MYH1G-AS is a chromatin-associated lncRNA that regulates skeletal muscle development in chicken.

BACKGROUND: Skeletal muscle development is pivotal for animal growth and health. Recently, long noncoding RNAs (lncRNAs) were found to interact with chromatin through diverse roles. However, little is known about how lncRNAs act as chromatin-associated RNAs to regulate skeletal muscle development. Here, we aim to investigate the regulation of chromatin-associated RNA (MYH1G-AS) during skeletal muscle development. METHODS: We provided comprehensive insight into the RNA profile and chromatin accessibility of different myofibers, combining RNA sequencing (RNA-seq) with an assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). The dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to analyze the transcriptional regulation mechanism of MYH1G-AS. ALKBH5-mediated MYH1G-AS N6-methyladenosine (m6A) demethylation was assessed by a single-base elongation and ligation-based qPCR amplification method (SELECT) assay. Functions of MYH1G-AS were investigated through a primary myoblast and lentivirus/cholesterol-modified antisense oligonucleotide (ASO)-mediated animal model. To validate the interaction of MYH1G-AS with fibroblast growth factor 18 (FGF18) protein, RNA pull down and an RNA immunoprecipitation (RIP) assay were performed. Specifically, the interaction between FGF18 and SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5 (SMARCA5) protein was analyzed by coimmunoprecipitation (Co-IP) and a yeast two-hybrid assay. RESULTS: A total of 45 differentially expressed (DE) lncRNAs, with DE ATAC-seq peaks in their promoter region, were classified as open chromatin-associated lncRNAs. A skeletal muscle-specific lncRNA (MSTRG.15576.9; MYH1G-AS), which is one of the open chromatin-associated lncRNA, was identified. MYH1G-AS transcription is coordinately regulated by transcription factors (TF) SMAD3 and SP2. Moreover, SP2 represses ALKBH5 transcription to weaken ALKBH5-mediated m6A demethylation of MYH1G-AS, thus destroying MYH1G-AS RNA stability. MYH1G-AS accelerates myoblast proliferation but restrains myoblast differentiation. Moreover, MYH1G-AS drives a switch from slow-twitch to fast-twitch fibers and causes muscle atrophy. Mechanistically, MYH1G-AS inhibits FGF18 protein stabilization to reduce the interaction of FGF18 to SMARCA5, thus repressing chromatin accessibility of the SMAD4 promoter to activate the SMAD4-dependent pathway. CONCLUSIONS: Our results reveal a new pattern of the regulation of lncRNA expression at diverse levels and help expound the regulation of m6A methylation on chromatin status.

Transcriptome and Metabolome Profiling Provide New Insights into Disuse Muscle Atrophy in Chicken: The Potential Role of Fast-Twitch Muscle Fibers.

Disuse muscle atrophy is a disease caused by restricted activity, affecting human health and animal protein quality. While extensive research on its mechanism has been studied in mammals, comparatively little is known about this process in chickens, which are a significant source of protein for human consumption worldwide. Understanding the mechanisms underlying skeletal muscle atrophy in chickens is crucial for improving poultry health and productivity, as well as for developing strategies to mitigate muscle loss. In this study, two groups of chickens were subjected to limb immobilization for two and four weeks, respectively, in order to induce disuse muscle atrophy and uniformly sampled gastrocnemius muscle at the fourth week. A combined analysis of the transcriptome and metabolome was conducted to investigate the mechanisms of disuse-induced muscle atrophy. Through H&E staining and immunofluorescence, we found that, compared to slow-twitch muscle fibers, the fast-twitch muscle fibers showed a greater reduction in cross-sectional area in the immobilized leg, and were also the main driver of changes in cross-sectional area observed in the non-immobilized leg. Integrated analysis revealed that differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were mainly enriched in pathways related to energy metabolism, such as fatty acid metabolism, oxidative phosphorylation (OXPHOS), and glycolysis. These results provide important insights for further research on disuse muscle atrophy.

Identifying driver pathways based on a parameter-free model and a partheno-genetic algorithm.

BACKGROUND: Tremendous amounts of omics data accumulated have made it possible to identify cancer driver pathways through computational methods, which is believed to be able to offer critical information in such downstream research as ascertaining cancer pathogenesis, developing anti-cancer drugs, and so on. It is a challenging problem to identify cancer driver pathways by integrating multiple omics data. RESULTS: In this study, a parameter-free identification model SMCMN, incorporating both pathway features and gene associations in Protein-Protein Interaction (PPI) network, is proposed. A novel measurement of mutual exclusivity is devised to exclude some gene sets with "inclusion" relationship. By introducing gene clustering based operators, a partheno-genetic algorithm CPGA is put forward for solving the SMCMN model. Experiments were implemented on three real cancer datasets to compare the identification performance of models and methods. The comparisons of models demonstrate that the SMCMN model does eliminate the "inclusion" relationship, and produces gene sets with better enrichment performance compared with the classical model MWSM in most cases. CONCLUSIONS: The gene sets recognized by the proposed CPGA-SMCMN method possess more genes engaging in known cancer related pathways, as well as stronger connectivity in PPI network. All of which have been demonstrated through extensive contrast experiments among the CPGA-SMCMN method and six state-of-the-art ones.

De Novo Assembly of 20 Chicken Genomes Reveals the Undetectable Phenomenon for Thousands of Core Genes on Microchromosomes and Subtelomeric Regions.

The gene numbers and evolutionary rates of birds were assumed to be much lower than those of mammals, which is in sharp contrast to the huge species number and morphological diversity of birds. It is, therefore, necessary to construct a complete avian genome and analyze its evolution. We constructed a chicken pan-genome from 20 de novo assembled genomes with high sequencing depth, and identified 1,335 protein-coding genes and 3,011 long noncoding RNAs not found in GRCg6a. The majority of these novel genes were detected across most individuals of the examined transcriptomes but were seldomly measured in each of the DNA sequencing data regardless of Illumina or PacBio technology. Furthermore, different from previous pan-genome models, most of these novel genes were overrepresented on chromosomal subtelomeric regions and microchromosomes, surrounded by extremely high proportions of tandem repeats, which strongly blocks DNA sequencing. These hidden genes were proved to be shared by all chicken genomes, included many housekeeping genes, and enriched in immune pathways. Comparative genomics revealed the novel genes had 3-fold elevated substitution rates than known ones, updating the knowledge about evolutionary rates in birds. Our study provides a framework for constructing a better chicken genome, which will contribute toward the understanding of avian evolution and the improvement of poultry breeding.

LncRNA-TBP mediates TATA-binding protein recruitment to regulate myogenesis and induce slow-twitch myofibers.

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. Video Abstract.

Local GHR roles in regulation of mitochondrial function through mitochondrial biogenesis during myoblast differentiation.

BACKGROUND: Myoblast differentiation requires metabolic reprogramming driven by increased mitochondrial biogenesis and oxidative phosphorylation. The canonical GH-GHR-IGFs axis in liver exhibits a great complexity in response to somatic growth. However, the underlying mechanism of whether local GHR acts as a control valve to regulate mitochondrial function through mitochondrial biogenesis during myoblast differentiation remains unknown. METHODS: We manipulated the GHR expression in chicken primary myoblast to investigate its roles in mitochondrial biogenesis and function during myoblast differentiation. RESULTS: We reported that GHR is induced during myoblast differentiation. Local GHR promoted mitochondrial biogenesis during myoblast differentiation, as determined by the fluorescence intensity of Mito-Tracker Green staining and MitoTimer reporter system, the expression of mitochondrial biogenesis markers (PGC1α, NRF1, TFAM) and mtDNA encoded gene (ND1, CYTB, COX1, ATP6), as well as mtDNA content. Consistently, local GHR enhanced mitochondrial function during myoblast differentiation, as determined by the oxygen consumption rate, mitochondrial membrane potential, ATP level and ROS production. We next revealed that the regulation of mitochondrial biogenesis and function by GHR depends on IGF1. In terms of the underlying mechanism, we demonstrated that IGF1 regulates mitochondrial biogenesis via PI3K/AKT/CREB pathway. Additionally, GHR knockdown repressed myoblast differentiation. CONCLUSIONS: In conclusion, our data corroborate that local GHR acts as a control valve to enhance mitochondrial function by promoting mitochondrial biogenesis via IGF1-PI3K/AKT/CREB pathway during myoblast differentiation. Video Abstract.

CircPLXNA2 Affects the Proliferation and Apoptosis of Myoblast through circPLXNA2/gga-miR-12207-5P/MDM4 Axis.

CircRNAs are newly identified special endogenous RNA molecules that covalently close a loop by back-splicing with pre-mRNA. In the cytoplasm, circRNAs would act as molecular sponges to bind with specific miRNA to promote the expression of target genes. However, knowledge of circRNA functional alternation in skeletal myogenesis is still in its infancy. In this study, we identified a circRNA-miRNA-mRNA interaction network in which the axis may be implicated in the progression of chicken primary myoblasts' (CPMs) myogenesis by multi-omics (i.e., circRNA-seq and ribo-seq). In total, 314 circRNA-miRNA-mRNA regulatory axes containing 66 circRNAs, 70 miRNAs, and 24 mRNAs that may be relevant to myogenesis were collected. With these, the circPLXNA2-gga-miR-12207-5P-MDM4 axis aroused our research interest. The circPLXNA2 is highly differentially expressed during differentiation versus proliferation. It was demonstrated that circPLXNA2 inhibited the process of apoptosis while at the same time stimulating cell proliferation. Furthermore, we demonstrated that circPLXNA2 could inhibit the repression of gga-miR-12207-5p to MDM4 by directing binding to gga-miR-12207-5p, thereby restoring MDM4 expression. In conclusion, circPLXNA2 could function as a competing endogenous RNA (ceRNA) to recover the function of MDM4 by directing binding to gga-miR-12207-5p, thereby regulating the myogenesis.

Galbase: a comprehensive repository for integrating chicken multi-omics data.

BACKGROUND: Multi-omics data can provide a stereoscopic view to explore potential causal variations and genes, as well as underlying genetic mechanisms of complex traits. However, for many non-mammalian species, including chickens, these resources are poorly integrated and reused, greatly limiting genetic research and breeding processes of the species. RESULTS: Here, we constructed Galbase, an easily accessible repository that integrates public chicken multi-omics data from 928 re-sequenced genomes, 429 transcriptomes, 379 epigenomes, 15,275 QTL entries, and 7,526 associations. A total of 21.67 million SNPs, 2.71 million InDels, and 488,583 cis-regulatory elements were included. Galbase allows users to retrieve genomic variations in geographical maps, gene expression profiling in heatmaps, and epigenomic signals in peak patterns. It also provides modules for batch annotation of genes, regions, and loci based on multi-layered omics data. Additionally, a series of convenient tools, including the UCSC Genome Browser, WashU Epigenome Browser, BLAT, BLAST, and LiftOver, were also integrated to facilitate search, visualization, and analysis of sequence features. CONCLUSION: Galbase grants new opportunities to research communities to undertake in-depth functional genomic studies on chicken. All features of Galbase make it a useful resource to identify genetic variations responsible for chicken complex traits. Galbase is publicly available at http://animal.nwsuaf.edu.cn/ChickenVar .

Growth hormone receptor gene influences mitochondrial function and chicken lipid metabolism by AMPK-PGC1α-PPAR signaling pathway.

BACKGROUND: Adipose tissue is an important endocrine and energy-storage organ in organisms, and it plays a crucial role in the energy-metabolism balance. Previous studies have found that sex-linked dwarf (SLD) chickens generally have excessively high abdominal fat deposition during the growing period, which increases feeding costs. However, the underlying mechanism of this fat deposition during the growth of SLD chickens remains unknown. RESULTS: The Oil Red O staining showed that the lipid-droplet area of SLD chickens was larger than that of normal chickens in E15 and 14d. Consistently, TG content in the livers of SLD chickens was higher than that of normal chickens in E15 and 14d. Further, lower ΔΨm and lower ATP levels and higher MDA levels were observed in SLD chickens than normal chickens in both E15 and 14d. We also found that overexpression of GHR reduced the expression of genes related to lipid metabolism (AMPK, PGC1α, PPARγ, FAS, C/EBP) and oxidative phosphorylation (CYTB, CYTC, COX1, ATP), as well as reducing ΔΨm and ATP levels and increasing MDA levels. In addition, overexpression of GHR inhibited fat deposition in CPPAs, as measured by Oil Red O staining. On the contrary, knockdown of GHR had the opposite effects in vitro. CONCLUSIONS: In summary, we demonstrate that GHR promotes mitochondrial function and inhibits lipid peroxidation as well as fat deposition in vivo and in vitro. Therefore, GHR is essential for maintaining the stability of lipid metabolism and regulating mitochondrial function in chicken.

PGC-1α affects skeletal muscle and adipose tissue development by regulating mitochondrial biogenesis.

The discovery and interpretation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) protein in mitochondrial biogenesis, skeletal muscle and adipose tissue development has broad research prospects, so it is important to review the related studies of PGC-1α in detail and comprehensively. PGC-1α is a protein composed of 798 amino acids (aa) with a molecular weight of about 91 kDa. PGC-1α is involved in the operation of the respiratory chain by combining with deacetylase and phosphorylase to bind some nuclear receptors. In addition, PGC-1α affects skeletal muscle and adipose metabolism by regulating mitochondrial oxidative phosphorylation. Recently, new data suggest that regulating mitochondrial metabolism in adipose tissue may be an effective adjunct to the treatment of obesity. In addition, dietary resveratrol, which has an effective anti-obesity effect, has been shown to promote mitochondrial biosynthesis by activating AMPK/PGC-1α axis, as well as to regenerate muscle damaged by obesity. In this review, we combined previous studies to explore the latest studies, showing that PGC-1α can regulate mitochondrial biogenesis and is regulated by AMPK and SIRT1. Furthermore, PGC-1α is a favored protein, which not only regulates muscle fiber type, inhibits muscle atrophy, but also participates in browning of white adipose tissue (WAT) and regulates body heat production. So, we concluded that PGC-1α is a key gene in mitochondrial biogenesis and plays an important role in the regulation and regulation of mitochondrial biogenesis along with other genes involved in the process. Meanwhile, PGC-1α acts as a core metabolic regulator in adipose tissue and skeletal muscle. This review comprehensively summarizes a large number of research findings. First, the role of PGC-1α in mitochondrial biogenesis was clarified, and then the key role of PGC-1α in the development of skeletal muscle and adipose tissue was reevaluated. Furthermore, the role of PGC-1α in some human diseases was discussed. Finally, the role of PGC-1α as a major gene in poultry was pointed out, and the future research direction was proposed.

Construction and Analysis of Disuse Atrophy Model of the Gastrocnemius Muscle in Chicken.

Disuse muscle atrophy is identified as the physiological, biochemical, morphological, and functional changes during restricted movement, immobilization, or weightlessness. Although its internal mechanism has been extensively studied in mammals and was thought to be mainly related to oxidative stress, it was unclear whether it behaved consistently in non-mammals such as chickens. In this study, we tried to construct a disuse atrophy model of the gastrocnemius muscle in chickens by limb immobilization, and collected the gastrocnemius muscles of the fixed group and the control group for RNA sequencing. Through analysis of muscle loss, HE staining, immunohistochemistry, and oxidative stress level, we found that limb immobilization could lead to loss of muscle mass, decrease in muscle fiber diameter, decrease in the proportion of slow muscle fibers, and increase in the proportion of fast muscle fibers, and also cause elevated levels of oxidative stress. In addition, a total of 565 different expression genes (DEGs) were obtained by RNA sequencing, which was significantly enriched in the biological processes such as cell proliferation and apoptosis, reactive oxygen species metabolism, and fast and slow muscle fiber transformation, and it showed that the FOXO signaling pathway, closely related to muscle atrophy, was activated. In brief, we initially confirmed that limb immobilization could induce disuse atrophy of skeletal muscle, and oxidative stress was involved in the process of disuse muscle atrophy.

LncRNA SMARCD3-OT1 Promotes Muscle Hypertrophy and Fast-Twitch Fiber Transformation via Enhancing SMARCD3X4 Expression.

Long noncoding RNA (lncRNA) plays a crucial part in all kinds of life activities, especially in myogenesis. SMARCD3 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3) is a member of the SWI/SNF protein complex and was reported to be required for cell proliferation and myoblast differentiation. In this study, we identified a new lncRNA named SMARCD3-OT1 (SMARCD3overlappinglncRNA), which strongly regulated the development of myogenesis by improving the expression of SMARCD3X4 (SMARCD3transcripts4). We overexpressed and knockdown the expression of SMARCD3-OT1 and SMARCD3X4 to investigate their function on myoblast proliferation and differentiation. Cell experiments proved that SMARCD3-OT1 and SMARCD3X4 promoted myoblast proliferation through the CDKN1A pathway and improved differentiation of differentiated myoblasts through the MYOD pathway. Moreover, they upregulated the fast-twitch fiber-related genes and downregulated the slow-twitch fiber-related genes, which indicated that they facilitated the slow-twitch fiber to transform into the fast-twitch fiber. The animals' experiments supported the results above, demonstrating that SMARCD3-OT1 could induce muscle hypertrophy and fast-twitch fiber transformation. In conclusion, SMARCD3-OT1 can improve the expression of SMARCD3X4, thus inducing muscle hypertrophy. In addition, SMARCD3-OT1 can facilitate slow-twitch fibers to transform into fast-twitch fibers.

PPARGC1A Is a Moderator of Skeletal Muscle Development Regulated by miR-193b-3p.

Meat production performance is one of the most important factors in determining the economic value of poultry. Myofiber is the basic unit of skeletal muscle, and its physical and chemical properties determine the meat quality of livestock and poultry to a certain extent. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) as a transcriptional coactivator has been found to be widely involved in a series of biological processes. However, PPARGC1A is still poorly understood in chickens. In this manuscript, we reported that PPARGC1A was highly expressed in slow-twitch myofibers. PPARGC1A facilitated mitochondrial biogenesis and regulated skeletal muscle metabolism by mediating the flux of glycolysis and the TCA cycle. Gain- and loss-of-function analyses revealed that PPARGC1A promoted intramuscular fatty acid oxidation, drove the transformation of fast-twitch to slow-twitch myofibers, and increased chicken skeletal muscle mass. Mechanistically, the expression level of PPARGC1A is regulated by miR-193b-3p. Our findings help to understand the genetic regulation of skeletal muscle development and provide a molecular basis for further research on the antagonism of skeletal muscle development and fat deposition in chickens.

CircDCLRE1C Regulated Lipopolysaccharide-Induced Inflammatory Response and Apoptosis by Regulating miR-214b-3p/STAT3 Pathway in Macrophages.

The immune cell inflammation response is closely related to the occurrence of disease, and much evidence has shown that circular RNAs (circRNAs) play vital roles in the occurrence of disease. However, the biological function and regulatory mechanisms of circRNAs in the immune cell inflammation response remain poorly understood. In this study, we constructed an inflammatory model using lipopolysaccharide (LPS)-stimulated chicken macrophage lines (also known as HD11) to verify the function and mechanism of the novel circDCLRE1C (ID: gga_circ_0001674), which was significantly upregulated in spleen tissues infected by coccidia and the macrophage cells exposed to LPS. The results showed that circDCLRE1C aggravated LPS-induced inflammation and apoptosis in HD11 cells. Systemically, circDCLRE1C acted as a sponge for miR-214b-3p binding sites thereby regulating the expression of STAT3. The overexpression of miR-214b-3p rescued the pro-inflammatory effect of circDCLRE1C in HD11 cells stimulated with LPS, and rescued the high expression of STAT3. In conclusion, our study showed that circDCLRE1C could aggravate LPS-induced inflammation and apoptosis through competitive adsorption of miR-214b-3p, thereby increasing the expression of STAT3.

Single-cell RNA sequencing of preadipocytes reveals the cell fate heterogeneity induced by melatonin.

Obesity is a global epidemic health disorder and associated with several diseases. Body weight-reducing effects of melatonin have been reported; however, no investigation toward examining whether the beneficial effects of melatonin are associated with preadipocyte heterogeneity has been reported. In this study, we profiled 25 071 transcriptomes of normal and melatonin-treated preadipocytes using scRNA-seq. By tSNE analysis, we present a cellular-state landscape for melatonin-treated preadipocytes that covers multiple-cell subpopulations, defined as cluster 0 to cluster 13. Cluster 0 and cluster 1 were the largest components of normal and melatonin-treated preadipocytes, respectively. G0S2, an inhibitor of adipose triglyceride lipase (ATGL), was significantly upregulated in cluster 0 and downregulated in cluster 1. We redefined cluster 0 as the G0S2-positive cluster (G0S2+ ) and cluster 1 as the G0S2-negative cluster (G0S2- ). Through pseudotime analysis, the G0S2- cluster cell differentiation trajectory was divided into three major structures, that is, the prebranch, the lipid catabolism branch, and the cell fate 2 branch. In vitro, G0S2 knockdown enhanced the expression levels of ATGL, BAT markers and fatty acid oxidation-related genes, but inhibited C/EBPα and PPARγ expression. In vivo, knockdown of G0S2 reduced the body weight gain in high-fat-fed mice. The beneficial effects of the G0S2- cell cluster in promoting lipolysis and inhibiting adipogenesis are dependent on two major aspects: first, downregulation of the G0S2 gene in the G0S2- cluster, resulting in activation of ATGL, which is responsible for the bulk of triacylglycerol hydrolase activity; and second, upregulation of FABP4 in the G0S2- cluster, resulting in inhibition of PPARγ and further reducing adipogenesis.

ALDH1A1 Inhibits Chicken Preadipocytes' Proliferation and Differentiation via the PPARγ Pathway In Vitro and In Vivo.

ALDH1A1 (aldehyde dehydrogenase 1A1) is a crucial protein in retinoids' metabolism, and the lack of ALDH1A1 inhibits the fat deposition in mice. However, whether ALDH1A1 has a similar effect on chickens' fat-depot is still unknown. In this study, we investigate the role of ALDH1A1 in chickens' adipogenesis. The immortalized chicken preadipocyte 1 (ICP1) cell line and chicken primary preadipocytes isolated from abdominal fat were used to perform a series of experiments in vitro to elucidate the effects of ALDH1A1. In addition, lentivirus was used to verify the results of cell experiments in vivo. The data showed that overexpression of ALDH1A1 significantly weakened the proliferation of preadipocytes and suppressed the differentiation of preadipocytes through the PPARγ pathway, and the knockdown experiments had the opposite results. Moreover, chickens injected with overexpression lentivirus had higher abdominal fat percentage, a bigger size of lipid droplets, and higher triglyceride content in abdominal fat, and chickens injected with interfering lentivirus had the opposite situation. We proved that ALDH1A1 not only inhibited the proliferation and differentiation of chickens' preadipocytes in vitro, but also inhibited the fat-depot of chickens in vivo, which was completely opposite the function of ALDH1A1 in mice, indicating that ALDH1A1 may have a different mechanism that is still unknown.

Identification of a novel antisense RNA that regulates growth hormone receptor expression in chickens.

Natural antisense transcripts (NATs) are widely present in mammalian genomes and act as pivotal regulator molecules of gene expression. However, studies on NATs in the chicken are relatively rare. We identified a novel antisense transcript in the chicken, designated GHR-AS-EST, transcribed from the growth hormone receptor (GHR) locus, which encodes a well-known regulatory molecule of muscle development and fat deposition. GHR-AS-EST is predominantly expressed in the chicken liver and muscle tissues. GHR-AS-EST sequence conservation among vertebrates is weak. GHR-AS-EST forms an RNA-RNA duplex with GHBP to increase its stability, and regulates the expression of GHR sense transcripts at both the mRNA and protein levels. Further, GHR-AS-EST promotes cell proliferation by stimulating the expression of signaling factors in the JAK2/STAT pathway, and contributes to fat deposition via downregulating the expression of signaling factors in the JAK2/SOCS pathway in LMH hepatocellular carcinoma cells. We expect that the discovery of a NAT for a regulatory gene associated with cell proliferation and lipolysis will further our understanding of the molecular regulation of both muscle development and fat deposition.

Integrative Analyses of mRNA Expression Profile Reveal the Involvement of IGF2BP1 in Chicken Adipogenesis.

Excessive abdominal fat deposition is an issue with general concern in broiler production, especially for Chinese native chicken breeds. A high-fat diet (HFD) can induce body weight gained and excessive fat deposition, and genes and pathways participate in fat metabolism and adipogenesis would be influenced by HFD. In order to reveal the main genes and pathways involved in chicken abdominal fat deposition, we used HFD and normal diet (ND) to feed a Chinese native chicken breed, respectively. Results showed that HFD can increase abdominal fat deposition and induce adipocyte hypertrophy. Additionally, we used RNA-sequencing to identify the differentially expressed genes (DEGs) between HFD and ND chickens in liver and abdominal fat. By analyzed these DEGs, we found that the many DEGs were enriched in fat metabolism related pathways, such as peroxisome proliferator-activated receptor (PPAR) signaling, fat digestion and absorption, extracellular matrix (ECM)-receptor interaction, and steroid hormone biosynthesis. Notably, the expression of insulin-like growth factor II mRNA binding protein 1 (IGF2BP1), which is a binding protein of IGF2 mRNA, was found to be induced in liver and abdominal fat by HFD. Ectopic expression of IGF2BP1 in chicken liver-related cell line Leghorn strain M chicken hepatoma (LMH) cell revealed that IGF2BP1 can regulate the expression of genes associated with fatty acid metabolism. In chicken preadipocytes (ICP cell line), we found that IGF2BP1 can promote adipocyte proliferation and differentiation, and the lipid droplet content would be increased by overexpression of IGF2BP1. Taken together, this study provides new insights into understanding the genes and pathways involved in abdominal fat deposition of Chinese native broiler, and IGF2BP1 is an important candidate gene for the study of fat metabolism and adipogenesis in chicken.

[The cell cycle pathway regulates chicken abdominal fat deposition as revealed by transcriptome sequencing].

With the improvement of growth traits and feed conversion rate, the abdominal fat rate of Chinese local breeds of broilers has been increasing. Excessive abdominal fat deposition not only reduces the slaughter rate and disease resistance of broiler chickens, but also produces waste due to the difficulty of fat treatment. In order to study the regulatory genes and pathways involved in abdominal fat deposition of broilers, we used high-fat diets to feed the Xinghua Chicken, which is a Chinese local breed. Two weeks after feeding, we found that the abdominal fat weight and rate of broilers in the high-fat diet group increased significantly, and the diameter and area of abdominal fat cells also increased significantly. Transcriptome sequencing of abdominal fat and livers showed that the differentially expressed genes in the abdominal fat were mainly enriched in the cell cycle, peroxisome proliferator- activated receptor (PPAR) and extracellular matrix (ECM) receptor signaling pathways. The differentially expressed genes in livers were also significantly enriched in the cell cycle pathway, as well as in the steroid biosynthesis and PPAR signaling pathway. By analyzing the common differentially expressed genes in abdominal fat and liver tissues, we found that these genes were also enriched in cell cycle. Finally, we used the chicken LMH (chicken hepatoma cell) cell line and chicken ICP (immortalized chicken preadipocytes) cell line to do the in vitro validation assays. We used high-fat and common medium to culture the cells. The results showed that after 48 hours, the high-fat medium could significantly promote cell cycle and increase the number of cells in S phase. Additionally, qRT-PCR results showed that the high-fat medium could significantly promote the expression of genes related to cell cycle. In conclusion, we found that high-fat diets activate the cell cycle progression of chicken hepatocytes and preadipocytes, promote cell proliferation, and then increase abdominal fat deposition.

Characterization of microRNA and mRNA expression profiles in skin tissue between early-feathering and late-feathering chickens.

BACKGROUND: Early feathering and late feathering in chickens are sex-linked phenotypes, which have commercial application in the poultry industry for sexing chicks at hatch and have important impacts on performance traits. However, the genetic mechanism controlling feather development and feathering patterns is unclear. Here, miRNA and mRNA expression profiles in chicken wing skin tissues were analysed through high-throughput transcriptomic sequencing, aiming to understand the biological process of follicle development and the formation of different feathering phenotypes. RESULTS: Compared to the N1 group with no primary feathers extending out, 2893 genes and 31 miRNAs displayed significantly different expression in the F1 group with primary feathers longer than primary-covert feathers, and 1802 genes and 11 miRNAs in the L2 group displayed primary feathers shorter than primary-covert feathers. Only 201 altered genes and 3 altered miRNAs were identified between the N1 and L2 groups (fold change > 2, q value < 0.01). Both sequencing and qPCR tests revealed that PRLR was significantly decreased in the F1 and L2 groups compared to the N1 group, whereas SPEF2 was significantly decreased in the F1 group compared to the N1 or L2 group. Functional analysis revealed that the altered genes or targets of altered miRNAs were involved in multiple biological processes and pathways related to feather growth and development, such as the Wnt signalling pathway, the TGF-beta signalling pathway, the MAPK signalling pathway, epithelial cell differentiation, and limb development. Integrated analysis of miRNA and mRNA showed that 14 pairs of miRNA-mRNA negatively interacted in the process of feather formation. CONCLUSIONS: Transcriptomic sequencing of wing skin tissues revealed large changes in F1 vs. N1 and L2 vs. N1, but few changes in F1 vs. L2 for both miRNA and mRNA expression. PRLR might only contribute to follicle development, while SPEF2 was highly related to the growth rate of primary feathers or primary-covert feathers and could be responsible for early and late feather formation. Interactions between miR-1574-5p/NR2F, miR-365-5p/JAK3 and miR-365-5p/CDK6 played important roles in hair or feather formation. In all, our results provide novel evidence to understand the molecular regulation of follicle development and feathering phenotype.