PDZ-Binding Kinase, a Novel Regulator of Vascular Remodeling in Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is high blood pressure in the lungs that originates from structural changes in small resistance arteries. A defining feature of PAH is the inappropriate remodeling of pulmonary arteries (PA) leading to right ventricle failure and death. Although treatment of PAH has improved, the long-term prognosis for patients remains poor, and more effective targets are needed. METHODS: Gene expression was analyzed by microarray, RNA sequencing, quantitative polymerase chain reaction, Western blotting, and immunostaining of lung and isolated PA in multiple mouse and rat models of pulmonary hypertension (PH) and human PAH. PH was assessed by digital ultrasound, hemodynamic measurements, and morphometry. RESULTS: Microarray analysis of the transcriptome of hypertensive rat PA identified a novel candidate, PBK (PDZ-binding kinase), that was upregulated in multiple models and species including humans. PBK is a serine/threonine kinase with important roles in cell proliferation that is minimally expressed in normal tissues but significantly increased in highly proliferative tissues. PBK was robustly upregulated in the medial layer of PA, where it overlaps with markers of smooth muscle cells. Gain-of-function approaches show that active forms of PBK increase PA smooth muscle cell proliferation, whereas silencing PBK, dominant negative PBK, and pharmacological inhibitors of PBK all reduce proliferation. Pharmacological inhibitors of PBK were effective in PH reversal strategies in both mouse and rat models, providing translational significance. In a complementary genetic approach, PBK was knocked out in rats using CRISPR/Cas9 editing, and loss of PBK prevented the development of PH. We found that PBK bound to PRC1 (protein regulator of cytokinesis 1) in PA smooth muscle cells and that multiple genes involved in cytokinesis were upregulated in experimental models of PH and human PAH. Active PBK increased PRC1 phosphorylation and supported cytokinesis in PA smooth muscle cells, whereas silencing or dominant negative PBK reduced cytokinesis and the number of cells in the G2/M phase of the cell cycle. CONCLUSIONS: PBK is a newly described target for PAH that is upregulated in proliferating PA smooth muscle cells, where it contributes to proliferation through changes in cytokinesis and cell cycle dynamics to promote medial thickening, fibrosis, increased PA resistance, elevated right ventricular systolic pressure, right ventricular remodeling, and PH.

BESST: a novel LncRNA knockout strategy with less genome perturbance.

Long noncoding RNAs (lncRNAs) are >200 nt RNA transcripts without protein-coding potential. LncRNAs can be categorized into intergenic, intronic, bidirectional, sense, and antisense lncRNAs based on the genomic localization to nearby protein-coding genes. The current CRISPR-based lncRNA knockout strategy works efficiently for lncRNAs distant from the protein-coding gene, whereas it causes genomic perturbance inevitably due to technical limitations. In this study, we introduce a novel lncRNA knockout strategy, BESST, by deleting the genomic DNA fragment from the branch point to the 3' splicing site in the last intron of the target lncRNA. The BESST knockout exhibited comparable or superior repressive efficiency to RNA silencing or conventional promoter-exon1 deletion. Significantly, the BESST knockout strategy minimized the intervention of adjacent/overlap protein-coding genes by removing an average of ∼130 bp from genomic DNA. Our data also found that the BESST knockout strategy causes lncRNA nuclear retention, resulting in decapping and deadenylation of the lncRNA poly(A) tail. Further study revealed that PABPN1 is essential for the BESST-mediated decay and subsequent poly(A) deadenylation and decapping. Together, the BESST knockout strategy provides a versatile tool for investigating gene function by generating knockout cells or animals with high specificity and efficiency.

The Long Noncoding RNA Cardiac Mesoderm Enhancer-Associated Noncoding RNA (Carmn) Is a Critical Regulator of Gastrointestinal Smooth Muscle Contractile Function and Motility.

BACKGROUND & AIMS: Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract that regulate GI motility. SMC contraction is regulated by posttranslational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality, but the mechanisms regulating SMC-specific contractile gene expression, including the role of long noncoding RNAs (lncRNAs), remain largely unexplored. Herein, we reveal a critical role of Carmn (cardiac mesoderm enhancer-associated noncoding RNA), an SMC-specific lncRNA, in regulating visceral SMC phenotype and contractility of the GI tract. METHODS: Genotype-Tissue Expression and publicly available single-cell RNA sequencing (scRNA-seq) data sets from embryonic, adult human, and mouse GI tissues were interrogated to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using novel green fluorescent protein (GFP) knock-in (KI) reporter/knock-out (KO) mice. Bulk RNA-seq and single nucleus RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms. RESULTS: Unbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is highly expressed in GI SMCs in humans and mice. Premature lethality was observed in global Carmn KO and inducible SMC-specific KO mice due to GI pseudo-obstruction and severe distension of the GI tract, with dysmotility in cecum and colon segments. Histology, GI transit, and muscle myography analysis revealed severe dilation, significantly delayed GI transit, and impaired GI contractility in Carmn KO vs control mice. Bulk RNA-seq of GI muscularis revealed that loss of Carmn promotes SMC phenotypic switching, as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes, including Mylk, a key regulator of SMC contraction. snRNA-seq further revealed SMC Carmn KO not only compromised myogenic motility by reducing contractile gene expression but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance, because silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression, including MYLK, and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program. CONCLUSIONS: Our data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.

Blockade of endothelial adenosine receptor 2 A suppresses atherosclerosis in vivo through inhibiting CREB-ALK5-mediated endothelial to mesenchymal transition.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and morbidity and mortality rates continue to rise. Atherosclerosis constitutes the principal etiology of CVDs. Endothelial injury, inflammation, and dysfunction are the initiating factors of atherosclerosis. Recently, we reported that endothelial adenosine receptor 2 A (ADORA2A), a G protein-coupled receptor (GPCR), plays critical roles in neovascularization disease and cerebrovascular disease. However, the precise role of endothelial ADORA2A in atherosclerosis is still not fully understood. Here, we showed that ADORA2A expression was markedly increased in the aortic endothelium of humans with atherosclerosis or Apoe-/- mice fed a high-cholesterol diet. In vivo studies unraveled that endothelial-specific Adora2a deficiency alleviated endothelial-to-mesenchymal transition (EndMT) and prevented the formation and instability of atherosclerotic plaque in Apoe-/- mice. Moreover, pharmacologic inhibition of ADORA2A with KW6002 recapitulated the anti-atherogenic phenotypes observed in genetically Adora2a-deficient mice. In cultured human aortic endothelial cells (HAECs), siRNA knockdown of ADORA2A or KW6002 inhibition of ADORA2A decreased EndMT, whereas adenoviral overexpression of ADORA2A induced EndMT. Mechanistically, ADORA2A upregulated ALK5 expression via a cAMP/PKA/CREB axis, leading to TGFß-Smad2/3 signaling activation, thereby promoting EndMT. In conclusion, these findings, for the first time, demonstrate that blockade of ADORA2A attenuated atherosclerosis via inhibition of EndMT induced by the CREB1-ALK5 axis. This study discloses a new link between endothelial ADORA2A and EndMT and indicates that inhibiting endothelial ADORA2A could be an effective novel strategy for the prevention and treatment of atherosclerotic CVDs.

Deficiency of the novel high mobility group protein HMGXB4 protects against systemic inflammation-induced endotoxemia in mice.

Sepsis is a major cause of mortality in intensive care units, which results from a severely dysregulated inflammatory response that ultimately leads to organ failure. While antibiotics can help in the early stages, effective strategies to curtail inflammation remain limited. The high mobility group (HMG) proteins are chromosomal proteins with important roles in regulating gene transcription. While HMGB1 has been shown to play a role in sepsis, the role of other family members including HMGXB4 remains unknown. We found that expression of HMGXB4 is strongly induced in response to lipopolysaccharide (LPS)-elicited inflammation in murine peritoneal macrophages. Genetic deletion of Hmgxb4 protected against LPS-induced lung injury and lethality and cecal ligation and puncture (CLP)-induced lethality in mice, and attenuated LPS-induced proinflammatory gene expression in cultured macrophages. By integrating genome-wide transcriptome profiling and a publicly available ChIP-seq dataset, we identified HMGXB4 as a transcriptional activator that regulates the expression of the proinflammatory gene, Nos2 (inducible nitric oxide synthase 2) by binding to its promoter region, leading to NOS2 induction and excessive NO production and tissue damage. Similar to Hmgxb4 ablation in mice, administration of a pharmacological inhibitor of NOS2 robustly decreased LPS-induced pulmonary vascular permeability and lethality in mice. Additionally, we identified the cell adhesion molecule, ICAM1, as a target of HMGXB4 in endothelial cells that facilitates inflammation by promoting monocyte attachment. In summary, our study reveals a critical role of HMGXB4 in exacerbating endotoxemia via transcriptional induction of Nos2 and Icam1 gene expression and thus targeting HMGXB4 may be an effective therapeutic strategy for the treatment of sepsis.

CARMN Is an Evolutionarily Conserved Smooth Muscle Cell-Specific LncRNA That Maintains Contractile Phenotype by Binding Myocardin.

BACKGROUND: Vascular homeostasis is maintained by the differentiated phenotype of vascular smooth muscle cells (VSMCs). The landscape of protein coding genes comprising the transcriptome of differentiated VSMCs has been intensively investigated but many gaps remain including the emerging roles of noncoding genes. METHODS: We reanalyzed large-scale, publicly available bulk and single-cell RNA sequencing datasets from multiple tissues and cell types to identify VSMC-enriched long noncoding RNAs. The in vivo expression pattern of a novel smooth muscle cell (SMC)-expressed long noncoding RNA, Carmn (cardiac mesoderm enhancer-associated noncoding RNA), was investigated using a novel Carmn green fluorescent protein knock-in reporter mouse model. Bioinformatics and quantitative real-time polymerase chain reaction analysis were used to assess CARMN expression changes during VSMC phenotypic modulation in human and murine vascular disease models. In vitro, functional assays were performed by knocking down CARMN with antisense oligonucleotides and overexpressing Carmn by adenovirus in human coronary artery SMCs. Carotid artery injury was performed in SMC-specific Carmn knockout mice to assess neointima formation and the therapeutic potential of reversing CARMN loss was tested in a rat carotid artery balloon injury model. The molecular mechanisms underlying CARMN function were investigated using RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. RESULTS: We identified CARMN, which was initially annotated as the host gene of the MIR143/145 cluster and recently reported to play a role in cardiac differentiation, as a highly abundant and conserved, SMC-specific long noncoding RNA. Analysis of the Carmn GFP knock-in mouse model confirmed that Carmn is transiently expressed in embryonic cardiomyocytes and thereafter becomes restricted to SMCs. We also found that Carmn is transcribed independently of Mir143/145. CARMN expression is dramatically decreased by vascular disease in humans and murine models and regulates the contractile phenotype of VSMCs in vitro. In vivo, SMC-specific deletion of Carmn significantly exacerbated, whereas overexpression of Carmn markedly attenuated, injury-induced neointima formation in mouse and rat, respectively. Mechanistically, we found that Carmn physically binds to the key transcriptional cofactor myocardin, facilitating its activity and thereby maintaining the contractile phenotype of VSMCs. CONCLUSIONS: CARMN is an evolutionarily conserved SMC-specific long noncoding RNA with a previously unappreciated role in maintaining the contractile phenotype of VSMCs and is the first noncoding RNA discovered to interact with myocardin.

Novel Myh11 Dual Reporter Mouse Model Provides Definitive Labeling and Identification of Smooth Muscle Cells-Brief Report.

OBJECTIVE: Myh11 encodes a myosin heavy chain protein that is specifically expressed in smooth muscle cells (SMCs) and is important for maintaining vascular wall stability. The goal of this study is to generate a Myh11 dual reporter mouse line for definitive visualization of MYH11+ SMCs in vivo. Approach and Results: We generated a Myh11 knock-in mouse model by inserting LoxP-nlacZ-4XpolyA-LoxP-H2B-GFP-polyA-FRT-Neo-FRT reporter cassette into the Myh11 gene locus. The nuclear (n) lacZ-4XpolyA cassette is flanked by 2 LoxP sites followed by H2B-GFP (histone 2B fused green fluorescent protein). Upon Cre-mediated recombination, nlacZ-stop cassette is removed thereby permitting nucleus localized H2B-GFP expression. Expression of the nuclear localized lacZ or H2B-GFP is under control of the endogenous Myh11 promoter. Nuclear lacZ was expressed specifically in SMCs at embryonic and adult stages. Following germline Cre-mediated deletion of nuclear lacZ, H2B-GFP was specifically expressed in the nuclei of SMCs. Comparison of nuclear lacZ expression with Wnt1Cre and Mef2cCre mediated-H2B-GFP expression revealed heterogenous origins of SMCs from neural crest and second heart field in the great arteries and coronary vessels adjacent to aortic root. CONCLUSIONS: The Myh11 knock-in dual reporter mouse model offers an exceptional genetic tool to visualize and trace the origins of SMCs in mice.

LOTUS domain is a novel class of G-rich and G-quadruplex RNA binding domain.

LOTUS domains are helix-turn-helix protein folds identified in essential germline proteins and are conserved in prokaryotes and eukaryotes. Despite originally predicted as an RNA binding domain, its molecular binding activity towards RNA and protein is controversial. In particular, the most conserved binding property for the LOTUS domain family remains unknown. Here, we uncovered an unexpected specific interaction of LOTUS domains with G-rich RNA sequences. Intriguingly, LOTUS domains exhibit high affinity to RNA G-quadruplex tertiary structures implicated in diverse cellular processes including piRNA biogenesis. This novel LOTUS domain-RNA interaction is conserved in bacteria, plants and animals, comprising the most ancient binding feature of the LOTUS domain family. By contrast, LOTUS domains do not preferentially interact with DNA G-quadruplexes. We further show that a subset of LOTUS domains display both RNA and protein binding activities. These findings identify the LOTUS domain as a specialized RNA binding domain across phyla and underscore the molecular mechanism underlying the function of LOTUS domain-containing proteins in RNA metabolism and regulation.

YAP1/TEAD1 upregulate platelet-derived growth factor receptor beta to promote vascular smooth muscle cell proliferation and neointima formation.

We have previously demonstrated that the transcription co-factor yes-associated protein 1 (YAP1) promotes vascular smooth muscle cell (VSMC) de-differentiation. Yet, the role and underlying mechanisms of YAP1 in neointima formation in vivo remain unclear. The goal of this study was to investigate the role of VSMC-expressed YAP1 in vascular injury-induced VSMC proliferation and delineate the mechanisms underlying its action. Experiments employing gain- or loss-of-function of YAP1 demonstrated that YAP1 promotes human VSMC proliferation. Mechanistically, we identified platelet-derived growth factor receptor beta (PDGFRB) as a novel YAP1 target gene that confers the YAP1-dependent hyper-proliferative effects in VSMCs. Furthermore, we identified TEA domain transcription factor 1 (TEAD1) as a key transcription factor that mediates YAP1-dependent PDGFRß expression. ChIP assays demonstrated that TEAD1 is enriched at a PDGFRB gene enhancer. Luciferase reporter assays further demonstrated that YAP1 and TEAD1 co-operatively activate the PDGFRB enhancer. Consistent with these observations, we found that YAP1 expression is upregulated after arterial injury and correlates with PDGFRß expression and VSMC proliferation in vivo. Using a novel inducible SM-specific Yap1 knockout mouse model, we found that the specific deletion of Yap1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation, largely due to inhibited PDGFRß expression and VSMC proliferation. Our study unravels a novel mechanism by which YAP1/TEAD1 promote VSMC proliferation via transcriptional induction of PDGFRß, thereby enhancing PDGF-BB downstream signaling and promoting neointima formation.

TEAD1 (TEA Domain Transcription Factor 1) Promotes Smooth Muscle Cell Proliferation Through Upregulating SLC1A5 (Solute Carrier Family 1 Member 5)-Mediated Glutamine Uptake.

RATIONALE: TEAD (TEA domain transcription factor) 1-a major effector of the Hippo signaling pathway-acts as an oncoprotein in a variety of tumors. However, the function of TEAD1 in vascular smooth muscle cells (VSMCs) remains unclear. OBJECTIVE: To assess the role of TEAD1 in vascular injury-induced smooth muscle proliferation and delineate the mechanisms underlying its action. METHODS AND RESULTS: We found that TEAD1 expression is enhanced in mouse femoral artery after wire injury and correlates with the activation of mTORC1 (mechanistic target of rapamycin complex 1) signaling in vivo. Using an inducible smooth muscle-specific Tead1 KO (knockout) mouse model, we found that specific deletion of Tead1 in adult VSMCs is sufficient to attenuate arterial injury-induced neointima formation due to inhibition of mTORC1 activation and VSMC proliferation. Furthermore, we found that TEAD1 plays a unique role in VSMCs, where it not only downregulates VSMC differentiation markers but also activates mTORC1 signaling, leading to enhanced VSMC proliferation. Using whole-transcriptome sequencing analysis, we identified Slc1a5 (solute carrier family 1 member 5)-a key glutamine transporter-as a novel TEAD1 target gene. SLC1A5 overexpression mimicked TEAD1 in promoting mTORC1 activation and VSMC proliferation. Moreover, depletion of SLC1A5 by silencing RNA or blocking SLC1A5-mediated glutamine uptake attenuated TEAD1-dependent mTORC1 activation and VSMC proliferation. CONCLUSIONS: Our study unravels a novel mechanism by which TEAD1 promotes VSMC proliferation via transcriptional induction of SLC1A5, thereby activating mTORC1 signaling and promoting neointima formation.

Mitochondrial membrane-based initial separation of MIWI and MILI functions during pachytene piRNA biogenesis.

PIWI-interacting RNAs (piRNAs) engage PIWI proteins to silence transposons and promote germ cell development in animals. In diverse species, piRNA biogenesis occurs near the mitochondrial surface, and involves mitochondrial membrane-anchored factors. In mice, two cytoplasmic PIWI proteins, MIWI and MILI, receive processed pachytene piRNAs at intermitochodrial cement (IMC). However, how MIWI and MILI are initially recruited to the IMC to engage multiple steps of piRNA processing is unclear. Here, we show that mitochondria-anchored TDRKH controls multiple steps of pachytene piRNA biogenesis in mice. TDRKH specifically recruits MIWI, but not MILI, to engage the piRNA pathway. It is required for the production of the entire MIWI-bound piRNA population and enables trimming of MILI-bound piRNAs. The failure to recruit MIWI to the IMC with TDRKH deficiency results in loss of MIWI in the chromatoid body, leading to spermiogenic arrest and piRNA-independent retrotransposon LINE1 de-repression in round spermatids. Our findings identify a mitochondrial surface-based scaffolding mechanism separating the entry and actions of two critical PIWI proteins in the same piRNA pathway to drive piRNA biogenesis and germ cell development.

Long noncoding RNA NEAT1 (nuclear paraspeckle assembly transcript 1) is critical for phenotypic switching of vascular smooth muscle cells.

In response to vascular injury, vascular smooth muscle cells (VSMCs) may switch from a contractile to a proliferative phenotype thereby contributing to neointima formation. Previous studies showed that the long noncoding RNA (lncRNA) NEAT1 is critical for paraspeckle formation and tumorigenesis by promoting cell proliferation and migration. However, the role of NEAT1 in VSMC phenotypic modulation is unknown. Herein we showed that NEAT1 expression was induced in VSMCs during phenotypic switching in vivo and in vitro. Silencing NEAT1 in VSMCs resulted in enhanced expression of SM-specific genes while attenuating VSMC proliferation and migration. Conversely, overexpression of NEAT1 in VSMCs had opposite effects. These in vitro findings were further supported by in vivo studies in which NEAT1 knockout mice exhibited significantly decreased neointima formation following vascular injury, due to attenuated VSMC proliferation. Mechanistic studies demonstrated that NEAT1 sequesters the key chromatin modifier WDR5 (WD Repeat Domain 5) from SM-specific gene loci, thereby initiating an epigenetic "off" state, resulting in down-regulation of SM-specific gene expression. Taken together, we demonstrated an unexpected role of the lncRNA NEAT1 in regulating phenotypic switching by repressing SM-contractile gene expression through an epigenetic regulatory mechanism. Our data suggest that NEAT1 is a therapeutic target for treating occlusive vascular diseases.

Genomic analysis of worldwide sheep breeds reveals PDGFD as a major target of fat-tail selection in sheep.

BACKGROUND: Fat tail is a unique trait in sheep acquired during domestication. Several genomic analyses have been conducted in sheep breeds from limited geographic origins to identify the genetic factors underlying this trait. Nevertheless, these studies obtained different candidates. The results of these regional studies were easily biased by the breed structures. RESULTS: To minimize the bias and distinguish the true candidates, we used an extended data set of 968 sheep representing 18 fat-tailed breeds and 14 thin-tailed breeds from around the world, and integrated two statistical tests to detect selection signatures, including Genetic Fixation Index (FST) and difference of derived allele frequency (ΔDAF). The results showed that platelet derived growth factor D (PDGFD) exhibited the highest genetic differentiation between fat- and thin-tailed sheep breeds. Analysis of sequence variation identified that a 6.8-kb region within the first intron of PDGFD is likely the target of positive selection and contains regulatory mutation(s) in fat-tailed sheep. Histological and gene expression analyses demonstrated that PDGFD expression is associated with maturation and hemostasis of adipocytes. Further retrospective analysis of public transcriptomic datasets revealed that PDGFD expression is down-regulated during adipogenesis in both human and mouse, and is higher in fat tissues of obese individuals than that in lean individuals. CONCLUSIONS: These results reveal that PDGFD is the predominant factor for the fat tail phenotype in sheep by contributing to adiopogenesis and maintaining the hemostasis of mature adipocytes. This study provides insights into the selection of fat-tailed sheep and has important application to animal breeding, as well as obesity-related human diseases.

Endogenous Avian Leukosis Virus in Combination with Serotype 2 Marek's Disease Virus Significantly Boosted the Incidence of Lymphoid Leukosis-Like Bursal Lymphomas in Susceptible Chickens.

In 2010, sporadic cases of avian leukosis virus (ALV)-like bursal lymphoma, also known as spontaneous lymphoid leukosis (LL)-like tumors, were identified in two commercial broiler breeder flocks in the absence of exogenous ALV infection. Two individual ALV subgroup E (ALV-E) field strains, designated AF227 and AF229, were isolated from two different breeder farms. The role of these ALV-E field isolates in development of and the potential joint impact in conjunction with a Marek's disease virus (MDV) vaccine (SB-1) were further characterized in chickens of an experimental line and commercial broiler breeders. The experimental line 0.TVB*S1, commonly known as the rapid feathering-susceptible (RFS) line, of chickens lacks all endogenous ALV and is fully susceptible to all subgroups of ALV, including ALV-E. Spontaneous LL-like tumors occurred following infection with AF227, AF229, and a reference ALV-E strain, RAV60, in RFS chickens. Vaccination with serotype 2 MDV, SB-1, in addition to AF227 or AF229 inoculation, significantly enhanced the spontaneous LL-like tumor incidence in the RFS chickens. The spontaneous LL-like tumor incidence jumped from 14% by AF227 alone to 42 to 43% by AF227 in combination with SB-1 in the RFS chickens under controlled conditions. RNA-sequencing analysis of the LL-like lymphomas and nonmalignant bursa tissues of the RFS line of birds identified hundreds of differentially expressed genes that are reportedly involved in key biological processes and pathways, including signaling and signal transduction pathways. The data from this study suggested that both ALV-E and MDV-2 play an important role in enhancement of the spontaneous LL-like tumors in susceptible chickens. The underlying mechanism may be complex and involved in many chicken genes and pathways, including signal transduction pathways and immune system processes, in addition to reported viral genes.IMPORTANCE Lymphoid leukosis (LL)-like lymphoma is a low-incidence yet costly and poorly understood disease of domestic chickens. The observed unique characteristics of LL-like lymphomas are that the incidence of the disease is chicken line dependent; pathologically, it appeared to mimic avian leukosis but is free of exogenous ALV infection; inoculation of the nonpathogenic ALV-E or MDV-2 (SB-1) boosts the incidence of the disease; and inoculation of both the nonpathogenic ALV-E and SB-1 escalates it to much higher levels. This study was designed to test the impact of two new ALV-E isolates, recently derived from commercial broiler breeder flocks, in combination with the nonpathogenic SB-1 on LL-like lymphoma incidences in both an experimental egg layer line of chickens and a commercial broiler breeder line of chickens under a controlled condition. Data from this study provided an additional piece of experimental evidence on the potency of nonpathogenic ALV-E, MDV-2, and ALV-E plus MDV-2 in boosting the incidence of LL-like lymphomas in susceptible chickens. This study also generated the first piece of genomic evidence that suggests host transcriptomic variation plays an important role in modulating LL-like lymphoma formation.

Marek's disease vaccines-induced differential expression of known and novel microRNAs in primary lymphoid organ bursae of White Leghorn.

Marek's disease (MD) is a contagious disease of domestic chickens caused by MD viruses. MD has been controlled primarily by vaccinations, yet sporadic outbreaks of MD take place worldwide. Commonly used MD vaccines include HVT, SB-1 and CVI988/Rispens and their efficacies are reportedly dependent of multiple factors including host genetics. Our previous studies showed protective efficacy of a MD vaccine can differ drastically from one chicken line to the next. Advanced understanding on the underlying genetic and epigenetic factors that modulate vaccine efficacy would greatly improve the strategy in design and development of more potent vaccines. Two highly inbred lines of White Leghorn were inoculated with HVT and CVI988/Rispens. Bursa samples were taken 26 days post-vaccination and subjected to small RNA sequencing analysis to profile microRNAs (miRNA). A total of 589 and 519 miRNAs was identified in one line, known as line 63, 490 and 630 miRNAs were identified in the other, known as line 72, in response to HVT or CVI988/Rispens inoculation, respectively. HVT and CVI988/Rispens induced mutually exclusive 4 and 13 differentially expressed (DE) miRNAs in line 63 birds in contrast to a non-vaccinated group of the same line. HVT failed to induce any DE miRNA and CVI988/Rispens induced a single DE miRNA in line 72 birds. Thousands of target genes for the DE miRNAs were predicted, which were enriched in a variety of gene ontology terms and pathways. This finding suggests the epigenetic factor, microRNA, is highly likely involved in modulating vaccine protective efficacy in chicken.

Exome sequencing reveals genetic differentiation due to high-altitude adaptation in the Tibetan cashmere goat (Capra hircus).

BACKGROUND: The Tibetan cashmere goat (Capra hircus), one of the most ancient breeds in China, has historically been a critical source of meat and cashmere production for local farmers. To adapt to the high-altitude area, extremely harsh climate, and hypoxic environment that the Tibetan cashmere goat lives in, this goat has developed distinct phenotypic traits compared to lowland breeds. However, the genetic components underlying this phenotypic adaptation remain largely unknown. RESULTS: We obtained 118,700 autosomal SNPs through exome sequencing of 330 cashmere goats located at a wide geographic range, including the Tibetan Plateau and low-altitude regions in China. The great majority of SNPs showed low genetic differentiation among populations; however, approximately 2-3% of the loci showed more genetic differentiation than expected under a selectively neutral model. Together with a combined analysis of high- and low-altitude breeds, we revealed 339 genes potentially under high-altitude selection. Genes associated with cardiovascular system development were significantly enriched in our study. Among these genes, the most evident one was endothelial PAS domain protein 1 (EPAS1), which has been previously reported to be involved in complex oxygen sensing and significantly associated with high-altitude adaptation of human, dog, and grey wolf. The missense mutation Q579L that we identified in EPAS1, which occurs next to the Hypoxia-Inducible Factor-1 (HIF-1) domain, was exclusively enriched in the high-altitude populations. CONCLUSIONS: Our study provides insights concerning the population variation in six different cashmere goat populations in China. The variants in cardiovascular system-related genes may explain the observed phenotypic adaptation of the Tibetan cashmere goat.

Identification of copy number variations in three Chinese horse breeds using 70K single nucleotide polymorphism BeadChip array.

Copy number variation (CNV), an essential form of genetic variation, has been increasingly recognized as one promising genetic marker in the analysis of animal genomes. Here, we used the Equine 70K single nucleotide polymorphism genotyping array for the genome-wide detection of CNVs in 96 horses from three diverse Chinese breeds: Debao pony (DB), Mongolian horse (MG) and Yili horse (YL). A total of 287 CNVs were determined and merged into 122 CNV regions (CNVRs) ranging from 199 bp to 2344 kb in size and distributed in a heterogeneous manner on chromosomes. These CNVRs were integrated with seven existing reports to generate a composite genome-wide dataset of 1558 equine CNVRs, revealing 69 (56.6%) novel CNVRs. The majority (69.7%) of the 122 CNVRs overlapped with 438 genes, whereas 30.3% were located in intergenic regions. Most of these genes were associated with common CNVRs, which were shared by divergent horse breeds. As many as 60, 42 and 91 genes overlapping with the breed-specific ss were identified in DB, MG and YL respectively. Among these genes, FGF11, SPEM1, PPARG, CIDEB, HIVEP1 and GALR may have potential relevance to breed-specific traits. These findings provide valuable information for understanding the equine genome and facilitating association studies of economically important traits with equine CNVRs in the future.

Epigenetic Factor MicroRNAs Likely Mediate Vaccine Protection Efficacy against Lymphomas in Response to Tumor Virus Infection in Chickens through Target Gene Involved Signaling Pathways.

Epigenetic factors, including microRNAs (miRNAs), play an important role in affecting gene expression and, therefore, are involved in various biological processes including immunity protection against tumors. Marek's disease (MD) is a highly contagious disease of chickens caused by the MD virus (MDV). MD has been primarily controlled by vaccinations. MD vaccine efficacy might, in part, be dependent on modulations of a complex set of factors including host epigenetic factors. This study was designed to identify differentially expressed miRNAs in the primary lymphoid organ, bursae of Fabricius, in response to MD vaccination followed by MDV challenge in two genetically divergent inbred lines of White Leghorns. Small RNA sequencing and bioinformatic analyses of the small RNA sequence reads identified hundreds of miRNAs among all the treatment groups. A small portion of the identified miRNAs was differentially expressed within each of the four treatment groups, which were HVT or CVI988/Rispens vaccinated line 63-resistant birds and line 72-susceptible birds. A direct comparison between the resistant line 63 and susceptible line 72 groups vaccinated with HVT followed by MDV challenge identified five differentially expressed miRNAs. Gene Ontology analysis of the target genes of those five miRNAs revealed that those target genes, in addition to various GO terms, are involved in multiple signaling pathways including MAPK, TGF-ß, ErbB, and EGFR1 signaling pathways. The general functions of those pathways reportedly play important roles in oncogenesis, anti-cancer immunity, cancer cell migration, and metastatic progression. Therefore, it is highly likely that those miRNAs may, in part, influence vaccine protection through the pathways.

A novel mouse model carrying a gene trap insertion into the Hmgxb4 gene locus to examine Hmgxb4 expression in vivo.

HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to regulate the structural architecture of DNA. HMGXB4 (also known as HMG2L1) is an HMG protein family member that contains a single HMG box domain. Our previous studies have demonstrated that HMGXB4 suppresses smooth muscle differentiation and exacerbates endotoxemia by promoting a systemic inflammatory response in mice. However, the expression of Hmgxb4 in vivo has not fully examined. Herein, we generated a mouse model that harbors a gene trap in the form of a lacZ gene insertion into the Hmgxb4 gene. This mouse enables the visualization of endogenous HMGXB4 expression in different tissues via staining for the ß-galactosidase activity of LacZ which is under the control of the endogenous Hmgxb4 gene promoter. We found that HMGXB4 is widely expressed in mouse tissues and is a nuclear protein. Furthermore, the Hmgxb4 gene trap mice exhibit normal cardiac function and blood pressure. Measurement of ß-galactosidase activity in the Hmgxb4 gene trap mice demonstrated that the arterial injury significantly induces Hmgxb4 expression. In summary, the Hmgxb4 gene trap reporter mouse described here provides a valuable tool to examine the expression level of endogenous Hmgxb4 in both physiological and pathological settings in vivo.

Coronary Artery Disease Risk Gene PRDM16 is Preferentially Expressed in Vascular Smooth Muscle Cells and a Potential Novel Regulator of Smooth Muscle Homeostasis.

Objective: Vascular smooth muscle cells (VSMCs) are the primary contractile component of blood vessels and can undergo phenotypic switching from a contractile to a synthetic phenotype in vascular diseases such as coronary artery disease (CAD). This process leads to decreased expression of SMC lineage genes and increased proliferative, migratory and secretory abilities that drive disease progression. Super-enhancers (SE) and occupied transcription factors are believed to drive expression of genes that maintain cell identify and homeostasis. The goal of this study is to identify novel regulator of VSMC homeostasis by screening for SE-regulated transcription factors in arterial tissues. Approach and Results: We characterized human artery SEs by analyzing the enhancer histone mark H3K27ac ChIP-seq data of multiple arterial tissues. We unexpectedly discovered the transcription factor PRDM16, a GWAS identified CAD risk gene with previously well-documented roles in brown adipocytes but with an unknown function in vascular disease progression, is enriched with artery-specific SEs. Further analysis of public bulk RNA-seq and scRNA-seq datasets, as well as qRT-PCR and Western blotting analysis, demonstrated that PRDM16 is preferentially expressed in arterial tissues and in contractile VSMCs but not in visceral SMCs, and down-regulated in phenotypically modulated VSMCs. To explore the function of Prdm16 in vivo, we generated Prdm16 SMC-specific knockout mice and performed histological and bulk RNA-Seq analysis of aortic tissues. SMC-deficiency of Prdm16 does not affect the aortic morphology but significantly alters expression of many CAD risk genes and genes involved in VSMC phenotypic modulation. Specifically, Prdm16 negatively regulates the expression of Tgfb2 that encodes for an upstream ligand of TGF-ß signaling pathway, potentially through binding to the promoter region of Tgfb2 . These transcriptomic changes likely disrupt VSMC homeostasis and predispose VSMCs to a disease state. Conclusions: Our results suggest that the CAD risk gene PRDM16 is preferentially expressed in VSMCs and is a novel regulator of VSMC homeostasis. Future studies are warranted to investigate its role in VSMCs under pathological conditions such as atherosclerosis.