Dynamic regulation of VEGF-inducible genes by an ERK-ERG-p300 transcriptional network
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Jason Fish
PROVIDER: E-MTAB-5207 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells (EC). We recently showed that DNA methyltransferase (DNMT)- and histone deacetylase (HDAC) inhibitors directly repress EC growth and tumor angiogenesis, suggesting that epigenetic modifications mediated by DNMTs and HDACs are involved in regulation of EC gene expression during tumor angiogenesis. To understand the mechanisms behind the epigenetic regulation of tumor angiogenesis, we used microarray analysis to perform a comprehensive screen to identify genes downregulated in tumor-conditioned versus quiescent EC, and re-expressed by 5-aza-2â-deoxycytidine and trichostatin A. Among the 81 genes identified, 77% harboured a promoter CpG island. Validation of mRNA levels of a subset of genes confirmed significant downregulation in tumor-conditioned EC and reactivation by treatment with a combination of 5-aza-2â-deoxycytidine and trichostatin A, as well as by both compounds separately. Silencing of these genes in tumor-conditioned EC correlated with promoter histone H3 deacetylation and loss of H3 lysine 4 methylation, however did not involve DNA methylation of promoter CpG islands. For six genes, downregulation in microdissected human tumor endothelium was confirmed. Functional validation by RNA interference revealed that clusterin, fibrillin 1 and quiescin Q6 are negative regulators of EC growth and angiogenesis. In summary, our data identify novel angiogenesis suppressing genes which become silenced in tumor-conditioned EC in association with promoter histone modifications and reactivated by DNMT- and HDAC inhibitors through reversal of these epigenetic modifications, providing a mechanism for epigenetic regulation of tumor angiogenesis. Experiment Overall Design: A commercial pool (a mixture of 32 donors) of HUVEC (Tebu-bio, Heerhugowaard, The Netherlands) was used for DNA microarray experiments. Total RNA was isolated using the RNeasy RNA isolation kit (Qiagen) according to the supplier's protocol. Possible genomic DNA contaminations were removed by on column DNase treatment with the RNase-free DNase set (Qiagen). The purified RNA was quantified using a Nanodrop spectrophotometer, and RNA quality was evaluated using the Agilent 2100 Bioanalyzer. cDNA synthesis was performed using the Agilent Fluorescent Direct Labelkit with direct incorporation of either cyanine 5 (Cy5) or Cy3 âdCTP nucleotides (Perkin Elmer) according to the manufacturerâs instructions. Labeled cDNA was purified using QIAquick PCR purification columns (Qiagen), followed by concentration by vacuum centrifugation. The Agilent human 1A cDNA microarray (Agilent Technologies, Amstelveen, The Netherlands) contained ~15000 cDNA probes. Labeled cDNA was resuspended in hybridisation buffer and hybridised to Agilent human 1A cDNA microarray for 17 h at 65°C, according to the Agilent protocols. All hybridisations were replicated with cy dyes switched. Two fluorescent microarray comparisons were performed: (1) A comparison of tumor-conditioned HUVEC and quiescent HUVEC and (2) a comparison of tumor-conditioned HUVEC treated with or without a combination of DAC and TSA. Experiment Overall Design: Microarray data processing and statistical analysis Experiment Overall Design: The image file was processed using Agilent's Feature Extraction software (version A.6.1.1, Agilent Technologies). This Feature Extraction program was used to identify pixels corresponding to fluorescent signal (as opposed to background) and to remove pixels with intensities that met the default criteria for outliers. The different normalisation routines applied (Local Background, Minimum signal (feature or background) & Average of all background areas) resulted in comparable results. For each identified area of signal and each of the two dyes, the basic measure of RNA abundance was taken to be the mean intensity over pixels in the identified signal area. The log ratio of the red to green intensities for each signal area was used for statistical analyses, with all subsequent analyses done using the R statistical software package (version 1.2). We selected fold change 1.5 as a threshold, since the 4 hybridisations increase the likelihood of statistical reliability.
2008-06-15 | E-GEOD-7132 | biostudies-arrayexpress
Project description:mRNA expression after Ezh2 knock down was analyzed to identify genes regulated by Ezh2. Human umbilical vein endothelial cells (HUVEC) were transfected with 25 nmol/L of control small interfering RNA (siRNA) (Silencer Select Negative Control Ambion, Austin, TX) or siRNA directed against Ezh2 (s4918; Ambion) using Oligofectamine (Invitrogen). Total RNA was harvested 72 hours after transfection.
2012-10-16 | E-GEOD-41610 | biostudies-arrayexpress
Project description:Transcriptional profiling of M.tuberculosis to 10 mM vitamin C at 8 h. This was compared to gene expression profile of untreated M. tuberculosis culture. Organism: Mycobacterium tuberculosis H37Rv, Genotypic Technology designed Custom Mycobacterium tuberculosis H37Rv Whole Genome 8x15k GE Microarray (AMADID-020181)
2014-10-01 | E-GEOD-60376 | biostudies-arrayexpress
Project description:The centromeric histone H3 variant CENP-A is overexpressed in many cancers. The mislocalization of CENP-A to noncentromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. However, pathways that promote or prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen for regulators of CENP-A localization which identified DNAJC9, a J-domain protein implicated in histone H3–H4 protein folding, as a factor restricting CENP-A mislocalization. Cells lacking DNAJC9 exhibit mislocalization of CENP-A throughout the genome, and CIN phenotypes. Global interactome analysis showed that DNAJC9 depletion promotes the interaction of CENP-A with the DNA-replication-associated histone chaperone MCM2. CENP-A mislocalization upon DNAJC9 depletion was dependent on MCM2, defining MCM2 as a driver of CENP-A deposition at ectopic sites when H3–H4 supply chains are disrupted. Cells depleted for histone H3.3, also exhibit CENP-A mislocalization. In summary, we have defined novel factors that prevent mislocalization of CENP-A, and demonstrated that the integrity of H3–H4 supply chains regulated by histone chaperones such as DNAJC9 restrict CENP-A mislocalization and CIN.
2024-04-11 | PXD043961 | Pride
Project description:BACKGROUND: This study was conducted to analyse M. leprae gene expression profiles in biopsy of leprosy patient and in environmental soil samples. Investigation was carried out to find out the expression profile for the minimal gene set necessary for growth of environmental M. leprae and its genes potentially require for infection and pathogenesis of leprosy. METHOD: RNA was extracted from human biopsy and environmental soil samples and total RNA obtained by subtractive hybridization of rRNA, was reverse transcribed and amplified using random primers. The whole genome was tiled at 10bp to obtain probes having 60 mer oligonucleotides in sense orientation. 179963 probes were designed in both sense and antisense orientation. Blast was performed against the mRNA sequence databases to check the specificity of the probes. Finally, 359926 probes were designed and 56579 specific probes were replicated to fill the remaining spots. RESULTS: Up regulation of several functional genes of M. leprae genome were observed from soil samples responsible for metabolism of Carbon compounds [9], Amino acids and amines [4] , Fatty acids synthesis [21], Lipid Biosynthesis [33], Phosphorous compounds [2]. Up regulation of genes were observed from soil samples in energy metabolism function such as Glycolysis [6], Pyruvate dehydrogenase [1], TCA cycle [12], Glyoxylate bypass [12]. In up regulation of respiration function [11] genes were observed and some of genes responsible for miscellaneous oxidoreductases and oxygenases [22] function, ATP-proton motive force [4] were also observed. Up regulation of genes for cell envelope function [65], conserved membrane proteins [107], cell processes [62], central intermediary metabolism [79], polyamine synthesis[38], biosynthesis of cofactors, prosthetic groups and carriers [36], broad regulatory functions [35], macromolecule metabolism[103], degradation of macromolecules [25], miscellaneous transferases [36], conserved hypothetical [204] and unknowns [101] were observed. Significant up regulation of Virulence genes [9], PE and PPE families [3] were also observed from soil samples. CONCLUSION: Transcriptome analysis indicated that up regulation of essential genes of M. leprae transcripts in environmental samples as compared to M. leprae from human biopsy samples which strongly suggested that several M. leprae genes are expressed more in environment for its survival and may act as source of infection. RNA was extracted from human biopsy and environmental soil samples and total RNA obtained by subtractive hybridization of rRNA, was reverse transcribed and amplified using random primers. The whole genome was tiled at 10bp to obtain probes having 60 mer oligonucleotides in sense orientation. 179963 probes were designed in both sense and antisense orientation. Blast was performed against the mRNA sequence databases to check the specificity of the probes. Finally, 359926 probes were designed and 56579 specific probes were replicated to fill the remaining spots.
2016-06-01 | E-GEOD-68192 | biostudies-arrayexpress
Project description:To shed light on the genetic events downstream of DELLA proteins, we have employed a microarray expression profiling of Arabidopsis thaliana seeds to identify target genes of the DELLA protein RGL2. Seeds of the germinating ga1-3 rga-t2 rgl2-1 and the non-germinating ga1-3 rga-t2 mutant were stratified, and RNA was extracted after five days. Transcript profiles of the non-germinating seeds closely resemble profiles of dormant seeds, and several transcription factors involved in light- and phytohormone-regulated signalling pathways appear to be up-regulated, suggesting that RGL2 controls various physiological aspects to inhibit seed germination. Gene expression five days after stratification was measured in ga1-3 rga-t2 seeds, using seeds of the ga1-3 rga-t2 rgl2-1 mutant as reference. Two biological replicates were performed for each sample.
2012-08-31 | E-GEOD-40485 | biostudies-arrayexpress
Project description:The mechanisms responsible for the molecular pathogenesis of the highly pathogenic avian influenza virus (HPAIV) or low pathogenic avian influenza virus (LPAIV) in avian species remain poorly understood. Thus, global immune response of chickens infected with HPAIV H5N1 (A/duck/India/02CA10/2011) and LPAIV H9N2 (A/duck/India/249800/2010) viruses was studied using microarray to identify crucial host genetic components responsive to these infection. HPAIV H5N1 induced excessive mRNA expression of cytokines (IFNA, OASL, MX1, RSAD2, IFITM5, GBP 1, IL1B, IL18, IL22, IL13, IL12B, CCL4, CCL9, CCL10, CX3CL1 etc) in lung tissues. This excessive cytokine response (cytokine storms) may cause tissue damage and high mortality in chickens. In contrast, the expression levels of most of the cytokines remained unchanged in the lungs of LPAIV H9N2 virus infected chickens. This study indicated the relationship between host cytokines response and their roles in pathogenesis in chickens infected with HPAIVs. Agilent Custom Chicken Gene Expression 8X60k (AMADID: G4102A_059389) designed by Genotypic Technology Private Limited , Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
2016-07-04 | E-GEOD-65231 | biostudies-arrayexpress
Project description:The underlying molecular mechanisms of pathogenesis and outcome of disease to different pathotypes of H5N1 influenza infection in ducks remain unclear. For that, we studied genome wide host gene expression of lung tissues infected with A/duck/India/02CA10/2011(AD2011) H5N1 virus and A/duck/Tripura/103597/2008 (AD2008) H5N1 virus in ducks using custom designed microarray. AD2011 is highly pathogenic whereas AD2008 is low pathogenic to ducks. Comparative analysis of differentially expressed genes revealed that 688 genes were commonly expressed, 877 and 1556 genes are uniquely expressed to infection with AD2011 and AD2008 virus isolate, respectively. The up-regulation of cytokines genes OAS, IL1B, IL17, IFITM2, CCL4, CXCR4, STAT3, TGFB1 and TGFB2 in the lungs tissues may cause high mortality in ducks infected with AD2011 virus. The expression of important antiviral immune genes IFIT5, IFITM5, RSAD2, EIF2AK2 (PKR), Mx, β-defensins, TRIM23 and SLC16A3 to AD2008 infection, but not in AD2011 infection, cause the host may fine-tune their innate immune responses and prevent from cytokines storms and tissue damage. Several immune related Gene ontology (GO) terms and immune pathways activated were qualitatively similar but quantitatively different to both virus infections. Based on these findings, we conclude that subtle differences in host immune responses may determine the different outcome of H5N1 infection in ducks. Agilent Custom Duck Gene Expression 8X60k (AMADID: G4102A_059612) designed by Genotypic Technology Private Limited , Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
2016-07-04 | E-GEOD-65230 | biostudies-arrayexpress
Project description:mRNA Transcripts expression profile of Plasmodium berghei infected host HepG2 cells with and without SUMO1 over expression. To check the changes in the transcript profiles upon SUMO1 over expression which is responsible for Plasmodium berghei parasite's growth arrest in host HepG2 cells. Agilent one-color experiment, Organism: Homo sapiens, Custom Agilent 8x60k Human Whole Genome Microarray Gene expression (AMADID: 039494), Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442).
2016-09-01 | E-GEOD-72049 | biostudies-arrayexpress
Project description:Whole genome expression of bone marrow deived hepatocytes after 1 and 5 months of transplantation are compared with that of primary hepatocytes and Lin- BM cells Agilent one-color experiment,Organism:Mouse, Agilent Whole Genome Mouse 8x60k (AMADID: 028005 & 026986) , Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
2016-07-01 | E-GEOD-73543 | biostudies-arrayexpress