Whole-genome expression profiling of embryonic and monocyte-derived Kupffer cells
Iron is an essential component of the erythrocyte protein hemoglobin and is crucial to oxygen transport in vertebrates. In the steady state, erythrocyte production is in equilibrium with erythrocyte removal1. In various pathophysiological conditions, erythrocyte life span is severely compromised, which threatens the organism with anemia and iron toxicity 2,3. Here we identify anon-demand mechanism specific to the liver that clears erythrocytes and recycles iron. We showthat Ly-6Chigh monocytes ingest stressed and senescent erythrocytes, accumulate in the liver, and differentiate to ferroportin 1 (FPN1)-expressing macrophages that can deliver iron to hepatocytes. Monocyte-derived FPN1+ Tim-4neg macrophages are transient, reside alongside embryonically-derived Tim-4high Kuppfer cells, and depend on Csf1 and Nrf2. The spleenlikewise recruits iron-loaded Ly-6Chigh monocytes, but they do not differentiate into ironrecycling macrophages due to the suppressive action of Csf2, and are instead shuttled to the livervia coordinated chemotactic cues. Inhibiting this mechanism by preventing monocyte recruitment to the liver leads to kidney failure and liver damage. These observations identify the liver as the primary organ supporting emergency erythrocyte removal and iron recycling, and uncover a mechanism by which the body adapts to fluctuations in erythrocyte integrity. Pregnant CX3CR1-CreERT+/+ Stopfl/fl tdTomato mice were injected subcutaneously with tamoxifen (8 mg/animal in 400 µl corn oil s.c.) on days 13.5and 15.5 p.c to induce tdTomato expression in embryonic monocytes/macrophages. At the age of 3 months, the progeny was transplanted with GFP bone marrow after busulfan pre-conditioning. After establishment of stable chimerism among blood leukocytes (3 weeks), mice were transfused with stressed red blood cells (sRBC). Embryonic (eKCs, CD45+ CD11b- F4/80+ GFP- tdTomato+) and monocyte-derived Kupffer cells (mKCs, CD45+ CD11b- F4/80+ GFP+ tdTomato-) of the liver were FACS-sorted 7 days after sRBC treatment and subjected to whole-genome expression profiling
Genome-wide identification of PPARG binding sites in NCI-H2347 and NCI-H1993 cell lines
While the regulation of metabolic enzymes by oncogenic drivers or tumor suppressors has been intensively studied over recent years, our understanding of how metabolic processes directly regulate cell proliferation has remained fragmentary. Here we show how the alteration of metabolism directly affects cell cycle progression in cancer cells. We found that activation of the nuclear receptor peroxisome-proliferation activated receptor gamma (PPARγ), a transcriptional master regulator of lipid metabolism, inhibits the growth of lung adenocarcinoma cells by triggering a metabolic switch that inhibits pyruvate oxidation and reduces glutathione levels. These PPARγ-induced metabolic changes result in a marked increase of reactive oxygen species (ROS) levels that lead to rapid hypophosphorylation of retinoblastoma protein (RB) and cell cycle arrest. Both of these changes can be prevented by suppressing pyruvate dehydrogenase kinase 4 (PDK4) or β-oxidation of fatty acids. Thus, we provide a mechanism that directly links metabolic changes to inhibition of cancer cell cycle progression by altering ROS levels.
Human TALL tumor vs Normal Human DNA
Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the cancer process. Here, we engineered lymphoma-prone mice with chromosomal instability to assess the utility of mouse models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number aberrations. Integrating with targeted re-sequencing, our comparative oncogenomic studies efficiently identified FBXW7 and PTEN as commonly deleted or mutated tumor suppressors in human T-cell acute lymphoblastic leukemia/lymphoma (T-ALL). More generally, the murine cancers acquire widespread recurrent clonal amplifications and deletions targeting loci syntenic to alterations present in not only human T-ALL but also diverse tumors of hematopoietic, mesenchymal and epithelial types. These results thus support the view that murine and human tumors experience common biological processes driven by orthologous genetic events as they evolve towards a malignant phenotype. The highly concordant nature of genomic events encourages the use of genome unstable murine cancer models in the discovery of biologically relevant driver events in human cancer. Keywords: comparative genomic hybridization
HDAC inhibitor SAHA reverses inflammatory gene expression in diabetic endothelial cells
While histone deacetylase (HDAC) inhibitors are thought to regulate gene expression by post-translational modification of histone as well as non-histone proteins. While histone hyperacetylation has long been considered the paradigmatic mechanism of action, recent genome-wide profiles indicate more complex interactions with the epigenome. In particular, HDAC inhibitors also induce histone deacetylation at the promoters of highly active genes, resulting in gene suppression. This was linked to the loss of histone acetyltransferase (HAT) binding. To illustrate pre-clinical utility of the HDAC inhibitor SAHA as a therapeutic, we show reversal of diabetes-associated EP300 target genes in diabetic HAECs of primary origin. These results were confirmed using SAHA, C646 (EP300/CREBBP inhibitor) or EP300 siRNA. These findings suggest the inhibition of gene expression by SAHA is mediated by EP300 function and provide a rationale for clinical trials of safety and efficacy in patients with diabetes.
Dynamic and coordinated expression changes of rice small RNAs in response to Xanthomonas oryzae pv. oryzae
Endogenous small RNAs are newly identified players in plant immune responses, yet their roles in rice (Oryza sativa) responding to pathogens are still less understood, especially for pathogens that can cause severe yield losses. Here, we examined the small RNA expression profiles of rice leaves at 2, 6, 12, and 24 hours post infection of Xanthomonas oryzae pv. oryzae (Xoo) virulent strain PXO99, the causal agent of rice bacterial blight disease. Dynamic expression changes of some miRNAs and trans-acting siRNAs (ta-siRNAs) were identified, together with a few novel miRNA targets, including a disease resistance gene targeted by osa-miR159a.1. Coordinated expression changes were observed among some miRNA and ta-siRNAs in response to Xoo infection, with small RNAs exhibiting the same expression pattern tended to regulate genes in the same or functional correlated signaling pathways, including auxin and GA signaling pathways, nutrition and defense related pathways, etc. Highly abundant small RNAs with pathogen-responsive expression changes were identified from the exonic region of a protein-coding gene, which may present a new class of functional small RNAs. These findings reveal the dynamic and complex roles of small RNAs in rice-pathogen interactions, and identified new targets for regulating plant immune responses.
Human Colon tumor vs Normal Human DNA
Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the cancer process. Here, we engineered lymphoma-prone mice with chromosomal instability to assess the utility of mouse models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number aberrations. Integrating with targeted re-sequencing, our comparative oncogenomic studies efficiently identified FBXW7 and PTEN as commonly deleted or mutated tumor suppressors in human T-cell acute lymphoblastic leukemia/lymphoma (T-ALL). More generally, the murine cancers acquire widespread recurrent clonal amplifications and deletions targeting loci syntenic to alterations present in not only human T-ALL but also diverse tumors of hematopoietic, mesenchymal and epithelial types. These results thus support the view that murine and human tumors experience common biological processes driven by orthologous genetic events as they evolve towards a malignant phenotype. The highly concordant nature of genomic events encourages the use of genome unstable murine cancer models in the discovery of biologically relevant driver events in human cancer. Keywords: comparative genomic hybridization
Slug-dependent upregulation of L1CAM is responsible for the increased invasion potential of pancreatic cancer cells following long-term 5-FU-treatment
In pancreatic cancer the survival rate is low, as the available treatment options usually only extend survival and seldom produce a cure. Drug resistance and disease reoccurrence is the typical reason for death after cancer diagnosis. 5-Fluorouracil (5-FU) is the main chemostatic used in first line therapy. However the majority of the tumors become resistant to treatment. To investigate acquired 5-FU resistance in pancreatic adenocarcinoma, we established chemoresistant monoclonal cell lines from the Panc03.27 cell line by long-term exposure to 5-FU. In addition to increased expression of markers associated with multidrug resistance, the 5-FU resistant clones showed alterations typical of the process of epithelial-to-mesenchymal transition (EMT), including upregulation of mesenchymal markers and increased invasiveness. Microarray analysis revealed the L1CAM pathway as one of the most upregulated pathways in the chemoresistant clones, which was confirmed on RNA and protein levels. Expression of the adhesion molecule L1CAM is associated with a chemoresistant and migratory phenotype of pancreatic cancer. Using esiRNA targeting L1CAM, or by blocking the extracellular part of L1CAM with monoclonal antibodies, we discovered that the increased invasiveness observed in the chemoresistant cells depends on L1CAM. Using esiRNA targeting β-catenin and/or Slug, we discovered that L1CAM expression depends on Slug rather than β-catenin in the 5-FU resistant cells. We demonstrate a functional link between Slug and the expression level of L1CAM in pancreatic cancer cells having undergone EMT following long-term exposure to 5-FU. Our findings provide further insight into the molecular mechanisms leading to a chemoresistant and migratory phenotype in pancreatic cancer cells and indicate the importance of Slug-induced L1CAM in refractory pancreatic cancer.
Expression profile of MCF7, CCD18 and Ramos human cell lines
To uncover the chromosome 16 associated proteome and to take advantage of the generated knowledge to make progress in human biology in health and disease, a consortium of 15 groups was organized in four working groups: SRM and protein sequencing, antibody and peptide standard, clinical healthcare and biobanking and bioinformatics. According to a preliminary in silico study integrating knowledge from Ensembl, UniProt and GPM, Ramos B lymphocyte cells, MCF-7 epitelial cells and CCD18 fibroblast were selected as it is theoretically expected that any chromosome 16 protein coding gene is expressed in at least one of them. To define in detail the transcriptome of the above mentioned cell lines Affymetrix microarray based analyses were performed. Upon hybridization in Human ST 1.0 arrays, raw data were processed with RMA algorithm for background correction and normalization. Chromosome 16 gene expression pattern was then defined in each cell line and comparative analysis was done with R package statistics. Biological functions involving chromosome 16 genes were analysed with GO and functional networks were studied with Ingenuity Pathway Analysis. Expressed genes were compared with data from shotgun proteomic experiments to find the degree of correlation mRNA-protein. Expression of genes coding for proteins with weak or none MS evidence is shown. The integration of this information in decision-making process of the mass spectrometry group is discussed.
Expression in Kir6.1-deficient heart following LPS challenge
KATP opposes depolarization of cells in the heart, smooth muscle, and other tissues by permitting the efflux of potassium ions and this efflux is evidently required to prevent unopposed vasoconstriction and insufficiency of coronary artery blood flow triggered by one or more cytokines induced in response to LPS. The cytokine(s) involved must elicit a dysfunctional response in the Kir6.1-deficient environment, and to gain further insight into the effects of the mutation, we examined the transcriptional status of whole heart, isolated from normal C57BL/6J mice or KcnJ8Md/Md mice, before and after injection of 1 μg of LPS Keywords: Time course, stress response, disease state analysis, genetic modification
Contribution of Veillonella parvula to Pseudomonas aeruginosa mediated pathogenicity in a murine tumor model system
We investigated the specific interactions of the most dominant bacterial CF-pathogen, Pseudomonas aeruginosa, and the anaerobic bacterium Veilllonella parvula, that has been recovered at comparable cell numbers in the respiratory tract of CF patients. We used our recently established in-vivo murine tumor model to investigate mutual influences of the two pathogens during a biofilm-associated infection process. We found that although P. aeruginosa and V. parvula colonized distinct niches within the tumor, in mice that were co-infected with both bacterial species significant higher cell numbers of P. aeruginosa were recovered from the tumor tissue. Concordantly, in vivo transcriptional profiling implied that the presence of V. parvula supports P. aeruginosa growth at the infected host site, and the higher P. aeruginosa load correlated with clinical deterioration.
Immune response of the Caribbean sea fan, Gorgonia ventalina exposed to an Aplanochytrium parasite as revealed by transcriptome sequencing
Background. Coral reef communities are undergoing marked declines due to a variety of stressors including climate change, eutrophication, sedimentation, and disease. The sea fan coral, Gorgonia ventalina, is a tractable study system to investigate the hypothesis that stressors compromise immunity and lead to onset of disease. Functional studies in Gorgonia ventalina immunity indicate that several key pathways and cellular responses are involved in response to natural microbial invaders, although to date the functional and regulatory pathways remain largely un-neffectors, the primary line of defense in invertebrates. This study used short-read sequencing (Illumina GAIIx) to identify genes involved in the response of G. ventalina to a naturally occurring Aplanochytrium spp. parasite. Results. De novo assembly of the G. ventalina transcriptome yielded 90,230 contigs of which 40, 142 were annotated. RNA-Seq analysis revealed 210 differentially expressed genes in sea fans exposed to the Aplanochytrium parasite. Differentially expressed genes involved in immunity include pattern recognition molecules, anti-microbial peptides, wound repair, and reactive oxygen species. Gene enrichment analysis indicated eight biological processes were enriched representing 36 genes, largely involved with protein translation and energy production. Conclusions. This is the first report using high-throughput sequencing to characterize the host response of a coral to a natural pathogen. Furthermore, we have generated the first transcriptome for a soft coral species. G. ventalina is a non-model species for which few sequences had been previously described, and we were able to annotate a large number of genes and describe their potential roles in immune function. Expression analysis revealed genes important in invertebrate innate immune pathways, as well as those whose role is previously un-described in cnidarians. This resource will be valuable in characterizing G. ventalina immune response to infection and co-infection of pathogens in the context of environmental change.
Comparative genomic hybridization (CGH) of human melanoma vs. normal human samples
Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the cancer process. Here, we engineered lymphoma-prone mice with chromosomal instability to assess the utility of mouse models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number aberrations. Integrating with targeted re-sequencing, our comparative oncogenomic studies efficiently identified FBXW7 and PTEN as commonly deleted or mutated tumor suppressors in human T-cell acute lymphoblastic leukemia/lymphoma (T-ALL). More generally, the murine cancers acquire widespread recurrent clonal amplifications and deletions targeting loci syntenic to alterations present in not only human T-ALL but also diverse tumors of hematopoietic, mesenchymal and epithelial types. These results thus support the view that murine and human tumors experience common biological processes driven by orthologous genetic events as they evolve towards a malignant phenotype. The highly concordant nature of genomic events encourages the use of genome unstable murine cancer models in the discovery of biologically relevant driver events in human cancer. Experiment Overall Design: 123 Melanoma samples were analyzed. Normal Human DNA was used as reference. Most samples were hybridized with dye-swap replica.
Cyclic mechanical stretch-induced gene expression
With gene expression profiling it was aimed to identify the differentially expressed genes associated with the regulation of the cytoskeleton to investigate the stretch-induced cell alignment mechanism. A whole genome microarray based analysis of the stretch-induced gene expression changes was done. Gene expression was measured at the beginning of the alignment process showing first reoriented cells after 5 h stretching and at the end after 24 h, where nearly all cells are aligned. Cyclic mechanical stretching of cells results in cellular alignment perpendicular to the stretch direction regulating cellular response. This stress response is assumed to be an adaptation mechanism to reduce extensive stretching but also acts as architectural restructuring changing performance and biomechanics of the tissue. Gene expression profiling of control vs. stretched primary human dermal fibroblasts after 5 h and 24 h demonstrated the regulation of differentially expressed genes associated with metabolism, differentiation and morphology.
Flow dependent gene expression in the rat aorta under physiological conditions
Objective: Shear forces play a key role in the maintenance of vessel wall integrity. Current understanding regarding shear-dependent gene expression is mainly based on in vitro or in vivo observations with experimentally deranged shear, hence reflecting acute molecular events in relation to flow. Our objective was to combine computational fluid dynamic (CFD) simulations with global microarray analysis to study flow-dependent vessel wall biology in portions of the entire aorta under physiological conditions. Methods and Results: Animal-specific WSS magnitude and vector direction were estimated using CFD based on aortic geometry and flow information acquired by MRI. Two distinct flow pattern regions were identified in the normal rat aorta; the distal part of the inner curvature being exposed to low WSS and a non-uniform vector direction, and a region along the outer curvature being subjected to markedly higher levels of WSS and a uniform vector direction. Microarray analysis identified numerous novel mechanosensitive genes, including Hand2, trpc4 and slain2, and confirmed well-known ones, such as klf2 and BMP4. Three genes were further validated for protein , including Hand2, which showed higher expression in the endothelium in regions exposed to disturbed flow. Gene ontology analysis revealed an over-representation of genes involved in transcriptional regulation.
RNA sequencing of circulating tumour cells implicates WNT signaling in pancreatic cancer metastasis (mouse data)
Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. While these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as enriched in CTCs. Expression of Wnt2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. The effect of Wnt2 is correlated with fibronectin upregulation, and it is mediated in part through non-canonical Wnt signaling and suppressed by inhibition of the Map3k7 (Tak1) kinase, an integrator of Wnt, BMP and TGF-beta signaling. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple Wnt genes, and pancreatic CTCs revealed significant enrichment for non-canonical Wnt signaling in 5 of 11 cases. Thus, molecular analysis of CTCs may identify novel therapeutic targets to prevent the distal spread of cancer.
Perivascular Progenitor Cells Derived from Human Embryonic Stem Cells Exhibit Functional Characteristics of Pericytes, and Improve the Retinal Vasculature in a Rodent Model of Diabetic Retinopathy
Diabetic retinopathy (DR) is the leading cause of blindness in working-age people. Pericyte loss is one of the pathologic cellular events in DR, which weakens the retinal microvessels. Damages to the microvascular networks are irreversible and permanent, thus further progression of DR is inevitable. In this study, we hypothesize that multipotent perivascular progenitor cells derived from human ESCs (hESC-PVPCs) improve the damaged retinal vasculature in the streptozotocin (STZ)-induced diabetic rodent models. We describe a highly efficient and feasible protocol to derive such cells using a natural selection method without cell sorting processes. As a cellular model of pericytes, hESC-PVPCs exhibited marker expressions such as CD140B, CD146, NG2, and functional characteristics of pericytes. Following a single intravitreal injection into diabetic Brown Norway (BN) rats, we demonstrate that the cells localized alongside typical perivascular regions of the retinal vasculature, and stabilized blood-retinal barrier (BRB) breakdown. Findings in this study highlight a therapeutic potential of hESC-PVPCs in DR by mimicking the role of pericytes in vascular stabilization.
Atm-/-,mTerc-/-,p53-/- triple knock-out lymphoma vs normal mouse DNA (GPL2872)
Highly rearranged and mutated cancer genomes present major challenges in the identification of pathogenetic events driving the cancer process. Here, we engineered lymphoma-prone mice with chromosomal instability to assess the utility of mouse models in cancer gene discovery and the extent of cross-species overlap in cancer-associated copy number aberrations. Integrating with targeted re-sequencing, our comparative oncogenomic studies efficiently identified FBXW7 and PTEN as commonly deleted or mutated tumor suppressors in human T-cell acute lymphoblastic leukemia/lymphoma (T-ALL). More generally, the murine cancers acquire widespread recurrent clonal amplifications and deletions targeting loci syntenic to alterations present in not only human T-ALL but also diverse tumors of hematopoietic, mesenchymal and epithelial types. These results thus support the view that murine and human tumors experience common biological processes driven by orthologous genetic events as they evolve towards a malignant phenotype. The highly concordant nature of genomic events encourages the use of genome unstable murine cancer models in the discovery of biologically relevant driver events in human cancer. Keywords: comparative genomic hybridization, genetic modification
Pseudomonas aeruginosa Ceftolozane-Tazobactam Resistance Development Requires Multiple Mutations Leading to Overexpression and Structural Modification of AmpC.
We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ∆mutS) P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64× MICs of each antibiotic in triplicate experiments. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64× MIC for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic, and strain. Ceftolozane-tazobactam-resistant mutants were further characterized by whole-genome analysis through RNA sequencing (RNA-seq). The development of high-level resistance was fastest for ceftazidime, followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed cross-resistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower, and high-level resistance was observed for the mutator strain only. PAO1 derivatives that were moderately resistant (MICs, 4 to 8 ug/ml) to ceftolozane-tazobactam showed only 2 to 4 mutations, which determined global pleiotropic effects associated with a severe fitness cost. High-level-resistant (MICs, 32 to 128 ug/ml) PAOMS derivatives showed 45 to 53 mutations. Major changes in the global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, which was caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1 to 4 mutations in the conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, compared to those in wild-type ampC. Therefore, the development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.
A permissive chromatin state regulated by ZFP281-AFF3 in controlling the imprinted Meg3 polycistron
Genomic imprinting is an epigenetic regulation which leads to gene expression in a parent-of-origin specific manner. Previously we have demonstrated that AFF3, the central component of Super Elongation Complex-like 3 (SEC-L3), can specifically bind both the intergenic differentially methylated region (IG-DMR) and the enhancer within the imprinted Dlk1-Dio3 locus to regulate the expression of the Meg3 polycistron. However, the mechanism underlying how AFF3 can interact with distinct chromatin regulatory elements remains unknown. Here, we demonstrate that AFF3 is associated with the Krüppel-like zinc finger protein ZFP281. ZFP281 is required for the recruitment of AFF3 to the enhancer and regulates the allele-specific expression of the Meg3 polycistron in mouse embryonic stem (ES) cells at the transcriptional elongation level. Our genome-wide analyses further identify ZFP281 as a critical factor generally associating with AFF3 at enhancers and functioning together with AFF3 in regulating the expression of a subset of gene. Our study suggests that different zinc finger proteins can function as molecular switchers to regulate the context-dependent function of AFF3 and set a balanced chromatin state for maintaining the allele specific expression pattern of the imprinted Dlk1-Dio3 locus. PLEASE BE AWARE THAT THE SUBMITTER REQUESTED DELETION OF BIGWIG AND PEAK-CALL FILES POST-PUBLICATION. PLEASE CONTACT SUBMITTER FOR FURTHER INFORMATION.
Natural antisense transcripts accumulate for over 70% of maize genes and exhibit genotypic-specific expression patterns
Natural Antisense Transcripts (NATs) can regulate gene expression by virtue of their ability to form double-stranded RNA duplexes. To investigate NATs in the maize transcriptome, cDNAs from seedling of two inbred lines (B73 and Mo17) were hybridized to an oligonucleotide microarray designed to validate the expression of in silico detected NATs and to screen for NATs that can anneal to a random set of 3’ UTRs and selected repeats found in 3’ UTRs regions. Quantitative Real-Time PCR experiments were conducted to determine the minimum detection threshold of microarray experiment and to thereby identify genes for which both sense and antisense transcripts accumulate to detectable levels. Two independent approaches, strand-specific RT-PCR and S1 nuclease assays were conducted to validate the results of the microarray experiment. Based on these conservative assays, NATs accumulate in seedlings that can anneal to over 70% of a random set of maize genes. In addition, both sense and antisense transcripts anneal to more than 80% of a set of maize repeats. Significantly, sense and antisense transcripts exhibit significant different expression patterns between the two genotypes. Based on these findings we hypothesize that interactions between sense and antisense transcripts may contribute to the differential patterns of gene expression in maize hybrids and to heterosis. Keywords: Global antisense transcripts profiling between two maize inbreds
KDM6 demethylase independent loss of histone H3 lysine 27 trimethylation during early embryonic development
H3K27me3 represses developmental genes at initial embryonic stages. The KDM6 family, comprised of UTX and JMJD3, are the only known proteins that demethylate H3K27me3 and they are hypothesized to catalyze the rapid removal of repressive chromatin in early mammalian development. However, we report that male embryos carrying mutations in both Utx and Jmjd3 survive to term and appear phenotypically normal at mid-gestation. We utilize several cell culture models to demonstrate that H3K27me3 is lost from repressed promoters in the absence of active KDM6 demethylation. Our data indicate that KDM6 H3K27me3 demethylation is not essential in the early embryo and that H3K27me3 loss from developmental genes occurs via novel mechanisms.
Global changes in the nuclear positioning of chromatin domains and genomic interactions that orchestrate B cell fate
The genome is folded into domains that are located in either transcriptionally inert or permissive compartments. Here we used genome-wide strategies to characterize domains during B cell development. Structured Interaction Matrix Analysis revealed that CTCF occupancy was primarily associated with intra-domain interactions, whereas p300, E2A, Pax5 and PU.1 were involved with intra- and inter-domain interactions that are developmentally regulated. We identified a spectrum of genes that switched nuclear location during early B cell development. In progenitors the transcriptionally inactive Ebf1 locus was sequestered at the nuclear lamina, thereby preserving multipotency, however upon development into the pro-B cell stage Ebf1 and other genes switched compartments to establish de novo intra- and inter-domain interactions that were associated with B lineage specific transcription signatures.
RNA sequencing of circulating tumour cells implicates WNT signaling in pancreatic cancer metastasis (human data)
Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. While these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as enriched in CTCs. Expression of Wnt2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. The effect of Wnt2 is correlated with fibronectin upregulation, and it is mediated in part through non-canonical Wnt signaling and suppressed by inhibition of the Map3k7 (Tak1) kinase, an integrator of Wnt, BMP and TGF-beta signaling. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple Wnt genes, and pancreatic CTCs revealed significant enrichment for non-canonical Wnt signaling in 5 of 11 cases. Thus, molecular analysis of CTCs may identify novel therapeutic targets to prevent the distal spread of cancer.
Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in self-gelling alginate discs reveals novel chondrogenic signature gene clusters
We have used a disc-shaped self-gelling alginate hydrogel as a scaffold for in vitro chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells. The comparison of monolayer cells and alginate embedded cells with or without differentiation medium allowed us to perform a detailed kinetic study of the expression of a range of genes and proteins known to be involved in chondrogenesis, using real-time polymerase chain reaction, fluorescence immunohistochemistry, and glycosaminoglycan measurement in the supernatant. mRNA encoding type II collagen (COL2), COL10, aggrecan, and SOX5, 6, and 9 were greatly elevated already at day 7, whereas COL1 and versican mRNA were gradually reduced. COL2 and aggrecan were dispersed throughout the extracellular matrix at day 21, whereas COL10 distribution was mainly intra/pericellular. COL1 seemed to be produced by only some of the cells. SOX proteins were predominantly localized in the nuclei. Then, using microarray analysis, we identified a signature cluster of extracellular matrix and transcription factor genes upregulated during chondrogenesis similar to COL2A1, and clusters of genes involved in immune responses, blood vessel development, and cell adhesion downregulated similar to the chemokine CXCL12. Analysis of the signature chondrogenic clusters, including novel potential marker genes identified here, may provide a better understanding of how the stem cell fate could be directed to produce perfect hyaline cartilage implants
Photomixotrophic growth protein A (PmgA) regulates carbohydrate metabolism through the non-coding RNA Ncr0700/PmgR1 in the cyanobacterium Synechocystis sp. PCC 6803
Carbohydrate metabolism is a tightly regulated process in photosynthetic organisms. The ability to utilize glucose for growth and to regulate storage carbohydrate (i.e. glycogen) metabolisms requires a number of regulatory elements. In the cyanobacterium Synechocystis sp. PCC 6803, the photomixotrophic growth protein A (PmgA), a putative serine/threonine kinase, is involved in governing these abilities; however its downstream regulatory elements are not well studied. Here a genome-wide microarray analysis of a ∆pmgA strain revealed altered transcript levels of several non-coding RNAs, among them Ncr0700 was the most strongly reduced transcript. Generally, Ncr0700 is highly conserved among cyanobacteria. In Synechocystis its expression is light-dependent, i.e. the abundance is reciprocal to the light intensity. Compared to WT and similar to a ΔpmgA mutant, a ∆ncr0700 deletion strain showed a ~2 fold increase in glycogen content under photoautotrophic conditions. Under these conditions ∆ncr0700 grew similarly to the wild type; in contrast its growth was arrested after a shift to photomixotrophic conditions. Ectopic expression of Ncr0700 in ∆ncr0700 as well as ∆pmgA restored both the glycogen and photomixotrophic growth to the wild type levels. These results indicate that Ncr0700 plays a role in regulation of carbohydrate metabolism, acts downstream of PmgA and therefore is renamed here to PmgR1 for photomixotrophic growth RNA 1.
Gene expression signature as a predictor of recurrence in stage II Colorectal cancer
We have been attempting to stratify the stage II CRC patients into good or poor prognoses based on gene expression signatures of resected specimen. We collected more CRC stage II cases up to 300, and analyzed gene expression for those samples. We selected a set of 3,023 probes (1,978 genes) for extracting metastasis-related discriminator genes. These genes consisted of 491 prognosis related genes identical to the probes on another platform array, and other probes for hybridization and quality control. Using these 3,023 probes, we attempted to select discriminator genes, with the 150 training sample set, resulting 55 markers. We then applied the discriminator set to the second, 150 samples set in a blind test for predictive accuracy and prognostic validity. The multivariate Cox analyses showed the significant parameters to predict the prognosis with a hazard ratio of 3.5 (95% confidence interval, 0.12-0.67; p=0.004). This personalized prognosis is crucial with CRC patients, the predictive performance may deserve serious consideration for possible application to clinic.
Halobacterium NRC-1, ZnSO4 Stress Response series 2
Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down regulation of Mn(II) uptake and up regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all these discoveries into a unified systems level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms. Keywords: stress response, dose response
Transcriptomic analysis of CV. ‘Shine Muscat’ (Vitis vinifera L.) buds during dormant and non dormant period
We used Illumina RNA-Seq technology to carry out digital gene expression profiling of Dormant and non dormant buds of Shine Muscat Cultivar.127 pathways were annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and metabolic, biosynthesis of secondry metabolite and. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that DEGs were involved in various metabolic processes, including phytohormone metabolism. The newly generated transcriptome and gene expression profiling data provide valuable genetic information for revealing transcriptomic variation during dormant and non dormant period in Grapevine and ultimately helpful in uplifting of table grape industry.
Halobacterium NRC-1, CoSO4 Stress Response series 2
Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down regulation of Mn(II) uptake and up regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all these discoveries into a unified systems level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms. Keywords: stress response, dose responseH
Discovery of a phenotypic switch regulating sexual mating in the opportunistic fungal pathogen Candida tropicalis
Sexual reproduction can promote genetic diversity in eukaryotes, and yet many pathogenic fungi have been labeled as obligate asexual species. It is becoming increasingly clear, however, that cryptic sexual programs may exist in some species, and that efficient mating requires the necessary developmental switch to be triggered. In this study we investigate Candida tropicalis, an important human fungal pathogen that has been reported to be asexual. Significantly, we demonstrate that C. tropicalis uses a phenotypic switch to regulate a cryptic program of sexual mating. Thus, diploid a and α cells must undergo a developmental transition to the mating-competent form, and only then does efficient cell-cell conjugation take place resulting in the formation of stable a/α tetraploids. We show that both the phenotypic switch and sexual mating depend on the conserved transcriptional regulator Wor1, which is regulated by temperature in other fungal species. In contrast, C. tropicalis mating occurs efficiently at both 25 °C and 37 °C, suggesting that it could occur in the mammalian host and have direct consequences for the outcome of an infection. Transcriptional profiling further reveals that ≈400 genes are differentially expressed between the two phenotypic states, including the regulatory factor Wor1. Taken together, our results demonstrate that C. tropicalis has a unique sexual program, and that entry to this program is controlled via a Wor1-mediated, metastable switch. These observations have direct implications for the regulation and evolution of cryptic sexual programs in related fungal pathogens.
Halobacterium NRC-1, NiSO4 Stress Response series 2
Given that transition metals are essential cofactors in central biological processes, misallocation of the wrong metal ion to a metalloprotein can have resounding and often detrimental effects on diverse aspects of cellular physiology. Therefore, in an attempt to characterize unique and shared responses to chemically similar metals we have reconstructed physiological behaviors of Halobacterium NRC-1, an archaeal halophile, in sub-lethal levels of Mn(II), Fe(II), Co(II), Ni(II), Cu(II) and Zn(II). Over 20% of all genes responded transiently within minutes of exposure to Fe(II), perhaps reflecting immediate large scale physiological adjustments to maintain homeostasis. At steady state, each transition metal induced growth arrest, attempts to minimize oxidative stress, toxic ion scavenging, increased protein turnover and DNA repair, and modulation of active ion transport. While several of these constitute generalized stress responses, up regulation of active efflux of Co(II), Ni(II), Cu(II), and Zn(II), down regulation of Mn(II) uptake and up regulation of Fe(II) chelation, confer resistance to the respective metals. We have synthesized all these discoveries into a unified systems level model to provide an integrated perspective of responses to six transition metals with emphasis on experimentally verified regulatory mechanisms. Finally, through comparisons across global transcriptional responses to different metals we provide insights into putative in vivo metal selectivity of metalloregulatory proteins and demonstrate that a systems approach can help rapidly unravel novel metabolic potential and regulatory programs of poorly studied organisms. Keywords: stress response, dose response
Dietary Genistein Prevents Denervation-Induced Muscle Atrophy in Male Rodents via Effects on Estrogen Receptor-α.
Genistein is one of the flabonoids which is included in high concentration in soy and has a high estrogenic activity. Beneficial effects of estrogen or hormone replacement therapy (HRT) on muscle mass or muscle atrophy have been demonstrated. We investigated the preventive effects and underlying mechanisms of genistein intake on denervation-induced muscle atrophy. Genistein intake significantly suppressed the loss of soleus muscle weight and the denervation-induced up-regulations of FOXO1 protein. The results of a DNA microarray showed that the estrogen receptor (ER) target genes are changed by genistein intake. Genistein suppressed the soleus muscle atrophy, and it was attenuated under the ER antagonist treatment. The administration of an ERα agonist suppressed the denervation-induced muscle atrophy and up-regulation of Atrogin1 gene expression, but the ERβ agonist had no effect.
Mesenchymal stem cells reduce inflammation while enhancing bacterial clearance and improving survival in sepsis.
Expression data from Total RNA extracted from murine spleen, liver, lungs, kidneys and hearts. Sepsis was induced in C57Bl/6J mice by cecal ligation and puncture (CLP), followed 6 hours later by an intravenous injection of Mesenchymal Stem Cell (MSC) or saline. Twenty-eight hours after CLP, plasma, bronchoalveolar lavage (BAL) fluid and tissues were collected for analyses. Total RNA was extracted using Trizol (as per manufactures' instruction) followed by clean-up procedure using Qiagen RNA easy Prep (as per manufactures instructions) In the following study we hypothesized that mesenchymal stem cells (MSCs), which have documented immunomodulatory properties, would reduce sepsis-associated inflammation and organ injury in a clinically relevant model of sepsis.
Phenotypic and transcriptional response to selection for alcohol sensitivity in Drosophila melanogaster
Alcoholism is a complex disorder determined by interactions between genetic and environmental risk factors. Drosophila represents a powerful model system to dissect the genetic architecture of alcohol sensitivity, as large numbers of flies can readily be reared in defined genetic backgrounds and under controlled environmental conditions. Furthermore, flies exposed to ethanol undergo physiological and behavioral changes that resemble human alcohol intoxication, including loss of postural control, sedation, and development of tolerance. We performed artificial selection for alcohol sensitivity for 25 generations and created duplicate selection lines that are either highly sensitive or resistant to ethanol exposure along with unselected control lines. We used whole genome expression analysis to identify 1,678 probe sets with different expression levels between the divergent lines, pooled across replicates, at a false discovery rate of q < 0.001. We assessed to what extent genes with altered transcriptional regulation might be causally associated with ethanol sensitivity by measuring alcohol sensitivity of 37 co-isogenic P-element insertional mutations in 35 candidate genes, and found that 32 of these mutants differed in sensitivity to ethanol exposure from their co-isogenic controls. Furthermore, 23 of these novel genes have human orthologues. Combining whole genome expression profiling with selection for genetically divergent lines is an effective approach for identifying candidate genes that affect complex traits, such as alcohol sensitivity. Because of evolutionary conservation of function, it is likely that human orthologues of genes affecting alcohol sensitivity in Drosophila may contribute to alcohol-associated phenotypes in humans. Keywords: artificial selection, whole genome expression profiling
Involvement of β-defensin 130 (DEFB130) in the macrophage microbicidal mechanisms for killing Plasmodium falciparum
Understanding the molecular defense mechanism of macrophages and identifying their effector molecules against malarial parasites may provide important clues for the discovery of new therapies. To analyze the immunological responses of malarial parasite-induced macrophages, we used DNA microarray technology to examine the gene profile of differentiated macrophages phagocytizing Plasmodium falciparum-parasitized erythrocytes (iRBCs). The transcriptional gene profile of macrophages in response to iRBCs represented 168 down-regulated genes, which were mainly involved in the cellular immune response, and 216 upregulated genes, which were involved in cellular proteolysis, growth, and adhesion. Importantly, the specific upregulation of β-defensin 130 (DEFB130) in these macrophages suggested a possible role for DEFB130 in malarial parasite elimination. Strikingly, differentiated macrophages phagocytizing iRBCs exhibited an increase in intracellular DEFB130 levels and DEFB130 appeared to accumulate at the site of iRBC engulfment. Furthermore, DEFB130 synthetic peptide exhibited a modest toxic effect on P. falciparum in vitro and P. yoelii in vivo, unlike scrambled DEFB130 peptide, which showed no antiplasmodial activity. Our data broaden our knowledge of the immunological response of macrophages to iRBCs and shed light on a new target for therapeutic intervention.
Gene expression analysis of human keratinocytes (HaCaTs) after non-thermal plasma treatment
Investigation of gene expression in human skin keratinocytes (HaCaT) following non-thermal plasma treatment for 20 s, 60 s, and 180 s compared to untreated and H2O2-treated controls. Microarrays were used to analyze and investigate the biological effects of non-thermal plasma on human keratinocyte cells. Using an argon plasma jet kinpen, regulated transcripts were analyzed and further described in Schmidt et al. (2014): “Transcript profiling identifies an important role for Nrf2/Keap1-pathway after non-thermal plasma treatment in human keratinocytes”.
Notch-mediated suppression of multiciliate differentiation promotes choroid plexus tumor initiation from progenitors in response to epithelium-derived Sonic Hedgehog
Aberrant Notch signaling has been linked to many cancers including choroid plexus (CP) tumors, a group of rare and predominantly pediatric brain neoplasms. We developed animal models of CP tumors by inducing sustained expression of Notch1 that recapitulate properties of human CP tumors with aberrant NOTCH signaling. Whole transcriptome and functional analyses showed that tumor cell proliferation is associated with Sonic Hedgehog (Shh) in the tumor microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumor cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate diffferentiation. A Shh-driven signaling cascade in the primary cilium occurs in tumor cells but not in epithelial cells. Lineage studies show that CP tumors arise from mono-ciliated progenitors in the roof plate characterized by elevated Notch signaling. Abnormal SHH signaling and distinct ciliogenesis are detected in human CP tumors, suggesting SHH pathway and cilia differentiation as potential therapeutic avenues.
Culturing Cytotrophoblasts Reverses Gene Dysregulation in Preeclampsia Revealing Possible Causes
During human pregnancy, a subset of placental cytotrophoblasts (CTBs) differentiates into cells that aggressively invade the uterus and its vasculature, anchoring the progeny and rerouting maternal blood to the placenta. In preeclampsia (PE), CTB invasion is limited, reducing placental perfusion and/or creating intermittent flow. This syndrome, affecting 4-8% of pregnancies, entails maternal vascular alterations (e.g., high blood pressure, proteinuria, and edema) ± fetal growth restriction. The only cure is removal of the faulty placenta, i.e., delivery. Previously we showed that defective CTB differentiation contributes to the placental component of PE, but the causes were unknown. Here, CTBs isolated from PE and control placentas were cultured for 48 h, enabling differentiation/invasion. In various severe forms of PE, transcriptomics revealed common aberrations in CTB gene expression immediately after isolation that resolved in culture. The upregulated genes included SEMA3B. Adding this protein to normal CTBs inhibited invasion and re-created aspects of the phenotype of these cells in PE. Additionally, SEMA3B downregulated VEGF signaling through the PI3K/AKT and GSK3 pathways, effects that were observed in PE CTBs. We propose that, in severe PE, the in vivo environment dysregulates CTB gene expression, the autocrine actions of the upregulated molecules, including SEMA3B, impair differentiation/invasion/signaling and patient-specific factors determine the signs.
Comparative Methylome Analyses Reveal Human Brain Specific Methylated Regions Implicated in Transcription Regulation
To gain further insights into human brain specific epigenetic changes, we generated whole genome bisulfite sequencing methylation maps (methylomes) of rhesus macaque brains. These were then compared to methylomes of human and chimpanzee brains that were previously generated. We then performed validation experiments using targeted bisulfite sequencing.
The lncRNA PCAT29 inhibits oncogenic phenotypes in prostate cancer
Long noncoding RNAs (lncRNAs) have recently been associated with the development and progression of a variety of human cancers. However, to date, the interplay between known oncogenic or tumor suppressive events and lncRNAs has not been well described. Here the novel lncRNA, Prostate Cancer-Associated Transcript 29 (PCAT29), is characterized along with its relationship to the androgen receptor (AR). PCAT29 is suppressed by dihydrotestosterone (DHT) and up-regulated upon castration therapy in a prostate cancer xenograft model. PCAT29 knockdown significantly increased proliferation and migration of prostate cancer cells, while PCAT29 overexpression conferred the opposite effect and suppressed growth and metastases of prostate tumors in chick chorioallantoic membrane (CAM) assays. Finally, in prostate cancer patient specimens, low PCAT29 expression correlated with poor prognostic outcomes. Taken together, these data expose PCAT29 as an androgen regulated tumor suppressor in prostate cancer
Chromatin interactions involving super-enhancers associate with specific expression patterns of target genes
Super-enhancers may regulate target genes through chromatin looping. We connected super-enhancers in the K562 chronic myelogenous leukemia cell line with chromatin interactions identified from Chromatin Interaction Analysis with Paired-End Tag (ChIA-PET) data. Gene expression at proximal elements that are connected with distal super-enhancers showed significantly higher cell-type specificity than at proximal elements connected with other elements or not involved in interaction. 4C and Episwitch analysis of chromatin interactions showed that certain chromatin interactions are cell-specific, but others are more general. While super-enhancers upstream of c-MYC at the MYC-335 element can be found in other cancers, only super-enhancers downstream of c-MYC can be found in K562. 4C analysis of the c-MYC promoter revealed no chromatin interactions that are directed upstream of c-MYC, but only downstream of c-MYC, in the PVT1 long non-coding RNA gene. Cell-specific usage of super-enhancers could explain why the MYC-335 element that is associated with many solid cancers such as colorectal cancer and breast cancer, but not with leukemia. Surprisingly, we found that a chromatin interaction between c-MYC and a c-MYC super-enhancer is lost in chronic myelogenous leukemia patient blood as compared with blood from individuals without the disease through Oxford Biodynamics’ Episwitch analysis. These results provide evidence for fine-tuning of expression patterns, such as cell-specific regulation of target genes by distal super-enhancers through chromatin interactions and an association between chromatin interactions and disease, and highlight that super-enhancers are more complex than previously described.
Novel biomarkers for distributed stem cells: asymmetric self-renewal associated (ASRA) genes identification by combined cDNA micro-array orthogonal intersection and sparse feature analysis
Biomarkers unique for distributed stem cells (DSCs) have proven elusive. Previous searches for proteins expressed specifically in DSCs were hampered by difficulty obtaining pure DSCs and challenges to successfully mining complex molecular expression data. To identify novel candidates for DSC biomarkers, we combined a sparse feature selection method with combinatorial molecular expression data focused on asymmetric self-renewal, a defining DSC property. Our analyses revealed reduced expression of the histone H2A variant H2A.Z as a superior discriminator for asymmetric self-renewal, which proved to be a novel pattern-specific biomarker of DSCs.
The mdx mutation in the 129/Sv background results in a milder phenotype: Transcriptome comparative analysis searching for the protective factors
Transcriptome analysis of hindlimb muscles from dystrophic mice. The mdx mouse is a good genetic and molecular murine model for Duchenne Muscular Dystrophy (DMD), a progressive and devastating muscle disease. However, this model is inappropriate for testing new therapies due to its mild phenotype. Here, we transferred the mdx mutation to the 129/Sv strain with the aim to create a more severe model for DMD. Unexpectedly, functional analysis of the first three generations of mdx129 showed a progressive amelioration of the phenotype, associated to less connective tissue replacement, and more regeneration than the original mdxC57BL. Transcriptome comparative analysis was performed to identify what is protecting this new model from the dystrophic characteristics. The mdxC57BL presents three times more differentially expressed genes (DEGs) than the mdx129 (371 and 137 DEGs, respectively). However, both models present more overexpressed genes than underexpressed, indicating that the dystrophic and regenerative alterations are associated with the activation rather than repression of genes. As to functional categories, the DEGs of both mdx models showed a predominance of immune system genes. Excluding this category, the mdx129 model showed a decreased participation of the endo/exocytic pathway and homeostasis categories, and an increased participation of the extracellular matrix and enzymatic activity categories. Spp1 gene overexpression was the most significant DEG exclusively expressed in the mdx129 strain. This was confirmed through relative mRNA analysis and osteopontin protein quantification. The amount of the 66 kDa band of the protein, representing the post-translational product of the gene, was about 4.8 times higher on Western blotting. Spp1 is a known DMD prognostic biomarker, and our data indicate that its upregulation can benefit phenotype. Modeling the expression of the DEGs involved in the mdx mutation with a benign course should be tested as a possible therapeutic target for the dystrophic process.
AP2 transcription factors regulate expression of CRABPII in hormone responsive breast carcinoma
BACKGROUND: The AP2 transcription factor family is a set of developmentally regulated, retinoic acid (RA) inducible genes, which regulate expression of estrogen receptor-alpha (ERalpha) in breast carcinoma. We hypothesized that AP2 factors regulate a set of genes characteristic of the hormone responsive breast cancer phenotype. To better understand the role of AP2 factors in hormone responsive breast cancer, we sought to identify AP2-target genes in breast epithelial cells. MATERIALS AND METHODS: Overexpression of AP2 factors was achieved in human mammary epithelial cells (HMECs) using adenoviral vectors. AP2 target genes were identified by comparative hybridization to cDNA microarrays containing 30,000 human genes. Expression patterns were confirmed by Northern and Western blot and by elimination of AP2 using siRNA. Potential regulatory elements in promoters of target genes were identified by DNase I hypersensitive site mapping. : Comparative cDNA microarray hybridization identified a set of genes induced by overexpression of AP2alpha and AP2gamma in HMECs. The up-regulation of cellular retinoic acid-binding protein 2 (CRABPII), EST-1, and ECM1 was induced by overexpression of AP2alpha, AP2gamma, or a chimeric AP2 factor in which the activation domain of AP2alpha was replaced by the activation domain of herpesvirus VP16. Interestingly, hormone unresponsive MDA-MB-231 cells were resistant to CRABPII induction by any of the AP2 factors. Elimination of AP2gamma in MCF7 cells resulted in a significant reduction in CRABPII expression. AP2alpha induced DNase I hypersensitive sites in the promoter of the CRABPII gene at -5000 bp, which corresponds to the site of action of RAR/RXR factors. CONCLUSIONS: AP2 factors regulate CRABPII expression in HMECs and breast cancer cells and accounts for the associated expression of ERalpha and CRABPII in hormone responsive breast cancer. Because CRABPII mediates growth suppressive effects of RA in breast cancer, the data suggest that AP2 factors have the ability to mediate RA responsiveness through the regulation of CRABP II expression. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
The blood transcriptional signature of chronic HCV [Affymetrix data]
This study characterizes the effects of chronic Hepatitis C virus (HCV) infection on gene expression by analyzing blood samples from 10 treatment-naive HCV patients and 6 healthy volunteers. Differential expression analysis of microarray data from peripheral blood mononuclear cells (PBMCs) identified a 136 gene signature, including 66 genes elevated in infected individuals. Most of the up-regulated genes were associated with interferon (IFN) activity (including members of the OAS and MX families, ISG15 and IRF7), suggesting an ongoing immune response. This HCV signature was also found to be consistently enriched in many other viral infection and vaccination datasets. Validation of these genes was carried out using a second cohort composed of 5 HCV patients and 5 healthy volunteers, confirming the up-regulation of the IFN signature. In summary, this is the first study to directly compare blood transcriptional profiles from HCV patients with healthy controls. The results show that chronic HCV infection has a pronounced effect on gene expression in PBMCs of infected individuals, and significantly elevates the expression of a subset of interferon-stimulated genes.
Translational validation of personalized treatment strategy based on genetic characteristics of glioblastoma [surgical samples]
Glioblastoma (GBM) heterogeneity in the genomic and phenotypic properties has potentiated personalized approach against specific therapeutic targets of each GBM patient. The Cancer Genome Atlas (TCGA) Research Network has been established the comprehensive genomic abnormalities of GBM, which sub-classified GBMs into 4 different molecular subtypes. The molecular subtypes could be utilized to develop personalized treatment strategy for each subtype. We applied a classifying method, NTP (Nearest Template Prediction) method to determine molecular subtype of each GBM patient and corresponding orthotopic xenograft animal model. The models were derived from GBM cells dissociated from patient's surgical sample. Specific drug candidates for each subtype were selected using an integrated pharmacological network database (PharmDB), which link drugs with subtype specific genes. Treatment effects of the drug candidates were determined by in vitro limiting dilution assay using patient-derived GBM cells primarily cultured from orthotopic xenograft tumors. The consistent identification of molecular subtype by the NTP method was validated using TCGA database. When subtypes were determined by the NTP method, orthotopic xenograft animal models faithfully maintained the molecular subtypes of parental tumors. Subtype specific drugs not only showed significant inhibition effects on the in vitro clonogenicity of patient-derived GBM cells but also synergistically reversed temozolomide resistance of MGMT-unmethylated patient-derived GBM cells. However, inhibitory effects on the clonogenicity were not totally subtype-specific. Personalized treatment approach based on genetic characteristics of each GBM could make better treatment outcomes of GBMs, although more sophisticated classifying techniques and subtype specific drugs need to be further elucidated.
Array CGH of Mexican Patient Isolates and BCS100 Strain
Array CGH of human H. pylori isolates from natrually infected Mexican patients and BCS-100 strain used for experimental infection of humans which is labeled as OVX-34 in this sample set(Graham et al., 2004, Gut, 53:1235-1243). Each Mexican isolate has a three digit number indicating the patient, a letter indicating biopsy site location and number indicating single colony clone number. Patient 249,259 and 251 are adults and patient 291,293,612 and 653 are children. Multiple single clonies were isolated from 2-4 gastric biopsy sites (a- antrum, c-corpus, f- fundus, i-incisura angularis). Genomic DNA prepared from each experimental strain (500 ng) was labeled with Cy5 (red channel) and co-hybridized with an equimolar mixture of genomic DNA from the reference strains 26659 and J99 (500 ng). Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
Genome-wide analysis of developmentally regulated microRNAs in Hami melon fruit
Background: MicroRNAs (miRNAs) represent a family of small endogenous, non-coding RNAs that play critical regulatory roles in plant growth, development, and environmental stress responses. Although Hami melon is an attractive model for valuable biological traits analysis, the role of miRNA action in the fruit development and ripening remains largely unknown. Here, we performed small RNA sequencing to investigate the Hami melon miRNA profiles at four fruit developmental stages Results: Small RNA sequencing yielded raw reads in eight libraries. miRNAs expression profiles were variable at different fruit developmental stages. The expression levels of five known miRNAs were validated by quantitative real-time PCR. Among the identified miRNAs, several miRNAs showed developmentally regulated and differentially expressed pattern during fruit development. Conclusions: Our results present a first comprehensive set of identification and characterization of Hami melon fruit miRNAs and their potential targets, which provide valuable basis for further research on the critical role of miRNAs in melon fruit development.
EPCR Expression Defines the Most Primitive Subset of Human HSPC and Is Required for Their In Vivo Activity
Cell purification technology combined with whole transcriptome sequencing and small molecule agonist of hematopoietic stem cell self-renewal has allowed us to identify the endothelial protein c receptor protein (EPCR) as a surface maker that defines a rare subpopulation of human cells which is highly enriched for stem cell activity in vivo. EPCR-positive cells exhibit a robust multi-lineage differentiation potential and serial reconstitution in immunocompromised mice. In culture, most if not all of the HSC activity is detected in the EPCR+ subset, arguing for the stability of this marker on the surface of cultured cells, a feature not found with more recently described markers such as CD49f. Functionally EPCR is essential for human HSC activity in vivo. Cells engineered to express low EPCR expression proliferate normally in culture but lack the ability to confer long-term reconstitution. EPCR is thus a stable marker for human HSC. Its exploitation should open new possibilities in our effort to understand the molecular bases behind HSC self-renewal.
Therapeutic potential of a novel HSP90 Inhibitor, NXD30001, for Neurofibromatosis type 2
Purpose: The growth and survival of NF2-deficient cells are enhanced by the activation of multiple signaling pathways including ErbB2/IGF-1R/Met, PI3K/Akt, and Ras/Raf/Mek/Erk1/2. The ubiquitously expressed chaperone protein HSP90 is known to be essential for the stabilization of these signaling molecules. We aim to evaluate the effect of the HSP90 inhibition on the signaling pathways activated in NF2-related tumors. We tested the efficacy of the newly synthesized small molecule NXD30001 which was shown to be more potent in HSP90 inhibition in vitro and less toxic in vivo than already existing HSP90 inhibitors. Experimental Design: The anti-proliferative activity of NXD30001 was tested in NF2-deficient mouse cells in vitro, and its anti-tumor efficacy was verified in an NF2-deficient allograft model in vivo. The underlying molecular alteration in vitro and in vivo was further characterized by a global transcriptome approach. Results: NXD30001 was found to induce degradation of client proteins and to suppress proliferation in NF2-deficient cells in vitro and in vivo. Differential expression analysis further identified subsets of genes implicated in cell proliferation, survival, vascularization, and Schwann cell differentiation, whose expression were altered by NXD30001 treatment. Conclusions: NXD30001 is a potent HSP90 inhibitor showing significant antitumor activity against NF2-related tumor cells in vitro and in vivo, and represents a promising option for novel NF2 therapies.
Translational validation of personalized treatment strategy based on genetic characteristics of glioblastoma [xenograft]
Glioblastoma (GBM) heterogeneity in the genomic and phenotypic properties has potentiated personalized approach against specific therapeutic targets of each GBM patient. The Cancer Genome Atlas (TCGA) Research Network has been established the comprehensive genomic abnormalities of GBM, which sub-classified GBMs into 4 different molecular subtypes. The molecular subtypes could be utilized to develop personalized treatment strategy for each subtype. We applied a classifying method, NTP (Nearest Template Prediction) method to determine molecular subtype of each GBM patient and corresponding orthotopic xenograft animal model. The models were derived from GBM cells dissociated from patient's surgical sample. Specific drug candidates for each subtype were selected using an integrated pharmacological network database (PharmDB), which link drugs with subtype specific genes. Treatment effects of the drug candidates were determined by in vitro limiting dilution assay using patient-derived GBM cells primarily cultured from orthotopic xenograft tumors. The consistent identification of molecular subtype by the NTP method was validated using TCGA database. When subtypes were determined by the NTP method, orthotopic xenograft animal models faithfully maintained the molecular subtypes of parental tumors. Subtype specific drugs not only showed significant inhibition effects on the in vitro clonogenicity of patient-derived GBM cells but also synergistically reversed temozolomide resistance of MGMT-unmethylated patient-derived GBM cells. However, inhibitory effects on the clonogenicity were not totally subtype-specific. Personalized treatment approach based on genetic characteristics of each GBM could make better treatment outcomes of GBMs, although more sophisticated classifying techniques and subtype specific drugs need to be further elucidated.
Array CGH after human challenge
Array CGH of H. pylori strain BCS100 after challenge to a new host (patients and experiment described in Graham et al., 2004, Gut, 53:1235-1243). Genomic DNA (500 ng) from the ouput from singel colony isolates obtained 15 or 90 days post innoculation were labeled with Cy5 (red) and cohybridized with 500 ng of genomic DNA from the innoculating strain BCS100 labeled with Cy3 (green). A reference experiement design type is where all samples are compared to a common reference. Keywords: reference_design
Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age (RNA-Seq)
Differentiating pluripotent cells from fibroblast progenitors is a potentially transformative tool in personalized medicine. We previously identified relatively greater success culturing dura-derived fibroblasts than scalp-derived fibroblasts from postmortem tissue. We hypothesized that these differences in culture success were related to epigenetic differences between the cultured fibroblasts by sampling location, and therefore generated genome-wide DNA methylation and transcriptome data on 11 intrinsically matched pairs of dural and scalp fibroblasts from donors across the lifespan (infant to 85 years). While these cultured fibroblasts were several generations removed from the primary tissue and morphologically indistinguishable, we found widespread epigenetic differences by sampling location at the single CpG (N=101,989), region (N=697), “block” (N=243), and global spatial scales suggesting a strong epigenetic memory of original fibroblast location. Furthermore, many of these epigenetic differences manifested in the transcriptome, particularly at the region-level. We further identified 7,265 CpGs and 11 regions showing significant epigenetic memory related to the age of the donor, as well as an overall increased epigenetic variability, preferentially in scalp-derived fibroblasts -83% of loci were more variable in scalp, hypothesized to result from cumulative exposure to environmental stimuli in the primary tissue. By integrating publicly available DNA methylation datasets on individual cell populations in blood and brain, we identified significantly increased inter-individual variability in our scalp- and other skin-derived fibroblasts on a similar scale as epigenetic differences between different lineages of blood cells. Lastly, these epigenetic differences did not appear to be driven by somatic mutation - while we identified 64 probable de-novo variants across the 11 subjects, there was no association between mutation burden and age of the donor (p=0.71). These results depict a strong component of epigenetic memory in cell culture from primary tissue, even after several generations of daughter cells, related to cell state and donor age.
Dynamic transcriptional response of Microcoleus vaginatus to hydration and dehydration in a desert biological soil crust
Biological soil crusts (BSCs) are cyanobacteria-dominated microbial communities that cover extensive portions of the world’s arid and semi-arid deserts. The infrequent periods of hydration are often too short to allow for dormancy strategies based on sporulation; consequently, survival is based on the unique capabilities of vegetative cells to resuscitate from and re-enter a stress resistant dormant state, one of which is migration within the crust layers in response to hydration. In this study, we sought to characterize the events that govern the emergence of the dominant cyanobacterium from dormancy, its subsequent growth, and the events triggered by re-desiccation and a transition back to dormant state. We performed a 48 hour laboratory wetting experiment of a desert BSC and tracked the response of Microcoleus vaginatus using a whole genome transcriptional time-course including night/day periods. This allowed the identification of genes with a diel expression pattern, genes involved uniquely in the signaling after hydration and those that contribute primarily to desiccation preparation.
M1 serotype (strain SF370): pharyngeal cell adherence vs. association
We developed spotted oligonucleotide arrays of the S. pyogenes SF370 (an M1 serotype) genome and compared the transcriptomes of streptococci that adhere to Detroit 562 human pharyngeal cells to non-adherent (“associated”) streptococci within the same experiment. We replicated experiments independently and used dye-swaps to incorporate biological and technical variation, biological replicate experiments. Following filtering and normalization, we analyzed data from four biological replicates with robust summary statistics, Bayesian statistics and permutation algorithms to identify genes differentially expressed with significance during pharyngeal cell adherence. This analysis identified 79 genes (4% of the genome) exhibiting statistically significant fold changes in expression (PF value < 0.05) during adherence from 1769 open reading frames represented on the array. The genes encoding several known virulence factors (e.g. cysteine protease SpeB and streptolysin O) were differentially expressed with significance; however, many were either never before associated with adherence (e.g. phage-encoded genes including speH), or of undefined function. Genes demonstrating up-regulation (n= 45) and down-regulation (n= 34) included virulence factors, prophage-encoded transcripts, metabolic genes and transcriptional regulators (Table 2). Undefined or hypothetical genes comprised 27% of differentially expressed genes (n= 21; 11 chromosomally-encoded, 10 phage-encoded). We conducted TaqMan (qRT-PCR) analysis of 11 differentially expressed genes to validate selected microarray hybridization results. We further analyzed the data with neighbor clustering, using a set of novel computational algorithms (termed “GenomeCrawler”) for evaluating bacterial microarray data, which couples the expression profiles of genes with their physical location on the chromosome. When applied to the microarray data from this study, neighbor clustering identified a greater number of differentially expressed genes, which facilitated the reconstruction of known multimeric proteins, complete metabolic pathways and intact virulence loci that would not have been possible without its application. These data sets and algorithms are freely available for download at www.rockefeller.edu/vaf. Keywords: Pharyngeal cell adherent vs. associated
MiR-193b promotes autophagy and non-apoptotic cell death in oesophageal cancer cells
Background: Successful treatment of oesophageal cancer is hampered by recurrent drug resistant disease. We have previously demonstrated the importance of apoptosis and autophagy for the recovery of oesophageal cancer cells following drug treatment. When apoptosis (with autophagy) is induced, these cells are chemosensitive and will not recover following chemotherapy treatment. In contrast, when cancer cells exhibit only autophagy and limited Type II cell death, they are chemoresistant and recover following drug withdrawal. Methods: MicroRNA (miRNA) expression profiling of an oesophageal cancer cell line panel was used to identify miRNAs that were important in the regulation of apoptosis and autophagy. The effects of miRNA overexpression on cell death mechanisms and recovery were assessed in the chemoresistant (autophagy inducing) KYSE450 oesophageal cancer cells. Results: MiR-193b was the most differentially expressed miRNA between the chemosensitive and chemoresistant cell lines with higher expression in chemosensitive apoptosis inducing cell lines. Colony formation assays showed that overexpression of miR-193b significantly impedes the ability of KYSE450 cells to recover following 5-fluorouracil (5-FU) treatment. The critical mRNA targets of miR-193b are unknown but target prediction and siRNA data analysis suggest that it may mediate some of its effects through stathmin 1 regulation. Apoptosis was not involved in the enhanced cytotoxicity. Overexpression of miR- 193b in these cells induced autophagic flux and non-apoptotic cell death. Conclusion: These results highlight the importance of miR-193b in determining oesophageal cancer cell viability and demonstrate an enhancement of chemotoxicity that is independent of apoptosis induction.
Genome-wide sequencing of hippocampal 5-hydroxymethylcytosine links plasticity genes to acute stress
We used an established chemical labeling and affinity purification method coupled with high-throughput sequencing technology to generate the first genome-wide profile of hippocampal 5hmC following exposure to acute restraint stress and a one-hour recovery. This approach found a genome-wide disruption in 5hmC associated with acute stress response, primarily in genic regions, and identified known and potentially novel stress-related targets that have a significant enrichment for neuronal ontological functions. Integration of these data with hippocampal gene expression data from these same mice found stress-related hydroxymethylation correlated to altered transcript levels and sequence motif predictions indicated that 5hmC may function by mediating transcription factor binding to these transcripts. Together, these data reveal an environmental impact on this newly discovered epigenetic mark in the brain and represent a critical step toward understanding stress-related epigenetic mechanisms that alter gene expression and can lead to the development of psychiatric disorders.
Ribosome Profiling in P. falciparum asexual blood stages
The description of the transcriptome and proteome datasets of Plasmodium falciparum, the deadliest strain of human malaria, has been a tremendous resource for the understanding of the molecular physiology of this parasite. However, the underlying translational control that links global measurements of steady-state mRNA with protein levels is not well understood. Our work bridges this disconnect by measuring translation on a whole genome scale using ribosome profiling, providing the first measurements of new protein synthesis through the asexual blood phase developmental cycle.
Comparative genomic hybridizations of Mycobacterium avium isolates obtained from multiple host species
A comparative genomic approach was used to identify large sequence polymorphisms among Mycobacterium avium isolates obtained from a variety of host species. DNA microarrays were used as a platform for comparing mycobacteria field isolates with the sequenced bovine isolate Mycobacterium avium subsp. paratuberculosis (Map) K10. ORFs were classified as present or divergent based on the relative fluorescent intensities of the experimental samples compared to Map K10 DNA. Map isolates cultured from cattle, bison, sheep, goat, avian, and human sources were hybridized to the Map microarray. Three large deletions were observed in the genomes of four Map isolates obtained from sheep and four clusters of ORFs homologous to sequences in the Mycobacterium avium subsp. avium (Maa) 104 genome were identified as being present in these isolates. One of these clusters encodes glycopeptidolipid biosynthesis enzymes. One of the Map sheep isolates had a genome profile similar to a group of Mycobacterium avium subsp. silvaticum (Mas) isolates which included four independent laboratory stocks of the organism traditionally identified as Maa strain 18. Genome diversity in Map appears to be mostly restricted to large sequence polymorphisms that are often associated with mobile genetic elements. Keywords: Comparative genomic hybridization
miRNA expression changes in small intestinal lamina propria leukocyte samples during the course of SIV infection of rhesus macaques
This study describes differential miRNA expression in small intestinal lamina propria leukocyte samples longitudinally during the course of SIV infection of rhesus macaques. Notably, the T-cell activation associated miR-15b, miR-142-3p, miR-142-5p and miR-150 expression was significantly downregulated at 90 and 180DPI. Further, reporter and overexpression assays validated IRAK1 as a direct miR-150 target. Furthermore, IRAK1 protein levels were markedly elevated in intestinal LPLs and epithelium. Finally, blockade of CD8+ T-cell activation/proliferation with delta-9 tetrahydrocannabinol (9-THC) significantly prevented miR-150 downregulation and IRAK1 upregulation. Our findings suggest that miR-150 downregulation during T-cell activation may disrupt the translational control of IRAK1 facilitating persistent GI inflammation.
FOXO4 knockdown in LNCaP prostate cancer cells
Compares shFOXO4 vs. Control in LNCaP grown in culture, or in nude mice as primary orthotopic tumors or lymph node metastases
Small molecule activation of PKM in cancer cells induces serine auxotrophy
Proliferating tumor cells use aerobic glycolysis to support their high metabolic demands. Paradoxically, increased glycolysis is often accompanied by expression of the lower activity PKM isoform, effectively constraining lower glycolysis. Here, we report the discovery of novel PKM activators with a unique allosteric binding mode. Characterization of how these compounds impact cancer cells revealed an unanticipated link between glucose and amino acid metabolism. PKM activation resulted in a metabolic rewiring of cancer cells manifested by a profound dependency on the non-essential amino acid serine for continued cell proliferation. Induction of serine auxotrophy by PKM activation was accompanied by reduced carbon flow into the serine biosynthetic pathway and increased expression of high affinity serine transporters. These data support the hypothesis that PKM expression confers metabolic flexibility to cancer cells that allows adaptation to nutrient stress.
Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age (Methylation)
Differentiating pluripotent cells from fibroblast progenitors is a potentially transformative tool in personalized medicine. We previously identified relatively greater success culturing dura-derived fibroblasts than scalp-derived fibroblasts from postmortem tissue. We hypothesized that these differences in culture success were related to epigenetic differences between the cultured fibroblasts by sampling location, and therefore generated genome-wide DNA methylation and transcriptome data on 11 intrinsically matched pairs of dural and scalp fibroblasts from donors across the lifespan (infant to 85 years). While these cultured fibroblasts were several generations removed from the primary tissue and morphologically indistinguishable, we found widespread epigenetic differences by sampling location at the single CpG (N=101,989), region (N=697), “block” (N=243), and global spatial scales suggesting a strong epigenetic memory of original fibroblast location. Furthermore, many of these epigenetic differences manifested in the transcriptome, particularly at the region-level. We further identified 7,265 CpGs and 11 regions showing significant epigenetic memory related to the age of the donor, as well as an overall increased epigenetic variability, preferentially in scalp-derived fibroblasts -83% of loci were more variable in scalp, hypothesized to result from cumulative exposure to environmental stimuli in the primary tissue. By integrating publicly available DNA methylation datasets on individual cell populations in blood and brain, we identified significantly increased inter-individual variability in our scalp- and other skin-derived fibroblasts on a similar scale as epigenetic differences between different lineages of blood cells. Lastly, these epigenetic differences did not appear to be driven by somatic mutation - while we identified 64 probable de-novo variants across the 11 subjects, there was no association between mutation burden and age of the donor (p=0.71). These results depict a strong component of epigenetic memory in cell culture from primary tissue, even after several generations of daughter cells, related to cell state and donor age.
Identification of a Mammalian Silicon Transporter
Silicon (Si) has long been known to play a major physiological role in certain organisms, including some sponges and many diatoms and higher plants, leading to the recent identification of multiple proteins responsible for silicon transport in a range of algal and plant species. In mammals, despite several convincing studies suggesting that silicon is an important factor in bone development and connective tissue health, there is a critical lack of understanding in biochemical pathways that enable silicon homeostasis. Here we report the identification of a mammalian efflux silicon transporter, namely Slc34a2 (also known as NaPiIIb), which was upregulated in the kidneys of rats following chronic dietary silicon deprivation. When heterologously expressed in Xenopus laevis oocytes, the protein displayed marked silicon transport activity, specifically efflux, comparable to plant OsLsi2 transfected in the same fashion and independent of sodium and/or phosphate influx. This is the first evidence for a specific active transporter protein for silicon in mammals and suggests an important role for silicon in vertebrates.
Upf2 in NMD pathway
Purpose: Probe the transcriptome-wide changes in the expression pattern between WT and Sertoli-specific Upf2 KO testes Methods: Total RNA were extracted from WT and Sertoli-specific Upf2 KO testes in triplicates and subject to deep-sequencing in Ion Torrent seq platform. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl−/− mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl−/− retina, with a fold change ≥1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study represents the first detailed analysis of Upf2-mediated NMD pathway in Sertoli cell development
Global gene expression analysis of Ncoa3 knockdown in mouse embryonic stem cells
Orphan nuclear receptor Esrrb is vital in maintaining ES cells and like Oct4, Sox2 and Nanog is essential for self-renewal and pluripotency. Esrrb functions in somatic cells via LBD/AF-2-dependent coactivator recruitment to target genes. Here we show that in ES cells coactivator recruitment is similarly required and identify Ncoa3 as the Esrrb coactivator needed for activation of its target genes. Ncoa3 is essential for self-renewal and the induction of pluripotency in reprogramming, and genome-wide analysis of Ncoa3 binding reveals extensive overlap with Esrrb and pluripotency factors along with marks of active genes. Mechanistically, we show Ncoa3 is specifically required to bridge RNApol2 to Esrrb. We thus identify a new member of the ES pluripotency network and describe Esrrb and Ncoa3 as key factors linking core pluripotency factors to the general transcription machinery.
Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age
This SuperSeries is composed of the SubSeries listed below.
24 h-fasting effects on the brown and white adipose tissue and liver
The functional balance between brown adipose tissue (BAT) and white adipose tissue (WAT) is important for metabolic homeostasis. We compared the effects of fasting on the gene expression profiles in BAT, WAT and liver, using DNA microarray analysis. Tissues were obtained from rats that had been fed or fasted for 24 h. Taking the false discovery rate (FDR) into account, we extracted the top 1,000 genes that were expressed differentially between fed and fasted rats. In all three tissues, Gene Ontology analysis revealed marked changes in the expression of ‘metabolism’ category genes and a hypergeometric test demonstrated that within this category, lipid and protein biosynthesis-related genes were down-regulated. These findings indicate simultaneous down-regulation of genes involved in energy-consuming pathways in the BAT, WAT and liver of fasted rats. In the BAT of fasted rats, there was marked up-regulation of genes in the ‘protein ubiquitination’ category, suggesting that the ubiquitin-proteasome system is involved in saving energy as an adaptation to food shortage. Keywords: treatment comparison
Large genomic alteration of 7q in two patients with multiple primary cancers, including triple negative breast cancer, and family history of malignant neoplasms
A great percentage of patients with multiple primary cancers (MPCs) and family history of cancer are suspected to have a hereditary cancer predisposition syndrome. However, only a small proportion of these cases are explained by mutations in high-penetrance genes, suggesting the involvement of undiscovered genes in cancer predisposition. In this study, we report the molecular and clinical characterization of two unrelated patients with MPCs, a positive family history of cancer, no germline pathogenic mutations in BRCA1, BRCA2 and TP53 genes and large genomic rearrangements mapped on chromosome 7q.
Expression Profiles of Monozygotic Twin
The expression level for 15 887 transcripts in lymphoblastoid cell lines from 19 monozygotic twin pairs (10 male, 9 female) were analysed for the effects of genotype and sex. On an average, the effect of twin pairs explained 31% of the variance in normalized gene expression levels, consistent with previous broad sense heritability estimates. The effect of sex on gene expression levels was most noticeable on the X chromosome, which contained 15 of the 20 significantly differentially expressed genes. A high concordance was observed between the sex difference test statistics and surveys of genes escaping X chromosome inactivation. Notably, several autosomal genes showed significant differences in gene expression between the sexes despite much of the cellular environment differences being effectively removed in the cell lines. A publicly available gene expression data set from the CEPH families was used to validate the results. The heritability of gene expression levels as estimated from the two data sets showed a highly significant positive correlation, particularly when both estimates were close to one and thus had the smallest standard error. There was a large concordance between the genes significantly differentially expressed between the sexes in the two data sets. Analysis of the variability of probe binding intensities within a probe set indicated that results are robust to the possible presence of polymorphisms in the target sequences. Keywords: Monozygotic twin pair Expression Profiles
Expression profile of lung adenocarcinoma, A549 cells following induction of non metastatic 2 (NME2/NM23 H2)
It is widely believed that reorganization of nucleosomes result in availability of binding sites that engage transcription factors during eukaryotic gene regulation. Recent findings, on the other hand, suggest that transcription factors induced as a result of physiological perturbations directly (or in association with chromatin modifiers) may alter nucleosome occupancy to facilitate DNA binding. Although, together these suggest a close relationship between transcription factor binding and nucleosome reorganization, the nature of the inter-dependency, or to what extent it influences regulatory transcription is not clear. Moreover, since most studies used physiolgical pertubations that induced multiple transcription factor chromatin modifiers, the relatively local (or direct) effect of transcription factor binding on nucleosome occupancy remains unclear. With these in mind, we used a single transcription factor to induce physiological changes, representing metastatic (aggressive cancer) and the corresponding non-metastatic state, in human cancer cells. Following characterization of the two states (before and after induction of the transcription factor) we determined: (a) genome wide binding sites of the transcription factor, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. Interestingly, we find only ~20% of TF binding results from nucleosome reorganization - however, almost all corresponding genes were transcriptionally altered. Whereas, in cases where TF-occupancy was independent of nucleosome repositioning (in close vicinity), or co-occurred with nucleosomes, only a small fraction of the corresponding genes were expressed/repressed. Together, these indicate a model where TF occupancy only when coupled with nucleosome repositioning in close proximity is transcriptionally active. This, to our knowledge, for the first time also helps explain why genome wide TF occupancy (e.g., from ChIP-seq) is typically associated with only a small fraction of genes that change expression.
Foxd3 binding at enhancers controls developmental timing of gene expression by regulation of histone acetylation [ChIP-seq]
Transcription factor/enhancer interactions determine cell specific gene expression. Here, we followed enhancers during differentiations of embryonic stem (ESCs) to epiblast like cells (EpiLCs). There were highly dynamic changes in histone lysine 27 acetylation at enhancer sites throughout the genome. These sites were enriched for a Foxd3 binding motif, a forkhead transcription factor essential in early embryonic development. Surprisingly, Foxd3 occupied largely mutually exclusive sites in the ESCs versus EpiLCs. Foxd3 bound to nucleosome occupied regions, simultaneously evicting the histones while inhibiting full gene expression through the recruitment of histone deacetylases. Knockout of Foxd3 resulted in hyperacetylation and transcriptional upregulation of neighboring genes, many of which were further upregulated at later stages of differentiation. These data show that Foxd3 primes enhancer sites during pregastrulation by removing nucleosomes, yet suppresses neighboring histone hyperacetylation. Such a mechanism may be common to many transcription factors that prepare enhancers for later gene activation during development.
Role of Dicer1-dependent microRNAs in the paracrine control of epididymal gene expression
Dicer1 is an endoribonuclease involved in the biogenesis of functional microRNAs (miRNAs). These small non-coding RNAs are important regulators of the posttranscriptional gene expression and participate to the control of male fertility. Knowing that 1) Dicer1-dependent factors are needed for proper sperm maturation in the epididymis, and that 2) miRNAs are potent mediators of intercellular communication in most biological systems, we investigated the role of Dicer1 dependent-miRNAs produced from the proximal epididymis (initial segment/caput) on the paracrine regulation of epididymal gene expression in the distal epididymis regions (i.e. corpus and cauda). To this aim, we performed comparative microarray and ANOVA analyses on control vs. Defb41iCre/wt;Dicer1fl/fl mice in which functional Dicer1 is absent from the principal cells of the proximal epididymis. We identified 21 and 16 transcripts, including Prostate And Testis Expressed 4 protein and the Zn-alpha 2-glycoprotein, that display significant expression level changes in the corpus and cauda regions, respectively (Fold change >2 or <-2; p2 or <-2; p<0.01). These miRNAs are secreted via extracellular vesicles derived from DC2 epididymal principal cell line, and their expression correlates with target transcripts involved in important biological pathways, as evidenced by in silico analysis. These observations suggest that Dicer1-dependent miRNAs could act as potent paracrine regulators of epididymal functions (i.e. control of sperm motility, ciliogenesis) and may contribute to the infertility phenotype observed in the Defb41iCre/wt;Dicer1fl/fl mouse model. These findings open new avenues for the identification of molecular targets important to male fertility control.
Genome wide analysis of RNA species present in SMN-containing RNPs
Several studies indicate that SMN-containing mRNP complexes could be involved in the axonal localization of a large number of mRNAs. We have used murine motor neuron-like NSC-34 cells and RNA Immuno-Precipitation experiments coupled to microarray analyses to perform a genome-wide analysis of RNA species present in mRNP complexes containing the full length SMN protein (flSMN). In situ hybridization and immuno-fluorescence experiments performed on several candidates indicate that these mRNAs colocalize with the SMN protein in neurites and axons of differentiated NSC-34 cells. Moreover, they localize in cell processes in a SMN-dependent manner. Thus, low SMN levels might result in localization deficiencies of mRNAs required for axonogenesis.
Effects of mycophenolic acid on human fibroblast proliferation, migration and adhesion in vitro and in vivo
Mycophenolic acid (MPA) is a potent inhibitor of the inosine monophosphate dehydrogenase and commonly used as an immunosuppressive drug in transplantation. MPA inhibits proliferation of both T- and B-lymphocytes by guansoin depletion. Since fibroblasts rely on the de novo synthesis of guanosin nucleotides, it is assumed that MPA interacts with fibroblasts causing an increased frequency of wound healing problems. We show a downregulation of the cytoskeletal proteins actin, vinculin and tubulin in human dermal fibroblasts exposed to pharmacologic doses of MPA using microarray technology and western blot. This reduction in protein content is accompanied by a substantial derangement of the cytoskeleton in MPA-treated fibroblasts as documented by confocal microscopy. The dysfunctional fibroblast growth was validated by scratch test documenting impaired migrational capacity. The results of the cultured dermal fibroblasts were applied to skin biopsies of renal transplant recipients. Skin biopsies of patients treated with MPA expressed less tubulin and actin as compared to control biopsies which could explain potential wound healing problems post transplantation. The perspective of MPA-induced cytoskeletal dysfunction may go beyond wound healing disturbances and has potential beneficial effects on (renal) allografts with respect to scarring. Keywords: Timecourse and MPA and/or Guanosin response
Bcl6 is Required for Somatic Hypermutation and Gene Conversion in Chicken DT40 Cells
The activation induced cytosine deaminase (AID) mediates diversification of B cell immunoglobulin genes by the three distinct yet related processes of somatic hypermutation (SHM), class switch recombination (CSR), and gene conversion (GCV). SHM occurs in germinal center B cells, and the transcription factor Bcl6 is a key regulator of the germinal center B cell gene expression program, including expression of AID. To test the hypothesis that Bcl6 function is important for the process of SHM, we compared WT chicken DT40 B cells, which constitutively perform SHM/GCV, to their Bcl6-deficient counterparts. We found that Bcl6-deficient DT40 cells were unable to perform SHM and GCV despite enforced high level expression of AID and substantial levels of AID in the nucleus of the cells. To gain mechanistic insight into the GCV/SHM dependency on Bcl6, transcriptional features of a highly expressed SHM target gene were analyzed in Bcl6-sufficient and -deficient DT40 cells. No defect was observed in the accumulation of single stranded DNA in the target gene as a result of Bcl6 deficiency. In contrast, association of Spt5, an RNA polymerase II (Pol II) and AID binding factor, was strongly reduced at the target gene body relative to the transcription start site in Bcl6-deficient cells as compared to WT cells. However, partial reconstitution of Bcl6 function substantially reconstituted Spt5 association with the target gene body but did not restore detectable SHM. Our observations suggest that in the absence of Bcl6, Spt5 fails to associate efficiently with Pol II at SHM targets, perhaps precluding robust AID action on the SHM target DNA. Our data also suggest, however, that Spt5 binding is not sufficient for SHM of a target gene even in DT40 cells with strong expression of AID.
The S. cerevisiae histone demethylase Jhd1 fine-tunes the distribution of H3K36me2
Histone methylation plays important roles in the regulation of chromatin dynamics and transcription. Steady state levels of histone lysine methylation are regulated by a balance between enzymes that catalyze either the addition or removal of methyl groups. Using an activity-based biochemical approach, we recently uncovered the JmjC domain as an evolutionarily conserved signature motif for histone demethylases. Furthermore, we demonstrated that Jhd1, a JmjC domain-containing protein in S. cerevisiae, is an H3K36-specific demethylase. Here we report further characterization of Jhd1. Similar to its mammalian homolog, Jhd1-catalyzed histone demethylation requires iron and alpha-ketoglutarate as cofactors. Mutation and deletion studies indicate that the JmjC domain and adjacent sequences are critical for Jhd1 enzymatic activity, while the N-terminal PHD domain is dispensable. Overexpression of JHD1 results in a global reduction of H3K36 methylation in vivo. Finally, chromatin immunoprecipitation coupled microarray (ChIP-chip) studies reveal subtle changes in the distribution of H3K36me2 upon overexpression or deletion of JHD1. Our studies establish Jhd1 as a histone demethylase in budding yeast and suggest that Jhd1 functions to maintain the fidelity of histone methylation patterns along transcription units. Keywords: ChIP-chip
Activation of proto-oncogenes by disruption of chromosome neighborhoods [5C-Seq]
Mutations such as gene fusion, translocation and focal amplification are a frequent cause of proto-oncogene activation during tumorigenesis, but such mutations do not explain all cases of proto-oncogene activation. Here we show that disruption of local chromosome conformation can also activate proto-oncogenes in human cells. We mapped chromosome structures in T-cell acute lymphoblastic leukemia (T-ALL), and found that active oncogenes and silent proto-oncogenes generally occur within insulated neighborhoods formed by the looping of two interacting CTCF sites co-occupied by cohesin. Recurrent microdeletions frequently overlap neighborhood boundary sites in T-ALL genomes, and we demonstrate that site-specific perturbation of loop boundaries is sufficient to activate the respective proto-oncogenes in non-malignant cells. We found somatic genomic rearrangements affecting loop boundaries in many cancers. These results suggest that chromosome structural organization is fundamental to identify functional somatic alterations in cancer genomes.
Evaluation of expression quantitative trait loci within two interacting blood pressure quantitative trait loci
Genetic dissection of the S rat genome has provided strong evidence for the presence of two interacting blood pressure (BP) quantitative trait loci (QTLs), termed QTL1 and QTL2, on rat chromosome 5. However, the identities of the underlying interacting genetic factors remain unknown. Further experiments targeted to identify the interacting genetic factors by the substitution mapping approach alone are difficult because of the interdependency of natural recombinations to occur at the two QTLs. We hypothesized that the interacting genetic factors underlying these two QTLs may interact at the level of gene transcription and thereby represent expression QTLs (eQTLs). To detect these interacting eQTLs, a custom QTL chip containing the annotated genes within QTL1 and QTL2 was developed and used to conduct a transcriptional profiling study of S and two congenic strains that retain either one or both the QTLs. The results uncovered an interaction between two transcription factors, DMRTA2 and NFIA. Further, the ‘biological signature’ elicited by these two transcription factors was differential between the congenic strain that retained LEW alleles at both QTL1 and 2 compared to the congenic strain that retained LEW alleles at QTL1 alone. A network of transcription factors potentially affecting BP could be traced, lending support to our hypothesis. Keywords: rat, hypertension, genetics, polygenic trait, microarray, gene expression
Foxc1 ablated mice are anhidrotic and recapitulate features of human miliaria sweat retention disorder
Sweat glands are critical for thermoregulation. The single tubular structure of sweat glands has a lower secretory portion and an upper reabsorptive duct leading to the secretory pore in the skin. Genes that determine sweat gland structure and function are largely unidentified. Here we report that a Fox family transcription factor, Foxc1, is obligate for appreciable sweat duct activity in mice. When Foxc1 was specifically ablated in skin, sweat glands appeared mature, but the mice were severely hypohidrotic. Morphological analysis revealed that ducts were blocked by hyperkeratotic or parakeratotic plugs. Consequently, lumens in ducts and secretory portions were dilated, and blisters and papules formed on the skin surface in the knockout mice. The phenotype was strikingly similar to human sweat retention disorder miliaria. We further found that Foxc1 deficiency ectopically induced the expression of keratinocyte terminal differentiation markers in the luminal duct cells, which most likely contributed to keratotic plug formation. In summary, Foxc1 regulates sweat duct luminal cell differentiation, and mutant mice may provide a model for miliaria.
Exosomal miR-1290 and miR-375 as prognostic markers in castrate resistant prostate cancer
Extracellular microRNAs (miRNAs) embedded in circulating exosomes may serves as prognostic biomarkers in cancer. This study was performed to identify and evaluate plasma exosomal miRNAs for prognostication in castration resistant prostate cancer (CRPC). RNA sequencing was performed to identify candidate exosomal miRNAs associated with overall survival in a screening cohort of 23 CRPC patients. Candidate miRNAs were further evaluated for prognosis using qRT-PCR in a follow-up cohort of 100 patients. Cox regression and Kaplan–Meier survival curve analysis were used to evaluate prognostic value of miRNA candidates with and without incorporation of clinical prognostic factors (age, Gleason score and time from androgen deprivation therapy to clinical progression). In the screening cohort, we obtained ~6.80 million mappable RNA reads per patient. Of those with normalized read counts ? 5, 43% were mapped to miRNAs for a total of 375 known and 57 novel miRNAs. Cox regression analysis identified an association of miR-1290, -1246, and -375 with overall survival (FDR<0.1). Of those, higher levels of miR-1290 and -375 were verified to be significantly associated with poor overall survival (p<0.004) in the follow-up cohort. The miR-1290/-375-based prediction model showed better performance with time-dependent area under the curve (AUC) =72% compared to clinical variable-based model with AUC=65%. Plasma exosomal miR-1290 and miR-375 are promising prognostic biomarkers for CRPC patients. Prospective validation is needed for further development of these candidate miRNAs.
Cell-specific nitrogen responses in the Arabidopsis root
The organs of multicellular species are comprised of cell types that must function together to perform specific tasks. One critical organ function is responding to internal or external change but little is known about how responses are tailored to specific cell types or coordinated among them on a global level. Here we use cellular profiling of five Arabidopsis root cell types in response to a limiting resource, nitrogen, to uncover a vast and predominantly cell-specific response that was largely undetectable using traditional methods. These methods reveal a new class of cell-specific nitrogen responses. As a proof-of-principle, we dissected one cell-specific response circuit that mediates nitrogen-induced changes in root branching from pericycle cells. Thus, cellular response profiling links gene modules to discrete functions in specific cell types. Keywords: cell type comparison, comparative genomic hybridization, genetic modification
Gene expression profiles of dextran sulfate sodium-induced colon tissues from Pla2g10 knockout mice and their wild-type littemates
Within the secreted phospholipase A2 (sPLA2) family, group X sPLA2 (sPLA2-X) has the highest capacity to hydrolyze cellular membranes and has long been thought to promote inflammation by releasing arachidonic acid (AA), a precursor of pro-inflammatory eicosanoids. Unexpectedly, we found that transgenic mice globally overexpressing human sPLA2-X (PLA2G10-Tg) displayed striking immunosuppressive and lean phenotypes with lymphopenia and increased M2-like macrophages, accompanied by marked elevation of free omega-3 polyunsaturated fatty acids (PUFAs) and their metabolites. Studies using Pla2g10-deficient mice revealed that endogenous sPLA2-X, which is highly expressed in the colon epithelium and spermatozoa, mobilized omega-3 PUFAs or their metabolites to protect against dextran sulfate (DSS)-induced colitis and to promote fertilization, respectively. In colitis, sPLA2-X deficiency increased colorectal expression of Th17 cytokines, and omega-3 PUFAs attenuated their production by lamina propria cells partly through the fatty acid receptor GPR120. In comparison, cytosolic phospholipase A2 (cPLA2alpha) protects from colitis by mobilizing w6 AA metabolites including prostaglandin E2. Thus, our results underscore a previously unrecognized role of sPLA2-X as an omega-3 PUFA mobilizer in vivo, segregated mobilization of omega-3 and omega-6 PUFA metabolites by sPLA2-X and cPLA2alpha, respectively, in protection against colitis, and the novel role of a particular sPLA2-X-driven PUFA in fertilization.
Transcriptomic profiling of Aspergillus flavus in response to 5-azacytidine & gallic acid
Aspergillus flavus is a common saprophyte and opportunistic pathogen producing aflatoxin (AF) and many other secondary metabolites. 5-Azacytidine (5-AC), a derivative of nucleoside cytidine, is widely used for studies in epigenetics and cancer biology as an inactivator of DNA methyltransferase and is also used for studying secondary metabolism in fungi. Our previous studies showed that 5-AC affects development and inhibits AF production in A. flavus, and that A. flavus lacks DNA methylation. How this common DNA methyltransferase inhibitor affects development and AF production is not clear. In this study, we applied an RNA-Seq approach to elucidate the mechanism of 5-AC’s effect on A. flavus. In our current study, we identified 240 significantly differently expressed (Q-value<0.05) genes after 5-AC treatment, including two backbone genes in secondary metabolite clusters #27 and #35, which are involved in development or survival of sclerotia. With 5-AC treatment, about three quarters of the genes in the AF biosynthetic gene cluster in A. flavus were down-regulated to a certain degree. Strikingly, at least two genes aflI and aflLa, were completely inhibited. Interestingly, several genes involved in fungal development were down-regulated, especially veA, which is a gene that encodes protein bridges VelB and LaeA. This result supports the hypothesis that 5-AC affects development and AF production through weakening or even interrupting the connection between VelB and LaeA and then causing dysregulation of the expression pattern of genes involved in development and secondary metabolism. Our results improved the A. flavus genome annotation, provided a comprehensive view of the transcriptome of A. flavus responding to 5-AC and confirmed that fungal development and secondary metabolism are co-regulated. In additon, the RNA-Seq data of another sample treated with gallic acid was used to improve A. flavus genome annotation.
Blood Transcriptional Signature of hyperinflammation in HIV-associated Tuberculosis
Patients with HIV-associated TB are known to experience systemic hyperinflammation, clinically known as immune reconstitution inflammatory syndrome (IRIS), following the commencement of antiretroviral therapy (ART). No prognostic markers or biomarkers have been identified to date and little is known about the mechanism mediating the hyperinflammation. We recruited a prospective cohort of 63 patients with HIV-associated TB, 33 of whom developed TB-IRIS. Of which transcriptomic profiling was performed using longitudinal whole blood RNA samples from 15 non-IRIS and 17 TB-IRIS patients. Transcriptomic signatures that distinguish patients who would eventually develop IRIS were identified as early as week 0.5 (2-5 days post-ART) and predicted a downstream activation of proinflammatory cytokines. At the peak of IRIS (week 2), transcriptomic signatures were overrepresented by innate receptor signaling pathways including toll-like receptor, IL-1 receptor and TREM-1.
Cutting Edge: IL-4, IL-21, and IFN-g Interact to Govern TBET and CD11c Expression in TLR-Activated B Cells
T-Box Expressed in T cells (TBET) and CD11c expression in B cells is linked with IgG2c isotype switching, virus-specific immune responses, and humoral autoimmunity. However, the activation requisites and regulatory cues governing TBET and CD11c expression remain poorly defined. In this article, we reveal a relationship among TLR engagement, IL-4, IL-21, and IFN-g that regulates TBET expression in B cells. We find that IL-21 or IFN-g directly promote TBET+ expression in the context of TLR engagement. Further, IL-4 antagonizes TBET induction. Finally, IL-21, but not IFN-g, promotes CD11c expression independent of TBET. Using influenza virus and Heligmosomoides polygyrus infections, we show that these interactions function in vivo to determine whether TBET+ and CD11c+ B cells are formed. These findings suggest that TBET+ B cells seen in health and disease share the common initiating features of TLR-driven activation within this circumscribed cytokine milieu.
Identification of discriminant genes and gene networks involved in pig ovarian follicular atresia
Folliculogenesis corresponds to the development of follicles leading to either ovulation or degeneration (a process called atresia). Even if atresia involves an apoptosis process, this mechanism is not well understood. The objective of this experiment was : 1) to analyse gene expression in pig granulosa cells of ovarian follicles during atresia using transcriptome analysis with a 9 024 cDNAs microarray, 2) the identification of gene networks involved in pig ovarian follicular atresia. Granulosa cells were isolated from atretic follicles (small, medium or large). Gene expression was analysed by hybridization of nylon cDNA microarrays. The images were quantified using Bzscan software and the data were managed with BASE software. Statistical analysis was performed using R software. Keywords: pig ovary, folliculogenesis, atresia, gene expression, cDNA microarray, bio-analysis
Lactate influences the gene expression profile of human mesenchymal stem cells (hMSC) in a dose dependent manner
Wounds, especially non healing wounds are characterised by elevated tissue lactate concentrations. Lactate is known for being able to stimulate collagen synthesis and vessel growth. Lately it has been shown that lactate, in vivo, plays an important role in homing of stem cells. With this work we aimed to show the influence of lactate on the gene expressionprofil of human mesenchymal stem cells (hMSC). hMSCs were obtained from bone marrow and characterised with fluorescence-activated cell sorting (FACS) analysis. Subsequently the hMSCs were treated with either 0, 5, 10 and 15 mM lactate (pH 7,4) for 24 hours. RNA Isolation from stimulated hMSCs and controls was performed. The Microarray analysis was performed using Affymetrix HuGene 1.0 ST Gene Chip. Selected targets were subsequently analysed using quantitative real time PCR (RTq-PCR). We were able to show that lactate in moderate concentrations of 5 respectively 10 mM leads to an anti-imflammatory, anti-apoptotic but growth and proliferation promoting gene expression after 24 h. In contrast, high latate concentrations of 15 mM leads to the opposed effect, namely promoting inflammation and apoptosis. Hypoxia induced genes did not not show any significant regulation. Contrary to expectation, we were not able to show any significant regulation of glycolysis associate candiadtes. We were able to show that lactate alters gene expression but does not change the cell phenotype, which might be helpful for further investigations of new treatment strategies for chronic non-healing wounds as well as tumor-therapy and neuronal plasticity.
Expression analysis of the effect of protoplasting and FACS sorting in roots
In order to estimate the effects of protoplasting and FACS sorting procedures on salt regulated gene expression we generated expression profiles for whole roots that had been treated with salt for 1 hour and for roots that were protoplasted and FACS sorted after the initial 1 hour salt treatment. Cells are amazingly adept at integrating both external and internal cues to regulate transcriptional states. While internal processes such as differentiation and cell-type specification are generally understood to have an important impact on gene expression, very little is known about how cells utilize these developmental cues to regulate responses to external stimuli. Here we use the response to a well characterized environmental stress, high salinity, to obtain a global view of the role that cell identity plays in guiding transcriptional responses in the root of Arabidopsis. Our analysis is based on three microarray data sets we have generated that explore transcriptional changes spatially among 6 cell layers and 4 longitudinal regions or temporally along 5 time points after salt treatment. We show that the majority of the response to salt stress is cell-type specific resulting in the differential regulation of unique biological functions in subsets of cell layers. To understand the regulatory mechanisms controlling these responses we have analyzed cis-element enrichment in the promoters of salt responsive genes and demonstrate that known stress regulatory elements likely control responses to salt occurring in multiple cell types. Despite the extensive shift in transcriptional state that salt stress elicits, we are able to identify several biological processes that consistently define each cell layer and find that transcriptional regulators of cell-identity tend to exhibit robust cell-type specific expression. Finally, using mutants that disrupt cell-type specification in the epidermis, we reveal cell autonomous and non-autonomous effects when cell identity is altered. Together, these data elucidate a novel intersection between physiology and development and expand our understanding of how transcriptional states are regulated in a multi-cellular context. Keywords: Expression analysis
Midgut-specific gene expression by MacoNPV baculovirus during infection of the bertha armyworm, Mamestra configurata
Infection of lepidopterans by baculoviruses has been traditionally studied using in vitro systems which enable efficient and highly synchronous infection. Many studies using varied virus-host combinations have yielded great insight into the molecular processes by which these large double-stranded DNA viruses achieve infection of host cells. However, a key difference in the virus strategy for infection between individual hosts, and within an individual host, lies with the production of two different forms of the virus; occlusion derived virus, which enables primary infection of insect gut tissues and budded virus, which efficiently infects a variety of different insect tissues throughout the host. To examine the primary infection of midgut cells specifically, we used MacoNPV infection of Mamestra configurata fourth instar larvae as our model and measured the expression of viral genes over a time course of infection. Both digital PCR and RNA sequencing methods showed the profile of transcription to be different from those typically seen with in vitro methods, having unique collections of genes expressed early, as well as much greater expression of p6.9 and much reduced expression of polh and p10 late, in comparison with in vitro studies. These differences likely reflect unique characteristics of midgut cell infection, and provide clues as to the processes these viruses use to regulate expression of different viral forms used to access different host tissues. Baculoviruses are versatile DNA viruses with great potential both as gene therapy vehicles and as biological control tools. Extensive study of their transcriptome in vitro has yielded valuable tools for use in protein expression systems, however it is critical that we obtain a fuller understanding of their in vivo activities before their full medical and agricultural potential can be realized. In these studies we have assessed the gene expression program from a group II alphabaculovirus in the midgut of its complementary larval host and confirmed that the in vivo activities of baculoviruses are unique from what is known of their in vitro transcriptome. These studies provide a first foray with next-generation molecular tools into the characterization of baculovirus biology in vivo.
Differential expression of genes and protein networks in the primary breast tumor that proceed to distant metastasis
The presence or absence of lymph node metastasis plays a major role in the prediction of prognosis and subsequent patient management. However, good proportion of patients who display lymph node positivity remain disease free for 3 years or more, after the initial treatment, while a third of those who were lymph node negative at presentation, develop distant metastasis within the same period. We performed gene expression profiling on a cohort Indian breast cancer patients followed up for a period of 3-5 years and in comparison with a previously published Caucasian cohort data, we identified gene signatures that are associated with distant metastasis. This association was irrespective of the hormone receptor status. Our results show that the genes that signify immune system development and response are repressed, while factors for DNA replication are up regulated in patients who develop distant metastasis. A large number of genes encoding proteins involved in the mitotic spindle formation that belong to the TRIM28 protein network, are differentially regulated in the metastatic tumors. Also, there was a significant overlap of genes reported in a mouse model of bone metastasis, with patients who developed bone metastasis in our cohort. In conclusion, we present for the first time probable gene signatures that correlate with distant metastasis in breast cancer patients irrespective of nodal or hormone receptor status
Gene expression analysis of CD11b+Gr-1+ myeloid cells and epithelial cells purified from Pten KO prostate tumours
Gr-1+ myeloid cells are recruited in prostate conditioanal Pten null tumours (Ptenpc-/-). To identify the secreted factors that may be released by Gr-1+ cells and epithelial cells in the tumour microenvironment, we compared, by gene expression analysis, the cytokine profile of Gr-1+ myeloid cells sorted from Ptenpc-/- tumours with the profile of immune cell-depleted Ptenpc-/- epithelial cells. qRT-PCR validation for IL-10, IL1rn, IL-1α, IL-6, VEGF, TNFα, CXCL1, CCL2 and CXCL12 was also performed.
Comparisons of toxicogenomic responses in zebrafish (Danio rerio) exposed to ZnO nanoparticles and ZnSO4: The impact of sublethal concentration (LC25) on global gene expression profiles
To investigate the patterns of global gene expression profiles modulated by the ZnO nanoparticles and ZnSO4, and to differentiate their modes of toxicity, zebrafish (Danio rerio) will be exposed to the ZnO nanoparticles and ZnSO4 (LC25), and used for microarray analysis by Agilent Zebrafish Oligo Microarray system. 244 genes overlaped between ZnO nanopartices and ZnSO4 treatments in a total of 1,586 differential expressed genes.G-protein coupled receptor protein (GPCR)-signaling pathways, cell-surface receptor-linked signal transduction and immune response are most significant GO terms enriched in genes up regulated by two treatments. Cytokine-cytokine receptor interactions pathway and intestinal immune network for IgA production pathway were enriched among ZnO nanoparticles activated genes, and specifically induced by ZnSO4 include cell adhesion molecules (CAMs) pathway.
Expression data from mitochondrial protease ClpP knock-out mice.
The peptidase ClpP is conserved from bacteria to human. In the mitochondrial matrix, it multimerizes and forms a macromolecular proteasome-like cylinder. Because of its known relevance for the mitochondrial unfolded protein response during cell stress, we characterized two ClpP knock-out mouse founder lines and documented similar phenotypes. Ubiquitously, ClpP absence led to accumulation of its interactor protein ClpX without transcript upregulation. Interestingly, most wild-type tissues with substantial ClpP amounts had no detectable ClpX. This inverse correlation suggests that ClpX levels and degradation are regulated by ClpP. The expectation of similar protein levels, in view of a reported association of heptameric ClpP rings with hexameric ClpX rings, was confirmed only in testis of wild-type animals. Germline tissue was exceptional also in its vulnerability to ClpP deletion, with both founder lines showing complete infertility for males and females. Otherwise, ubiquitous mitochondrial dysfunction was apparent from severe growth retardation and reduced spontaneous motor activity of the animals, and from a pronounced decrease in pre-/postnatal survival. Spermatogenesis was found aborted at the spermatid stage, acrosomes and axonemes were not formed. Overall, tissue-specific roles of ClpP were evident by this massive effect for germ cells, mild bioenergetic deficits in muscle and liver tissues, and excellent compensation in brain. ClpX was previously reported to chaperone unfolded proteins and also DNA condensation in mitochondria, so it is likely that this pathway is particularly susceptible in germ cells. In conclusion, our study indicates that the role of ClpP in quality control is indispensable during development for cells with rapid changes of mitochondrial numbers, and is relevant during aging for growth and survival of the organism.
Target repression induced by endogenous microRNAs: large differences, small effects
MicroRNAs are small RNAs that regulate protein levels. It is commonly assumed that the expression level of a microRNA is directly correlated with its repressive activity – that is, highly expressed microRNAs will repress their target mRNAs more. Here we investigate the quantitative relationship between endogenous microRNA expression and repression for 32 mature microRNAs in Drosophila melanogaster S2 cells. In general, we find that more abundant microRNAs repress their targets to a greater degree. However, the relationship between expression and repression is nonlinear, such that a 10-fold greater microRNA concentration produces only a 10% increase in target repression. The expression/repression relationship is the same for both dominant guide microRNAs and minor mature products (so-called passenger strands/microRNA* sequences). However, we find examples of microRNAs whose cellular concentrations differ by several orders of magnitude, yet induce similar repression of target mRNAs. Likewise, microRNAs with similar expression can have very different repressive abilities. We show that the association of microRNAs with Argonaute proteins does not explain this variation in repression. The observed relationship is consistent with the limiting step in target repression being the association of the microRNA/RISC complex with the target site. These findings argue that modest changes in cellular microRNA concentration will have minor effects on repression of targets.
JeB001: Elucidation of the distal convoluted tubule transcriptome identified new candidate players in magnesium balance
The distal convoluted tubule (DCT) plays a key role in the fine-tuning of renal Mg2+ reabsorption. In recent years, hereditary magnesium (Mg2+) transport disorders have helped to identify important players in DCT Mg2+ homeostasis. Here, we report the Mg2+-sensitive transcriptional expression of the DCT transcriptome using gene expression microarray analysis. Mice expressing eGFP under a DCT-specific promoter were subjected to Mg2+-deficient or Mg2+-enriched diets. Subsequently, the Complex Object Parametric Analyzer and Sorter (COPAS) allowed isolation of eGFP-positive DCT cells. RNA extracts were subjected to pair-wise microarray analysis, high vs. low Mg2+. Of all genes in the genome, 46 were shown to be differentially expressed with a minimal fold-change of 2. Several known magnesiotropic genes, such as Trpm6 and Parvalbumin, were upregulated in the low Mg2+ fraction. Moreover, new genes were identified that are potentially involved in renal Mg2+ homeostasis. To confirm that the selected candidate genes are regulated by dietary Mg2+ availability, the expression levels of Slc41a3, Pcbd1, Tbc1d4 and Umod were determined by RT-PCR analysis. Indeed, all four genes were shown to be significantly upregulated in mice fed the Mg2+-deficient diet. In conclusion, by elucidating the Mg2+-sensitive DCT transcriptome, new candidate players in renal Mg2+ homeostasis were identified.
Gene expression regulated by transcription factor MiT in Drosophila
To understand the role of MiT in Drosophila, we set out to identify critical gene targets by looking at changes in the WT transcriptome induced by either gain or loss of MiT function. Mutant hindgut and malpighian tubules provided loss-of function tissue and nub-Gal4-driven expression of MiT in the wing epithelium was used for gain-of-function. In the wing disc experiment, 543 genes were upregulated by exogenous MiT, and 359 genes were downregulated (>1.4 fold; P value 1.4 fold; P value < 0.01) after MiT. Among these genes, 85 were both upregulated in wing discs and downregulated in mutant HG+MT, and are the common genes that regulated by MiT in both tissues.
Expression data from VND7 induction line
Plants typically contain two different types of cell walls: a primary wall that is being deposited around all growing cells, and a secondary wall that is produced in cells with specialized functions once they have ceased to grow. In Arabidopsis, VND7 is a transcription factor that is sufficient to activate secondary cell wall synthesis. To artificially turn on the secondary cell wall synthesis, VND7 was fused to the activation domain of the herpes virus VP16 protein and the glucocorticoid receptor (GR) domain. Thus, the transgenic plants harbouring the constructs can then be treated with dexamethasone (DEX), a glucocorticoid derivative, to induce the secondary cell wall formation. We used microarrays to investigate the global program of gene expression during the secondary cell wall development and identified up- and down-regulated genes associated with this process. Cultures were harvested at indicated time points after induction (1, 3, 6, 9, 12, 24, 30, 48 h) and at time point zero (0). Uninduced cultures (DMSO instead of DEX) served as controls for each time point, and thus, 48 microarray analyses for 8 time points (3 biological reps) for +/- induced VND7-VP16-GR cultures, and 3 arrays for time 0h. To ensure that the DEX did not influence the measurements, an empty vector construct (EV), i.e., without the VND7 gene, VP16-GR, was used at three time points, i.e., VP16-GR +/- DEX at 9h, 12h, and 24h after DEX induction.
Regulatory impact of RNA secondary structure across the Arabidopsis thaliana transcriptome
The secondary structure of an RNA molecule plays an integral role in its maturation, regulation, processing, and functionality. However, the global influence of this feature on plant gene expression is still for the most part unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach in conjunction with transcriptome-wide sequencing of polyA+-selected (RNA-seq), small (smRNA-seq), and ribosome-bound (ribo-seq) RNA populations to investigate the impact of RNA secondary structure on gene expression regulation in Arabidopsis. From this analysis, we find that highly unpaired and paired RNAs are strongly correlated with euchromatic and heterochromatic epigenetic histone modifications, respectively, providing further evidence that secondary structure is necessary for RNA-mediated posttranscriptional regulatory pathways. Additionally, we uncover key structural patterns across protein-coding transcripts that indicate RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in this model plant. We also reveal that RNA folding is significantly anti-correlated with overall transcript abundance, which is likely due to the increased propensity of highly structured mRNAs to be degraded and/or processed into smRNAs. Finally, we find that secondary structure affects mRNA translation, suggesting that this feature regulates plant gene expression at multiple levels. Overall, our findings provide the first global assessment of RNA folding and its significant regulatory effects in a plant transcriptome.
Expression analysis of root developmental zones after treatment with salt
To gain a genome-scale understanding of the role that developmental processes play in regulating stimulus response, we examined the effect of salt stress on gene expression along the longitudinal axis of the root. Since roots grow from stem cells located near the tip, the position of cells along the longitudinal axis can be used as a proxy for developmental time, with distance from the root tip correlating with increased differentiation. To estimate the role developmental stage plays in regulating salt response, roots were dissected into four longitudinal zones (LZ data set) after transfer to standard or salt media and transcriptionally profiled. Cells are amazingly adept at integrating both external and internal cues to regulate transcriptional states. While internal processes such as differentiation and cell-type specification are generally understood to have an important impact on gene expression, very little is known about how cells utilize these developmental cues to regulate responses to external stimuli. Here we use the response to a well characterized environmental stress, high salinity, to obtain a global view of the role that cell identity plays in guiding transcriptional responses in the root of Arabidopsis. Our analysis is based on three microarray data sets we have generated that explore transcriptional changes spatially among 6 cell layers and 4 longitudinal regions or temporally along 5 time points after salt treatment. We show that the majority of the response to salt stress is cell-type specific resulting in the differential regulation of unique biological functions in subsets of cell layers. To understand the regulatory mechanisms controlling these responses we have analyzed cis-element enrichment in the promoters of salt responsive genes and demonstrate that known stress regulatory elements likely control responses to salt occurring in multiple cell types. Despite the extensive shift in transcriptional state that salt stress elicits, we are able to identify several biological processes that consistently define each cell layer and find that transcriptional regulators of cell-identity tend to exhibit robust cell-type specific expression. Finally, using mutants that disrupt cell-type specification in the epidermis, we reveal cell autonomous and non-autonomous effects when cell identity is altered. Together, these data elucidate a novel intersection between physiology and development and expand our understanding of how transcriptional states are regulated in a multi-cellular context. Keywords: Tissue specific analysis