Dual-RPA assay for rapid detection and differentiation of E.granulosus and E.multilocularis.

Echinococcus granulosus (Eg) and Echinococcus multilocularis (Em) are the two most widely prevalent types of echinococcosis. Several diagnostic methods have been developed for detecting Eg and Em. However, some limitations, such as being time-consuming, needing expensive instruments, or exhibiting low sensitivity, make these methods unsuitable for on-site detection. In this study, a dual-RPA assay was established to detect and differentiate Eg and Em. The primer concentration ratio, reaction time, and reaction temperature of the dual-RPA were optimized. The result showed that the primer concentration ratio of Eg:Em was 400 nM:400 nM, and the best amplification efficiency was obtained by reacting at 38 °C for 20 min. The sensitivity, specificity, and repeatability of the assay were also tested. The assay's detection limit for both Eg and Em was 10 copies/µL. The assay showed reasonable specificity by testing ten parasitic nucleic acids. The assay's intra- and inter-batch coefficients of variation were below 10%, which indicates robust reproducibility of the assay. Finally, to validate the performance of the dual-RPA assay, it was compared with real-time PCR by using 86 clinical nucleic acid samples. The coincidence rate of Eg between dual-RPA and TaqMan real-time PCR was 96.51%, and the coincidence rate of Em between dual-RPA and TaqMan real-time PCR was 98.84%, indicating its potential for accurate clinical diagnosis. Therefore, this study established a rapid and sensitive dual-RPA assay that can rapidly detect and differentiate Eg and Em in one reaction tube and provided a new assay for the detection of echinococcosis in the field.

Functional SNPs in SYISL promoter significantly affect muscle fiber density and muscle traits in pigs.

Our previous studies showed that SYISL is a negative regulator of muscle growth and regeneration in mice, pigs and humans. SYISL knockout resulted in an increase in the density of muscle fibers and muscle growth. However, it is unclear whether there are natural mutations in pig SYNPO2 intron sense-overlapping lncRNA (pSYISL) that affect the expression of pSYISL and muscle growth traits. In this study, three SNPs in exons and six SNPs within the promoter of pSYISL were identified. Association analysis showed that the two SNPs in exons are significantly associated with loin muscle area (p < 0.05); the six SNPs in the promoter that show complete linkage are significantly associated with live backfat thickness and live loin muscle area in American Large White pigs. Bioinformatics and luciferase reporter assays as well as in vitro binding experiments indicated that the mutation of SNP rs702045770 (g.539G>A) leads to the loss of YY1 binding to the promoter, thus affecting the expression level of pSYISL, and we found that Jiangshan Black pigs with genotype GG have a higher expression level of pSYISL than genotype AA individuals, but the muscle fiber density was significantly lower than in genotype AA individuals. Furthermore, the association analysis showed that the carcass backfat thickness of genotype GG of SNP rs702045770 was significantly higher than that of other genotypes in (Pietrain × Duroc) × (Landrace × Yorkshire) crossbred pigs (p < 0.05). The glycolytic potential of genotype GG was significantly higher than that of other genotypes (p < 0.05). These results provide novel insight into the identification of functional SNPs in non-coding genomic regions.

DOCK2 Deficiency Attenuates Abdominal Aortic Aneurysm Formation-Brief Report.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a potentially lethal disease that lacks pharmacological treatment. Degradation of extracellular matrix proteins, especially elastin laminae, is the hallmark for AAA development. DOCK2 (dedicator of cytokinesis 2) has shown proinflammatory effects in several inflammatory diseases and acts as a novel mediator for vascular remodeling. However, the role of DOCK2 in AAA formation remains unknown. METHODS: Ang II (angiotensin II) infusion of ApoE-/- (apolipoprotein E deficient) mouse and topical elastase-induced AAA combined with DOCK2-/- (DOCK2 knockout) mouse models were used to study DOCK2 function in AAA formation/dissection. The relevance of DOCK2 to human AAA was examined using human aneurysm specimens. Elastin fragmentation in AAA lesion was observed by elastin staining. Elastin-degrading enzyme MMP (matrix metalloproteinase) activity was measured by in situ zymography. RESULTS: DOCK2 was robustly upregulated in AAA lesion of Ang II-infused ApoE-/- mice, elastase-treated mice, as well as human AAA lesions. DOCK2-/- significantly attenuated the Ang II-induced AAA formation/dissection or rupture in mice along with reduction of MCP-1 (monocyte chemoattractant protein-1) and MMP expression and activity. Accordingly, the elastin fragmentation observed in ApoE-/- mouse aorta infused with Ang II and elastase-treated aorta was significantly attenuated by DOCK2 deficiency. Moreover, DOCK2-/- decreased the prevalence and severity of aneurysm formation, as well as the elastin degradation observed in the topical elastase model. CONCLUSIONS: Our results indicate that DOCK2 is a novel regulator for AAA formation. DOCK2 regulates AAA development by promoting MCP-1 and MMP2 expression to incite vascular inflammation and elastin degradation.

FHL3 promotes the formation of fast glycolytic muscle fibers by interacting with YY1 and muscle glycolytic metabolism.

The proportions of the various muscle fiber types are important in the regulation of skeletal muscle metabolism, as well as animal meat production. Four-and-a-half LIM domain protein 3 (FHL3) is highly expressed in fast glycolytic muscle fibers and differentially regulates the expression of myosin heavy chain (MyHC) isoforms at the cellular level. Whether FHL3 regulates the transformation of muscle fiber types in vivo and the regulatory mechanism is unclear. In this study, muscle-specific FHL3 transgenic mice were generated by random integration, and lentivirus-mediated gene knockdown or overexpression in muscles of mice or pigs was conducted. Functional analysis showed that overexpression of FHL3 in muscles significantly increased the proportion of fast-twitch myofibers and muscle mass but decreased muscle succinate dehydrogenase (SDH) activity and whole-body oxygen consumption. Lentivirus-mediated FHL3 knockdown in muscles significantly decreased muscle mass and the proportion of fast-twitch myofibers. Mechanistically, FHL3 directly interacted with the Yin yang 1 (YY1) DNA-binding domain, repressed the binding of YY1 to the fast glycolytic MyHC2b gene regulatory region, and thereby promoted MyHC2b expression. FHL3 also competed with EZH2 to bind the repression domain of YY1 and reduced H3K27me3 enrichment in the MyHC2b regulatory region. Moreover, FHL3 overexpression reduced glucose tolerance by affecting muscle glycolytic metabolism, and its mRNA expression in muscle was positively associated with hemoglobin A1c (HbA1c) in patients with type 2 diabetes. Therefore, FHL3 is a novel potential target gene for the treatment of muscle metabolism-related diseases and improvement of animal meat production.

Long noncoding RNA lncMREF promotes myogenic differentiation and muscle regeneration by interacting with the Smarca5/p300 complex.

Long noncoding RNAs (lncRNAs) play important roles in the spatial and temporal regulation of muscle development and regeneration. Nevertheless, the determination of their biological functions and mechanisms underlying muscle regeneration remains challenging. Here, we identified a lncRNA named lncMREF (lncRNA muscle regeneration enhancement factor) as a conserved positive regulator of muscle regeneration among mice, pigs and humans. Functional studies demonstrated that lncMREF, which is mainly expressed in differentiated muscle satellite cells, promotes myogenic differentiation and muscle regeneration. Mechanistically, lncMREF interacts with Smarca5 to promote chromatin accessibility when muscle satellite cells are activated and start to differentiate, thereby facilitating genomic binding of p300/CBP/H3K27ac to upregulate the expression of myogenic regulators, such as MyoD and cell differentiation. Our results unravel a novel temporal-specific epigenetic regulation during muscle regeneration and reveal that lncMREF/Smarca5-mediated epigenetic programming is responsible for muscle cell differentiation, which provides new insights into the regulatory mechanism of muscle regeneration.

Conservative analysis of Synaptopodin-2 intron sense-overlapping lncRNA reveals its novel function in promoting muscle atrophy.

BACKGROUND: Dissection of the regulatory pathways that control skeletal muscle development and atrophy is important for the treatment of muscle wasting. Long noncoding RNA (lncRNA) play important roles in various stages of muscle development. We previously reported that Synaptopodin-2 (SYNPO2) intron sense-overlapping lncRNA (SYISL) regulates myogenesis through an interaction with enhancer of zeste homologue 2 (EZH2). However, it remains unclear whether SYISL homologues exist in humans and pigs, and whether the functions and mechanisms of these homologues are conserved among species. METHODS: Bioinformatics, cell fractionation, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were used for the identification and molecular characterization of SYISL homologues in humans and pigs. Effects on myogenesis and muscle atrophy were determined via loss-of-function or gain-of-function experiments using C2C12 myoblasts, myogenic progenitor cells, dexamethasone (DEX), and aging-induced muscle atrophy models. RNA pulldown, RNA immunoprecipitation, dual luciferase reporting, and co-transfection experiments were used to explore the mechanisms of SYISL interactions with proteins and miRNAs. RESULTS: We identified SYISL homologues in humans (designated hSYISL) and pigs (designated pSYISL). Functional experiments demonstrated that hSYISL and pSYISL regulate myogenesis through interactions with EZH2. Interestingly, we showed that SYISL functions to regulate muscle atrophy and sarcopenia through comparative analysis. SYISL is significantly up-regulated after muscle atrophy (P < 0.01); it significantly promotes muscle atrophy in DEX-induced muscle atrophy models (P < 0.01). SYISL knockdown or knockout alleviates muscle atrophy and sarcopenia in DEX-induced and aged mice. The tibialis anterior (TA) muscle weight of 3-month-old wild-type (WT) mice decreased by 33.24% after DEX treatment (P < 0.001), while the muscle weight loss of 3-month-old SYISL knockout mice was only 18.20% after DEX treatment (P < 0.001). SYISL knockout in 18-month-old WT mice significantly increased the weights of quadriceps (Qu), gastrocnemius (Gas), and TA muscles by 10.45% (P < 0.05), 13.95% (P < 0.01), and 24.82% (P < 0.05), respectively. Mechanistically, SYISL increases the expression levels of the muscle atrophy genes forkhead box protein O3a (FoxO3a), muscle ring finger 1 (MuRF1), and muscle atrophy-related F-box (Atrogin-1) via sponging of miR-23a-3p/miR-103-3p/miR-205-5p and thus promotes muscle atrophy. Additionally, we verified that human SYISL overexpression in muscles of 18-month-old WT mice significantly decreased the weights of Gas, Qu, and TA muscles by 7.76% (P < 0.01), 12.26% (P < 0.05), and 13.44% (P < 0.01), respectively, and accelerates muscle atrophy through conserved mechanisms. CONCLUSIONS: Our results identify SYISL as a conserved lncRNA that modulates myogenesis in mice, pigs, and humans. We also demonstrated its previously unknown ability to promote muscle atrophy.

Single Nucleotide Polymorphisms of Porcine lncMGPF Regulate Meat Production Traits by Affecting RNA Stability.

lncMGPF is a novel positive regulator of myogenic differentiation, muscle growth and regeneration in mouse, pig, and human. But whether natural mutations within lncMGPF gene regulate animal meat production traits is unclear. In this study, ten single nucleotide polymorphisms (SNPs) of pig lncMGPF (plncMGPF) gene were identified among commercial pig breeds and Chinese local pig breeds. These SNPs are highly linked and constructed into multiple haplotypes, and haplotype ATTCATGTTC (H1) mainly exists in commercial pig breeds while haplotype GCCTGCACCT (H3) is more frequent in Chinese local pig breeds. Association analysis indicated that all SNPs are significantly associated with the backfat thickness and loin muscle area (P < 0.05), respectively, and homologous H1 individuals have higher loin muscle area and lower backfat thickness than H3 pigs. Bioinformatics and functional analysis showed that haplotype H1 has a longer half-life and more stable RNA secondary structure than haplotype H3. plncMGPF haplotype H1 has stronger effects on pig primary myogenic progenitor cells differentiation and muscle growth than haplotype H3. Further experiments showed that two SNPs (rs81403974 and rs325492834) function together to confer plncMGPF stability and function. Our observation suggested that the SNPs in lncMGPF can change the RNA stabilities and lncMGPF function, thereby affecting the porcine meat production traits.

Functional Non-coding RNA During Embryonic Myogenesis and Postnatal Muscle Development and Disease.

Skeletal muscle is a highly heterogeneous tissue that plays a crucial role in mammalian metabolism and motion maintenance. Myogenesis is a complex biological process that includes embryonic and postnatal development, which is regulated by specific signaling pathways and transcription factors. Various non-coding RNAs (ncRNAs) account for the majority of total RNA in cells and have an important regulatory role in myogenesis. In this review, we introduced the research progress in miRNAs, circRNAs, and lncRNAs related to embryonic and postnatal muscle development. We mainly focused on ncRNAs that regulate myoblast proliferation, differentiation, and postnatal muscle development through multiple mechanisms. Finally, challenges and future perspectives related to the identification and verification of functional ncRNAs are discussed. The identification and elucidation of ncRNAs related to myogenesis will enrich the myogenic regulatory network, and the effective application of ncRNAs will enhance the function of skeletal muscle.

lncMGPF is a novel positive regulator of muscle growth and regeneration.

BACKGROUND: Long non-coding RNAs (lncRNAs) play critical regulatory roles in diverse biological processes and diseases. While a large number of lncRNAs have been identified in skeletal muscles until now, their function and underlying mechanisms in skeletal myogenesis remain largely unclear. METHODS: We characterized a novel functional lncRNA designated lncMGPF (lncRNA muscle growth promoting factor) using RACE, Northern blot, fluorescence in situ hybridization and quantitative real-time PCR. Its function was determined by gene overexpression, interference, and knockout experiments in C2C12 myoblasts, myogenic progenitor cells, and an animal model. The molecular mechanism by which lncMGPF regulates muscle differentiation was mainly examined by cotransfection experiments, luciferase reporter assay, RNA immunoprecipitation, RNA pull-down, and RNA stability analyses. RESULTS: We report that lncMGPF, which is highly expressed in muscles and positively regulated by myoblast determination factor (MyoD), promotes myogenic differentiation of muscle cells in vivo and in vitro. lncMGPF knockout in mice substantially decreases growth rate, reduces muscle mass, and impairs muscle regeneration. Overexpression of lncMGPF in muscles can rescue the muscle phenotype of knockout mice and promote muscle growth of wild-type mice. Mechanistically, lncMGPF promotes muscle differentiation by acting as a molecular sponge of miR-135a-5p and thus increasing the expression of myocyte enhancer factor 2C (MEF2C), as well as by enhancing human antigen R-mediated messenger RNA stabilization of myogenic regulatory genes such as MyoD and myogenin (MyoG). We confirm that pig lncRNA AK394747 and human lncRNA MT510647 are homologous to mouse lncMGPF, with conserved function and mechanism during myogenesis. CONCLUSIONS: Our data reveal that lncMGPF is a novel positive regulator of myogenic differentiation, muscle growth and regeneration in mice, pigs, and humans.

RIP-Seq of EZH2 Identifies TCONS-00036665 as a Regulator of Myogenesis in Pigs.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of polycomb repressive complex 2 and contains a SET domain that catalyzes histone H3 trimethylation on lysine 27 (H3K27me3) to generate an epigenetic silencing mark. EZH2 interacts with transcription factors or RNA transcripts to perform its function. In this study, we applied RNA immunoprecipitation sequencing and long intergenic non-coding RNA (lincRNA) sequencing methods to identify EZH2-binding lincRNAs. A total of 356 novel EZH2-binding lincRNAs were identified by bioinformatics analysis and an EZH2-binding lincRNA TCONS-00036665 was characterized. TCONS-00036665 promoted pig skeletal satellite cell proliferation but inhibited cell differentiation, and this function was conserved between pigs and mice. Further mechanistic studies indicated that TCONS-00036665 can bind to EZH2 and recruits EZH2 to the promoters of the target genes p21, MyoG, and Myh4, which leads to the enrichment of H3K27me3 and the repression of target gene expression and pig myogenesis. In conclusion, the lincRNA TCONS-00036665 regulates pig myogenesis through its interaction with EZH2.

Activated FXR promotes xenobiotic metabolism of T-2 toxin and attenuates oxidative stress in broiler chicken liver.

The transmission of T-2 toxin and its metabolites into the edible tissues of poultry has potential effects on human health. The bile acid and xenobiotic system composes an intricate physiological network of chemoprotective and transporter-related functions, which ensures the detoxification and removal of harmful xenobiotic and endobiotic compounds from the body. This study revealed that cholic acid (CA), as one of the bile acids, promoted the metabolism of T-2 toxin in vivo by inducing the xenobiotic metabolism enzymes expression, thereby increasing the stress resistance and attenuating the oxidative stress. This study also indicated that dietary supplementation of 1% CA alleviated the mortality caused by T-2 toxin. Liver histology results demonstrated that CA supplementation significantly reduced inflammatory cell infiltration, sinusoidal expansion and congestion. Biochemistry results showed that the elevations of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and the increase in concentration of hydrogen peroxide (H2O2) in liver induced by the T-2 toxin were decreased by dietary supplementation of 1% CA. Additionally, CA supplementation led to the increase in superoxide dismutase (SOD) activity, but the decrease in catalase (CAT) activity in broiler chicken livers. Based on these findings, we propose that activation of FXR promotes T-2 toxin xenobiotic metabolism, and FXR plays a hepatoprotection role in liver injury induced by T-2 toxin.

Functions and Regulatory Mechanisms of lncRNAs in Skeletal Myogenesis, Muscle Disease and Meat Production.

Myogenesis is a complex biological process, and understanding the regulatory network of skeletal myogenesis will contribute to the treatment of human muscle related diseases and improvement of agricultural animal meat production. Long noncoding RNAs (lncRNAs) serve as regulators in gene expression networks, and participate in various biological processes. Recent studies have identified functional lncRNAs involved in skeletal muscle development and disease. These lncRNAs regulate the proliferation, differentiation, and fusion of myoblasts through multiple mechanisms, such as chromatin modification, transcription regulation, and microRNA sponge activity. In this review, we presented the latest advances regarding the functions and regulatory activities of lncRNAs involved in muscle development, muscle disease, and meat production. Moreover, challenges and future perspectives related to the identification of functional lncRNAs were also discussed.

Long noncoding RNA Neat1 modulates myogenesis by recruiting Ezh2.

Neat1 is widely expressed in many tissues and cells and exerts pro-proliferation effects on many cancer cells. However, little is known about the function of Neat1 in myogenesis. Here we characterized the roles of Neat1 in muscle cell formation and muscle regeneration. Gain- or loss-of-function studies in C2C12 cells demonstrated that Neat1 accelerates myoblast proliferation but suppresses myoblast differentiation and fusion. Further, knockdown of Neat1 in vivo increased the cross-sectional area of muscle fibers but impaired muscle regeneration. Mechanically, Neat1 physically interacted with Ezh2 mainly through the core binding region (1001-1540 bp) and recruited Ezh2 to target gene promoters. Neat1 promoted myoblast proliferation mainly by decreasing the expression of the cyclin-dependent kinase inhibitor P21 gene but inhibited myoblast differentiation by suppressing the transcription of myogenic marker genes, such as Myog, Myh4, and Tnni2. Altogether, we uncover a previously unknown function of Neat1 in muscle development and the molecular mechanism by which Neat1 regulates myogenesis.

Analysis of differential gene expression of the transgenic pig with overexpression of PGC1α in muscle.

In order to better understand the key regulatory mechanisms of PGC1α in muscle fiber type transition, the RNA-seq was used to compare the change of gene expression in gastrocnemius muscles between wild type pigs and transgenic pigs with overexpression of PGC1α gene in muscle. 371 differentially expressed genes (P ≤ 0.05 and Ratio ≥ 2), including 184 up-regulated genes and 187 down-regulated genes, were identified. Five main signaling pathways including metabolic pathways, ECM-receptor interaction, PPAR signaling pathway, adipocytokine signaling pathway and insulin signaling pathway, were authenticated using KEGG pathway analysis. Our results indicate that the fat metabolism pathway plays an important role in the transformation of muscle fiber types regulated by PGC1α.

The nuclear protein-coding gene ANKRD23 negatively regulates myoblast differentiation.

Muscle fiber formation is a complex process and subject to fine regulation of a variety of protein-coding genes and non-coding RNA. In this study, we identified a nuclear protein-coding gene ANKRD23 which was highly expressed in muscle. Quantitative real-time PCR, western blotting and immunofluorescence were used to detect the expression change of myoblast differentiation marker genes after knockdown and overexpression of ANKRD23. The results showed that the expression of myoblast differentiation marker genes were increased by interference and reduced by ANKRD23 overexpression, indicating that ANKRD23 played a negative role in the myoblast differentiation. Interestingly, we discovered a long non-coding RNA-AK004293 which was overlapped with the 3'UTR of ANKRD23 gene. Then we detected the effect of AK004293 on the expression of ANKRD23 and myoblast differentiation marker genes in C2C12 myoblasts. The results showed that AK004293 had no significant effect on the expression of myoblast differentiation maker genes and ANKRD23. In conclusion, our results established the foundation for further studies about the regulation mechanism of ANKRD23 in muscle development.

DOCK2 deficiency mitigates HFD-induced obesity by reducing adipose tissue inflammation and increasing energy expenditure.

Obesity is the major risk factor for type 2 diabetes, cardiovascular disorders, and many other diseases. Adipose tissue inflammation is frequently associated with obesity and contributes to the morbidity and mortality. Dedicator of cytokinesis 2 (DOCK2) is involved in several inflammatory diseases, but its role in obesity remains unknown. To explore the function of DOCK2 in obesity and insulin resistance, WT and DOCK2-deficient (DOCK2-/-) mice were given chow or high-fat diet (HFD) for 12 weeks followed by metabolic, biochemical, and histologic analyses. DOCK2 was robustly induced in adipose tissues of WT mice given HFD. DOCK2-/- mice with HFD showed decreased body weight gain and improved metabolic homeostasis and insulin resistance compared with WT mice. DOCK2 deficiency also attenuated adipose tissue and systemic inflammation accompanied by reduced macrophage infiltration. Moreover, DOCK2-/- mice exhibited increased expression of metabolic genes in adipose tissues with greater energy expenditure. Mechanistically, DOCK2 appeared to regulate brown adipocyte differentiation because increased preadipocyte differentiation to brown adipocytes in interscapular and inguinal fat was observed in DOCK2-/- mice, as compared with WT. These data indicated that DOCK2 deficiency protects mice from HFD-induced obesity, at least in part, by stimulating brown adipocyte differentiation. Therefore, targeting DOCK2 may be a potential therapeutic strategy for treating obesity-associated diseases.

Identification of MyoD-Responsive Transcripts Reveals a Novel Long Non-coding RNA (lncRNA-AK143003) that Negatively Regulates Myoblast Differentiation.

Myogenic differentiation factor (MyoD) is a master transcription factor in muscle development and differentiation. Although several long non-coding RNAs (lncRNAs) linked to MyoD have been found to influence muscle development, the functions of many lncRNAs have not been explored. Here we utilized lncRNA and mRNA microarray analysis to identify potential lncRNAs regulated by MyoD in muscle cells. A total of 997 differentially expressed lncRNAs (335 up-regulated and 662 down-regulated) and 1,817 differentially expressed mRNAs (148 up-regulated and 1,669 down-regulated) were identified after MyoD knockdown in C2C12 cells. Functional predictions suggested that most lncRNAs are involved in the biological pathways related to muscle differentiation and cell cycle with co-expressed genes. To gain further insight into the MyoD-mediated lncRNA expression in muscle differentiation, tissue expression profiles and MyoD overexpression were performed, and we found one of the candidate lncRNAs-AK143003 was significantly regulated by MyoD. Further analyses showed its noncoding ability and cytoplasmic localisation. Silencing of AK143003 stimulated the accumulation of myogenic marker genes, whereas AK143003 overexpression led to their decreased synthesis. This study identified a multitude of MyoD-mediated lncRNAs for further investigation and identified a novel lncRNA, lnc-AK143003, which plays a role in controlling muscle differentiation.

FHL3 differentially regulates the expression of MyHC isoforms through interactions with MyoD and pCREB.

In skeletal muscle, muscle fiber types are defined by four adult myosin heavy chain (MyHC) isoforms. Four and a half LIM domain protein 3 (FHL3) regulates myoblasts differentiation and gene expression by acting as a transcriptional co-activator or co-repressor. However, how FHL3 regulates MyHC expression is currently not clear. In this study, we found that FHL3 down-regulated the expression of MyHC 1/slow and up-regulated the expression of MyHC 2a and MyHC 2b, whereas no significant effect was found on MyHC 2x expression. MyoD and phosphorylated cAMP response element binding protein (pCREB) played important roles in the regulation of MyHC 1/slow and MyHC 2a expression by FHL3, respectively. FHL3 could interact with MyoD, CREB and pCREB in vivo. pCREB had stronger interaction with the cyclic AMP-responsive elements (CRE) of the MyHC 2a promoter compared with CREB, and FHL3 significantly affected the binding capacity of pCREB to CRE. We established a model in which FHL3 promotes the expression of MyHC 2a through CREB-mediated transcription and inhibits the expression of MyHC 1/slow by inhibiting MyoD transcription activity during myogenesis. Our data support the notion that FHL3 plays important roles in the regulation of muscle fiber type composition.

The Functional SNPs in the 5' Regulatory Region of the Porcine PPARD Gene Have Significant Association with Fat Deposition Traits.

Peroxisome proliferator-activated receptor delta (PPARD) is a key regulator of lipid metabolism, insulin sensitivity, cell proliferation and differentiation. In this study, we identified two Single Nucleotide Polymorphisms (SNPs, g.1015 A>G and g.1018 T>C) constituting four haplotypes (GT, GC, AC and AT) in the 5' regulatory region of porcine PPARD gene. Functional analysis of the four haplotypes showed that the transcriptional activity of the PPARD promoter fragment carrying haplotype AC was significantly lower than that of the other haplotypes in 3T3-L1, C2C12 and PK-15 cells, and haplotype AC had the lowest binding capacities to the nuclear extracts. Transcription factor 7-like 2 (TCF7L2) enhanced the transcription activities of promoter fragments of PPARD gene carrying haplotypes GT, GC and AT in C2C12 and 3T3-L1 cells, and increased the protein expression of PPARD gene in C2C12 myoblasts. TCF7L2 differentially bound to the four haplotypes, and the binding capacity of TCF7L2 to haplotype AC was the lowest. There were significant associations between -655A/G and fat deposition traits in three pig populations including the Large White × Meishan F2 pigs, France and American Large White pigs. Pigs with genotype GG had significantly higher expression of PPARD at both mRNA and protein level than those with genotype AG. These results strongly suggested that the SNPs in 5' regulatory region of PPARD genes had significant impact on pig fat deposition traits.