Gene expression data on human keratinocytes with knockdown or overexpression of SNAI2
Ontology highlight
ABSTRACT: 1. Keratinocytes infected with retroviruses expressing control or SNAI2 shRNAs were cultured in growth medium and Affymetrix HG-U133 plus 2.0 arrays were used to determine global gene expression profiles. 2. Keratinocytes infected with retroviruses overexpressing LACZ or SNAI2 were cultured in growth medium and Affymetrix HG-U133 plus 2.0 arrays were used to determine global gene expression profiles. Keratinocytes with knockdown or overexpression of SNAI2 were used to determine gene expression profiles
Project description:To determine if fibroblasts could be reprogrammed to a keratinocyte phenotype p63+KLF4 or LacZ expressing retroviruses were transduced into primary human neonatal fibroblasts. Global gene expression profiling using U133 plus 2.0 arrays were used to deteremine the extent of reprogramming to a keratinocyte phenoypte upon transduction with p63+KLF4. Fibroblasts transduced with p63+KLF4 were also treated +/- high calcium to determine if treatment with calcium could induce differentiation of these cells. Microarray analysis was also performed on cells treated +/- calcium. For gene expression profiling, cultured human fibroblasts were infected with LacZ or p63+KLF4 expressing retroviruses. p63+KLF4 cells were also treated +/- calcium. Microarray analysis using Affymetrix HG-U133 2.0 plus arrays was performed on duplicate samples.
Project description:Stem and progenitor cells maintain the tissue they reside in for life by regulating the balance between proliferation and differentiation. How this is done is not well understood. Here, we report that the human exosome maintains progenitor cell function. The expression of several subunits of the exosome were found to be enriched in epidermal progenitor cells, which were required to retain proliferative capacity and to prevent premature differentiation. Loss of PM/Scl-75 also known as EXOSC9, a key subunit of the exosome complex, resulted in loss of cells from the progenitor cell compartment, premature differentiation, and loss of epidermal tissue. EXOSC9 promotes self-renewal and prevents premature differentiation by maintaining transcript levels of a transcription factor necessary for epidermal differentiation, GRHL3, at low levels through mRNA degradation. These data demonstrate that control of differentiation specific transcription factors through mRNA degradation is required for progenitor cell maintenance in mammalian tissue. Refer to publication (Mistry et. Cell Stem Cell 2012) for more detail For gene expression profiling, cultured primary human keratinocytes were knocked down for EXOSC9, EXOSC9 and GRHL3, or control. RNA was harvested from the cells 5 days after knockdown. Microarray analysis using Affymetrix HG-U133 2.0 plus arrays was performed on duplicate samples. Significantly changed genes were identified as previously described(Sen et al., 2010).
Project description:NFkB is a family of transcriptional factors that are responsible for inflammatory and immune gene expression. RelA, RelB and cRel are the transactivation domain containing family members. P50 encoded by Nfkb1 is the primary dimerization partner. Many NFkB deficient mice are embryonic lethal. In order to identify NFkB dependent genes, MEF were isolated from Rela-/-cRel-/-, Rela-/-cRel-/-Nfkb1-/- and Rela-/-Relb-/-cRel-/- embryos at E12.5. They were treated with 100ng/ml LPS for 1hr and then profiled gene expression by microarray.
Project description:The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between TOC1 and CCA1/LHY. CCA1 and LHY are MYB transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data supports that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNA binding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the Pseudoreceiver (PR) domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify new potential TOC1 targets and output pathways. Together these results define the biochemical action of the core clock protein TOC1 and refine our perspective on how plant clocks function. Keywords: Expression profiling by array wild type (Col-0) and ALC::TOC1 were sown on Murashige-Skoog with 0.8% agar, stratified for 48 hours and grown in12:12 light:dark (LD) for 12 days and either left in LD or transferred to constant light (LL) and then grown for one more day before the start of the experiment. Tissue was submerged in Murashige-Skoog media supplemented with 2.5% ethanol or no ethanol (mock) and with 20mM MG132 for 3 hours and harvested at ZT1. Three replicates each of the seedlings were collected and frozen in liquid nitrogen.
Project description:1. Keratinocytes infected with retroviruses expressing control or SNAI2 shRNAs were cultured in growth medium and Affymetrix HG-U133 plus 2.0 arrays were used to determine global gene expression profiles. 2. Keratinocytes infected with retroviruses overexpressing LACZ or SNAI2 were cultured in growth medium and Affymetrix HG-U133 plus 2.0 arrays were used to determine global gene expression profiles.
Project description:Plasmodium and Toxoplasma are parasites of major medical importance that belong to the Apicomplexa phylum of protozoa. These parasites transform into various stages during their life cycle and express a specific set of proteins at each stage. Although still little is known of how gene expression is controlled in Apicomplexa, histone modifications, particularly acetylation, are emerging as key regulators of parasite differentiation and stage conversion. Here, we investigated the anti-Apicomplexa effect of FR235222, a histone deacetylase (HDAC) inhibitor. We show that FR235222 is active against a variety of Apicomplexa genera, including Plasmodium and Toxoplasma, and is more potent than other HDACi such as TSA and the clinically relevant compound, pyrimethamine. We identify TgHDAC3 as the target of FR235222 in Toxoplasma tachyzoites and demonstrate the crucial role of the conserved and Apicomplexa HDAC-specific residue TgHDAC3 T99 in the inhibitory activity of the drug. We also show that FR235222 induces differentiation of the tachyzoite (replicative) into the bradyzoite (non replicative) stage. Additionally, via its anti-TgHDAC3 activity, FR235222 influences the expression of ~370 genes, a third of which are stage-specifically expressed. These results identify FR235222 as a potent HDAC inhibitor of Apicomplexa, and establish HDAC3 as a central regulator of gene expression and stage conversion in Toxoplasma and likely other Apicomplexa. Freshly released tachyzoites were needle-passed, and filtered using a 3-µm nucleopore membrane. Parasites were resuspended into fresh DMEM supplemented with 10% (v/v) FBS and 25 mM HEPES buffer pH7.2. Parasites were incubated in the presence of FR235222 (40 nM) or DMSO (0.1%) for 4 h at 37°C with 5% CO2. For ChIP-chip experiments freshly released tachyzoites (~5 x 109 at ~12 x 107 parasites/mL) were fixed for 15 min in 1% formaldehyde. Increase in AcH4 signals was verified by immunoblot to verify that FR235222 treatment was effective. To prepare chromatin samples, fixed parasites were lysed in MNase buffer (0.32 M Sucrose, 50 mM Tris-HCl pH7.8, 4 mM MgCl2, 3 mM CaCl2, 100 mM NaCl, 0.25% (v/v) NP40, 5% (v/v) glycerol, protease inhibitor EDTA-free cocktail (Roche)) and DNA was digested for 4 min at 37°C by MNase (2 units/mL). Digestion was stopped with 20 mM EDTA and chromatin was recovered in the soluble fraction after centrifugation at 10,000 g at 4°C; this constituted the S1 fractions. Pelleted materials were resuspended in dialysis buffer (1mM Tris-HCl pH7.8, 0.2 mM EDTA) containing 1 mM PMSF and protease inhibitor cocktail (Roche®) and dialyzed overnight at 4°C against the same solution. Then dialyzed materials were centrifuged and supernatant were harvested; this constitutes the S2 fractions. For chromatin immunoprecipitations, fractions S1 and S2 were pooled and DNA quality was verified by electrophoresis on 2% agarose gels; oligonucleosome ladder of 100-1000 bp were obtained. The histone-DNA complexes were immunoprecipitated with anti-acetyl histone H4 (Upstate®, catalog # 06-866) antibodies according to NimbleGenâs protocol (http://www.genomecenter.ucdavis.edu/expression_analysis/documents).
Project description:High-fat diet (HFD) in normoxia causes a worsened phenotype in adult female flies compared to regular diet (RD). We used microarrays to examine the transcriptional changes occuring on a HFD compared to RD in normoxia. Adult female Drosophila were collected at d3-5 and placed on RD or HFD and in normoxia for 1 week. Following this week,flies were immediately frozen on dry ice, RNA was extracted and hybridized on Affymetrix microarrays.
Project description:Coordinated regulation of protection mechanisms against environmental abiotic stress and pathogen attack is essential for plant adaptation and survival. Initial abiotic stress can interfere with disease resistance signaling. Conversely, initial plant immune signaling may interrupt subsequent ABA signal transduction. However, the processes involved in cross talk between these signaling networks have not been determined. By screening a 9,600 compound chemical library, we identified a small molecule [5-(3,4-Dichlorophenyl)Furan-2-yl]-Piperidin-1-ylMethanethione that rapidly down-regulates ABA-dependent gene expression and also inhibits ABA-induced stomatal closure. Transcriptome analyses show that DFPM also stimulates expression of plant defense-related genes. Plate grown 12-day-old seedlings were transferred into 6 well plates with 1:5000 (V/V) DMSO in water as a control, 30uM DFPM, and 10uM ABA in water as a treatment for 6 hours. DFPM was added 30 min prior to ABA treatment. RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA) and further purified using RNeasy Plant RNA purification kit (QIAgen, Valencia, CA, USA). Three biological replicates of ATH1 oligonucleotide arrays were hybridized with labeled samples from 1) wild-type Columbia (WT) untreated, 2) WT with 30uM DFPM treatment, 3) WT with 10uM ABA treatment, 4) WT with 30uM DFPM and 10uM ABA treatment. Each biological replicate was prepared by combining 7 independently-treated samples. Seedling treatments, 4 agent, 1 genotype/variation sets.
Project description:Examination of genome-wide, gene expression level differences among adult and fetal mammary sub-populations including fetal CD24high cells and adult CD49fhighCD24medium cells which harbor enriched mammary stem cell activity. A genome wide 12-plex expression array using linear-amplified (Eberwine) mRNA from dissociated and FACS sorted fetal and adult mammary gland populations. Samples represent independent pooled biological replicates.
Project description:We find that epithelial-specific deletion of Nfatc1 decreases skin tumor susceptibility in mice subjected to DMBA/TPA carcinogenesis. To probe the molecular mechanisms by which Nfatc1 may regulate tumorigenesis, we identify gene expression changes associated with constitutive NFATc1 activation in primary mouse keratinocytes. Constitutively active NFATc1 activity in primary mouse keratinocytes to investigate how Nfatc1 regulates tumor initiation in the skin. Primary mouse keratinocytes were infected with retroviruses encoding a recombinant human NFATc1 containing mutations in the autoinhibitory domain that render it constitutively active (CAC1). Keratinocytes infected with retroviruses generated from an empty MSCV reotroviral vector were used as a control. Three biological replicates were performed.