Genome-wide Gene Expression Research Articles

Scaffold protein MAPK8IP2 expression is a robust prognostic factor in prostate cancer associated with AR signaling activity.

Mitogen-activated protein kinase-8-interacting protein 2 (MAPK8IP2) is a scaffold protein that modulates MAPK signal cascades. Although MAPK pathways were heavily implicated in prostate cancer progression, the regulation of MAPK8IP2 expression in prostate cancer is not yet reported. We assessed MAPK8IP2 gene expression in prostate cancer related to disease progression and patient survival outcomes. MAPK8IP2 expression was analyzed using multiple genome-wide gene expression datasets derived from The Cancer Genome Atlas (TCGA) RNA-sequence project and complementary DNA (cDNA) microarrays. Multivariable Cox regressions and log-rank tests were used to analyze the overall survival outcome and progression-free interval. MAPK8IP2 protein expression was evaluated using the immunohistochemistry approach. The quantitative PCR and Western blot methods analyzed androgen-stimulated MAPK8IP2 expression in LNCaP cells. In primary prostate cancer tissues, MAPK8IP2 mRNA expression levels were significantly higher than those in the case-matched benign prostatic tissues. Increased MAPK8IP2 expression was strongly correlated with late tumor stages, lymph node invasion, residual tumors after surgery, higher Gleason scores, and preoperational serum prostate-specific antigen (PSA) levels. MAPK8IP2 upregulation was significantly associated with worse overall survival outcomes and progression-free intervals. In castration-resistant prostate cancers, MAPK8IP2 expression strongly correlated with androgen receptor (AR) signaling activity. In cell culture-based experiments, MAPK8IP2 expression was stimulated by androgens in AR-positive prostate cancer cells. However, MAPK8IP2 expression was blocked by AR antagonists only in androgen-sensitive LNCaP but not castration-resistant C4-2B and 22RV1 cells. These results indicate that MAPK8IP2 is a robust prognostic factor and therapeutic biomarker for prostate cancer. The potential role of MAPK8IP2 in the castration-resistant progression is under further investigation.

An Integrated Approach of Learning Genetic Networks From Genome-Wide Gene Expression Data Using Gaussian Graphical Model and Monte Carlo Method.

Global genetic networks provide additional information for the analysis of human diseases, beyond the traditional analysis that focuses on single genes or local networks. The Gaussian graphical model (GGM) is widely applied to learn genetic networks because it defines an undirected graph decoding the conditional dependence between genes. Many algorithms based on the GGM have been proposed for learning genetic network structures. Because the number of gene variables is typically far more than the number of samples collected, and a real genetic network is typically sparse, the graphical lasso implementation of GGM becomes a popular tool for inferring the conditional interdependence among genes. However, graphical lasso, although showing good performance in low dimensional data sets, is computationally expensive and inefficient or even unable to work directly on genome-wide gene expression data sets. In this study, the method of Monte Carlo Gaussian graphical model (MCGGM) was proposed to learn global genetic networks of genes. This method uses a Monte Carlo approach to sample subnetworks from genome-wide gene expression data and graphical lasso to learn the structures of the subnetworks. The learned subnetworks are then integrated to approximate a global genetic network. The proposed method was evaluated with a relatively small real data set of RNA-seq expression levels. The results indicate the proposed method shows a strong ability of decoding the interactions with high conditional dependences among genes. The method was then applied to genome-wide data sets of RNA-seq expression levels. The gene interactions with high interdependence from the estimated global networks show that most of the predicted gene-gene interactions have been reported in the literatures playing important roles in different human cancers. Also, the results validate the ability and reliability of the proposed method to identify high conditional dependences among genes in large-scale data sets.

The rates of adult neurogenesis and oligodendrogenesis are linked to cell cycle regulation through p27-dependent gene repression of SOX2

Cell differentiation involves profound changes in global gene expression that often has to occur in coordination with cell cycle exit. Because cyclin-dependent kinase inhibitor p27 reportedly regulates proliferation of neural progenitor cells in the subependymal neurogenic niche of the adult mouse brain, but can also have effects on gene expression, we decided to molecularly analyze its role in adult neurogenesis and oligodendrogenesis. At the cell level, we show that p27 restricts residual cyclin-dependent kinase activity after mitogen withdrawal to antagonize cycling, but it is not essential for cell cycle exit. By integrating genome-wide gene expression and chromatin accessibility data, we find that p27 is coincidentally necessary to repress many genes involved in the transit from multipotentiality to differentiation, including those coding for neural progenitor transcription factors SOX2, OLIG2 and ASCL1. Our data reveal both a direct association of p27 with regulatory sequences in the three genes and an additional hierarchical relationship where p27 repression of Sox2 leads to reduced levels of its downstream targets Olig2 and Ascl1. In vivo, p27 is also required for the regulation of the proper level of SOX2 necessary for neuroblasts and oligodendroglial progenitor cells to timely exit cell cycle in a lineage-dependent manner.

Study on the mechanism of cholic acid derivatives in traditional Chinese medicine based on the regulation of gene expression

To investigate the pharmacological action and mechanism of cholic acid derivatives in traditional Chinese medicine (TCM) based on the regulation of gene expression. Genome-wide gene expression profiles of Michigan Cancer Foundation-7 (MCF-7) cells treated with or without 4 cholic acid derivatives were detected by gene chip technology. Similarities in upregulated and downregulated genes were analyzed using the Connectivity Map (CMap) database. The affinity between cholic acid derivatives and the potential target was confirmed by molecular docking. The cholic acid derivative-regulated pathway enrichment analysis was performed by the STRING database, and the potential pathway was confirmed by in vitro experiments on MD Anderson-Metastatic Breast-231 (MDA-MB-231) cells. Compared with the reference genome in the CMap database, the gene expression profiles of cholic acid derivatives were similar to those of antipsychotic, anticancer, anti-inflammatory, and anti-infective drugs. Among them, 4 derivatives were associated with antianxiety drugs, and molecular docking results showed that these compounds may act by binding to the ligand-binding site of gamma-aminobutyric acid (GABA) receptors. Moreover, the cytoskeletal pathway is one of the pathways enriched in the derivatives. Of them, ursodeoxycholic acid showed significant inhibitory activity on the cytoskeleton formation of MDA-MB-231 cells. The gene expression detection method, combined with CMap and pathway enrichment analysis, could be used to study the mechanism of the active ingredients of TCM. In addition, our research showed that cholic acid derivatives have a potential affinity for membrane receptors, where they can exert anxiolytic activity by modulating opioid receptor, GABA receptor, and dopamine receptor. Moreover, ursodeoxycholic and chenodeoxycholic acid inhibit cytoskeleton formation, probably by acting on membrane proteins to activate the corresponding cytoskeletal pathways.

Host Transcriptome Analysis of Ferret Tissues Following Henipavirus Infection.

Ferrets are commonly used as experimental models of infection for a variety of viruses due to their susceptibility to human respiratory viruses and the close resemblance of pathological outcomes found in human infections. Even though ferret-specific reagents are limited, the use of ferrets as a preclinical experimental model of infection has gained considerable interest since the publication of the ferret transcriptome and draft ferret genome. These advances have made it feasible to easily perform whole-genome gene expression analysis in the ferret infection model. Here, we describe methods for genome-wide gene expression analysis using RNA sequence (RNAseq) data obtained from the lung and brain tissues obtained from experimental infections of Hendra (HeV) and Nipah (NiV) viruses in ferrets. We provide detailed methods for RNAseq and representative data for host gene expression profiles of the lung tissues that show early activation of interferon pathways and later activation of inflammation-related pathways.

Development and validation of a prognostic 15-gene signature for stratifying HER2+/ER+ breast cancer

BackgroundHuman epidermal growth receptor 2-positive (HER2+) breast cancer (BC) is a heterogeneous subgroup. Estrogen receptor (ER) status is emerging as a predictive marker within HER2+ BCs, with the HER2+/ER+ cases usually having better survival in the first 5 years after diagnosis but have higher recurrence risk after 5 years compared to HER2+/ER-. This is possibly because sustained ER signaling in HER2+ BCs helps escape the HER2 blockade. Currently HER2+/ER+ BC is understudied and lacks biomarkers. Thus, a better understanding of the underlying molecular diversity is important to find new therapy targets for HER2+/ER+ BCs. MethodsIn this study, we performed unsupervised consensus clustering together with genome-wide Cox regression analyses on the gene expression data of 123 HER2+/ER+ BC from The Cancer Genome Atlas Breast Invasive Carcinoma (TCGA-BRCA) cohort to identify distinct HER2+/ER+ subgroups. A supervised eXtreme Gradient Boosting (XGBoost) classifier was then built in TCGA using the identified subgroups and validated in another two independent datasets (Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and Gene Expression Omnibus (GEO) (accession number GSE149283)). Computational characterization analyses were also performed on the predicted subgroups in different HER2+/ER+ BC cohorts. ResultsWe identified two distinct HER2+/ER+ subgroups with different survival outcomes using the expression profiles of 549 survival-associated genes from the Cox regression analyses. Genome-wide gene expression differential analyses found 197 differentially expressed genes between the two identified subgroups, with 15 genes overlapping the 549 survival-associated genes.XGBoost classifier, using the expression values of the 15 genes, achieved a strong cross-validated performance (Area under the curve (AUC) = 0.85, Sensitivity = 0.76, specificity = 0.77) in predicting the subgroup labels. Further investigation partially confirmed the differences in survival, drug response, tumor-infiltrating lymphocytes, published gene signatures, and CRISPR-Cas9 knockout screened gene dependency scores between the two identified subgroups. ConclusionThis is the first study to stratify HER2+/ER+ tumors. Overall, the initial results from different cohorts showed there exist two distinct subgroups in HER2+/ER+ tumors, which can be distinguished by a 15-gene signature. Our findings could potentially guide the development of future precision therapies targeted on HER2+/ER+ BC.

Unbiased Transcriptomic Analysis of Chondrocyte Differentiation in a High-Density Cell Culture Model

In the developing vertebrate skeleton, cartilage is an important precursor to the formation of bones. Cartilage is produced by chondrocytes, which derive from embryonic mesoderm and undergo a stereotypical program of differentiation and maturation. Here we modeled this process in vitro, using primary fetal mouse rib chondrocytes in a high-density cell culture model of cartilage differentiation, and performed genome-wide gene expression profiling over the course of culture. The overarching goal of this study was to characterize the molecular pathways involved in cartilage differentiation and maturation. Our results also enable a comprehensive appraisal of distinctions between common in vitro models for cartilage differentiation, and of differences in their molecular resemblance to cartilage formation in vivo.

Chromatin modifier developmental pluripotency associated factor 4 (DPPA4) is a candidate gene for alcohol-induced developmental disorders

BackgroundPrenatal alcohol exposure (PAE) affects embryonic development, causing a variable fetal alcohol spectrum disorder (FASD) phenotype with neuronal disorders and birth defects. We hypothesize that early alcohol-induced epigenetic changes disrupt the accurate developmental programming of embryo and consequently cause the complex phenotype of developmental disorders. To explore the etiology of FASD, we collected unique biological samples of 80 severely alcohol-exposed and 100 control newborns at birth.MethodsWe performed genome-wide DNA methylation (DNAm) and gene expression analyses of placentas by using microarrays (EPIC, Illumina) and mRNA sequencing, respectively. To test the manifestation of observed PAE-associated DNAm changes in embryonic tissues as well as potential biomarkers for PAE, we examined if the changes can be detected also in white blood cells or buccal epithelial cells of the same newborns by EpiTYPER. To explore the early effects of alcohol on extraembryonic placental tissue, we selected 27 newborns whose mothers had consumed alcohol up to gestational week 7 at maximum to the separate analyses. Furthermore, to explore the effects of early alcohol exposure on embryonic cells, human embryonic stem cells (hESCs) as well as hESCs during differentiation into endodermal, mesodermal, and ectodermal cells were exposed to alcohol in vitro.ResultsDPPA4, FOXP2, and TACR3 with significantly decreased DNAm were discovered—particularly the regulatory region of DPPA4 in the early alcohol-exposed placentas. When hESCs were exposed to alcohol in vitro, significantly altered regulation of DPPA2, a closely linked heterodimer of DPPA4, was observed. While the regulatory region of DPPA4 was unmethylated in both control and alcohol-exposed hESCs, alcohol-induced decreased DNAm similar to placenta was seen in in vitro differentiated mesodermal and ectodermal cells. Furthermore, common genes with alcohol-associated DNAm changes in placenta and hESCs were linked exclusively to the neurodevelopmental pathways in the enrichment analysis, which emphasizes the value of placental tissue when analyzing the effects of prenatal environment on human development.ConclusionsOur study shows the effects of early alcohol exposure on human embryonic and extraembryonic cells, introduces candidate genes for alcohol-induced developmental disorders, and reveals potential biomarkers for prenatal alcohol exposure.

Genome-wide screening and expression of glutathione S-transferase genes reveal that GSTe4 contributes to sensitivity against β-cypermethrin in Zeugodacus cucurbitae

Glutathione S-transferases (GSTs) are an essential multifunctional protein family with common detoxifying enzymes. In this study, 34 GST genes were identified from the melon fly, Zeugodacus cucurbitae, one of the most destructive pests worldwide. These GSTs include 32 cytosolic genes and two microsomal genes. Furthermore, these cytosolic GSTs were classified into six classes: 11 delta, 13 epsilon, three theta, one sigma, two zeta, and two omega. Most of these showed dynamic expression during the developmental stage, some of which showed stage-specific expression. The expression in various adult tissues showed that most of them were expressed in anti-stress-related tissues. The transcriptional response of the delta and epsilon families was determined when Z. cucurbitae was exposed to three insecticides, abamectin, dinotefuran, and β-cypermethrin. Seven genes were significantly up-regulated by abamectin exposure. Moreover, five and four genes were significantly up-regulated with dinotefuran and β-cypermethrin exposure, respectively, demonstrating their involvement in the detoxification of these such toxic substances in Z. cucurbitae. One example of these genes, ZcGSTe4, was randomly selected to explore its function in response to β-cypermethrin exposure. Over-expressed ZcGSTe4 in E. coli showed significant tolerance to β-cypermethrin, and RNAi-mediated suppression of ZcGSTe4 also increased the sensitivity of melon fly to this agent. This study provides a foundation for further studies on the mechanism of detoxification metabolism in the melon fly.

Analysis of the response regulatory network of pepper genes under hydrogen peroxide stress.

Hydrogen peroxide (H2O2) is a regulatory component related to plant signal transduction. To better understand the genome-wide gene expression response to H2O2 stress in pepper plants, a regulatory network of H2O2 stress-gene expression in pepper leaves and roots was constructed in the present study. We collected the normal tissues of leaves and roots of pepper plants after 40 days of H2O2 treatment and obtained the RNA-seq data of leaves and roots exposed to H2O2 for 0.5-24 h. By comparing the gene responses of pepper leaves and roots exposed to H2O2 stress for different time periods, we found that the response in roots reached the peak at 3h, whereas the response in leaves reached the peak at 24h after treatment, and the response degree in the roots was higher than that in the leaves. We used all datasets for K-means analysis and network analysis identified the clusters related to stress response and related genes. In addition, CaEBS1, CaRAP2, and CabHLH029 were identified through a co-expression analysis and were found to be strongly related to several reactive oxygen species-scavenging enzyme genes; their homologous genes in Arabidopsis showed important functions in response to hypoxia or iron uptake. This study provides a theoretical basis for determining the dynamic response process of pepper plants to H2O2 stress in leaves and roots, as well as for determining the critical time and the molecular mechanism of H2O2 stress response in leaves and roots. The candidate transcription factors identified in this study can be used as a reference for further experimental verification.

Genome-wide identification and gene expression analysis of the 14–3-3 gene family in potato (Solanum tuberosum L.)

Background14–3-3 proteins are essential in regulating various biological processes and abiotic stress responses in plants. Although 14–3-3 proteins have been studied in model plants such as Arabidopsis thaliana and Oryza sativa, there is a lack of research on the 14–3-3 gene family in potatoes (Solanum tuberosum L.).ResultsA total of 18 14–3-3 genes encoding proteins containing a typical conserved PF00244 domain were identified by genome-wide analysis in potatoes. The St14–3-3 gene family members were unevenly distributed across the chromosomes, and gene structure analysis showed that gene length and intron number varied greatly among the members. Phylogenetic analysis of 14–3-3 proteins in potatoes and other plant species showed that they could be divided into two distinct groups (ε and non-ε). Members in the ε group tended to have similar exon-intron structures and conserved motif patterns. Promoter sequence analysis showed that the St14–3-3 gene promoters contained multiple hormone-, stress-, and light-responsive cis-regulatory elements. Synteny analysis suggested that segmental duplication events contributed to the expansion of the St14–3-3 gene family in potatoes. The observed syntenic relationships between some 14–3-3 genes from potato, Arabidopsis, and tomato suggest that they evolved from a common ancestor. RNA-seq data showed that St14–3-3 genes were expressed in all tissues of potatoes but that their expression patterns were different. qRT-PCR assays revealed that the expression levels of nearly all tested St14–3-3 genes were affected by drought, salt, and low-temperature stresses and that different St14–3-3 genes had different responses to these stresses.ConclusionsIn summary, genome-wide identification, evolutionary, and expression analyses of the 14–3-3 gene family in potato were conducted. These results provide important information for further studies on the function and regulation of St14–3-3 gene family members in potatoes.

CHANGES IN DIFFERENTIAL GENE EXPRESSION PATTERN IN FLY HEADS AS THE RESPONSE TO AN EXPERIMENTALLY EVOLVED X CHROMOSOME

Sexually antagonistic (SA) mutations that increase fitness in one sex and decrease it in the other sex (also known as intralocus sexual conflict) are central to the sexual antagonism hypothesis of sex chromosome evolution. It was suggested long ago (by Fisher, in 1931) that tight linkage to a sex-determining locus facilitates the accumulation of SA mutations even when their detrimental effect in one sex exceeds its benefit in the other. Due to the difficulties in detecting SA alleles directly, we know very little about the nature of X-linked SA mutations and their role in the evolution of differential gene expression between sexes. To investigate how X-linked SA mutations affect the transcriptome profile in both sexes, we analyzed changes in genome-wide gene expression pattern in Drosophila melanogaster head tissue that had undergone 95 generations of a female-limited X-chromosome experimental evolution. We found an evidence of change in gene expression towards to the female optimum, which we interpret as the result of resolution of sexual conflict over X-linked SA mutations. In addition, we also found a potential unknown gene expression effect of the balancer chromosome on gene expression (that was necessary to use to limit the inheritance of target X chromosome to the female line). All these findings indicate that a highly polymorphic nature of the X chromosome with an important role in genome-wide gene expression profile.

Orelabrutinib and venetoclax synergistically induce cell death in double-hit lymphoma by interfering with the crosstalk between the PI3K/AKT and p38/MAPK signaling.

Double-hit lymphoma (DHL) is a rare and aggressive mature B-cell malignancy with concurrentMYCandBCL2rearrangements. When DHL becomes relapsed or refractory, it becomes resistant to the majority of therapeutic approaches and has subpar clinical results. Therefore, innovative therapeutics for this particular patient population are urgently needed. Orelabrutinib, a new oral BTK inhibitor, combined with the Bcl-2 inhibitor venetoclax, was used to confirm the antitumor effect of DHL. Cell counting kit-8 and Annexin V-FITC/PI assays were used to examine the interaction of this combined regimen on DHL cell lines and primary lymphoma cells. RNA sequencing, EdU incorporation assay, mitochondrial membrane potential assay, and western blotting were employed to explore the molecule mechanism for the cytotoxicity of orelabrutinib with or without venetoclax against DHL cell lines. In this study, orelabrutinib combined with venetoclax synergistically induced DHL cell death, as evidenced by the inhibition of cell proliferation, the induct of cell cycle arrest, and the promotion of cell apoptosis via the mitochondrial pathway. Orelabrutinib treatment alters genome-wide gene expression in DHL cells. The combined regimen decreases the expression of BTK and Mcl-1, potentially interfering with the activity and crosstalk of PI3K/AKT signaling and p38/MAPK signaling. In addition, the combination of orelabrutinib and venetoclax shows cytotoxic activity in primary B-lymphoma cells. In summary, these findings reveal a novel therapy targeting BCR signaling and the Bcl-2 family for DHL patients with a poor prognosis.

Development of innovative marker detection methods for high-fertility ducks (Anas plastyrhynchos)

We previously analyzed the genome-wide gene expression at the transcription level in pre-hierarchical ovarian follicles (approximate 5 mm in diameter) between two groups of ducks representing high and low fertility. Orthodenticle homeobox 2 (otx2) was identified with significantly differential expression in the high-fertility group versus the low-fertility group. To identify the relationships between genotypes and phenotypes, we recorded the reproductive performance in advance, including fertility, hatchability, and fertile period of female ducks. To ensure coverage of the entire duration of the fertile period, we extended the egg collection period after artificial insemination. Naturally, sperm cannot survive after a certain period of time in the female reproductive tract (sperm is not immortal); therefore, lower average values for fertility were observed in this study than that observed after a normal egg collection period, i.e., the lower average values of fertility (18 days after artificial insemination), were not due to the effect of otx2. The otx2 genomic sequence of Tsaiya ducks was firstly amplified with a primer pair of i3F and i3R for polymerase chain reaction based on Pekin duck sequence and a resultant 444-base pair fragment was obtained for DNA sequencing. Using multiple sequence alignment, new single-nucleotide polymorphisms g.366T > C and g.182G > T were discovered in the otx2 gene. With respect to g.366T > C, ducks were classified into CC, CT, and TT genotypes. For g.182G > T, three genotypes (GG, GT, and TT) were identified. Ducks were genotyped using novel specific primers and probes to rapidly screen their single-nucleotide polymorphisms. The results indicated that ducks with the CC genotype of g.366T > C exhibited the highest fertility among the CC, CT, and TT genotypes (p < 0.05). No significant difference was found in the fertile period and hatchability among three genotypes of g.366T > C. Moreover, no association was found between g.182G > T genotypes and the three reproductive phenotypes examined in this study. Collectively, the otx2 g.366T > C genotype is associated with duck females, and can be used as a marker for farming a flock of ducks with high fertility, as well as for genetic selection of breeders.

SpatialCorr identifies gene sets with spatially varying correlation structure

Recent advances in spatially resolved transcriptomics technologies enable both the measurement of genome-wide gene expression profiles and their mapping to spatial locations within a tissue. A first step in spatial transcriptomics data analysis is identifying genes with expression that varies spatially, and robust statistical methods exist to address this challenge. While useful, these methods do not detect spatial changes in the coordinated expression within a group of genes. To this end, we present SpatialCorr, a method for identifying sets of genes with spatially varying correlation structure. Given a collection of gene sets pre-defined by a user, SpatialCorr tests for spatially induced differences in the correlation of each gene set within tissue regions, as well as between and among regions. An application to cutaneous squamous cell carcinoma demonstrates the power of the approach for revealing biological insights not identified using existing methods.

Adenosine signalling in T-cell activation favours development of IL-17 positive cells with suppressive properties.

Evidence suggests that the anti-inflammatory nucleoside adenosine can shape immune responses by shifting the regulatory (Treg )/helper (Th17) T-cell balance in favour of Treg . Since this observation is based on in vivo and in vitro studies mostly confined to murine models, we comprehensively analysed effects of adenosine on human T-cells. Proliferation, phenotype and cytokine production of stimulated T-cells were assessed by flow cytometry, multiplex assay and ELISA, gene expression profiling was determined by microarray. We found that the pan-adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) skews human CD3+ T-cell responses towards non-inflammatory Th17 cells. Addition of NECA during T-cell activation increased the development of IL-17+ cells with a CD4+ RORγt+ phenotype and enhanced CD161 and CD196 surface expression. Remarkably, these Th17 cells displayed non-inflammatory cytokine and gene expression profiles including reduced Th1/Th17 transdifferentiation, a stem cell-like molecular signature and induced surface expression of the adenosine-producing ectoenzymes CD39 and CD73. Thus, T-cells cultured under Th17-inducing conditions together with NECA were capable of suppressing responder T-cells. Finally, genome-wide gene expression profiling revealed metabolic quiescence previously associated with non-pathogenic Th17 cells in response to adenosine signalling. Our data suggest that adenosine induces non-inflammatory Th17 cells in human T-cell differentiation, potentially through regulation of metabolic pathways.

Autotetraploidization Gives Rise to Differential Gene Expression in Response to Saline Stress in Rice.

Plant polyploidization represents an effective means for plants to perpetuate their adaptive advantage in the face of environmental variation. Numerous studies have identified differential responsiveness to environmental cues between polyploids and their related diploids, and polyploids might better adapt to changing environments. However, the mechanism that underlies polyploidization contribution during abiotic stress remains hitherto obscure and needs more comprehensive assessment. In this study, we profile morphological and physiological characteristics, and genome-wide gene expression between an autotetraploid rice and its diploid donor plant following saline stress. The results show that the autotetraploid rice is more tolerant to saline stress than its diploid precursor. The physiological characteristics were rapidly responsive to saline stress in the first 24 h, during which the elevations in sodium ion, superoxide dismutase, peroxidase, and 1-aminocyclopropane-1-carboxylic acid were all significantly higher in the autotetraploid than in the diploid rice. Meanwhile, the genome-wide gene expression analysis revealed that the genes related to ionic transport, peroxidase activity, and phytohormone metabolism were differentially expressed in a significant manner between the autotetraploid and the diploid rice in response to saline stress. These findings support the hypothesis that diverse mechanisms exist between the autotetraploid rice and its diploid donor plant in response to saline stress, providing vital information for improving our understanding on the enhanced performance of polyploid plants in response to salt stress.

A Noncoding Germline Variant Creates a Gain-of-Function De Novo Enhancer That up-Regulates IL5 Transcription to Cause Familial Hypereosinophilia, Revealing a Previously Undescribed Mechanism for Human Disease

Introduction: Familial eosinophilia (FE) is a rare inherited condition characterized by lifelong idiopathic hypereosinophilia (absolute eosinophil count >1.5 x 109/L) in multiple family members with or without clinical manifestations. Although the gene underlying FE in one large kindred was mapped to chromosome 5q31-33 using genomewide linkage analysis, targeted sequencing of genes in this region, including interleukin 5 (IL5), was unrevealing. Despite more recent studies demonstrating selective dysregulation of IL5 transcription, the underlying genetic abnormality in FE has remained elusive to date. Methods: Whole genome sequencing (WGS) was performed using peripheral blood mononuclear cell (PBMC) DNA from 5 affected and 1 unaffected family members of the above-described kindred followed by targeted Sanger sequencing of candidate variants. After identifying a single nucleotide variant common to all affected family members in a noncoding region upstream of IL5, RNA sequencing (RNAseq), including long-read PacBio sequencing, was performed to examine IL5 gene expression and to identify IL5 isoforms. To predict potential transcription factor binding sites, we applied motif analyses around the variant and performed chromatin immunoprecipitation (ChIP)-PCR to assess chromatin occupancy of the predicted transcriptional regulators (HMGA1, FOXO3) and of RNA Polymerase II (Poll II). The variant of interest was introduced into a B-lymphoblast cell line (NCI-BL1184) via CRISPR/Cas9, after which clones derived from single cells were compared with respect to IL5 transcription (via quantitative PCR) and chromatin occupancy of HMGA1, FOXO3, and Pol II (by ChIP-PCR). Chromatin marks associated with active gene expression at enhancers (H3K4me1, H3K27Ac) and promoters (H3K4me3) were assessed by ChIP-PCR. Integration of genomewide ChIP-Seq, ATAC-Seq and gene expression analyses are underway to further assess the role of the variant as the primary driver of aberrant IL5 expression. Results: WGS revealed a novel single nucleotide variant in all 5 affected family members located 13.19 kb from the transcription start site (TSS) of the IL5 gene and 250 bp from the TSS of the RAD50 gene. Targeted Sanger sequencing confirmed the presence of this variant in 4/4 affected and 0/4 unaffected family members. Bulk RNA-seq revealed abnormally long transcripts spanning the entire intergenic region between the variant and the IL5 TSS in affected PBMCs, while long-read RNA-seq identified novel full-length IL5 isoforms, including one containing all 5 exons of the IL5 gene. By contrast, RAD50 transcription was unaffected by the variant. In silico motif analysis predicted that the variant generates potential binding sites for transcriptional regulators, including the High Mobility Group A1 (HMGA1) chromatin regulator and the forkhead transcription factor, FOXO3. ChIP-PCR confirmed robust chromatin binding of both HMGA1 and FOXO3 at the variant site in PBMCs from affected family members, but not in PBMCs lacking the variant. Introduction of the single nucleotide variant into B-lymphoblast cells via CRISPR/Cas9 recapitulates the findings in affected family members, with up-regulation of IL5 expression and protein levels and no effects on IL4, IL13, or RAD50 gene expression. In edited lymphoblasts with the variant, HMGA1 chromatin binding and that of H3K4me3/H3K27Ac are enriched in the 200 bp region encompassing the variant, although FOXO3 occupancy was not enriched in this setting nor were there changes in occupancy of control histone H3 in that region. Conclusions: We describe a previously unknown mechanism of human disease: a non-coding single nucleotide germline variant that results in a de novo enhancer and up-regulation of IL5 production in a family with autosomal dominant hypereosinophilia. Orthogonal methods indicate that this variant enables HMGA1 to bind DNA, recruit active histone marks, and induce IL5 expression from a de novo TSS. This finding was validated by CRISPR/Cas9-mediated introduction of the variant into a B cell lymphoblast cell line that does not normally produce IL-5. This mechanism highlights the importance of assessing non-coding regions in the study of Mendelian disorders and may be relevant to other human diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Genome-Wide Identification, Structural Characterization, and Gene Expression Analysis of BES1 Transcription Factor Family in Tartary Buckwheat (Fagopyrum tataricum)

The transcription factor (TFs) BES1, which mediates brassinosteroid (BR) signaling, regulates plant growth and development. However, BES1 genes have not yet been reported in Tartary buckwheat. Here, ten FtBES1 genes were identified in the Tartary buckwheat genome, and they were named FtBES1-1 to FtBES1-10. These genes were divided into four groups according to the classification in Arabidopsis thaliana. Multiple sequence alignment indicated that all BES1 gene members contained the BES1_N structural domain. Phylogenetic relationship FtBES1 genes in the same group had similar gene structures and motifs. An analysis of cis-acting elements demonstrated that the BES1 TFs contains many light-responsive, hormonal, and abiotic stress-responsive elements, etc. The 10 FtBES1 genes were located on four chromosomes of Tartary buckwheat, and gene distribution and synteny analysis revealed that segmental duplications have played important roles in FtBES1 gene family expansion. Tissue specificity revealed that all of the ten FtBES1 members expressed highly in two periods, and relatively high expression levels were observed in mature leaves. Gene expression profiles under different hormone treatments demonstrated that FtBES1 gene family participated in the hormone stress response. This study enriches our knowledge of the Tartary buckwheat BES1 gene family and provides a theoretical basis for analyzing the biological functions and stress tolerance mechanisms of the Tartary buckwheat BES1 transcription factors.

Transcriptomic Analysis in Human 3D Skin Model Injected with Resorbable Hyaluronic Acid Fillers Reveals Foreign Body Response.

Usage of injectable dermal fillers applied for aesthetic purposes has extensively increased over the years. As such, the number of related adverse reactions has increased, including patients showing severe complications such as product migration, topical swelling and inflammatory reactions of the skin. In order to understand the underlying molecular events of these adverse reactions we performed a genome-wide gene expression study on the multi-cell type human Phenion® Full-Thickness Skin Model exposed to five experimental hyaluronic acid (HA) preparations with increasing cross-linking degree, four commercial fillers from Perfectha®, and non-resorbable filler Bio-Alcamid®. In addition, we evaluated whether cross-linking degree or particle size of the HA-based fillers could be associated with the occurrence of adverse effects. In all cases, exposure to different HA fillers resulted in a clearly elevated gene expression of cytokines and chemokines related to acute inflammation as part of the foreign body response. Furthermore, for one experimental filler genes of OXPHOS complexes I-V were significantly down-regulated (adjusted p-value &lt; 0.05), resulting in mitochondrial dysfunction which can be linked to over-expression of pro-inflammatory cytokines TNFα and IL-1β and chemokine CCL2. Our hypothesis that cross-linking degree or particle size of the HA-based fillers is related to the biological responses induced by these fillers could only partially be confirmed for particle size. In conclusion, our innovative approach resulted in gene expression changes from a human 3D skin model exposed to dermal fillers that mechanistically substantiate aforementioned adverse reactions, and thereby adds to the weight of evidence that these fillers may induce inflammatory and fibrotic responses.