Down-regulated Gene Clusters Research Articles

Thyroidal Transcriptomic Profiles of Pathoadaptive Responses to Congenital Hypothyroidism in XB130 Knockout Mice.

Congenital hypothyroidism is a genetic condition in which the thyroid gland fails to produce sufficient thyroid hormone (TH), resulting in metabolic dysfunction and growth retardation. Xb130−/− mice exhibit perturbations of thyrocyte cytoskeleton and polarity, and develop postnatal transient growth retardation due to congenital hypothyroidism, leading ultimately to multinodular goiter. To determine the underlying mechanisms, we performed transcriptomic analyses on thyroid glands of mice at three age points: week 2 (W2, before visible growth retardation), W4 (at the nadir of growth); and W12 (immediately before full growth recovery). Using gene set enrichment analysis, we compared a defined set of thyroidal genes between Xb130+/+ and Xb130−/− mice to identify differentially enriched gene clusters. At the earliest postnatal stage (W2), the thyroid glands of Xb130−/− mice exhibited significantly downregulated gene clusters related to cellular metabolism, which continued to W4. Additionally, mutant thyroids at W4 and W12 showed upregulated gene clusters related to extracellular matrix, angiogenesis, and cell proliferation. At W12, despite nearly normal levels of serum TH and TSH and body size, a significantly large number of gene clusters related to inflammatory response were upregulated. Early postnatal TH deficiency may suppress cellular metabolism within the thyroid gland itself. Upregulation of genes related to extracellular matrix and angiogenesis may promote subsequent thyroid growth. Chronic inflammatory responses may contribute to the pathogenesis of multinodular goiter in later life. Some of the pathoadaptive responses of Xb130−/− mice may overlap with those from other mutations causing congenital hypothyroidism.

Bioinformatics analysis deciphering the transcriptomic signatures associated with signalling pathways and prognosis in the myelodysplastic syndromes

ABSTRACT Background: Several studies scatteredly identified the myelodysplastic syndromes’ transcriptomic profiles (MDS). However, the exploration of transcriptional signatures, key signalling pathways, and their association with prognosis and diagnosis in the integrated multiple datasets remains lacking. Methods: We integrated the GSE4619, GSE19429, GSE30195, and GSE58831 microarray datasets of CD34 + cells for identifying the differentially expressed genes (DEGs) in the MDS. The series of bioinformatics methods are applied to identify the key hub genes, gene clusters, prognostic hub genes, and genes associated with diagnostic efficacy. Finally, we validated the expression differences of hub genes in the GSE114922 dataset. Results: We explored the DEGs related to gene ontology enrichment and KEGG pathways. We identified significant hub genes, including 168 upregulated hub genes (such as STAT1, IFIH1, EPRS, GRB2, RAC2, MAPK14, CASP1, and SPI1) and 52 downregulated hub genes (such as CREBBP, HIF1A, PIK3CA, EZH2, PIK3R1, MDM2, IRF4, CXCR4, PCNA, and CD19) in the MDS. In addition, we identified six significant molecular complex detection (MCODE)-derived upregulated gene clusters and one downregulated gene cluster, respectively. Moreover, we found that the higher expression level of MX2, GBP2, PXN, IFI44, FDXR, PLCB2, ASS1, ERCC4, PML, and RRAGD and the lower expression level of CD19, PAX5, TCF3, LEF1, NUSAP1, and TIMELESS hub genes are significantly correlated with shorter survival times of MDS patients. Furthermore, the area value under the ROC curve (AUC) of PXN, FDXR, PLCB2, PML, CD19, PAX5, and LEF1 prognostic genes are more than 0.80, indicating that these genes could be effectively used for the diagnostic efficacy of MDS patients. Conclusions: Identifying key hub genes and their association with the prognosis and diagnostic efficacy may provide substantial clues for the treatment and diagnosis of MDS patients.

Striatal Transcriptome Reveals Differences Between Cognitively Impaired and Unimpaired Aged Male Rats.

Cognitive processes require striatal activity. The underlying molecular mechanisms are widely unknown. For this reason the striatal transcriptome of young (YM), aged cognitively impaired (OMB), and unimpaired (OMG) male rats was analyzed. The global comparison of transcripts reveal a higher number of differences between OMG and YM as compared to OMB and YM. Hierarchical clustering detects differences in up- and down-regulated gene clusters in OMG and OMB when compared to YM. In OMG we found more single genes to be specifically regulated in this group than in OMB when compared to young. These genes were considered as cognition specific, whereas genes shared in OMG and OMB were considered as age specific. OMB specific up-regulated genes are related to negative control of cell differentiation and transcription (Hopx), to phagocytosis (Cd202) and cell adhesion (Pcdhb21), whereas down-regulated genes are related to associative learning, behavioral fear response and synaptic transmission (Gabra5). OMG specific up-regulated genes are in the context of maintenance of transcription and estrogen receptor signaling (Padi2, Anxa3), signal transduction [Rassf4, Dock8)], sterol regulation (Srebf1), and complement activity (C4a, C4b). Down-regulated genes are related to lipid oxidation reduction processes (Far2) and positive regulation of axon extension (Islr2). These relations were supported by pathway analysis, which reveals cholesterol metabolism processes in both aged group and cholesterol biosynthesis specifically in OMG; adipogenesis and focal adhesion in OMB. In OMG glucuronidation, estrogen metabolism, inflammatory responses and TGF beta signaling where detected as specific for this group. Signal transduction of the sphingosine-1-phospate-receptor (S1P) receptor was the main pathway difference in the comparison of OMB and OMG with downregulated genes in the first group. This difference could also be observed in the OMB vs. YM comparison but not in the OMG vs. YM analysis. Thus, an up-regulation of cognition related genes could be observed in OMG compared to OMB rats. The S1P pathway discriminated between OMB and OMG as well as between OMB and OMG. Since this pathway has been described as essential for cognitive processes in the striatum of mice, it may, among steroid hormone signaling, significantly contribute to the maintenance of cognitive processes in OMG.

Identification of Pathways Associated with Placental Adaptation to Maternal Nutrient Restriction in Sheep.

Maternal nutrient restriction impairs placental growth and development, but available evidence suggests that adaptive mechanisms exist, in a subset of nutrient restricted (NR) ewes, that support normal fetal growth and do not result in intrauterine growth restriction (IUGR). This study utilized Affymetrix GeneChip Bovine and Ovine Genome 1.0 ST Arrays to identify novel placental genes associated with differential fetal growth rates within NR ewes. Singleton pregnancies were generated by embryo transfer and, beginning on Day 35 of pregnancy, ewes received either a 100% National Research Council (NRC) (control-fed group; n = 7) or 50% NRC (NR group; n = 24) diet until necropsy on Day 125. Fetuses from NR ewes were separated into NR non-IUGR (n = 6) and NR IUGR (n = 6) groups based on Day 125 fetal weight for microarray analysis. Of the 103 differentially expressed genes identified, 15 were upregulated and 88 were downregulated in NR non-IUGR compared to IUGR placentomes. Bioinformatics analysis revealed that upregulated gene clusters in NR non-IUGR placentomes associated with cell membranes, receptors, and signaling. Downregulated gene clusters associated with immune response, nutrient transport, and metabolism. Results illustrate that placentomal gene expression in late gestation is indicative of an altered placental immune response, which is associated with enhanced fetal growth, in a subpopulation of NR ewes.

Abstract 962: The dipeptidyl peptidase inhibitor BXCL701 activates innate immunity followed by adaptive immunity on a molecular and cellular level in a mouse model of pancreatic cancer

Abstract The oral DPP8/9 inhibitor BXCL701 in combination with an anti-PD-1 antibody (aPD-1) [AACR 2017] or in triple combination with aPD-1 and pegylated IL-2 (NKTR-214) [ASCO 2018] has demonstrated inhibition of tumor growth or complete regression respectively in animal models of pancreatic cancer. In the present study, the mechanism of action of BXCL701 has been elucidated in a sub-chronic pharmacokinetic / pharmacodynamic study at molecular and cellular level, by administering BXCL701 as a single agent. BXCL701 (20 μg) was administered orally every day for 14 days in tumor (Pan02) bearing mice. Tumor and serum samples were harvested after 0, 1, 8 and 16 hrs after BXCL701 administration on day 1, 7 and 14. Serum samples were analysed for cytokines and tumor tissues were analysed for infiltrating immune cells and gene expression. BXCL701 is known to inhibit regulatory proteases DPP8/9, consequently activating Nlrp1b inflammasome, which in turn activates pro-caspase-1 to mediate pyroptosis in mouse macrophages [Okondo et al, 2018]. Caspase-1 is involved in cleavage of pro-IL-1β and pro-IL-18 to their active forms, IL-1β and IL-18 respectively [Walle et al, 2016,]. IL-18 was observed to be significantly (p<0.05) upregulated (>50 fold) at 8 hrs on day 1 and achieved steady state levels by day 7 in BXCL701-treated animals. Other cytokines like IL-1β, IFN-γ, G-CSF, IL-5, IL-6, CXCL9, MCP-1, KC and Eotaxin were also observed to be upregulated in BXCL701-treated animals at the 8 hr timepoint on day 1, 7 and 14 in comparison to day 1, 0 hr. BXCL701 significantly upregulates T cells (total T cells, CD4+ T helper cells and CD8+ T cells) infiltration along with NK cells within the tumor microenvironment. It also appears to enhance antigen presentation by upregulating MHC class I genes and MHC class I expressing cells within the tumor. The mechanism of action of BXCL701 was evaluated at the molecular level as well. The comparison of genes in tumor tissues from BXCL701-treated animals vs respective vehicle-treated animals on Day 7 and 14 demonstrated that the upregulated gene clusters were innate and adaptive immune response genes, T-cell receptor genes and MHC genes. Also, genes associated with T cell and NK cell mediated apoptosis and cytolysis e.g. FasL, GzmA, were upregulated and indicates enhanced cell death (e.g. upregulation of pdcd1) within tumor. On the other hand, downregulated gene clusters belonged to functional categories like cell cycle, DNA repair, several genes associated with cancer progression (GPCRs and Olfactory receptors) and extra cellular matrix (ECM) modification (collagen and metalloproteases). In conclusion, BXCL701-treatment induces innate and adaptive immune responses that leads to tumor growth inhibition probably via inducing cell death and reducing ECM modification. Citation Format: Veena Agarwal, John MacDougall, shubhendu Trivedi, Dimple Bhatia, Zeenia Jagga, Hemant Banga, Diane Healy, Sreenivas Adurthi, Vince O'Neill. The dipeptidyl peptidase inhibitor BXCL701 activates innate immunity followed by adaptive immunity on a molecular and cellular level in a mouse model of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 962.

Enrichment of Up-regulated and Down-regulated Gene Clusters Using Gene Ontology, miRNAs and lncRNAs in Colorectal Cancer.

It is interesting to find the gene signatures of cancer stages based on the omics data. The aim of study was to evaluate and to enrich the array data using gene ontology and ncRNA databases in colorectal cancer. The human colorectal cancer data were obtained from the GEO databank. The downregulated and up-regulated genes were identified after scoring, weighing and merging of the gene data. The clusters with high-score edges were determined from gene networks. The miRNAs related to the gene clusters were identified and enriched. Furthermore, the long non-coding RNA (lncRNA) networks were predicted with a central core for miRNAs. Based on cluster enrichment, genes related to peptide receptor activity (1.26E-08), LBD domain binding (3.71E-07), rRNA processing (2.61E-34), chemokine (4.58E-19), peptide receptor (1.16E-19) and ECM organization (3.82E-16) were found. Furthermore, the clusters related to the non-coding RNAs, including hsa-miR-27b-5p, hsa-miR-155-5p, hsa-miR-125b-5p, hsa-miR-21-5p, hsa-miR-30e-5p, hsa-miR-588, hsa-miR-29-3p, LINC01234, LINC01029, LINC00917, LINC00668 and CASC11 were found. The comprehensive bioinformatics analyses provided the gene networks related to some non-coding RNAs that might help in understanding the molecular mechanisms in CRC.

Mechanism of salt-inhibited early seed germination analysed by transcriptomic sequencing

AbstractSeed germination, the first and critical step of the plant's life cycle, is affected by salt stress. However, the underlying mechanism of salt tolerance during early seed germination remains elusive. Here, a comparative RNA-seq analysis was performed using early germinating seeds either under normal conditions or in 100 and 150 mM sodium chloride. A total of 575 genes were up-regulated and 913 genes were down-regulated in the presence of 100 mM NaCl. Under the 150 mM NaCl treatment 1921 genes were up-regulated and 3501 genes were down-regulated. A total of 379 or 863 genes were up-regulated or down-regulated in both 100 and 150mM NaCl. These co-regulated genes were further analysed by GO enrichment. Genes in the categoriesabscisic acid signaling and synthesisandnutrient reservoir activitywere significantly enriched in the up-regulated genes. Transcription factors responsive to gibberellin and auxin were significantly down-regulated by salinity stress. Genes related toanti-oxidant activitywere significantly enriched in the down-regulated gene clusters by NaCl treatment. Our results suggest that salt stress inhibits seed germination by activating ABA synthesis and signalling, and depressing GA and auxin signalling, while preserving nutrition and down-regulated anti-oxidant activity. Our study provides more insight into the molecular mechanism of salt tolerance during early seed germination.

P066 INTESTINAL EPITHELIAL MICROVILLI ARE ABNORMAL IN CROHN’S DISEASE

Crohn’s disease (CD) presents as chronic and often progressive intestinal inflammation, but the contributing pathogenic mechanisms are unclear. Our goal was to identify underlying alterations in intestinal host cells that could contribute to the chronic and progressive course of CD. We performed RNA-seq with RNA extracted from formalin-fixed, paraffin-embedded (FFPE) ileal tissue sections obtained from CD subjects (n=32) and controls without inflammatory bowel disease (n=36). This method allowed us to select relatively uninflamed samples prior to molecular profiling and to perform histological validation with serial tissue sections. Accurate sampling of microvillar length was achieved by measuring 5 cells per villi and 10 villi per sample. We developed new methods to visualize an overlapping modular network of dysregulated genes in CD. With partitional clustering, we identified multiple down-regulated gene clusters present in nearly all of the CD samples. One cluster of 1,056 down-regulated genes was functionally associated with formation and stabilization of microvilli, leading us to quantify microvillar length. CD samples had a significant reduction in microvillar length relative to controls and this directly correlated to average microvillar gene cluster expression, validating our results. Network analysis supported a common regulatory mechanism for several of the common down-regulated gene clusters associated with altered metabolism in the intestinal tissue of CD subjects and this super-cluster was linked to the microvillar cluster. The FFPE transcriptomic approach can identify and link transcriptional alterations to histological alterations in CD tissue, such as a previously unrecognized alteration in microvillar length. We propose that decreased enterocyte microvillar length and gene expression represents an ongoing epithelial malfunction that is independent of active inflammation or local tissue damage.

P066 INTESTINAL EPITHELIAL MICROVILLI ARE ABNORMAL IN CROHN'S DISEASE

Crohn’s disease (CD) presents as chronic and often progressive intestinal inflammation, but the contributing pathogenic mechanisms are unclear. Our goal was to identify underlying alterations in intestinal host cells that could contribute to the chronic and progressive course of CD. We performed RNA-seq with RNA extracted from formalin-fixed, paraffin-embedded (FFPE) ileal tissue sections obtained from CD subjects (n=32) and controls without inflammatory bowel disease (n=36). This method allowed us to select relatively uninflamed samples prior to molecular profiling and to perform histological validation with serial tissue sections. Accurate sampling of microvillar length was achieved by measuring 5 cells per villi and 10 villi per sample. We developed new methods to visualize an overlapping modular network of dysregulated genes in CD. With partitional clustering, we identified multiple down-regulated gene clusters present in nearly all of the CD samples. One cluster of 1,056 down-regulated genes was functionally associated with formation and stabilization of microvilli, leading us to quantify microvillar length. CD samples had a significant reduction in microvillar length relative to controls and this directly correlated to average microvillar gene cluster expression, validating our results. Network analysis supported a common regulatory mechanism for several of the common down-regulated gene clusters associated with altered metabolism in the intestinal tissue of CD subjects and this super-cluster was linked to the microvillar cluster. The FFPE transcriptomic approach can identify and link transcriptional alterations to histological alterations in CD tissue, such as a previously unrecognized alteration in microvillar length. We propose that decreased enterocyte microvillar length and gene expression represents an ongoing epithelial malfunction that is independent of active inflammation or local tissue damage.

Identification and characterization of large DNA deletions affecting oil quality traits in soybean seeds through transcriptome sequencing analysis.

Key messageIdentification and characterization of a 254-kb genomic deletion on a duplicated chromosome segment that resulted in a low level of palmitic acid in soybean seeds using transcriptome sequencing.A large number of soybean genotypes varying in seed oil composition and content have been identified. Understanding the molecular mechanisms underlying these variations is important for breeders to effectively utilize them as a genetic resource. Through design and application of a bioinformatics approach, we identified nine co-regulated gene clusters by comparing seed transcriptomes of nine soybean genotypes varying in oil composition and content. We demonstrated that four gene clusters in the genotypes M23, Jack and N0304-303-3 coincided with large-scale genome rearrangements. The co-regulated gene clusters in M23 and Jack mapped to a previously described 164-kb deletion and a copy number amplification of the Rhg1 locus, respectively. The coordinately down-regulated gene clusters in N0304-303-3 were caused by a 254-kb deletion containing 19 genes including a fatty acyl-ACP thioesterase B gene (FATB1a). This deletion was associated with reduced palmitic acid content in seeds and was the molecular cause of a previously reported nonfunctional FATB1a allele, fapnc. The M23 and N0304-304-3 deletions were located in duplicated genome segments retained from the Glycine-specific whole genome duplication that occurred 13 million years ago. The homoeologous genes in these duplicated regions shared a strong similarity in both their encoded protein sequences and transcript accumulation levels, suggesting that they may have conserved and important functions in seeds. The functional conservation of homoeologous genes may result in genetic redundancy and gene dosage effects for their associated seed traits, explaining why the large deletion did not cause lethal effects or completely eliminate palmitic acid in N0304-303-3.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-016-2725-z) contains supplementary material, which is available to authorized users.

Genome-wide Analysis of Acute Inflammatory and Anti-Inflammatory Responses in RAW264 Cells Suggests cis-Elements Associated with Translational Regulation

Translational regulation plays pivotal roles in mediating inflammatory responses. Glucocorticoids, represented in this study by dexamethasone (DEX), are widely recognized anti-inflammatory agents that exert significant inhibitory effects on the translation of diverse gene groups, including inflammatory genes. However, their regulation is highly complex and diverse, involving transcriptional and translational regulation. Although transcriptional regulation has been investigated by genome-wide transcriptome analyses, translational regulation has been studied by only a few specific gene targets (e.g., Tumor necrosis factor) and the global impact of glucocorticoids on translation levels has scarcely been studied, mainly due to its technical difficulty. Here, using ribosome profiling coupled with high-throughput mRNA sequencing (mRNA-Seq) in which footprints of translating ribosomes can be captured, we conducted a genomewide transcriptional and translational analysis of the acute inflammatory or anti-inflammatory responses of RAW264 cells stimulated by lipopolysaccharide (LPS) or LPS coupled with DEX (LPS + DEX). We showed that the majority of the differential regulation between LPS alone and LPS + DEX were predominated by translation levels rather than transcription levels. Further analysis on the up- and down-regulated gene clusters revealed putative cis regulatory elements exclusively enriched in the 3'-UTR of either up- or down-regulated genes induced by LPS + DEX. The results imply an alternative to the currently recognized mechanisms of glucocorticoid-induced translational regulation in the acute inflammatory response of RAW264 cells.

Molecular Profile of Mitochondrial Dysfunction in Kidney Transplant Biopsies Is Associated With Poor Allograft Outcome

BackgroundIn kidney transplantation (KT), progression of chronic histological damage with subclinical inflammation is associated with poor long-term allograft survival. The role of nonimmunological pathways in chronic allograft injury has not been fully assessed. MethodsWe analyzed a public microarray dataset that used 1-year protocol kidney transplant biopsy specimens to investigate whether nonimmunological genes and pathways might influence long-term allograft outcome. The selected microarray dataset included 3 patient/sample groups based on their histological findings: normal histology (n = 25), interstitial fibrosis alone (IF alone, n = 24), and interstitial fibrosis with inflammation (IF+i, n = 16). The IF+i group had lower death-censored graft survival and renal function in patients with a mean follow-up of 4 years. We performed statistical analysis comparing gene expression patterns in the 3 group samples. ResultsGene cluster enrichment and group-specific expression patterns demonstrated a divergent pattern between mitochondrial and immune response genes, with downregulation of mitochondrial genes in the IF+i group. Gene ontological analysis of the downregulated mitochondrial genes identified generation of precursor metabolite and energy, and response to oxidative stress as the most significant biological processes. The transcription regulation pathway analysis of downregulated gene cluster demonstrated transcription factors involved in mitochondrial biogenesis. ConclusionsThe molecular signature of mitochondrial dysfunction reflects mitochondrial energetic insufficiency, and inadequate antioxidant response involved in mitochondria biogenesis pathways is associated with IF+i and worse long-term allograft survival. Thus, mitochondria function impairment appears to be an important nonimmune factor involved in chronic allograft injury.

CoBi: Pattern Based Co-Regulated Biclustering of Gene Expression Data

Co-regulation is a common phenomenon in gene expression. Finding positively and negatively co-regulated gene clusters from gene expression data is a real need. Existing techniques based on global similarity are unable to detect true up- and down-regulated gene clusters. This paper presents an expression pattern based biclustering technique, CoBi, for grouping both positively and negatively regulated genes from microarray expression data. Regulation pattern and similarity in degree of fluctuation are accounted for while computing similarity between two genes. Unlike traditional biclustering techniques, which use greedy iterative approaches, it uses a BiClust tree that needs single pass over the entire dataset to find a set of biologically relevant biclusters. Biclusters determined from different gene expression datasets by the technique show highly enriched functional categories.

Identification of a Gene Regulatory Network Necessary for the Initiation of Oligodendrocyte Differentiation

Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes requires extensive changes in gene expression, which are partly mediated by post-translational modifications of nucleosomal histones. An essential modification for oligodendrocyte differentiation is the removal of acetyl groups from lysine residues which is catalyzed by histone deacetylases (HDACs). The transcriptional targets of HDAC activity within OPCs however, have remained elusive and have been identified in this study by interrogating the oligodendrocyte transcriptome. Using a novel algorithm that allows clustering of gene transcripts according to expression kinetics and expression levels, we defined major waves of co-regulated genes. The initial overall decrease in gene expression was followed by the up-regulation of genes involved in lipid metabolism and myelination. Functional annotation of the down-regulated gene clusters identified transcripts involved in cell cycle regulation, transcription, and RNA processing. To define whether these genes were the targets of HDAC activity, we cultured rat OPCs in the presence of trichostatin A (TSA), an HDAC inhibitor previously shown to inhibit oligodendrocyte differentiation. By overlaying the defined oligodendrocyte transcriptome with the list of ‘TSA sensitive’ genes, we determined that a high percentage of ‘TSA sensitive’ genes are part of a normal program of oligodendrocyte differentiation. TSA treatment increased the expression of genes whose down-regulation occurs very early after induction of OPC differentiation, but did not affect the expression of genes with a slower kinetic. Among the increased ‘TSA sensitive’ genes we detected several transcription factors including Id2, Egr1, and Sox11, whose down-regulation is critical for OPC differentiation. Thus, HDAC target genes include clusters of co-regulated genes involved in transcriptional repression. These results support a de-repression model of oligodendrocyte lineage progression that relies on the concurrent down-regulation of several inhibitors of differentiation.

Gene expression patterns in bone following mechanical loading

The advent of high-throughput measurements of gene expression and bioinformatics analysis methods offers new ways to study gene expression patterns. The primary goal of this study was to determine the time sequence for gene expression in a bone subjected to mechanical loading during key periods of the bone-formation process, including expression of matrix-related genes, the appearance of active osteoblasts, and bone desensitization. A standard model for bone loading was employed in which the right forelimb was loaded axially for 3 minutes per day, whereas the left forearm served as a nonloaded contralateral control. We evaluated loading-induced gene expression over a time course of 4 hours to 32 days after the first loading session. Six distinct time-dependent patterns of gene expression were identified over the time course and were categorized into three primary clusters: genes upregulated early in the time course, genes upregulated during matrix formation, and genes downregulated during matrix formation. Genes then were grouped based on function and/or signaling pathways. Many gene groups known to be important in loading-induced bone formation were identified within the clusters, including AP-1-related genes in the early-response cluster, matrix-related genes in the upregulated gene clusters, and Wnt/β-catenin signaling pathway inhibitors in the downregulated gene clusters. Several novel gene groups were identified as well, including chemokine-related genes, which were upregulated early but downregulated later in the time course; solute carrier genes, which were both upregulated and downregulated; and muscle-related genes, which were primarily downregulated. © 2011 American Society for Bone and Mineral Research.

Analysis of gene expression profiles of liver stellate cells during liver regeneration in rats.

This study performed a large-scale, high-throughput analysis of transcriptional profiling of liver stellate cells (LSCs) at the cellular level to investigate changes in the biological activity of LSCs during rat liver regeneration (LR) and the relation of these changes to LR. First, a rat liver regeneration model was established by partial hepatectomy (PH). Stellate cells were isolated in high purity and yield from the regenerating rat liver by Percoll density gradient centrifugation and immunomagnetic bead sorting. The changes in gene expression of LSCs after PH were examined using a rat genome 230 2.0 array composed of 24622 genes. The results indicated that 10241 of the 24622 genes investigated on the array were differentially expressed in LSCs. Of the 10241 genes, 1563 known genes were related to LR, which were grouped into three major gene expression clusters according to three-fold cut-off threshold: the upregulated gene cluster, the down-regulated gene cluster, and the cluster composed of genes showing complex changes in expression. Additionally, the genes were grouped into those involved in transcription regulation, signal transduction, transport, cellular metabolism, inflammation and immunity by functional analysis. When gene expression profiles were combined with the results of gene functional analysis, most of the genes involved in cytokine secretion and retinol metabolism in LSCs were significantly enriched in the cluster characterized by decreased expression, whereas genes involved in lipid metabolism were mostly enriched in the cluster showing increased expression. Based on further analysis of genes expressed in a phase-dependent manner during LR, it was suggested that lipid metabolism in LSCs was enhanced in the whole regeneration process, and that immune response and cytokine secretion were impaired during all three regenerative phases.

Critical Role for GATA3 in Mediating Tie2 Expression and Function in Large Vessel Endothelial Cells

Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.

Gene Ontology analysis in multiple gene clusters under multiple hypothesis testing framework

Gene Ontology (GO) has become a routine resource for functional analysis of gene lists. Although a number of tools have been provided to identify enriched GO terms in one or two gene lists, two technical challenges remain. First, how to handle multiple hypothesis testing in the analysis given that the tests are heavily correlated; second, how to identify GO terms that are enriched in a gene cluster, as compared to multiple other gene clusters. We provide a statistical procedure to rigorously treat these problems and offer a software tool for applying GO to the analysis of gene clusters. We previously introduced a statistical procedure that handles hypothesis testing in a two-group comparison scenario. In this paper we extend the two-group comparison procedure into a general procedure that enables the analysis of any number of gene lists/clusters. This new procedure enables identification of GO terms enriched in any gene cluster, while it controls for multiple hypothesis testing. This procedure is implemented into a user-friendly analysis tool: GoSurfer. The current version of GoSurfer takes one or several gene lists as input, and it identifies the GO terms that are enriched in any of the input gene lists. GoSurfer estimates a conservative false discovery rate (FDR) for every GO term. The FDR estimation procedure in GoSurfer has two advantages: it does not rely on independence assumption, and it does not assume all the hypotheses are null hypothesis (complete null). Thus GoSurfer's FDR estimates are mildly conservative rather than overly conservative. We implemented the new procedure for GO analysis in multiple gene clusters into the GoSurfer software. We provide three examples on using GoSurfer to analyze time course gene expression data sets on the differentiation of embryonic stem cells. In the example of analysis of multiple gene clusters, we first used a typical clustering algorithm and identified five gene clusters, representing up-regulation, down-regulation and other patterns in the differentiation time course. Taking all the five gene clusters as input data, GoSurfer reports "cell adhesion" and "muscle contraction" as significant GO terms for the up-regulated cluster, "amino acids metabolism" as a significant GO term for the down-regulated gene cluster, and GoSurfer reports a number of GO terms related to RNA processing and RNA transport as significant terms to a cluster that is up-regulated in both early and late time points. This may suggest that genes for RNA processing and genes for RNA transport are coregulated in the differentiation process of embryonic stem cells. The GoSurfer software is provided to analyze multiple gene clusters and identify GO terms that are enriched in any gene cluster. Gosurfer is available at: www.gosurfer.org.

Activity-dependent neuroprotective protein (ADNP) differentially interacts with chromatin to regulate genes essential for embryogenesis

Complete deficiency in activity-dependent neuroprotective protein (ADNP) results in neural tube closure defects and death at days 8.5–9.5 of gestation in the mouse (E8.5–9.5). To elucidate ADNP associated pathways, Affymetrix 22,690-oligonucleotide-based microarrays were used on ADNP knockout and control mouse embryos (E9) separated completely from extra embryonic tissue. Marked differences in expression profiles between ADNP-deficient embryos and ADNP-expressing embryos were discovered. Specifically, a group of dramatically up-regulated gene transcripts in the ADNP-deficient embryos were clustered into a family encoding for proteins enriched in the visceral endoderm such as apolipoproteins, cathepsins and methallotionins. In contrast, a down regulated gene cluster associated with ADNP-deficiency in the developing embryo consisted of organogenesis markers including neurogenesis ( Ngfr, neurogenin1, neurod1) and heart development ( Myl2). The pluripotent P19 cells were used for ADNP-chromatin-immunoprecipitation, showing direct interactions with multiple relevant gene promoters including members of the up-regulated as well as the down-regulated gene clusters. A comparison between non-differentiated and neuro-differentiated P19 cells revealed increased chromatin interaction of ADNP with chromatin from differentiated cells. These results place ADNP at a crucial point of gene regulation, repressing potential endoderm genes and enhancing genes associated with organogenesis/neurogenesis.

Global Transcriptional Analysis Details Prior Knowledge and Identifies New Genes and Processes Associated with T-Cell Activation in the Context of Ex Vivo Expansion.

Introduction. Ex vivo expanded T cells are increasingly employed in a variety of immunotherapy protocols. The success of such protocols requires large numbers of biologically active T cells. Several activation and culture protocols have been examined, but none in significant or molecular detail to allow more targeted protocol interventions. In this study, our aim was to better understand early T-cell activation using a comprehensive, microarray-based transcriptional analysis, in conjunction with targeted protein-level and functional analyses.Methods. Three biological experiments were transcriptionally analyzed. T cells were activated with anti-CD3/anti-CD28 Mab, cultured for 96 hours, and samples collected at 0, 4, 10, 48 and 96 hours. mRNA levels at each time point were compared to that of 0 hour. Genes were selected as follows: a minimum 1.8-fold difference in at least 6 time points of the 12 (3x4) time points. Data were subject to Gene Ontology (GO) analysis, EASE, to discover enriched biological themes. Interferon-γ (IFNG) and CCL20 ELISA assays and NF-κB p65 (active complex) assay by flow cytometry were carried out to confirm select findings of the transcriptional analysis.Results. 2340 genes passed the gene-selection criteria. GO analysis showed that the GO groups Immune Response, Regulation of Apoptosis, and Mitochondrion are among the most over-represented groups among differentially expressed genes during T-cell activation. The upregulated Immune Response group contained many genes coding cytokines typically produced by helper T cells. Highly upregulated genes in this category include IL2, CCL20, CXCL11, CXCL9, CXCL10, CD83, IFNG, CCL3, CSF2, IL23A, XCL2, TNF, CCL4, TNFSF6 and ICOS. Although several of these findings would have been expected, our data include a more extensive gene list and suggest potential use of these cytokines as T-cell ex vivo culture supplements. We singled out CCL20, sharing a very similar transcription pattern with IL2, and IFNG, for further analysis. ELISA assays of IFNG and CCL20 showed that the kinetics of secreted protein levels were in agreement with transcriptional data. In the GO group Regulation of Apoptosis, the downregulated gene cluster was enriched by pro-apoptotic genes, while the upregulated cluster was enriched by anti-apoptotic genes. Our data indicated that NF-κB complex activity changed upon T-cell activation. This was verified by NF-κB p65 Phosflow assay, showing that phosphorylated NF-κB p65 was suppressed up to 10 hours, and then increased until hour 96. Although it is known that mitochondrial hyperpolarization is important in T-cell activation, our transcriptional data suggest more profound changes in mitochondrial biology. Genes in this category include several having oxidoreductase activity, such as SOD2, SCO1, NDUFAB1, GLUD2, consistent with reported increases in intracellular ROS levels upon T-cell activation, and suggest the potential use of anti-oxidants in ex vivo T-cell expansion.Conclusion. Our data and analysis provide a better understanding of T-cell activation and identify several potential targets that can be explored to benefit T-cell expansion protocols for immunotherapy applications. Although there is much known about T-cell activation, modern genomic tools provide an extraordinary opportunity to verify, extend and enrich prior knowledge, and, discover new players and processes not previously associated with T-cell activation.