Microarray of GFP-positive rat germ cells after exposure to ethynilestradiol or genistein
Ontology highlight
ABSTRACT: We took advantage of a transgenic rat expressing the green fluorescent protein (GFP) specifically in germ cells allowing purification of perinatal GFP-positive germ cells. Timed-pregnant rats were exposed to ethinylestradiol (EE2, 2 µg/kg/d), genistein (GE, 10 mg/kg/d) or vehicle by gavage, from gestational days (GD) 13 to 19; testes were sampled at GD20 or post-natal (PND) 5 for sorting of GFP-positive cells. Gene expression was assessed in GFP-positive cells using Affymetrix Rat Gene 2.0 ST microarrays.
Project description:Protein deficiency and intestinal parasite infection during pregnancy impair fetal growth through passage of signals from the maternal environment which signal impairment of fetal growth. The placenta is an important regulator of the transfer of these signals through differential expression of key placental genes. We used microarrays to examine placental gene expression responses to maternal protein deficiency (6% vs. 24% protein) and Heligmosomoides bakeri infection. 2x2 factorial.
Project description:The implication of alveolar macrophages (AM) in asthma, a Th2 disease, has not been well characterized. Thus, the goal of this study is to better characterize AM phenotype of allergic asthmatic compared with normal subjects using genomic expression analyses. Microarray analyses were performed with AM isolated from bronchoalveolar lavage. Robust multiarray analysis (RMA) normalization and Smythâs moderated t test were used to select differentially expressed genes. Fifty differentially expressed genes were identified. Nineteen have been classified in categories linked to stress or immune responses and among them; nine are part of the heat shock protein (HSP) family. Difference of expression for three (HSPD1, PRNP, SERPINH1) of the five selected genes were validated using real-time reverse transcriptionâpolymerase chain reaction. Enzyme linked immunosorbent assay was used to measure the protein level of heat shock protein 60 (HSP60), the protein encoded by HSPD1, and showed difference in AM protein level between allergic asthmatic and control subjects. In summary, this study suggests that HSP gene family, particularly HSP60, is involved in AM functions in a context of allergic asthma. These results also support the involvement of AM immune functions in the development of an allergic asthmatic response. Ten alveolar macrophage samples from bronchoalveolar lavages have been studied. Five of them are from allergic asthmatic subjects and five from control subjects (without allergy and asthma).
Project description:Tha altered biological pathways in Epidermolysis bulloda simplex, a rare monogenetic skin disease, have not been well characterized. Thus, the goal of this study is to characterize the expression profile of EBS patients compared with normal subjects using genomic expression analyses. Microarray analyses were performed with RNA isolated from skin biopsies. Robust multiarray analysis (RMA) normalization and Smyth’s moderated t test were used to select differentially expressed genes. Expression profiling comparisons show that 28 genes are differentially expressed in EBS patients compared to control subjects and 41 genes in EBS-DM compared to their matched controls. Nine genes involved in fatty acid metabolism and 2 genes in epidermal keratinisation are common altered expressed genes between the two subgroups. These two biological pathways contribute both to the formation of the cell envelope barrier and seem to be defective in the severe EBS phenotype. This study demonstrates, for the first time, the relevance of metabolic cluster, specifically fatty acid metabolism in EBS biology. Difference of expression for three (AWAT2, ELOVL , and SPRR4 ) of the five selected genes were validated using real-time reverse transcription–polymerase chain reaction. To our knowledge, the distinctive pattern of gene expression that characterizes EBS versus healthy skin tissue has never been reported. RNA used for the microarrays analysis was isolated from superficial 2mm punch biopsies composed of mainly epidermis with minimal dermis amounts of normal-appearing skin of six EBS patients ( two females and four males) and six healthy volunteers.
Project description:The goal of this study is to characterize the expression profile of Epidermolysis bullosa simplex-mottled pigmentation (EBS-MP) patient compared with normal subjects using genomic expression analyses. Microarray analyses were performed with RNA isolated from skin biopsies. Robust multiarray analysis (RMA) normalization and Smyth’s moderated t test were used to select differentially expressed genes. Expression profiling comparisons show that 52 genes are differentially expressed in EBS-MP patients compared to control subjects. Difference of expression for three genes (TYR, CCL22 , and ACVR1C ) was validated using real-time reverse transcription–polymerase chain reaction. Twelve genes were related to lipid biosynthesis process, two to keratinisation and skin pigmentation, Nineteen to cell growth and apoptosis, five to immune response and fourteen to predicted or less known function cluster. To our knowledge, the distinctive pattern of gene expression that characterizes EBS-MP versus healthy skin tissue has never been reported. RNA used for the microarrays analysis was isolated from superficial 2mm punch biopsies composed of mainly epidermis with minimal dermis amounts of normal-appearing skin of one EBS-MP patient and seven healthy volunteers.
Project description:Myocyte enhancer factor 2B (MEF2B) is a transcription factor with somatic mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). The recurrence of these mutations indicates that they may drive lymphoma development. However, inferring the mechanisms by which they may drive lymphoma development was complicated by our limited understanding of MEF2B’s normal functions. To expand our understanding of the cellular activities of wildtype (WT) and mutant MEF2B, I developed and addressed two hypotheses: (1) identifying genes regulated by WT MEF2B will allow identification of cellular phenotypes affected by MEF2B activity and (2) contrasting the DNA binding sites, effects on gene expression and effects on cellular phenotypes of mutant and WT MEF2B will help refine hypotheses about how MEF2B mutations may contribute to lymphoma development. To address these hypotheses, I first identified genome-wide WT MEF2B binding sites and transcriptome-wide gene expression changes mediated by WT MEF2B. Using these data I identified and validated novel MEF2B target genes. I found that target genes of MEF2B included the cancer genes MYC, TGFB1, CARD11, NDRG1, RHOB, BCL2 and JUN. Identification of target genes led to findings that WT MEF2B promotes expression of mesenchymal markers, promotes HEK293A cell migration, and inhibits DLBCL cell chemotaxis. I then investigated how K4E, Y69H and D83V mutations change MEF2B’s activity. I found that K4E, Y69H and D83V mutations decreased MEF2B DNA binding and decreased MEF2B’s capacity to promote gene expression in both HEK293A and DLBCL cells. These mutations also reduced MEF2B’s capacity to alter HEK293A and DLBCL cell movement. From these data, I hypothesize that MEF2B mutations may promote DLBCL and FL development by reducing expression of MEF2B target genes that would otherwise function to help confine germinal centre B-cells to germinal centres. Overall, my research demonstrates how observations from genome-scale data can be used to identify cellular effects of candidate driver mutations. Moreover, my work provides a unique resource for exploring the role of MEF2B in cell biology: I map for the first time the MEF2B ‘regulome’, demonstrating connections between a relatively understudied transcription factor and genes significant to oncogenesis. We analysed Affymetrix Human Exon 1.0 ST array data from HEK293A cells stably transfected with WT or mutant (K4E, Y69H or D83V) MEF2B-V5 and untransfected control cells. Data was collected in triplicate from all cell types under DMSO treatment, as well as from triplicates of WT and untransfected cells that were ionomycin treated (1.5uM for 6hr).
Project description:The tumour suppressor p14/19ARF regulates ribosomal RNA (rRNA) synthesis by controlling the nucleolar localization of Transcription Termination Factor 1 (TTF1). However, the role played by TTF1 in regulating the rRNA genes and in potentially controlling growth has remained unclear. We now show that TTF1 expression regulates cell growth by determining the cellular complement of ribosomes. Unexpectedly, it achieves this by acting as a “roadblock” to synthesis of the non-coding LncRNA and pRNA that we show are generated from the “Spacer Promoter” duplications present upstream of the 47S pre-rRNA promoter on the mouse and human ribosomal RNA genes. Unexpectedly, the endogenous generation of these non-coding RNAs does not induce CpG methylation or gene silencing. Rather, it acts in cis to suppress 47S preinitiation complex formation and hence de novo pre-rRNA synthesis by a mechanism reminiscent of promoter interference or occlusion. Taken together, our data delineate a pathway from p19ARF to cell growth suppression via the regulation of ribosome biogenesis by non-coding RNAs and validate a key cellular growth law in mammalian cells.
Project description:The tumour suppressor p14/19ARF regulates ribosomal RNA (rRNA) synthesis by controlling the nucleolar localization of Transcription Termination Factor 1 (TTF1). However, the role played by TTF1 in regulating the rRNA genes and in potentially controlling growth has remained unclear. We now show that TTF1 expression regulates cell growth by determining the cellular complement of ribosomes. Unexpectedly, it achieves this by acting as a “roadblock” to synthesis of the non-coding LncRNA and pRNA that we show are generated from the “Spacer Promoter” duplications present upstream of the 47S pre-rRNA promoter on the mouse and human ribosomal RNA genes. Unexpectedly, the endogenous generation of these non-coding RNAs does not induce CpG methylation or gene silencing. Rather, it acts in cis to suppress 47S preinitiation complex formation and hence de novo pre-rRNA synthesis by a mechanism reminiscent of promoter interference or occlusion. Taken together, our data delineate a pathway from p19ARF to cell growth suppression via the regulation of ribosome biogenesis by non-coding RNAs and validate a key cellular growth law in mammalian cells.
Project description:Synthesis and accumulation of seed storage proteins (SSPs) is an important aspect of the seed maturation program. Genes encoding SSPs are specifically and highly expressed in the seed during maturation. However, the mechanisms that repress the expression of these genes in leaf tissue are not well understood. To gain insight into the repression mechanisms, we have performed a transgenic screening for mutants that express SSPs in leaves. Here we show that mutations of BRAHMA (BRM), a SNF2 chromatin remodelling ATPase, cause the ectopic expression of a subset of SSPs and other embryogenesis related genes in leaf tissue. Consistent with the notion that such SNF2-like ATPases form protein complexes in vivo, we observed similar phenotypes for mutations of AtSWI3C, a BRM interacting partner, and BSH, a SNF5 homolog and essential SWI/SNF subunit. Further, we present chromatin immunoprecipitation evidence that BRM is recruited to the promoters of a number of embryogenesis genes including the 2S genes, which are expressed/elevated in brm leaves. Consistent with its role in nucleosome remodelling, BRM appears to control the chromatin structure of the At2S2 promoter. These results show that a BRM-containing chromatin remodelling ATPase complex is involved in the direct repression of SSPs in leaf tissue. A matrix comprising the signal intensity value of each gene per replicate hybridization and the averaged data of each gene generated from three replicate hybridizations of the wild type and mutant samples, respectively, is linked below as a supplementary file. Experiment Overall Design: Total RNA was isolated from three independent biological replicates of essp3 mutant and Wild type (Pro?CG:GUS)) respectively. Three ATH1 chips were used for the mutant and three for the wild type.
Project description:Analysis of a SigX knockout mutant of Pseudomonas aeruginosa H103 strain in minimal medium with glucose as carbon source (M9G). SigX, one of the 19 extra-cytoplasmic function sigma factors of P. aeruginosa, was only known to be involved in transcription of the gene encoding the major outer membrane protein OprF in Pseudomonas aeruginosa. Deletion of the ECF sigma factor sigX gene provide insights into the SigX role in several virulence and biofilm- related phenotypes in Pseudomonas aeruginosa. To better understand the cellular function of SigX, a deletion mutant of the sigX gene (PAOSX) was generated and its expression profile was compared with parental strain Pseudomonas aeruginosa H103. To this end, H103 and a sigX mutant were cultured in M9G, in which their growth are similar. Three independant biological replicate were taken for the RNA extraction and hybridization on affymetrix array in the middle of the exponential growth phase.
Project description:The seed maturation program occurs only during late phase of embryo development and repression of the maturation genes is pivotal for seedling development. However, mechanisms that repress the expression of this program in vegetative tissues are not well understood. A genetic screen was performed for mutants that express maturation genes in leaves. Here, it is shown that mutations affecting SDG8 (SET DOMAIN GROUP 8), a putative histone methyltransferase, cause ectopic expression of a subset of maturation genes in leaves. Further, to investigate the relationship between SDG8 and the Polycomb Group (PcG) proteins, which are known to repress many developmentally important genes including seed maturation genes, double mutants was made and formation of somatic embryos was observed on mutant seedlings with mutations in both SDG8 and EMF2 (EMBRYONIC FLOWER 2). Interestingly, double mutant of sdg8 and mutations in VRN2 (VERNALIZATION 2), a paralog of EMF2, grow and develop normally to maturity. Analysis of histone methylation status at chromatins of a number of maturation loci revealed synergistic effect of emf2 and sdg8 on the deposition of the active histone mark, trimethylation of lysine 4 on histone 3 (H3K4me3), which is consistent with high expression of these genes (formation of somatic embryos) in emf2 sdg8 double mutants. These observations demonstrate a functional cooperative interplay between SDG8 and an EMF2-containing PcG complex in maintaining vegetative cell identity by repressing seed genes to promote seedling development. The work also indicates the functional specificities of PcG complexes in Arabidopsis. Total RNA was isolated from three independent biological replicates of Wild type (ProM-NM-2CG:GUS) and two independent biological replicates of sdg8-2 and essp4/sdg8-5, respectively. Four ATH1 chips were used for the two mutants and three for the wild type. Matrices comprising the signal intensity value of each gene per replicate hybridization and the averaged data of each gene generated from three replicate hybridizations of the wild type and two replicate hybridizations of mutant samples, respectively, are linked below as supplementary files.