Leveraging transcriptome and epigenome landscapes to infer regulatory networks during the onset of sexual maturation.

BACKGROUND: Despite sexual development being ubiquitous to vertebrates, the molecular mechanisms underpinning this fundamental transition remain largely undocumented in many organisms. We designed a time course experiment that successfully sampled the period when Atlantic salmon commence their trajectory towards sexual maturation. RESULTS: Through deep RNA sequencing, we discovered key genes and pathways associated with maturation in the pituitary-ovarian axis. Analyzing DNA methylomes revealed a bias towards hypermethylation in ovary that implicated maturation-related genes. Co-analysis of DNA methylome and gene expression changes revealed chromatin remodeling genes and key transcription factors were both significantly hypermethylated and upregulated in the ovary during the onset of maturation. We also observed changes in chromatin state landscapes that were strongly correlated with fundamental remodeling of gene expression in liver. Finally, a multiomic integrated analysis revealed regulatory networks and identified hub genes including TRIM25 gene (encoding the estrogen-responsive finger protein) as a putative key regulator in the pituitary that underwent a 60-fold change in connectivity during the transition to maturation. CONCLUSION: The study successfully documented transcriptome and epigenome changes that involved key genes and pathways acting in the pituitary - ovarian axis. Using a Systems Biology approach, we identified hub genes and their associated networks deemed crucial for onset of maturation. The results provide a comprehensive view of the spatiotemporal changes involved in a complex trait and opens the door to future efforts aiming to manipulate puberty in an economically important aquaculture species.

Chromatin accessibility and regulatory vocabulary across indicine cattle tissues.

BACKGROUND: Spatiotemporal changes in the chromatin accessibility landscape are essential to cell differentiation, development, health, and disease. The quest of identifying regulatory elements in open chromatin regions across different tissues and developmental stages is led by large international collaborative efforts mostly focusing on model organisms, such as ENCODE. Recently, the Functional Annotation of Animal Genomes (FAANG) has been established to unravel the regulatory elements in non-model organisms, including cattle. Now, we can transition from prediction to validation by experimentally identifying the regulatory elements in tropical indicine cattle. The identification of regulatory elements, their annotation and comparison with the taurine counterpart, holds high promise to link regulatory regions to adaptability traits and improve animal productivity and welfare. RESULTS: We generate open chromatin profiles for liver, muscle, and hypothalamus of indicine cattle through ATAC-seq. Using robust methods for motif discovery, motif enrichment and transcription factor binding sites, we identify potential master regulators of the epigenomic profile in these three tissues, namely HNF4, MEF2, and SOX factors, respectively. Integration with transcriptomic data allows us to confirm some of their target genes. Finally, by comparing our results with Bos taurus data we identify potential indicine-specific open chromatin regions and overlaps with indicine selective sweeps. CONCLUSIONS: Our findings provide insights into the identification and analysis of regulatory elements in non-model organisms, the evolution of regulatory elements within two cattle subspecies as well as having an immediate impact on the animal genetics community in particular for a relevant productive species such as tropical cattle.

Cloning and tissue distribution of novel splice variants of the ovine ghrelin gene.

BACKGROUND: The ghrelin axis is involved in the regulation of metabolism, energy balance, and the immune, cardiovascular and reproductive systems. The manipulation of this axis has potential for improving economically valuable traits in production animals, and polymorphisms in the ghrelin (GHRL) and ghrelin receptor (GHSR) genes have been associated with growth and carcass traits. Here we investigate the structure and expression of the ghrelin gene (GHRL) in sheep, Ovis aries. RESULTS: We identify two ghrelin mRNA isoforms, which we have designated Δex2 preproghrelin and Δex2,3 preproghrelin. Expression of Δex2,3 preproghrelin is likely to be restricted to ruminants, and would encode truncated ghrelin and a novel C-terminal peptide. Both Δex2 preproghrelin and canonical preproghrelin mRNA isoforms were expressed in a range of tissues. Expression of the Δex2,3 preproghrelin isoform, however, was restricted to white blood cells (WBC; where the wild-type preproghrelin isoform is not co-expressed), and gastrointestinal tissues. Expression of Δex2 preproghrelin and Δex2,3 preproghrelin mRNA was elevated in white blood cells in response to parasitic worm (helminth) infection in genetically susceptible sheep, but not in resistant sheep. CONCLUSIONS: The restricted expression of the novel preproghrelin variants and their distinct WBC expression pattern during parasite infection may indicate a novel link between the ghrelin axis and metabolic and immune function in ruminants.

The sheep genome illuminates biology of the rumen and lipid metabolism.

Sheep (Ovis aries) are a major source of meat, milk, and fiber in the form of wool and represent a distinct class of animals that have a specialized digestive organ, the rumen, that carries out the initial digestion of plant material. We have developed and analyzed a high-quality reference sheep genome and transcriptomes from 40 different tissues. We identified highly expressed genes encoding keratin cross-linking proteins associated with rumen evolution. We also identified genes involved in lipid metabolism that had been amplified and/or had altered tissue expression patterns. This may be in response to changes in the barrier lipids of the skin, an interaction between lipid metabolism and wool synthesis, and an increased role of volatile fatty acids in ruminants compared with nonruminant animals.

RNF14 is a regulator of mitochondrial and immune function in muscle.

BACKGROUND: Muscle development and remodelling, mitochondrial physiology and inflammation are thought to be inter-related and to have implications for metabolism in both health and disease. However, our understanding of their molecular control is incomplete. RESULTS: In this study we have confirmed that the ring finger 14 protein (RNF14), a poorly understood transcriptional regulator, influences the expression of both mitochondrial and immune-related genes. The prediction was based on a combination of network connectivity and differential connectivity in cattle (a non-model organism) and mice data sets, with a focus on skeletal muscle. They assigned similar probability to mammalian RNF14 playing a regulatory role in mitochondrial and immune gene expression. To try and resolve this apparent ambiguity we performed a genome-wide microarray expression analysis on mouse C2C12 myoblasts transiently transfected with two Rnf14 transcript variants that encode 2 naturally occurring but different RNF14 protein isoforms. The effect of both constructs was significantly different to the control samples (untransfected cells and cells transfected with an empty vector). Cluster analyses revealed that transfection with the two Rnf14 constructs yielded discrete expression signatures from each other, but in both cases a substantial set of genes annotated as encoding proteins related to immune function were perturbed. These included cytokines and interferon regulatory factors. Additionally, transfection of the longer transcript variant 1 coordinately increased the expression of 12 (of the total 13) mitochondrial proteins encoded by the mitochondrial genome, 3 of which were significant in isolated pair-wise comparisons (Mt-coxII, Mt-nd2 and mt-nd4l). This apparent additional mitochondrial function may be attributable to the RWD protein domain that is present only in the longer RNF14 isoform. CONCLUSIONS: RNF14 influences the expression of both mitochondrial and immune related genes in a skeletal muscle context, and has likely implications for the inter-relationship between bioenergetic status and inflammation.

Proteomic analysis of the abomasal mucosal response following infection by the nematode, Haemonchus contortus, in genetically resistant and susceptible sheep.

Sheep have a variable ability to resist gastrointestinal nematode infection, but the key factors mediating this response are poorly defined. Here we report the first large-scale application of quantitative proteomic technologies to define proteins that are differentially abundant between sheep selectively bred to have an enhanced (resistant) or reduced (susceptible) ability to eliminate nematodes. Samples were collected from the abomasal mucosa three days after experimental challenge with the nematode, Haemonchus contortus. This timing reflects the initial interaction of host and parasite, and the tissue represents the immediate interface. We identified and quantified more than 4400 unique proteins, of which 158 proteins showed >1.5 fold difference between the resistant and susceptible sheep. Trefoil factor 2, a member of RAS oncogene family (RAP1A) and ring finger protein 126 were amongst the proteins found to be highly abundant in the abomasal surface of resistant sheep, whereas adenosine deaminase and the gastrokine-3 like precursor were found at higher levels in susceptible sheep. Construction of gut proteome interaction networks identified mitochondrial function and energetic partitioning as important components of an effective nematode eliminating response. The differentially abundant proteins may be useful targets for phenotypic tests that aim to identify sheep with an enhanced ability to resist nematode infection.

Divergent ghrelin expression patterns in sheep genetically resistant or susceptible to gastrointestinal nematodes.

Gastrointestinal nematodes are a major problem for pastoral ruminant production systems. This problem could be reduced by the application of breeding strategies that select for nematode resistant sheep, but no suitable molecular markers are available. Research selection flocks containing lines that are resistant (R) or susceptible (S) to gastrointestinal nematodes provide an excellent resource for discovering selectable markers, and for studying the underlying mechanisms of an effective anti-nematode response. In this study we have used a combination of quantitative real time PCR assays and ELISA to determine if nematode challenge impacts on the expression of the satiety-regulating hormone ghrelin. The expression responses were then compared between the selection flock R and S lines. The results show that the basal levels of ghrelin in plasma were greater than 2-fold higher in nematode naïve S line sheep. Three days after a primary nematode challenge divergent ghrelin expression patterns were observed between the selection lines, with levels increasing in R sheep while decreasing in S sheep. After a secondary challenge this trend was repeated, but following a third challenge ghrelin expression levels rose in both R and S sheep, by which time the S animals had acquired an effective immune response to the nematodes, as measured by a significant reduction in faecal egg output. Importantly, this phenomenon was observed in gene expression studies in gut tissues and also in ELISA measurements of ghrelin peptide levels in plasma. A regression analysis showed that ghrelin transcript expression in the gut accounted for >40% of the variation in faecal egg count measured following Haemonchus or Trichostrongylus infection. We therefore hypothesise that the direction of ghrelin expression (up or down) immediately following nematode exposure may play an important role in regulating the differing anti-nematode responses that occur in the R and S lines. Such differences identify ghrelin as a previously unrecognized factor influencing the acquisition of immunity to nematodes.

Identification of a dieldrin resistance-associated mutation in Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae), is a major vector of tick fever organisms affecting cattle in many parts of the world, including Australia, Africa, and South America. Control of the southern cattle tick through acaricide use is an important approach in disease management. Resistance has emerged to many of the acaricides currently and previously used, including the cyclodienes. Although cyclodiene resistance mechanisms have been characterized in many insect species, this report is the first to identify mutations associated with dieldrin resistance in the cattle tick. A novel two base pair mutation in the GABA-gated chloride channel gene has been identified at position 868-9 and causes a codon change from threonine to leucine. Analysis of a small number of field-collected samples resistant to dieldrin shows this mutation has been maintained without selection pressure since the withdrawal of dieldrin in Australia > 20 yr ago. The mutation is not found in other laboratory-maintained strains of R. microplus that were subject to selection pressure with various acaricides.

Gene expression profiles of BMP4, FGF10 and cognate inhibitors, in the skin of foetal Merino sheep, at the time of secondary follicle branching.

The high concentration of secondary branched follicles is a distinctive feature of the Merino sheep. These follicles initiate from 100 days of gestation. Here, we report a transition in abundance of the BMP4 and FGF10 morphogens occurring at this time. At 103 days of gestation, FGF10 gene expression dropped steadily from maximal levels, in a trend that continued until day 143. Conversely, from day 105, BMP4 transcript levels rapidly increased to maximal levels that were maintained until 131 days, before declining. This profile closely matches reported changes in branched follicle numbers, which peak in density at day 134. SPRY4, a known regulator of FGF10, increased to maximal levels concomitant with the fall in FGF10, suggesting a relationship. Levels of the BMP4 inhibitor NOG matched the initial rise of BMP4, with a fivefold spike at 108 days; but consistent with the rise in BMP4, this high level was not sustained.

Gastrointestinal nematode challenge induces some conserved gene expression changes in the gut mucosa of genetically resistant sheep.

Sheep have a varying ability to resist infection with gastrointestinal nematodes. This ability is due in part to genetic differences that exist between individuals. In order to define these differences we have used real-time PCR to quantify gene expression responses in the gut mucosal surface of genetically resistant and susceptible sheep, following a nematode challenge. Expression profiles were determined in response to two different nematode species, Haemonchus contortus and Trichostrongylus colubriformis, and in divergent sheep originating from two different genetic backgrounds. Results show that the response generated differs between resistant and susceptible animals and is further impacted by the origin of the sheep and nematode species used for challenge. However, some conserved features of a response mounted by a resistant or a susceptible animal were identified. Genes found to be more abundantly expressed in resistant animals include markers of an early inflammatory response, several Toll-like receptors (TLR2, 4, 9) and free radical producing genes (DUOX1 and NOS2A). Conversely, genes differentiating susceptible animals indicate a prolonged response and development of a chronic inflammatory state, characterised by elevated expression of members of the NF-kappabeta signalling pathway (IKBKB and NFKBIA) together with delayed expression of regulatory markers such as IL2RA (CD25), IL10 and TGFbeta2. While multiple nematode response pathways were identified, the identification of conserved aspects of the response which associate with resistance provides evidence that alternative nematode control strategies, such as breeding for resistant animals, may be feasible.

Identification and expression of Toll-like receptors 1-10 in selected bovine and ovine tissues.

Members of the Toll-like receptor (TLR) family are vital to immune function through the sensing of pathogenic agents and initiation of an appropriate immune response. More specifically, tissue and cell specific TLR expression patterns have been correlated with the ability to respond to various pathogenic challenges. Bovine sequence exists for 4 of the 10 human TLR Reference Sequences and no ovine TLR sequence has been reported. The main goal of this study was to determine if homologues of human TLRs 1-10 exist within the cattle and sheep. Subsequent to this, quantitative real time PCR assays were to be developed to produce transcript expression profiles in cattle skin and sheep gut-associated lymphoid tissue, as these epithelial tissues are the primary sites of host/pathogen interactions for numerous pathogens. Our findings show that homologues of human TLRs 1-10 do indeed exist within both cattle and sheep, with respective bovine and ovine homologues sharing at least 95% nucleotide sequence identity and 83-90% identity to the corresponding human Reference Sequences. Conservation of the amino acid sequence between homologous ruminant and human TLRs ranged between 84 and 97%. Quantitative real time PCR (qPCR) assays confirmed expression of all 10 TLRs within ovine jejunum, Peyer's patch and mesenteric lymph nodes. While in bovine skin all TLRs apart from TLR6 were detected. The most abundant TLR transcripts within the ovine jejunum were TLRs 3, 5 and 6, while TLRs 6, 7 and 10 were abundant in both ovine Peyer's patch and mesenteric lymph node. In bovine skin TLRs 2 and 7 were most abundant. In all tissues tested TLR4 expression was at the lower limit of detection.

A novel antigen from Anaplasma marginale: characterization, expression and preliminary evaluation of the recombinant protein.

Through a process of protein fractionation and vaccination we previously identified four native antigens that confer a degree of protection against challenge with Anaplasma marginale. One of these, Ana 29 has been successfully cloned and sequenced using degenerate primers designed to N-terminal and internal peptide sequences. The full-length gene codes for a protein with a theoretical molecular weight of 27 kDa and pI 8.6. The sequence is highly conserved, showing 99% identity between two Australian and an American isolate of A. marginale. The gene sequences from these isolates also share 99% identity with the strain of Anaplasma centrale used in the commercial Australian vaccine. Protein prediction algorithms suggest the native protein is an integral membrane protein. This protein has been over-expressed and purified from Escherichia coli and used in vaccination trials in cattle using two adjuvants. The initial results from the trial show a significant level of protection was obtained with one adjuvant; in comparison, the second adjuvant slightly aggravated the disease. Preliminary data suggests a good correlation between the induction of an IgG2 response and protection.

Ex vivo profiling of CD8+-T-cell responses to human cytomegalovirus reveals broad and multispecific reactivities in healthy virus carriers.

Human cytomegalovirus (HCMV) can establish both nonproductive (latent) and productive (lytic) infections. Many of the proteins expressed during these phases of infection could be expected to be targets of the immune response; however, much of our understanding of the CD8(+)-T-cell response to HCMV is mainly based on the pp65 antigen. Very little is known about T-cell control over other antigens expressed during the different stages of virus infection; this imbalance in our understanding undermines the importance of these antigens in several aspects of HCMV disease pathogenesis. In the present study, an efficient and rapid strategy based on predictive bioinformatics and ex vivo functional T-cell assays was adopted to profile CD8(+)-T-cell responses to a large panel of HCMV antigens expressed during different phases of replication. These studies revealed that CD8(+)-T-cell responses to HCMV often contained multiple antigen-specific reactivities, which were not just constrained to the previously identified pp65 or IE-1 antigens. Unexpectedly, a number of viral proteins including structural, early/late antigens and HCMV-encoded immunomodulators (pp28, pp50, gH, gB, US2, US3, US6, and UL18) were also identified as potential targets for HCMV-specific CD8(+)-T-cell immunity. Based on this extensive analysis, numerous novel HCMV peptide epitopes and their HLA-restricting determinants recognized by these T cells have been defined. These observations contrast with previous findings that viral interference with the antigen-processing pathway during lytic infection would render immediate-early and early/late proteins less immunogenic. This work strongly suggests that successful HCMV-specific immune control in healthy virus carriers is dependent on a strong T-cell response towards a broad repertoire of antigens.