Abstract
Background
Skeletal muscle is comprised of heterogeneous myofibers that differ in their physiological and metabolic parameters. Of these, slow-twitch (type I; oxidative) myofibers have more myoglobin, more mitochondria, and higher activity of oxidative metabolic enzymes compared to fast-twitch (type II; glycolytic) myofibers.
Methods
In our previous study, we found a novel LncRNA-TBP (for “LncRNA directly binds TBP transcription factor”) is specifically enriched in the soleus (which has a higher proportion of slow myofibers). The primary myoblast cells and animal model were used to assess the biological function of the LncRNA-TBP in vitro or in vivo. Meanwhile, we performed a RNA immunoprecipitation (RIP) and pull-down analysis to validate this interaction between LncRNA-TBP and TBP.
Results
Functional studies demonstrated that LncRNA-TBP inhibits myoblast proliferation but promotes myogenic differentiation in vitro. In vivo, LncRNA-TBP reduces fat deposition, activating slow-twitch muscle phenotype and inducing muscle hypertrophy. Mechanistically, LncRNA-TBP acts as a regulatory RNA that directly interacts with TBP protein to regulate the transcriptional activity of TBP-target genes (such as KLF4, GPI, TNNI2, and CDKN1A).
Conclusion
Our findings present a novel model about the regulation of LncRNA-TBP, which can regulate the transcriptional activity of TBP-target genes by recruiting TBP protein, thus modulating myogenesis progression and inducing slow-twitch fibers.
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Background
Skeletal muscles constitute approximately 35% of the body weight and main resources of animal protein for human consumption. Abnormal regulation of skeletal muscle‐specific genes leads to various muscle diseases that development directly influences animal meat quantity and quality [1,2,3]. Myogenesis is a highly ordered process during which muscle stem cell proliferation, migration, differentiation, and fusion are activated to form myofibers [4].
Skeletal muscle is composed of different types of myofibers, which are mainly differentiated by the expression of heavy chain myosin and the reliance on oxidative phosphorylation [5]. Compared to fast-twitch (type II; glycolytic) myofibers, slow-twitch (type I; oxidative) myofibers have more myoglobin, more mitochondria, and higher activity of oxidative metabolic enzymes [6, 7]. Under certain conditions, fast-twitch myofibers and slow-twitch myofibers can transform into each other [8].
Transcriptional activation is a major step in the regulation of gene expression during development. TATA-binding protein (TBP) and 12–15 associated factors (TAFs) together form the pre-initiation complex (PIC), which as an obligate step leading to transcription [9, 10]. In mice, a large polyQ repeat in TBP causes primary muscle degeneration and decreases the association of MyoD with TBP and DNA promoters [11]. Recent studies have demonstrated that the TBP associated factor (TAF9b) plays an important role in neurodevelopmental and mental disorders. As a coactivator that stabilized the structure of P53, it also participates in P53-mediated apoptosis and cell cycle regulation [12,13,14,15].
Long noncoding RNAs (lncRNAs), a novel class of regulatory RNAs, are commonly defined as transcribed RNAs with sizes ranging from 200 bp to > 100 kb and not translated to protein [16]. Substantial evidences have shown that lncRNAs play critical regulatory roles in diverse biological processes and diseases, such as skeletal muscle development and muscle disorders [17, 18]. Based on our previous RNA-sequencing (RNA-seq) analysis, we screened and identified an lncRNA (MSTRG.6038.1), which differentially expressed between pectoralis major (PEM, which is mainly composed of fast-twitch fibers) and soleus (SOL, which has a higher proportion of slow muscle fibers) in 7-week-old Xinghua chicken, was named “LncRNA-TBP” (for “LncRNA directly binds TBP transcription factor”). Functional studies demonstrated that LncRNA-TBP inhibits myoblast proliferation but promotes myogenic differentiation as well as reduces fat deposition, activating slow-twitch muscle phenotype and reducing muscle atrophy. Mechanistically, LncRNA-TBP acts as a regulatory RNA that directly interacts with TBP protein to regulate the transcriptional activity of TBP-target genes (such as KLF4, GPI, TNNI2, and CDKN1A).
Materials and methods
Ethics statement
The experimental animals were chickens of Chinese local breeds. All animal studies were sanctioned by the Institutional Animal Care and Use Committee at the South China Agricultural University. All the experiments were performed according to the regulations and guidelines established by the committee and international standards for animal welfare (approval ID: SCAU#2021c008). We made every effort to reduce the suffering of animals.
Experimental animals and tissues
Four 7-week-old Xinghua female chickens were received from the Zhicheng Poultry Breeding Co., Ltd. (Guangdong, China). The tissues (including the cerebrum, cerebellum, hypothalamus, heart, liver, spleen, lung, kidney, muscular stomach, glandular stomach, breast muscle, and leg muscle) were collected, quickly frozen into liquid nitrogen, and then stored at − 80 °C.
Cell culture and transfection
CPMs were isolated from E11 chicken leg muscles as previously described [19] and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, USA) with 20% fetal bovine serum (Gibco). All transient transfections were performed using Lipofectamine 3000 reagent (Invitrogen, USA) according to the manufacturer’s instructions.
Lentivirus assay
Chinese native breeds (XH chickens) were used for the in vivo experiment in this study. For the construction of animal models of LncRNA-TBP overexpression and knockdown.
1-day-old chicks were randomly divided into two groups (Lv-LncRNA-TBP and Lv-NC; n = 30), respectively. Chicks received two intramuscular doses of lentivirus (106 titers) in two different sites of the gastrocnemius. Thirteen days after the initial injection, chick gastrocnemius samples were collected from the above two groups.
7-days-old chickens were randomly divided into two groups (Chol-ASO-LncRNA-TBP and Chol-ASO-NC; n = 15) respectively. Chicks received two intramuscular doses of modified ASOs (40 nmol) by intramuscular injection on days 14 and 18. The chickens were euthanized at 21 days old, and the gastrocnemius muscles were detached and stored at − 80 °C.
Rapid-amplification of cDNA ends (RACE)
The full-length of LncRNA-TBP was amplified by using a SMARTer RACE cDNA Amplification Kit (Clontech, Japan), following the manufacturer’s instructions. The primer pairs used in RACE are listed in Additional file 10: Table S4.
RNA isolation, complementary DNA (cDNA) synthesis, and real-time (RT) PCR analysis
Total RNA was extracted from tissues or cells using RNAiso plus reagent (TaKaRa, Japan). cDNA synthesis was obtained by using a PrimeScript RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Japan). Real-time quantitative PCR (qRT-PCR) reactions were performed on a QuantStudio 5 Real-Time PCR Systems (Thermo Fisher, Waltham, MA, USA) by using an ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). All primers for RT-PCR and real-time qPCR are listed in Additional file 10: Table S4.
Plasmid construction and RNA oligonucleotides
For Flag fusion protein construction, twelve ORFs of LncRNA-TBP were amplified and cloned into pcDNA3.1-3xFlag (SiDanSai, Shanghai, China), and pcDNA3.1-3xFlag-β-actin was used as a positive control.
For overexpression vectors construction, the full-length sequence of LncRNA-TBP was amplified and cloned into reconstructive pcDNA3.1 vector (Promega, Madison, WI, USA), which with GFP tag at the C-terminus.
For viral vectors constructed, the full-length sequence of LncRNA-TBP was amplified and then cloned into the lentiviral vector (pLVX-mCMV-ZsGreen-IRES-Puro; Addgene, Cambridge, MA, USA).
The antisense oligonucleotide (ASO) with Cy3-modified that was used for the specific knockdown of LncRNA-TBP was designed and synthesized by Guangzhou RiboBio (Guangzhou, China). The siRNA against TBP (NCBI Reference Sequence: NM_205103.1) was also designed and synthesized.
The primers and oligonucleotide sequences used in this study are shown in Additional file 10: Table S4 and S5.
Flow cytometry, 5-ethynyl-2’-deoxyuridine (EdU) and cell counting kit-8 (CCK-8) assays
The Cell Cycle Analysis Kit (Thermo Fisher Scientific, USA), C10310 EdU Apollo In Vitro Imaging Kit (RiboBio, China) and TransDetect Cell Counting Kit (TransGen, Beijing, China) were used for flow cytometry, EdU, and CCK-8 assay, as the manufacturer’s protocol.
Immunofluorescence, immunohistochemistry and hematoxylin and eosin staining
Immunofluorescence was performed using anti-MyHC (B103; DHSB, USA; 2.5 mg/mL), and images were captured using a fluorescence microscope (DMi8; Leica, Germany). The area of cells labeled with anti-MyHC was measured and calculated as previously described [20].
Immunohistochemistry was carried out using an SP-POD Kit (SP0041; Solarbio, China) with primary antibodies including anti-MYH1A (F59, DSHB, 1:100) and anti-MYH7B (S58, DSHB, 1:300). The number of myofibers labeled with anti-MYH1A or anti-MYH7B was calculated.
Hematoxylin and eosin (H&E) staining was performed using muscle tissues embedded in paraffin and cut into 4-mm-thick transverse sections. Subsequently, the sections were stained with H&E.
Mitochondrial DNA (mtDNA) content and fatty acid oxidation (FAO) rate assay
Total DNA was extracted using the Tissue DNA Kit (D3396, Omega, GA, USA) according to the manufacturer’s instructions. The amount of mitochondrial DNA was determined by quantification of cytochrome c oxidase subunit II (COX2). The nuclear-encoded β-globin gene was used as an internal control. Primers used in this study can be found in Additional file 10: Table S4.
The mitochondria of the myoblast and gastrocnemius were isolated using the Cell/Tissue Mitochondria Isolation Kit (C3601/C3606, Beyotime, China). After measuring the mitochondrial protein concentration, freshly isolated mitochondria were subjected to FAO rate assay with the Colorimetric Fatty Acid Oxidation Rate Assay Kit (HL50679, Haling, Shanghai, China), according to the manufacturer’s protocol.
Metabolite and enzyme activities assays
Content of TG, FFA, and glycogen as well as enzyme activity of LDH and SDH in skeletal muscle were measured using commercially available kits (BC0625, BC0595, BC0345, BC0685, BC0955, respectively; Solarbio, China), according to the manufacturer’s instructions.
Central carbon metabolic profiling
LncRNA-TBP overexpression gastrocnemius samples (n = 6) were used for metabolites extraction and then performed on HPIC-MS/MS analysis. The high-performance ion-exchange liquid chromatography (HPIC) separation was carried out using a Thermo Scientific Dionex ICS-6000 HPIC System (Thermo Fisher Scientific, IL, USA). An AB SCIEX 6500 QTRAP+ triple quadrupole mass spectrometer (AB Sciex, USA), equipped with electrospray ionization (ESI) interface, was applied for assay development.
Metabolic hierarchical clustering analysis (HCA) was performed using Cluster3.0 software as previously described [21].
Western blot analysis
Western blot analysis was performed as previously described [44]. The primary antibodies used were anti-FLAG (AF519, Beyotime,1:1,000), anti-MYOD (ABP53067, Abbkine, 1:500), anti-MyHC (B103, DHSB, 0.5 mg/mL), anti-FASN (10624-2-AP, Proteintech, 1:200), anti-CPT1 (bs-23779R, Bioss, 1:500), anti-ULK1 (bs-3602R, Bioss,1:500), anti-LC3B (NB100-2220, Novus, 2.0 mg/mL), anti-P62 (18420-1-AP, Proteintech, 1:1,000), anti-TBP (44059, Cell signaling, 1:1000), anti-KLF4 (bs-1064R, Bioss,1:500), anti-GPI (GTX113203, GeneTex, 1:500), anti-TNNI2 (bs-10617R, Bioss, 1:500), anti-CDKN1A (GTX112898, GeneTex, 1:500), and anti-GAPDH (60004-1-Ig, Proteintech, 1:5,000). ProteinFind goat anti-mouse IgG(H + L), HRP conjugate (HS201-01, TransGen, 1:1,000) and ProteinFind goat anti-rabbit IgG(H + L), HRP conjugate (HS101-01, TransGen, 1:500) were used as secondary antibodies. The original images of western blot are shown in Additional file 1.
RNA pull-down assay and RIP assay
Biotinylated RNAs were harvested by using a Ribo RNAmax-T7 biotin-labeled transcription kit (RiboBio, China). A Pierce Magnetic RNA–Protein Pull-Down Kit (Thermo Fisher Scientific) was used in RNA–protein pull-down experiments according to the manufacturer’s instructions. The eluted products were identified by mass spectrometry with a Q Exactive mass spectrometer (Thermo Fisher) or western blot.
Immunoprecipitation Kit (Millipore, USA) according to the manufacturer’s instructions. The antibody used for RIP assays was anti-TBP (44059, Cell Signaling, 1:100).
Chromatin immunoprecipitation (CHIP)
TBP ChIP was performed with ChIP Kit (Millipore, Bedford, MA) according to the manufacturer’s instructions. Briefly, the ChIPed DNA was eluted, reverse X-linked, purified, and analyzed by qRT-PCR. All primers used in ChIP-qPCR are presented in Additional file 10: Table S4.
Luciferase reporter assay
For luciferase reporter assay, reporter plasmids with the promoter region of KLF4, GPI, TNNI2, and CDKN1A were transfected into CPMs by Lipofectamine 3000 (Invitrogen, USA) in 96-well plates. The luciferase activities were measured 48 h after differentiation by using Dual-Luciferase®Reporter Assay System (Promega, Madison, WI, USA). Firefly activity was normalized to Renilla luciferase activity.
Statistical analysis
In this study, all experiments were repeated at least three times, and results were represented as mean ± SEM. Where applicable, the statistical significance of the data was tested using independent sample t-test or ANOVA followed by Dunnett’s test. The types of tests and the P-values, when applicable, are indicated in the figure legends.
Results
LncRNA-TBP is a novel lncRNA associated with myogenesis
Our previous RNA-seq study found a muscle-related lncRNA (LncRNA-TBP) was highly expressed in SOL (Fig. 1A, B). 5′ and 3′ ends of LncRNA-TBP were identified by RACE analysis (Fig. 1C). The NCBI BLAST indicated that LncRNA-TBP located on Chromosome 3 and spanned from 82341588 to 82342330, and 82369736 to 82370049 with 1057 nt long, relatively conserved in Meleagris gallopavo, Apteryx mantelli mantelli, and Numida meleagris (Additional file 2: Fig. S1 and Additional file 10: Table S1). LncRNA-TBP highly expressed in polyadenylated RNA (Fig. 1D). LncRNA-TBP upregulated during myogenic differentiation, and enriched in leg muscles and breast muscles (Fig. 1E, F), implying that it may play an important role in skeletal muscle development. In addition, cell-fractionation assays demonstrated that LncRNA-TBP is mainly present in the nucleus of chicken primary myoblasts (CPMs) (Fig. 1G). To further prove the coding potential of LncRNA-TBP, we analyzed the twelve potential ORFs of LncRNA-TBP by western blot. The results show that LncRNA-TBP is a lncRNA without protein-encoding potential (Fig. 1H).
LncRNA-TBP inhibits myoblast proliferation and promotes myoblast differentiation
LncRNA-TBP was predominantly expressed in breast muscle and leg muscle (Fig. 1F), implying that LncRNA-TBP plays an important role in myogenesis. To assess the effect of LncRNA-TBP on proliferation and differentiation of myoblast, the overexpression vector and inhibitor of LncRNA-TBP were transfected into CPMs (Fig. 2A and Additional file 3: Fig. S2A). Overexpression of LncRNA-TBP increased the expression of cell cycle-inhibiting genes like CDKN1A and CDKN1B while decreasing the expression level of cell cycle-promoting genes like PCNA. The opposite result was observed with LncRNA-TBP knockdown (Fig. 2B and Additional file 3: Fig. S2B). The 5-ethynyl-2’-deoxyuridine (EdU) staining and cell counting kit-8 (CCK-8) assay demonstrated that LncRNA-TBP overexpression significantly inhibited myoblast proliferation and viability (Fig. 2C–E). Conversely, interference with LncRNA-TBP promoted EdU incorporation and myoblast proliferation (Additional file 3: Fig. S2C–E). At the same time, overexpression of LncRNA-TBP significantly increased the number of G0/G1 cells, and the number of S phase cells was lower than the control group, whereas myoblast division was inhibited with LncRNA-TBP interference (Fig. 2F and Additional file 3: Fig. S2F).
To further investigate the potential function of LncRNA-TBP in myoblast differentiation, immunofluorescence staining was performed after overexpression and inhibition of LncRNA-TBP. The results showed that overexpression of LncRNA-TBP increased the total areas of myotubes, while myotube formation was facilitated (Fig. 2G–I). In contrast, LncRNA-TBP interference suppressed myoblast differentiation (Additional file 3: Fig. S2G–I). Moreover, the expressions level of myoblast differentiation marker genes, including MYOD, MYOG, and MyHC were significantly upregulated with LncRNA-TBP overexpression (Fig. 2J, K). Conversely, LncRNA-TBP interference repressed their expression (Additional file 3: Fig. S2J, K).
LncRNA-TBP accelerates fatty acid oxidation, and enhances TCA cycle flux in skeletal muscle
To verify whether LncRNA-TBP regulates skeletal muscle development in vivo, lentiviral-mediated LncRNA-TBP overexpression (LV-LncRNA-TBP) or cholesterol-modified antisense oligonucleotide (Chol-ASO-LncRNA-TBP) were injected to the gastrocnemius of Xinghua chicken (Fig. 3A and Additional file 4: Fig. S3A). LncRNA-TBP overexpressed increased mitochondrial DNA content, which potentially contributed to the acceleration of fatty acid oxidation (FAO) and inhibited the accumulation of free fatty acid (FFA) and triglyceride (TG) (Fig. 3B–D). In contrast, mitochondrial DNA content and fatty acid β-oxidation were reduced after the LncRNA-TBP knockdown (Additional file 4: Fig. S3B–D). Besides, the qPCR and western blotting analyses showed that knockdown of LncRNA-TBP downregulated FAO-related genes like CPT1 and upregulating key genes involved in fatty acid synthesis (such as FASN), while opposite results were shown with LncRNA-TBP overexpression (Fig. 3E, F and Additional file 4: Fig. S3E, F).
Mitochondria switch between lipid and glucose oxidation through the TCA cycle to generate ATP, which is pivotal for maintaining systemic energy homeostasis [19,20,21,22]. Given that overexpression of LncRNA-TBP promoted the content of mitochondrial DNA (Fig. 3B), we performed a comparative metabolome analysis to study whether LncRNA-TBP functions muscle metabolism. The result of hierarchical clustering analysis (HCA) separated controls and overexpression of LncRNA-TBP (Fig. 3G and Additional file 11: Table S2). For example, compared with control, glycolytic metabolites such as fructose 6-phosphate and glucose 6-phosphate were significantly decreased with LncRNA-TBP overexpression (Fig. 3H and Additional file 11: Table S2). In the meantime, metabolites of the TCA cycle, including malic acid, isocitric acid, and fumaric acid were significantly promoted (Fig. 3H and Additional file 11: Table S2). Altogether, our results indicated that LncRNA-TBP decreases the end products of glycolysis and elevates metabolites of the TCA cycle by promoting mitochondrial function, leading to reduction of lipid accumulation.
LncRNA-TBP activates slow-twitch muscle phenotype and induces muscle hypertrophy
Skeletal muscle development is primarily regulated by fiber type composition and muscle fiber size. The composition of myofiber types is closely related to the way muscles are metabolized [23, 24]. Given that LncRNA-TBP is highly expressed in SOL and mediated the flux of glycolysis and TCA cycle, we further examined whether LncRNA-TBP could affect the conversion of skeletal muscle fiber types in vivo. As expected, the activity of lactate dehydrogenase (LDH) was suppressed, while the activity of succinate dehydrogenase (SDH) was enhanced with LncRNA-TBP overexpression (Fig. 4A). Meanwhile, glycogen content was increased and expression of glycogenolytic and glycolytic genes was downregulated with overexpression of LncRNA-TBP (Fig. 4B, C). The opposite results were shown with the knockdown of LncRNA-TBP (Additional file 5: Fig. S4A-C). The expression levels of fast-twitch myofiber genes like SOX6 and slow-twitch myofiber genes (such as TNNC1, TNNI1 and TNNT1) were further tested. It was found that overexpression of LncRNA-TBP promoted expressions of slow-twitch myofiber genes (Fig. 4D). More importantly, results of immunohistochemistry showed that LncRNA-TBP overexpression promoted the expression level of MYH7B/slow-twitch protein and suppressed the expression level of MYH1A/fast-twitch protein (Fig. 4E, F). On the contrary, LncRNA-TBP knockdown upregulated the fast-twitch protein level and drove the transformation of slow-twitch to fast-twitch myofibers (Additional file 5: Fig. S4D-F).
Recent evidences have revealed that remodeling of skeletal muscle fiber types can affect muscle mass, and induce muscle hypertrophy and muscle atrophy by anabolic and catabolic signaling pathways, respectively [25]. LncRNA-TBP overexpression leads to increased muscle mass and cross-sectional area (CSA), while the opposite result occurred upon LncRNA-TBP knockdown (Fig. 3G and H and Additional file 5: Fig. S4G, H), suggesting that LncRNA-TBP regulates skeletal muscle hypertrophy. Autophagy is a highly conserved homeostatic process carrying out degradation of cytoplasmic components including damaged organelles, toxic protein aggregates, and intracellular pathogen [26]. Maintaining basal autophagy flux is essential to clear damaged organelles or recycle macromolecules in muscles during metabolic stress [27]. To further explore the regulatory mechanism of LncRNA-TBP in inducing muscle hypertrophy, we detected expressions of autophagy-related genes. LncRNA-TBP overexpression upregulated the expression level of SQSTM1, whereas expressions of autophagy-related genes (such as MAP1LC3B, and ULK1) and content of LC3BII were downregulated (Fig. 4I, J). Conversely, LncRNA-TBP knockdown activated autophagy (Additional file 5: Fig. S4I, J), suggesting that LncRNA-TBP may promote muscle hypertrophy by decreasing basal autophagy flux.
LncRNA-TBP directly interacts with TBP
Recent studies have found that many nuclear lncRNAs perform their functions through interaction with proteins [28]. The nuclear localization of LncRNA-TBP suggested that this lncRNA may modulates the transcriptional regulation of target genes. Thus, we attempted to identify the protein partners of LncRNA-TBP. First, the potential LncRNA-TBP-binding proteins were predicted using the RNA–protein interaction prediction (RPISeq), and TBP was found may interact with LncRNA-TBP (Additional file 6: Fig. S5A). To validate this interaction between LncRNA-TBP and TBP, we performed a RNA immunoprecipitation (RIP) analysis in CPMs. As expected, reverse transcription-polymerase chain reaction (RT-PCR) analysis of antibody-enriched RNA revealed that TBP antibody pulled down significantly more LncRNA-TBP than the IgG control (Fig. 5A), suggesting that TBP interacts with LncRNA-TBP. To determine the core protein-binding domain of LncRNA-TBP, we constructed a series of truncated LncRNA-TBP fragments. We found that like full-length LncRNA-TBP, all of the truncated fragments could physically bind TBP (Fig. 5B, C). Collectively, these findings showed that LncRNA-TBP directly interacts with TBP.
LncRNA-TBP regulates transcriptional activity of TBP-target genes by binding to TBP
The general transcription factor (TBP) is a key initiation factor involved in transcription by all three eukaryotic RNA polymerases, is required for every single transcription event in eukaryotes [29,30,31,32]. Through our previous ATAC sequencing analysis, a total of 20 target genes (e.g., glycolysis-related genes (GPI), cell proliferation-related genes (CDKN1A and KLF4) and fast muscle-related genes (TNNI2)) were predicted be regulated by TBP (Additional file 6: Fig. S5B and Additional file 12: f S3). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis found that these TBP-target genes were mainly enriched in biological processes such as cellular process, metabolic process, cellular component organization or biogenesis, and biological regulation, as well as participated in biological processes including metabolic pathways, carbon metabolism and so on (Additional file 6: Fig. S5C, D). By performing ChIP-qPCR, we validated that TBP can bind and regulate the promoter of KLF4, GPI, TNNI2, and CDKN1A (Fig. 5D).
Because LncRNA-TBP specifically interacts with TBP, we investigated whether there is a mutual regulation relationship between LncRNA-TBP and TBP in CPMs. The results showed that LncRNA-TBP knockdown or overexpression did not significantly influence TBP mRNA and protein expression (Additional file 7: Fig. S6A–D). These results suggested that LncRNA-TBP may regulate myogenesis through its interaction with TBP rather than by regulating TBP gene expression. Given that TBP functions in regulate the promoter activity of its target genes, we also performed ChIP-qPCR to elucidate whether LncRNA-TBP affects the capacity of TBP to bind the promoters of its target genes. LncRNA-TBP overexpression significantly increased the enrichment of TBP to the promoter of KLF4, GPI, TNNI2, and CDKN1A, whereas the results were reversed after LncRNA-TBP knockdown (Fig. 5E and Additional file 8: Fig. S7A). Next, to determine whether LncRNA-TBP regulates promoter activity of TBP-target genes such as KLF4, GPI, TNNI2, and CDKN1A, luciferase reporter assays were performed. Overexpression of LncRNA-TBP promoted the promoter activity of KLF4 and CDKN1A while inhibiting the promoter activity of GPI and TNNI2 (Fig. 5F–I). Consistently, the knockdown of LncRNA-TBP had opposite effects in CPMs (Additional file 8: Fig. S7B-E). We further examined the mRNA and protein expressions of KLF4, GPI, TNNI2, and CDKN1A. As expected, LncRNA-TBP would promote the expression of KLF4 and CDKN1A, while decreasing the expression of GPI and TNNI2 (Fig. 5J, K and Additional file 8: Fig. S7F, G), suggesting that LncRNA-TBP can modulate transcriptional activity of TBP-target genes by binding to TBP protein.
TBP is involved in myogenesis
TBP was upregulated during myoblast differentiation (Additional file 9: Fig. S8A), implying that it may play an important role in skeletal muscle development. Moreover, Subcellular location annotation showed that TBP protein exists in the nucleus (Additional file 9: Fig. S8B). To explore the potential biological functions of TBP in myogenesis, we examined the effects of TBP in myoblasts proliferation and differentiation in vivo. TBP was successfully overexpressed or knockdown in CPMs (Fig. 6A, L). Overexpression of TBP reduced cell-cycle-promoting genes while increasing the expression of cell-cycle-inhibiting genes. The EdU and CCK-8 assays showed that overexpression of TBP decreased EdU incorporation and repressed myoblast viability, whereas its inhibition promoted myoblast proliferation (Fig. 6B–E, N–P). Moreover, flow cytometric analysis revealed that TBP overexpression reduced the number of S-phase cells (Fig. 6F). Conversely, TBP inhibition resulted in a greater number of S-phase cells (Fig. 6Q).
Next, immunofluorescence staining was performed to detected the role of TBP in myogenetic differentiation. TBP overexpression significantly facilitated myoblast differentiation, increased the total areas of myotubes and myotube formation (Fig. 6G–I). Meanwhile, qPCR and western blotting showed that expressions of myoblast differentiation marker genes were upregulated with TBP overexpression (Fig. 6J, K). In contrast, TBP interference repressed myoblast differentiation (Fig. 6R–V). Taken together, these data indicated that TBP suppresses myoblast proliferation and promotes myoblast differentiation, which is similar to LncRNA-TBP in function.
Discussion
Myogenesis is a highly ordered process including myoblast proliferation and differentiation, myotube formation and maturity, is controlled by a series of myogenic regulatory factors [33]. Many studies have suggested the important role of lncRNAs in skeletal muscle myogenesis while highlighting the necessity to systematically identify lncRNAs altered in skeletal muscle development [20, 34, 35]. The composition of myofiber types may influence meat quality by affecting the content of metabolites postmortem in livestock such as pH, meat color, and drip loss [36, 37]. Compare to the fast-twitch muscle phenotype, the proportion of type slow-muscle fibers is proportional to the content of intramuscular fat, which have higher tenderness, flavor, and juiciness [38, 39]. Here, we found that LncRNA-TBP is highly expressed in skeletal muscle, and its expression gradually increased with the stage of myoblast differentiation. Functional analyses showed that LncRNA-TBP suppresses myoblast proliferation and induces myogenic differentiation in vivo, LncRNA-TBP activated the slow-twitch muscle phenotype and reduces fat deposition.
Skeletal muscle can maintain systemic energy homeostasis in response to various metabolic stresses through regulating glucose uptake, lipid storage, and energy balance [40]. In this study, we found that LncRNA-TBP increases cellular mitochondrial DNA content and facilitates fatty acid oxidation in skeletal muscle, resulting in inhibiting the deposition of intramuscular fat. In the meantime, LncRNA-TBP reduced glycolytic capacity and increase oxidative capacity of skeletal muscle, which suppressed the autophagy pathway and reduced muscle atrophy.
TATA-binding protein (TBP) is a key component of the general transcription machinery that is involved in transcription by all three eukaryotic RNA polymerases [27,28,29,30]. Recent studies have found that mutant of TBP decreased its association with MyoD, which is a muscle-specific transcription factor, and caused a dramatic shrink in skeletal muscle mass [41, 42]. As a TATA-binding protein (TBP) associated factors, TAF9b was found can act a coactivator to stabilize the structure of P53 and promote P53 activation, thus reducing glycolysis, increasing superoxide levels, and inhibiting autophagy [12,13,14,15]. Notably, recent evidences have revealed that LncRNAs are able to be widely involved in a variety of biological processes through recruit RNA-binding proteins to regulate the transcription of target genes [43,44,45]. Here, we found LncRNA-TBP is a novel player in TBP-regulating network that can regulate the transcriptional activity of TBP-target genes by recruiting TBP protein, thus modulating myogenesis progression and inducing slow-twitch fibers.
Conclusion
In conclusion, we identify an lncRNA, LncRNA-TBP, and propose a mechanistic model to elucidate its role in the regulation of myogenesis and myofiber transformation through TBP-mediated transcriptional regulation (Fig. 7). Our findings present a novel model about the regulation of lncRNA in myogenesis, and will contribute to the development of further research.
Availability of data and materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Abbreviations
- CPMs:
-
Chicken primary myoblasts
- CSA:
-
Cross-section area
- FAO:
-
Fatty acid oxidation
- FFA:
-
Free fatty acid
- HCA:
-
Hierarchical clustering analysis
- HE:
-
Hematoxylin and eosin
- LDH:
-
Lactic dehydrogenase
- mtDNA:
-
Mitochondrial DNA
- PEM:
-
Pectoralis major
- SDH:
-
Succinate dehydrogenase
- SOL:
-
Soleus
- TCA cycle:
-
Tricarboxylic acid cycle
- TG:
-
Triglyceride
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This work was supported by National Key R&D Program of China (2021YFD1300100), the Natural Scientific Foundation of China (U1901206 and 31761143014), Local Innovative and Research Teams Project of Guangdong Province (2019BT02N630), China Agriculture Research System (CARS-41-G03), Guangdong Basic and Applied Basic Research Foundation (2021A1515111069), and China Postdoctoral Science Foundation (2022M710052).
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QN, HX and XZ conceived and designed the study. MM and BC performed the experiments, interpreted the data and wrote the paper. ZZ, SK and JZ performed the experiments. All authors read and approved the final manuscript.
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All animal experimental protocols were conformed to “The Instructive Notions with Respect to Caring for Laboratory Animals” issued by the Ministry of Science and Technology of the People’s Republic of China, and approved by the Institutional Animal Care and Use Committee at the South China Agricultural University (approval ID: SCAU#2021c008).
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Supplementary Information
Additional file 1.
Original data of WB.
Additional file 2: Figure S1
. Conservative analysis of LncRNA-TBP performed by using the NCBI’s BLAST. A total of eighteen species, including Anas platyrhynchos, Anser cygnoides, Apteryx mantelli mantelli, Aquila chrysaetos, Bos taurus, Coturnix japonica, Gallus gallus, Geospiza fortis, Homo sapiens, Meleagris gallopavo, Melopsittacus undulatus, Mus musculus, Numida meleagris, Ovis aries, Pan troglodytes, Rattus norvegicus, Sus scrofa and Zebra finch were used for Nucleotide BLAST. Top 4 most conservative results were listed above.
Additional file 3: Figure S2
. Interference of LncRNA-TBP promotes myoblast proliferation but inhibits myogenic differentiation. (A-K) Relative LncRNA-TBP expression (n = 6) (A), relative mRNA levels of several cell cycle genes (n = 6) (B), EdU proliferation assays (n = 3) (C), proliferation rate of myoblasts (n = 8) (D), CCK-8 assays (n = 6) (E), cell cycle analysis (n = 4) (F), MyHC immunostaining (n = 3) (G), myotube area (n = 8) (H), myoblast fusion index (n = 8) (I), relative mRNA (n = 6) (J) and protein (n = 3) (K) expression levels of myoblast differentiation marker genes with LncRNA-TBP interference in vitro. In panel (K), the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. Results are presented as mean ± SEM. In panels (A-B, D-F, and H-J), the statistical significance of differences between means was assessed using an independent sample t-test.
Additional file 4: Figure S3
. Interference of LncRNA-TBP inhibits fatty acid oxidation in skeletal muscle. (A-F) Relative LncRNA-TBP expression (n = 4) (A), relative mtDNA content (n = 4) (B), relative fatty acid β-oxidation rate (n = 4) (C), relative FFA and TG content (n = 4) (D), relative mRNA (n = 6) (E) and protein (n = 3) (F) expression levels of fatty acid oxidation or synthesis related-genes in gastrocnemius with LncRNA-TBP interference in vivo. In panel (F), the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. Results are shown as mean ± SEM. In panels (A-E), the statistical significance of differences between means was assessed using an independent sample t-test.
Additional file 5: Figure S4
. Interference of LncRNA-TBP activates fast-twitch muscle phenotype and reduces muscle hypertrophy. (A-N) relative enzymes activity of LDH and SDH (n = 4) (A), Relative glycogen content (n = 5) (B), relative mRNA expression levels of glycogenolytic and glycolytic genes (n = 6) (C), relative mRNA expression levels of several fast-/slow-twitch myofiber genes (n = 6) (D), immunohistochemistry analysis of MYH1A/MYH7B (n = 3) (E), MYH1A/MYH7B protein content (n = 8) (F), relative gastrocnemius muscle weight (n = 6) (G), H&E staining (n = 3) (H), relative mRNA (n = 6) (I), and the protein (n = 3) (J) expression levels of the atrophy and autophagy-related genes of in gastrocnemius with LncRNA-TBP interference in vivo. In panel (J), the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. Results are shown as mean ± SEM. In panels (A-D, F and H-I), the statistical significance of differences between means was assessed using an independent sample t-test.
Additional file 6: Figure S5
. TBP specific target genes identified by ATAC-seq. (A) The RPISeq results showed that the TBP was predicted to interact with LncRNA-TBP. (B) Analysis of TBP-targeted binding target genes by ATAC-seq. (C) GO functions analysis of TBP specific binding target genes identified by ATAC-seq. (D) KEGG pathways analysis of TBP specific binding target genes identified by ATAC-seq.
Additional file 7: Figure S6
. Overexpression and knockdown of LncRNA-TBP did not change the mRNA and protein expression level of TBP. (A and B) The mRNA level of TBP with LncRNA-TBP overexpression (n = 6) (A) and knockdown (n = 6) (B) in vitro. (C and D) The protein level of TBP with LncRNA-TBP overexpression (n = 3) (C) and knockdown (n = 3) (D) in vitro. In panel (C, D), the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. In panels (A, B), the statistical significance of differences between means was assessed using an independent sample t-test.
Additional file 8: Figure S7
. Interference of LncRNA-TBP inhibit the transcriptional activity of TBP-target genes. (A-G) TBP enrichment at the KLF4, GPI, TNNI2, and CDLN1A promoter enrichment (n = 3) (A), relative promoter activity of KLF4 (B), GPI (C), TNNI2 (D), and CDKN1A (E) (n = 4), relative mRNA (n = 4) (F) and protein (n = 3) (G) of KLF4, GPI, TNNI2, and CDKN1A with LncRNA-TBP interference in vitro. In panel (G), the numbers shown below the bands were folds of band intensities relative to control. Band intensities were quantified by ImageJ and normalized to GAPDH. Data are expressed as a fold-change relative to the control. Results are shown as mean ± SEM. In panels (A-F), the statistical significance of differences between means was assessed using an independent sample t-test.
Additional file 9: Figure S8
. The expression and location analysis of TBP. (A) Relative TBP expression during the proliferation and differentiation of CPM isolated from XH chicken (n = 4). (B) Subcellular location of TBP protein annotated by UniProt Knowledgebase (https://www.uniprot.org/). In panels (A), results are presented as mean ± SEM.
Additional file 10.
Supplementary Information.
Additional file 11: Table S2.
Comparative metabolome analysis of control group versus LncRNA-TBP overexpression gastrocnemius.
Additional file 12: Table S3.
TBP specific target genes identified by ATAC-seq analysis.
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Ma, M., Cai, B., Zhou, Z. et al. LncRNA-TBP mediates TATA-binding protein recruitment to regulate myogenesis and induce slow-twitch myofibers. Cell Commun Signal 21, 7 (2023). https://doi.org/10.1186/s12964-022-01001-3
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DOI: https://doi.org/10.1186/s12964-022-01001-3