Coding complete genome of a strain of ovine herpesvirus-2 associated with a clinical case of malignant catarrhal fever in a domestic lamb.

Ovine herpesvirus-2 causes sheep-associated malignant catarrhal fever, a fatal disease of ruminants and pigs. The virus is carried by sheep, and infection is typically subclinical. Here, we report the coding complete genome sequence of a strain of OvHV-2 obtained from a clinically affected domestic lamb.

A DNA Regulatory Element Haplotype at Zinc Finger Genes Is Associated with Host Resilience to Small Ruminant Lentivirus in Two Sheep Populations.

Small ruminant lentivirus (SRLV) causes Maedi-Visna or Ovine Progressive Pneumonia in sheep and creates insidious livestock production losses. This retrovirus is closely related to human immunodeficiency virus and currently has no vaccines or cure. Genetic marker assisted selection for sheep disease resiliency presents an attractive management solution. Previously, we identified a region containing a cluster of zinc finger genes that had association with ovine SRLV proviral concentration. Trait-association analysis validated a small insertion/deletion variant near ZNF389 (rs397514112) in multiple sheep breeds. In the current study, 543 sheep from two distinct populations were genotyped at 34 additional variants for fine mapping of the regulatory elements within this locus. Variants were selected based on ChIP-seq annotation data from sheep alveolar macrophages that defined active cis-regulatory elements predicted to influence zinc finger gene expression. We present a haplotype block of variants within regulatory elements that have improved associations and larger effect sizes (up to 4.7-fold genotypic difference in proviral concentration) than the previously validated ZNF389 deletion marker. Hypotheses for the underlying causal mutation or mutations are presented based on changes to in silico transcription factor binding sites. These variants offer alternative markers for selective breeding and are targets for future functional mutation assays.

Genes involved in immune, gene translation and chromatin organization pathways associated with Mycoplasma ovipneumoniae presence in nasal secretions of domestic sheep.

Mycoplasma ovipneumoniae contributes to polymicrobial pneumonia in domestic sheep. Elucidation of host genetic influences of M. ovipneumoniae nasal detection has the potential to reduce the incidence of polymicrobial pneumonia in sheep through implementation of selective breeding strategies. Nasal mucosal secretions were collected from 647 sheep from a large US sheep flock. Ewes of three breeds (Polypay n = 222, Rambouillet n = 321, and Suffolk n = 104) ranging in age from one to seven years, were sampled at three different times in the production cycle (February, April, and September/October) over four years (2015 to 2018). The presence and DNA copy number of M. ovipneumoniae was determined using a newly developed species-specific qPCR. Breed (P<0.001), age (P<0.024), sampling time (P<0.001), and year (P<0.001) of collection affected log10 transformed M. ovipneumoniae DNA copy number, where Rambouillet had the lowest (P<0.0001) compared with both Polypay and Suffolk demonstrating a possible genetic component to detection. Samples from yearlings, April, and 2018 had the highest (P<0.046) detected DNA copy number mean. Sheep genomic DNA was genotyped with the Illumina OvineHD BeadChip. Principal component analysis identified most of the variation in the dataset was associated with breed. Therefore, genome wide association analysis was conducted with a mixed model (EMMAX), with principal components 1 to 6 as fixed and a kinship matrix as random effects. Genome-wide significant (P<9x10-8) SNPs were identified on chromosomes 6 and 7 in the all-breed analysis. Individual breed analysis had genome-wide significant (P<9x10-8) SNPs on chromosomes 3, 4, 7, 9, 10, 15, 17, and 22. Annotated genes near these SNPs are part of immune (ANAPC7, CUL5, TMEM229B, PTPN13), gene translation (PIWIL4), and chromatin organization (KDM2B) pathways. Immune genes are expected to have increased expression when leukocytes encounter M. ovipneumoniae which would lead to chromatin reorganization. Work is underway to narrow the range of these associated regions to identify the underlying causal mutations.

Characterizing Genetic Regulatory Elements in Ovine Tissues.

The Ovine Functional Annotation of Animal Genomes (FAANG) project, part of the broader livestock species FAANG initiative, aims to identify and characterize gene regulatory elements in domestic sheep. Regulatory element annotation is essential for identifying genetic variants that affect health and production traits in this important agricultural species, as greater than 90% of variants underlying genetic effects are estimated to lie outside of transcribed regions. Histone modifications that distinguish active or repressed chromatin states, CTCF binding, and DNA methylation were used to characterize regulatory elements in liver, spleen, and cerebellum tissues from four yearling sheep. Chromatin immunoprecipitation with sequencing (ChIP-seq) was performed for H3K4me3, H3K27ac, H3K4me1, H3K27me3, and CTCF. Nine chromatin states including active promoters, active enhancers, poised enhancers, repressed enhancers, and insulators were characterized in each tissue using ChromHMM. Whole-genome bisulfite sequencing (WGBS) was performed to determine the complement of whole-genome DNA methylation with the ChIP-seq data. Hypermethylated and hypomethylated regions were identified across tissues, and these locations were compared with chromatin states to better distinguish and validate regulatory elements in these tissues. Interestingly, chromatin states with the poised enhancer mark H3K4me1 in the spleen and cerebellum and CTCF in the liver displayed the greatest number of hypermethylated sites. Not surprisingly, active enhancers in the liver and spleen, and promoters in the cerebellum, displayed the greatest number of hypomethylated sites. Overall, chromatin states defined by histone marks and CTCF occupied approximately 22% of the genome in all three tissues. Furthermore, the liver and spleen displayed in common the greatest percent of active promoter (65%) and active enhancer (81%) states, and the liver and cerebellum displayed in common the greatest percent of poised enhancer (53%), repressed enhancer (68%), hypermethylated sites (75%), and hypomethylated sites (73%). In addition, both known and de novo CTCF-binding motifs were identified in all three tissues, with the highest number of unique motifs identified in the cerebellum. In summary, this study has identified the regulatory regions of genes in three tissues that play key roles in defining health and economically important traits and has set the precedent for the characterization of regulatory elements in ovine tissues using the Rambouillet reference genome.

Association of TMEM8B and SPAG8 with Mature Weight in Sheep.

Signature of selection studies have identified many genomic regions with known functional importance and some without verified functional roles. Multiple studies have identified Transmembrane protein 8B (TMEM8B)rs426272889 as having been recently under extreme selection pressure in domesticated sheep, but no study has provided sheep phenotypic data clarifying a reason for extreme selection. We tested rs426272889 for production trait association in 770 U.S. Rambouillet, Targhee, Polypay, and Suffolk sheep. TMEM8Brs426272889 was associated with mature weight at 3 and 4 years (p < 0.05). This suggested selection for sheep growth and body size might explain the historical extreme selection pressure in this genomic region. We also tested Sperm-associated antigen 8 (SPAG8) rs160159557 encoding a G493C substitution. While this variant was associated with mature weights at ages 3 and 4, it was not as strongly associated as TMEM8Brs426272889. Transmembrane protein 8B has little functional information except as an inhibitor of cancer cell proliferation. To our knowledge, this is the first study linking TMEM8B to whole organism growth and body size under standard conditions. Additional work will be necessary to identify the underlying functional variant(s). Once identified, such variants could be used to improve sheep production through selective breeding.

Assay to compare cell- and antibody-mediated immune responses in domestic sheep and goats.

Assessment of immune fitness is valuable in many aspects of livestock management and research. Determining immune consequences of selection for increased disease resistance or inhabiting various environments or climates can lead to different management decisions. The ability to measure immune responses due to different diets, pregnancy status, or aging will increase insight about how these factors contribute to overall immune health. The main objective of these experiments was to adapt a methodology used in cattle and pigs to measure both the humoral and cell-mediated immune response in sheep and goats. The route of administration of two antigens, Candida albicans and hen egg white lysozyme, were compared in sheep to determine differences in antibody or cell-mediated immune response. Subcutaneous injection produced a larger (P < 0.001) cell-mediated response compared to intramuscular injection. Inoculation in the axillary space produced a larger (P = 0.0031) antibody response compared to neck region. Finally, methodology was confirmed in goats. Complete blood cell counts were compared and lymphocytes were highest in low cell-mediated responders while eosinophils were highest in average antibody-mediated responders. This work provides a means to measure immune fitness in sheep and goats allowing for future experiments examining environmental or genetic effects on the immune response.

Genome-Wide Histone Modifications and CTCF Enrichment Predict Gene Expression in Sheep Macrophages.

Alveolar macrophages function in innate and adaptive immunity, wound healing, and homeostasis in the lungs dependent on tissue-specific gene expression under epigenetic regulation. The functional diversity of tissue resident macrophages, despite their common myeloid lineage, highlights the need to study tissue-specific regulatory elements that control gene expression. Increasing evidence supports the hypothesis that subtle genetic changes alter sheep macrophage response to important production pathogens and zoonoses, for example, viruses like small ruminant lentiviruses and bacteria like Coxiella burnetii. Annotation of transcriptional regulatory elements will aid researchers in identifying genetic mutations of immunological consequence. Here we report the first genome-wide survey of regulatory elements in any sheep immune cell, utilizing alveolar macrophages. We assayed histone modifications and CTCF enrichment by chromatin immunoprecipitation with deep sequencing (ChIP-seq) in two sheep to determine cis-regulatory DNA elements and chromatin domain boundaries that control immunity-related gene expression. Histone modifications included H3K4me3 (denoting active promoters), H3K27ac (active enhancers), H3K4me1 (primed and distal enhancers), and H3K27me3 (broad silencers). In total, we identified 248,674 reproducible regulatory elements, which allowed assignment of putative biological function in macrophages to 12% of the sheep genome. Data exceeded the FAANG and ENCODE standards of 20 million and 45 million useable fragments for narrow and broad marks, respectively. Active elements showed consensus with RNA-seq data and were predictive of gene expression in alveolar macrophages from the publicly available Sheep Gene Expression Atlas. Silencer elements were not enriched for expressed genes, but rather for repressed developmental genes. CTCF enrichment enabled identification of 11,000 chromatin domains with mean size of 258 kb. To our knowledge, this is the first report to use immunoprecipitated CTCF to determine putative topological domains in sheep immune cells. Furthermore, these data will empower phenotype-associated mutation discovery since most causal variants are within regulatory elements.

Forkhead box O member FOXO1 regulates the majority of follicle-stimulating hormone responsive genes in ovarian granulosa cells.

FSH promotes maturation of ovarian follicles. One pathway activated by FSH in granulosa cells (GCs) is phosphatidylinositol-3 kinase/AKT. The AKT target FOXO1 is reported to function primarily as a repressor of FSH genes, including Ccnd2 and Inha. Based on its broad functions in other tissues, we hypothesized that FOXO1 may regulate many more GC genes. We transduced GCs with empty adenovirus or constitutively active FOXO1 followed by treatment with FSH for 24 h, and conducted RNA deep sequencing. Results show that FSH regulates 3772 genes ≥2.0-fold; 60% of these genes are activated or repressed by FOXO1. Pathway Studio Analysis revealed enrichment of genes repressed by FOXO1 in metabolism, signaling, transport, development, and activated by FOXO1 in signaling, cytoskeletal functions, and apoptosis. Gene regulation was verified by q-PCR (eight genes) and ChIP analysis (two genes). We conclude that FOXO1 regulates the majority of FSH target genes in GCs.

Maximal expression of Foxl2 in pituitary gonadotropes requires ovarian hormones.

Gonadotropin-releasing hormone (GnRH) and activin regulate synthesis of FSH and ultimately fertility. Recent in vivo studies cast SMAD4 and FOXL2 as master transcriptional mediators of activin signaling that act together and independently of GnRH to regulate Fshb gene expression and female fertility. Ovarian hormones regulate GnRH and its receptor (GNRHR) through negative and positive feedback loops. In contrast, the role of ovarian hormones in regulating activin, activin receptors, and components of the activin signaling pathway, including SMAD4 and FOXL2, remains understudied. The widespread distribution of activin and many of its signaling intermediates complicates analysis of the effects of ovarian hormones on their synthesis in gonadotropes, one of five pituitary cell types. We circumvented this complication by using a transgenic model that allows isolation of polyribosomes selectively from gonadotropes of intact females and ovariectomized females treated with or without a GnRH antagonist. This paradigm allows assessment of ovarian hormonal feedback and distinguishes responses that are either independent or dependent on GnRH. Surprisingly, our results indicate that Foxl2 levels in gonadotropes decline significantly in the absence of ovarian input and independently of GnRH. Expression of the genes encoding other members of the activin signaling pathway are unaffected by loss of ovarian hormonal feedback, highlighting their selective effect on Foxl2. Expression of Gnrhr, a known target of FOXL2, also declines upon ovariectomy consistent with reduced expression of Foxl2 and loss of ovarian hormones. In contrast, Fshb mRNA increases dramatically post-ovariectomy due to increased compensatory input from GnRH. Together these data suggest that ovarian hormones regulate expression of Foxl2 thereby expanding the number of genes controlled by the hypothalamic-pituitary-gonadal axis that ultimately dictate reproductive fitness.

GnRH regulation of Jun and Atf3 requires calcium, calcineurin, and NFAT.

GnRH binds to its receptor on gonadotropes and activates multiple members of the MAPK signaling family that in turn regulates the expression of several immediate early genes (IEGs) including Jun, Fos, Atf3, and Egr1. These IEGs confer hormonal responsiveness to gonadotrope-specific genes including Gnrhr, Cga, Fshb, and Lhb. In this study we tested the hypothesis that GnRH specifically regulates the accumulation of Jun and Atf3 mRNA through a pathway that includes intracellular Ca²âº, calcineurin, and nuclear factor of activated T cells (NFAT). Our results indicate that pretreatment of murine LßT2 cells with 1, 2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)-ester, a Ca²âº chelator, reduced the expression of all the IEGs to varying degrees, whereas treatment with thapsigargin, an intracellular Ca²âº protein pump inhibitor, increased the expression of the IEG. Furthermore, cyclosporin A, a calcineurin-specific inhibitor, reduced the ability of GnRH to regulate accumulation of Jun and Atf3 mRNA and to a lesser extent Fos. In contrast, Egr1 mRNA was unaffected. NFATs are transcription factors regulated by calcineurin and were detected in LßT2 cells. GnRH increased luciferase activity of an NFAT-dependent promoter reporter that was dependent on intracellular Ca²âº and calcineurin activity. Additionally, although small interfering RNA specific for Nfat4 only marginally reduced GnRH regulation of Jun, Fos, and Atf3 mRNA accumulation, activity of an activator protein-1-responsive reporter construct was reduced by 48%. Together these data suggest that calcineurin and NFAT are new members of the gonadotrope transcriptional network that confer hormonal responsiveness to several key genes required for gonadotropin synthesis and secretion.

Cold-shock-domain protein A (CSDA) contributes posttranscriptionally to gonadotropin-releasing hormone-regulated expression of Egr1 and indirectly to Lhb.

Gonadotropin-releasing hormone (GnRH), a hypothalamic neurohormone, regulates transcription of Lhb in gonadotrophs indirectly through transient induction and accumulation of EGR1, a zinc finger transcription factor. AlphaT3 and LbetaT2 cell lines model gonadotrophs at two distinct stages of development, prenatal and postnatal expression of Lhb. Although GnRH induces EGR1 in both cell lines, the levels of the DNA-binding protein are lower and disappear more quickly in alphaT3 than in LbetaT2 cells. Herein we show that overexpression of Egr1 in alphaT3 cells rescues activity of a transfected LHB promoter-reporter, suggesting that its transcription is dependent on EGR1 crossing a critical concentration threshold. We also show that Csda, a gene that encodes an RNA-binding protein and is a member of the cold-shock-domain (CSD) family, is expressed at higher levels in LbetaT2 compared to alphaT3 cells. Transient expression studies indicate that at least one Csd element, residing in the 3' untranslated region of Egr1 mRNA, increases activity of a chimeric pGL3 luciferase reporter vector in LbetaT2 cells. Additional experiments indicate that CSDA physically interacts with Egr1 mRNA. Furthermore, siRNA-mediated reduction of endogenous Csda mRNA attenuates GnRH regulation of a transiently transfected LHB reporter vector. Taken together, these studies suggest that CSDA contributes posttranscriptionally to GnRH-regulated expression of Egr1, thereby enabling the transcription factor to cross a critical concentration threshold necessary for maximal accumulation of Lhb mRNA in response to the neurohormone.