, MSCs)，哺乳动物出生后，肌纤维数量不再增加，后续肌肉量的增加主要依赖肌纤维的肥大。当骨骼肌受损时，位于肌纤维肌浆膜和基底膜之间的肌卫星细胞 (Muscle Satellite Cells, MuSCs) 被激活，启动自我更新程序，在经历增殖、分化和融合后形成新的肌纤维，最终修复受损的肌肉组织。肌肉生长发育是一个多阶段的复杂过程，涉及细胞周期调控、肌肉特异性基因转录程序启动、细胞融合等多个关键生物学事件，这一复杂的调控过程是多种调控因子和信号通路相互作用的结果。TG结合因子1 (TG-interacting factor 1, Tgif1) 是三氨基酸环延伸家族的成员，其分子结构特征包含碱性螺旋环螺旋 (basic Helix-Loop-Helix, bHLH) 结构域。在鸽子与猪骨骼肌的研究中发现，Tgif1是影响骨骼肌发育和生长的候选基因。而课题组前期单细胞转录组测序分析发现，Tgif1在猪MuSCs中高表达。所以，我们推测Tgif1是一个影响肌肉生长发育的关键候选因子，但其在调控肌生成过程的具体分子机理仍有待研究。因此，本研究以猪MuSCs和小鼠稳定成肌细胞系 (C2C12) 为模型，研究Tgif1基因在肌生成过程中的作用和分子调控机理。

本研究首先通过公共数据库分析了Tgif1基因在猪不同组织和细胞中的表达模式；然后利用CRISPR/Cas9技术在猪MuSCs中沉默Tgif1基因，并利用免疫荧光 (Immunofluorescence, IF)、蛋白质印迹 (Western blot, WB)、荧光定量PCR (Real Time quantitative Polymerase Chain Reaction, RT-qPCR)、细胞计数试剂盒-8 (Cell Counting Kit-8, CCK-8) 和5’-乙炔基-2’-脱氧尿苷 (5-Ethynyl-2'-deoxyuridine, EdU) 标记细胞增殖检测法，系统评价了Tgif1基因沉默对猪MuSCs增殖与分化能力的影响；进一步利用RNA-seq技术分析了Tgif1基因沉默对猪MuSCs基因表达变化的影响，根据基因表达变化发现了一系列Tgif1基因的潜在下游靶基因。为了深入研究Tgif1基因在肌生成中的具体调控机理，我们以C2C12细胞为模型，利用多种分子生物学技术开展了分子机理研究。首先通过数据库分析和RT-qPCR检测了Tgif1基因在小鼠不同组织和细胞中的表达模式；随后，分别利用慢病毒载体系统和CRISPR/Cas9技术建立了Dox诱导过表达Tgif1基因和Tgif1基因敲除的稳定细胞系，利用IF、WB、RT-qPCR、CCK-8和EdU等方法检测了Tgif1基因对成肌细胞增殖与分化能力的影响；进一步，基于CUT&Tag和RNA-seq的联合分析，鉴定了Tgif1直接作用的下游靶基因，胆碱能受体烟碱α1亚单位 (Cholinergic receptor nicotinic alpha 1 subunit, Chrna1) 基因，利用siRNA干扰技术在成肌细胞中敲低Chrna1基因，并利用IF、WB、RT-qPCR和EdU等方法系统评价Chrna1基因对成肌细胞增殖与分化能力的影响；最后，利用WB和RT-qPCR检测了Tgif1基因在成肌细胞分化过程中的表达模式，并利用流式细胞仪分析了Tgif1基因对细胞周期的影响。本研究的主要结果如下：

1. Tgif1是猪MuSCs和小鼠成肌细胞分化的正调控因子

表达模式分析结果显示，Tgif1基因在猪和小鼠肌肉组织，猪MuSCs和小鼠成肌细胞中均高表达，提示Tgif1基因参与肌生成这一过程。在Tgif1基因沉默的猪MuSCs、Tgif1基因超表达和Tgif1基因敲除的小鼠成肌细胞系开展的细胞分化能力评价试验中发现，超表达Tgif1基因可以显著提升小鼠成肌细胞的分化能力；沉默或敲除Tgif1基因可以显著抑制猪MuSCs和小鼠成肌细胞的肌管生成。

2. CUT&Tag和RNA-seq联合分析鉴定Tgif1基因的下游靶基因

Tgif1基因沉默的猪MuSCs (sTG-KO) 与对照组 (sCrtl) 转录组分析结果显示，Chrna1、肌源性分化因子1 (Myoblast determination protein 1, Myod1)、早期生长反应基因1 (Early growth response 1, Egr1) 和双特异性磷酸酶1 (Dual specificity phosphatases 1, Dusp1) 等基因差异表达，京都基因与基因组百科全书 (Kyoto Encyclopedia of Genes and Genomes, KEGG) 和基因本体分析 (Gene Ontology Analysis, GO) 分析表明差异基因主要富集在cAMP信号通路、Pi3k-Akt信号通路和钙信号通路等肌肉分化相关过程中。

Tgif1基因敲除的小鼠成肌细胞 (mTG-KO) 与对照组 (mCrtl) 在分化阶段和增殖阶段的转录组分析结果显示，差异表达基因 (Differentially Expressed Genes, DEGs) 主要富集在钙信号通路、Rap1信号通路、MAPK信号通路、Pi3k-Akt信号通路等肌肉分化相关过程中。Tgif1抗体在小鼠成肌细胞中的CUT&Tag富集结果显示，Tgif1主要结合下游靶基因的转录起始位点 (Transcription Start Site, TSS) 区域，且靶基因主要富集在Wnt信号通路、mTor信号通路、AMPK信号通路、MAPK信号通路、Notch信号通路中。RNA-seq和CUT&Tag联合分析筛选到细胞周期蛋白依赖性激酶1 (Cyclin-dependent kinase 1，Cdk1)、信号转导和转录激活因子3 (Signal Transducer and Activator of Transcription 3, Stat3)、Dusp1、Egr1和Chrna1等潜在靶基因。其中，Chrna1基因是在增殖和分化阶段都被Tgif1直接结合其TSS区的靶基因，并且也是猪MuSCs中Tgif1基因沉默后的差异表达基因。而先前有研究报道，Chrna1基因参与肌肉分化的过程。因此，本研究将Chrna1基因作为Tgif1基因的下游关键靶基因进行下一步验证。

3. Tgif1-Chrna1基因轴促进小鼠成肌细胞分化的调控机理

在小鼠成肌细胞中的试验结果显示，Tgif1基因超表达后，ChIP富集到更多Chrna1基因TSS位点附近的序列。启动子活性分析试验表明，Chrna1基因启动子驱动的荧光素酶活性更高。细胞分化能力评价试验结果显示，Chrna1基因敲低显著抑制了小鼠成肌细胞的分化能力，表明了Tgif1可靶定Chrna1基因的TSS区域进而促进肌管生成。增殖能力评价试验结果显示，Tgif1和Chrna1基因对小鼠成肌细胞的增殖能力并没有显著影响。另外，基因表达模式检测发现，Tgif1和Chrna1基因在小鼠成肌细胞分化的早期阶段，尤其是肌管形成时具有相对高丰度的表达。细胞周期试验发现，在分化24 h时，超表达Tgif1基因可增强G1期细胞的积累，而敲除Tgif1基因会显著增加S期细胞，从而影响细胞周期阻滞。试验表明，敲除Tgif1基因可造成小鼠成肌细胞无法进入分化程序，从而降低成肌细胞分化能力。

综上所述，Tgif1在骨骼肌中高度表达，是一个影响肌细胞分化的关键调节因子。Tgif1基因可增强G1期细胞的积累，促进细胞退出细胞周期，进而调控成肌细胞分化启动，最终促进成肌细胞分化。Chrna1基因是Tgif1基因的关键下游靶标，本研究阐明了Tgif1靶向Chrna1基因TSS区域促进肌细胞分化的作用机理。本研究不仅加深了人们对肌肉生长发育的理解，而且为肌肉相关疾病治疗提供了潜在的靶点。

Muscle weight and quality are critical to the economic efficiency of pig farming. Skeletal muscle is the main source of pork, and the identification of key regulators and mechanisms of skeletal muscle growth and development can provide a theoretical basis and potential targets for precise genetic improvement of pork production traits. Mammalian skeletal muscle is derived from Mesenchymal Stem Cells (MSCs). After birth, the number of myofibers does not increase, and the subsequent increase in muscle mass depends on the hypertrophy of myofibers. When skeletal muscle is damaged, Muscle Satellite Cells (MuSCs) located between the plasma membrane and the basement membrane of myofibers are activated, initiating a self-renewal program that undergoes proliferation, differentiation, and fusion to form new muscle fibers, and ultimately repairing the damaged muscle tissue. Muscle growth and development is a multi-stage and complex process involving multiple key biological events such as cell cycle regulation, initiation of muscle-specific gene transcription programs, and cell fusion. This complex regulatory process is the result of the interaction of multiple regulatory factors and signaling pathways. TG-interacting factor 1 (Tgif1) is a member of a family of three amino acid loop extensions whose molecular structure is characterized by a basic Helix-Loop-Helix (bHLH) domain. Previous studies in pigeon and pig skeletal muscle revealed that Tgif1 is a candidate gene that affects skeletal muscle development and growth. And the group's previous single-cell transcriptome sequencing analysis found that Tgif1 was highly expressed in pig MuSCs. Therefore, we hypothesized that Tgif1 is a key candidate factor affecting muscle growth and development, but its specific molecular mechanism in myogenesis remains to be investigated. Therefore, in this study, we investigated the role and potential molecular regulatory mechanisms of Tgif1 gene in the myogenesis using pig MuSCs and mouse stable myoblast cell line (C2C12) as models.

In this study, we first analyzed the expression pattern of Tgif1 gene in different tissues and cells of pigs through public databases; then we silenced the Tgif1 gene in pig MuSCs using CRISPR/Cas9 technology, and systematically evaluated the Tgif1 gene silencing on the proliferation and differentiation ability of pig MuSCs using Immunofluorescence (IF), Western blot (WB), Real Time quantitative Polymerase Chain Reaction (RT-qPCR), Cell Counting Kit-8 (CCK-8) and 5'-ethynyl-2'-deoxyuridine (EdU)-labeled cell proliferation assay. We further analyzed the effects of Tgif1 gene silencing on gene expression changes in pig MuSCs using RNA-seq technology, and identified a series of potential downstream target genes of Tgif1 gene based on gene expression changes. In order to deeply investigate the specific regulatory mechanism of Tgif1 gene in myogenesis, we carried out molecular mechanistic studies using various molecular biology techniques with mouse stable myogenic cell line (C2C12) as a model. Firstly, we detected the expression pattern of Tgif1 gene in different tissues and cells of mice by database analysis and RT-qPCR; subsequently, Dox-induced overexpression of Tgif1 gene and Tgif1 gene knockout stable cell lines were established by using the lentiviral vector system and CRISPR/Cas9 technology, respectively. The effects of Tgif1 gene on the proliferation and differentiation ability of myoblasts were detected using IF, WB, RT-qPCR, CCK-8, and EdU. Further, based on the combined analysis of CUT&Tag and RNA-seq, the downstream target gene of Tgif1 direct action, cholinergic receptor nicotinic alpha 1 subunit (Chrna1), was identified. Knockdown of Chrna1 gene in C2C12 cells using siRNA interference and evaluation of the effect of Chrna1 gene on proliferation and differentiation ability using IF, WB, RT-qPCR, and EdU. Finally, the expression pattern of Tgif1 gene during differentiation of myoblasts was detected by WB and RT-qPCR, and the effect of Tgif1 gene on cell cycle was analyzed by flow cytometry. The main results of this study are as follows:

1. Tgif1 is a positive regulator of differentiation of pig MuSCs and mouse myoblasts

Expression pattern analysis showed that Tgif1 gene was highly expressed in both pig and mouse muscle tissues, pig MuSCs and mouse myoblasts, suggesting that Tgif1 gene is involved in the process of myogenesis. In the cell differentiation evaluation of Tgif1-silenced pig MuSCs, Tgif1 gene overexpression and Tgif1 gene knockout mouse myoblast cell lines, it was found that overexpression of Tgif1 gene significantly enhanced the differentiation ability of mouse myoblasts, and that silencing or knocking out of Tgif1 gene significantly inhibited myotubular myogenesis in both pig MuSCs and mouse myoblasts.

2. Combined CUT&Tag and RNA-seq analysis to identify downstream target genes of Tgif1 gene

Transcriptome analysis of Tgif1-silenced pig MuSCs (sTG-KO) and controls (sCrtl) showed that Chrna1, myogenic differentiation factor 1 (Myod1), early growth response 1 (Egr1), and dual specificity phosphatases 1 (Dusp1) were differentially expressed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology Analysis (GO) analyses showed that the differentially expressed genes were mainly enriched in the cAMP signaling pathway, Pi3k-Akt signaling pathway, and calcium signaling pathway, which are related to muscle differentiation.

Transcriptome analysis of Tgif1-knockout C2C12 cells (mTG-KO) and control cells (mCrtl) at differentiation and proliferation stages showed that Differentially Expressed Genes (DEGs) were mainly enriched in the calcium signaling pathway, the Rap1 signaling pathway, MAPK signaling pathway, and the Pi3k−Akt signaling pathway. The CUT&Tag enrichment of Tgif1 antibody in mouse myoblasts showed that Tgif1 mainly binds to the Transcription Start Site (TSS) region of the downstream target genes, and the target genes were mainly enriched in Wnt signaling, mTor signaling, AMPK signaling, MAPK signaling, and Notch signaling pathways. RNA-seq and CUT&Tag combined analysis screened for Cyclin-dependent kinase 1 (Cdk1), Dusp1, Egr1, Signal Transducer and Activator of Transcription 3 (Stat3) and Chrna1 are potential target genes. Among them, the Chrna1 geneis a target gene that is directly bound to its TSS region by Tgif1 at both the proliferation and differentiation stages, and is also a DEG after silencing Tgif1 gene in pig MuSCs. Therefore, the Chrna1 gene was next validated as a key target gene downstream of Tgif1 in this study.

3. Mechanisms by which the Tgif1-Chrna1 gene axis promotes differentiation of mouse myoblasts

Validation assays in mouse myoblasts showed that ChIP was enriched to more sequences near the TSS site of the Chrna1 gene after Tgif1 gene overexpression. Promoter activity analysis assay showed higher luciferase activity driven by Chrna1 gene promoter. The results of the cell differentiation ability evaluation assay showed that Chrna1 knockdown significantly inhibited the differentiation ability of mouse myoblasts, suggesting that Tgif1 can target the TSS region of the Chrna1 gene and thus promote myotube generation. The results of proliferative capacity evaluation assay showed that Tgif1 and Chrna1 genes did not significantly affect the proliferative capacity of mouse myoblasts. In addition, gene expression patterns revealed that Tgif1 and Chrna1 genes were expressed in high relative abundance at the early stage of myoblast differentiation, especially during myotube formation. Cell cycle assay revealed that overexpression of Tgif1 gene enhanced the accumulation of G1-phase cells at 24 h of differentiation, whereas knockout of Tgif1 gene significantly increased the S-phase cells, which affected the cell cycle block and might result in the inability of mouse myoblasts to enter the differentiation program, thus reducing the differentiation capacity of myoblasts.

In summary, Tgif1 is highly expressed in skeletal muscle and is a key regulator affecting myoblast differentiation. Tgif1 gene enhances the accumulation of G1-phase cells and promotes cell exit from the cell cycle, which in turn regulates the initiation of myoblast differentiation and ultimately promotes myoblast differentiation. Chrna1 gene is a key downstream target of Tgif1 gene, and this study elucidated the reciprocal mechanism by which Tgif1 targets the TSS region of Chrna1 gene to promote myoblast differentiation. This study not only deepens the understanding of muscle growth and development, but also provides potential targets for the treatment of muscle-related diseases.

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