Identification of genes differentially expressed during prenatal development of skeletal muscle in two pig breeds differing in muscularity

<p>Abstract</p> <p>Background</p> <p>Postnatal muscle growth is largely depending on the number and size of muscle fibers. The number of myofibers and to a large extent their metabolic and contractile properties, which also influence their size, are determined prenatally during the process of myogenesis. Hence identification of genes and their networks governing prenatal development of skeletal muscles will provide insight into the control of muscle growth and facilitate finding the source of its variation. So far most of the genes involved in myogenesis were identified by <it>in vitro </it>studies using gene targeting and transgenesis. Profiling of transcriptome changes during the myogenesis <it>in vivo </it>promises to obtain a more complete picture. In order to address this, we performed transcriptome profiling of prenatal skeletal muscle using differential display RT-PCR as on open system with the potential to detect novel transcripts. Seven key stages of myogenesis (days 14, 21, 35, 49, 63, 77 and 91 <it>post conception</it>) were studied in two breeds, Pietrain and Duroc, differing markedly in muscularity and muscle structure.</p> <p>Results</p> <p>Eighty prominent cDNA fragments were sequenced, 43 showing stage-associated and 37 showing breed-associated differences in the expression, respectively. Out of the resulting 85 unique expressed sequence tags, EST, 52 could be assigned to known genes. The most frequent functional categories represented genes encoding myofibrillar proteins (8), genes involved in cell adhesion, cell-cell signaling and extracellular matrix synthesis/remodeling (8), genes regulating gene expression (8), and metabolism genes (8). Some of the EST that showed no identity to any known transcripts in the databases are located in introns of known genes and most likely represent novel exons (e.g. <it>HMGA2</it>). Expression of thirteen transcripts along with five reference genes was further analyzed by means of real-time quantitative PCR. Nine of the target transcripts showed higher than twofold differences in the expression between the two breeds (<it>GATA3</it>, <it>HMGA2</it>, <it>NRAP</it>, <it>SMC6L1</it>, <it>SPP1</it>, <it>RAB6IP2, TJP1 </it>and two EST).</p> <p>Conclusion</p> <p>The present study revealed several genes and novel transcripts not previously associated with myogenesis and expands our knowledge of genetic factors operating during myogenesis. Genes that exhibited differences between the divergent breeds represent candidate genes for muscle growth and structure.</p

Dual effect of a single nucleotide polymorphism in the first intron of the porcine Secreted phosphoprotein 1 gene: allele-specific binding of C/EBP beta and activation of aberrant splicing

<p>Abstract</p> <p>Background</p> <p>Secreted phosphoprotein 1 (SPP1 or Osteopontin, OPN) is a multifunctional matricellular glycoprotein involved in development and regeneration of skeletal muscle. Previously, we have demonstrated that porcine <it>SPP1 </it>shows breed-related differential mRNA expression during myogenesis. With the aim to identify putative contributing <it>cis</it>-regulatory DNA variation we resequenced the 5' upstream region of the gene in the respective breeds Pietrain and Duroc. We found two single nucleotide polymorphisms (SNP; [GenBank:<ext-link ext-link-id="M84121" ext-link-type="gen">M84121</ext-link>]: g.1804C>T and g.3836A>G). We focused our investigation on the SNP g.3836A>G, because <it>in silico </it>analysis and knowledge about the regulation of <it>SPP1 </it>suggested an effect of this SNP on a CCAAT/enhancer binding protein beta (C/EBPβ) responsive transcriptional enhancer.</p> <p>Results</p> <p>Using electrophoretic mobility shift assay we demonstrated that, similar to human <it>SPP1</it>, the 3' terminal end of the first intron of porcine <it>SPP1 </it>harbors a C/EBPβ binding site and showed that this binding site is negatively affected by the mutant G allele. Genotyping of 48 fetuses per breed revealed that the G allele segregated exclusively in Duroc fetuses with a frequency of 57 percent. Using real-time quantitative PCR we showed that, consistent with its negative effect on a transcriptional enhancer element, the G allele tends to decrease mRNA abundance of <it>SPP1 </it>in the fetal <it>musculus longissimus dorsi </it>(~1.3 fold; P ≥ 0.1).</p> <p>Moreover, we showed that the SNP g.3836A>G leads to ubiquitous aberrant splicing of the first intron by generating a <it>de novo </it>and activating a cryptic splice acceptor site. Aberrantly spliced transcripts comprise about half of the <it>SPP1 </it>messages expressed by the G allele. Both aberrant splice variants differ from the native transcript by insertions in the leader sequences which do not change the reading frame of <it>SPP1</it>.</p> <p>Conclusion</p> <p>At the 3' terminal end of the first intron of the porcine <it>SPP1 </it>we identified a unique, dually functional SNP g.3836A>G. This SNP affects the function of the <it>SPP1 </it>gene at the DNA level by affecting a C/EBPβ binding site and at the RNA level by activating aberrant splicing of the first intron, and thus represents an interesting DNA-marker to study phenotypic effects of <it>SPP1 </it>DNA-variation.</p

Host-Microbiota Interactions in Ileum and Caecum of Pigs Divergent in Feed Efficiency Contribute to Nutrient Utilization

peer-reviewedThe composition of the intestinal microbiota plays an important role in the digestion and utilization of nutrients and for gut health. Low-fiber diets stimulate digestion and absorption processes, predominantly in the upper region of the gastrointestinal tract, thereby increasing the conversion of feed into body weight. As a consequence, the chemical composition of digesta after duodenal and jejunal absorption processes and passage has a limited complexity affecting colonization and molecular profiles of enterocytes in the hind gut. To decipher ileal and caecal microbial ecosystems and host transcriptional profiles that are beneficial for effective use of the remaining nutrients, pigs differing in feeding efficiency were studied. Biological functions that were consistently enriched at both the gene and microbiota levels comprise immunity-related processes, which ensure the integrity of the gastrointestinal tract. In addition, the differential abundance of certain genera, including Rothia, Subdoligranulu, Leeia and Cellulosilyticum, reflects the establishment of a microbial profile that supports the digestion of endogenously indigestible dietary components in highly feed-efficient pigs. Overall, the results indicate the potential to promote these beneficial functions and further improve feed efficiency through manipulation of dietary and probiotic strategies

Association of HPA axis-related genetic variation with stress reactivity and aggressive behaviour in pigs

<p>Abstract</p> <p>Background</p> <p>Stress, elicited for example by aggressive interactions, has negative effects on various biological functions including immune defence, reproduction, growth, and, in livestock, on product quality. Stress response and aggressiveness are mutually interrelated and show large interindividual variation, partly attributable to genetic factors. In the pig little is known about the molecular-genetic background of the variation in stress responsiveness and aggressiveness. To identify candidate genes we analyzed association of DNA markers in each of ten genes (<it>CRH </it>g.233C>T, <it>CRHR1 </it>c.*866_867insA, <it>CRHBP </it>c.51G>A, <it>POMC </it>c.293_298del, <it>MC2R </it>c.306T>G, <it>NR3C1 </it>c.*2122A>G, <it>AVP </it>c.207A>G, <it>AVPR1B </it>c.1084A>G, <it>UCN </it>g.1329T>C, <it>CRHR2 </it>c.*13T>C) related to the hypothalamic-pituitary-adrenocortical (HPA) axis, one of the main stress-response systems, with various stress- and aggression-related parameters at slaughter. These parameters were: physiological measures of the stress response (plasma concentrations of cortisol, creatine kinase, glucose, and lactate), adrenal weight (which is a parameter reflecting activity of the central branch of the HPA axis over time) and aggressive behaviour (measured by means of lesion scoring) in the context of psychosocial stress of mixing individuals with different aggressive temperament.</p> <p>Results</p> <p>The SNP <it>NR3C1 </it>c.*2122A>G showed association with cortisol concentration (p = 0.024), adrenal weight (p = 0.003) and aggressive behaviour (front lesion score, p = 0.012; total lesion score p = 0.045). The SNP <it>AVPR1B </it>c.1084A>G showed a highly significant association with aggressive behaviour (middle lesion score, p = 0.007; total lesion score p = 0.003). The SNP <it>UCN </it>g.1329T>C showed association with adrenal weight (p = 0.019) and aggressive behaviour (front lesion score, p = 0.029). The SNP <it>CRH </it>g.233C>T showed a significant association with glucose concentration (p = 0.002), and the polymorphisms <it>POMC </it>c.293_298del and <it>MC2R </it>c.306T>G with adrenal weight (p = 0.027 and p < 0.0001 respectively).</p> <p>Conclusions</p> <p>The multiple and consistent associations shown by SNP in <it>NR3C1 </it>and <it>AVPR1B </it>provide convincing evidence for genuine effects of their DNA sequence variation on stress responsiveness and aggressive behaviour. Identification of the causal functional molecular polymorphisms would not only provide markers useful for pig breeding but also insight into the molecular bases of the stress response and aggressive behaviour in general.</p

Identification and Functional Characterization of Cis-Regulatory Elements Controlling Expression of the Porcine ADRB2 Gene

The beta-2 adrenergic receptor (beta-2 AR) modulates metabolic processes in skeletal muscle, liver, and adipose tissue in response to catecholamine stimulation. We showed previously that expression of the porcine beta-2 AR gene (ADRB2) is affected by cis-regulatory polymorphisms. These are most likely responsible for the association of ADRB2 with economically relevant muscle-related traits in pigs. The present study focused on characterization of promoter elements involved in basal transcriptional regulation of the porcine ADRB2 in different cell types to aid identification of its cis-regulatory polymorphisms. Based on in silico analysis, luciferase reporter gene assays and gel shift assays were performed using COS-7, HepG2, C2C12, and 3T3-L1 cells. Deletion mapping of the 5´ flanking region (-1324 to +33) of ADRB2 revealed the region between -307 and -269 to be the minimal promoter, including regulatory elements essential for the basal transcriptional activity in all four tested cell types. Directly upstream (-400 to -323) we identified an important enhancer element required for maximal promoter activity. In silico analysis and gel shift assays revealed that this GC-rich element harbors two evolutionarily conserved binding sites of Sp1, a constitutive transcriptional activator. Significant transcriptional activation of the porcine ADRB2 promoter was demonstrated by overexpression of Sp1. Our results demonstrate, for the first time, an important role of Sp1 and of the responsive enhancer element in the regulation of ADRB2 expression. Polymorphisms located in this domain of the porcine ADRB2 promoter represent candidate causal cis-regulatory variants

Elucidating Molecular Networks That Either Affect or Respond to Plasma Cortisol Concentration in Target Tissues of Liver and Muscle

Cortisol is a steroid hormone with important roles in regulating immune and metabolic functions and organismal responses to external stimuli are mediated by the glucocorticoid system. Dysregulation of the afferent and efferent axis of glucocorticoid signaling have adverse effects on growth, health status, and well-being. Glucocorticoid secretion and signaling show large interindividual variation that has a considerable genetic component; however, little is known about the underlying genetic variants. Here, we used trait-correlated expression analysis, screening for expression quantitative trait loci (eQTL), genome-wide association (GWA) studies, and causality modeling to identify candidate genes in porcine liver and muscle that affect or respond to plasma cortisol levels. Through trait-correlated expression, we characterized transcript activities in many biological functions in liver and muscle. Candidates from the list of trait-correlated expressed genes were narrowed using only those genes with an eQTL, and these were further prioritized by determining whether their expression was predicted to be related to variation in plasma cortisol levels. Using network edge orienting (NEO), a causality modeling algorithm, 26 of 990 candidates in liver were predicted to affect and 70 to respond to plasma cortisol levels. Of 593 candidates in muscle that were correlated with cortisol levels and were regulated by eQTL, 2 and 25 were predicted as effective and responsive, respectively, to plasma cortisol levels. Comprehensive data integration has helped to elucidate the complex molecular networks contributing to cortisol levels and thus its subsequent metabolic effects. The discrimination of up- and downstream effects of transcripts affecting or responding to plasma cortisol concentrations improves the understanding of the biology of complex traits related to growth, health, and well-being

Genetic variants of major genes contributing to phosphate and calcium homeostasis and their association with serum parameters in pigs

Calcium and phosphorus are irreplaceable components of life. Tracking the fate of calcium and phosphorus in organisms deserves high attention due to their relevance in bone metabolism and subsequently animal health. Indeed, bone serves as reservoir for calcium and phosphorus, whose formation and resorption follow specific molecular routes including hormones, receptors, and transcription factors. The objective of the study was to analyze the genetic variation of major components driving mineral utilization such as calcitonin receptor, calcium sensing receptor, fibroblast growth factor 23 (FGF23), parathyroid hormone receptor, osteopontin, stanniocalcin 1, RAF-type zinc finger domain containing 1 (TRAFD1), and vitamin D receptor. A German Landrace pig population (n = 360) was used to perform an association analysis between selected single nucleotide polymorphisms (SNP) and relevant serum parameters (calcium, phosphorus, calcium/phosphorus ratio, alkaline phosphatase). Analyzed SNPs in FGF23 (rs710498025) and TRAFD1 (rs345195312) were significantly (p ≤ 0.05) associated with the serum calcium/phosphorus ratio and serum phosphorus levels, respectively. This might represent a modulation of the homeostatic balance between calcium and phosphorus. Furthermore, TRAFD1 is known to be involved in skeletal disorders which emphasize its link to phosphorus utilization and immune system. However, none of the analyzed genetic variants of these major regulators of phosphate and calcium homeostasis showed significant associations after correction for multiple testing (q value &gt; 0.05). Thus, minor contributors as well as unknown and yet to be elucidated regulators of mineral homeostasis need to be characterized towards the implementation of improved phosphorus efficiency in pig breeding programs

A naturally hypersensitive glucocorticoid receptor elicits a compensatory reduction of hypothalamus–pituitary–adrenal axis activity early in ontogeny

We comprehensively characterized the effects of a unique natural gain-of-function mutation in the glucocorticoid receptor (GR), GRAla610Val, in domestic pigs to expand current knowledge of the phenotypic consequences of GR hypersensitivity. Cortisol levels were consistently reduced in one-week-old piglets, at weaning and in peripubertal age, probably due to a reduced adrenal capacity to produce glucocorticoids (GC), which was indicated by an adrenocortical thinning in GRAla610Val carriers. Adrenocorticotrophic hormone (ACTH) levels were significantly reduced in one-week-old piglets only. Expression analyses in peripubertal age revealed significant downregulation of hypothalamic expression of CRH and AVP, the latter only in females, and upregulation of hepatic expression of SERPINA6, by GRAla610Val. Transcriptional repression of proinflammatory genes in peripheral blood mononuclear cells (PBMCs) from GRAla610Val carriers was more sensitive to dexamethasone treatment ex vivo. However, no significant effects on growth, body composition, blood chemistry or cell counts were observed under baseline conditions. These results suggest that GRAla610Val-induced GR hypersensitivity elicits a compensatory reduction in endogenous, bioactive glucocorticoid levels via readjustment of the hypothalamus–pituitary–adrenal (HPA) axis early in ontogeny to maintain an adequate response, but carriers are more sensitive to exogenous GC. Therefore, GRAla610Val pigs represent a valuable animal model to explore GR-mediated mechanisms of HPA axis regulation and responses to glucocorticoid-based drugs