Project description:To explore global gene expression using microarray analysis in mTEC's isolated from MBD1 WT and MBD1 KO mice mTECs were sorted by flow cytometry from 30 day old MBD1 WT and KO Mice. 4 biological replicates were performed for both MBD1 WT and KO mice (1 replicate of each failed quality control and wasd removed from the analysis).
Project description:HEK 293 cells were transiently transfected with plasmids expressing Vector only(PCMV), Aire, or MBD-VP16 with the goal of comparing the global gene expression profiles in the Aire and MBD-VP16 groups We used microarrays to detail the global gene expression profile. HEK 293 cells were transiently transfected with plasmids expressing Vector only(PCMV), Aire, or MBD-VP16 and 72 hrs post transfection total RNA was collected for microarray analysis.
Project description:The aim of this study is to discover genes regulated by miR-204. Differential gene expression in HEK-293 cells transfected with miR-204-mimic compared to HEK-293 cells transfected with control oligo (HEK-293 control) was analyzed using the Agilent Human Whole Genome 4x44K gene expression array (Agilent Technologies, Santa Clara, CA). HEK-293 cells were transfected with either miR-204 or a control, and gene expression was analyzed using the Agilent Human Whole Genome 4x44K array. A dye-swap was performed.
Project description:This SuperSeries is composed of the following subset Series: GSE17342: The role of miRNA in Wilms' tumorigenesis GSE28397: Copy number alteration in Wilms' tumor with custom-designed miRNA probes GSE28400: MIR-204 target gene Refer to individual Series
Project description:Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. We used sequence conservation analysis and ChIP-seq against the enhancer-associated histone mark H3K27ac to identify a candidate Aire cis-regulatory element. There is enrichment of H3K27ac near this element, ACNS1, in mTECs and the element also has characteristics of being NF-κB-responsive. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance. Two experimental groups (GFP neg mTECs and GFP pos mTECs), each with three samples, and one control sample (D10 Th2 cells).
Project description:MicroRNAs (miRNAs) regulate many basic aspects of cell biology including neuronal plasticity, but little is known of their roles in drug addiction. Extended access to cocaine can trigger the emergence of compulsive drug-seeking behaviors, but molecular mechanisms regulating this process remain unclear. Here we report that microRNA-212 (miR-212) is upregulated in the dorsal striatum of rats with extended access to cocaine. Striatal overexpression of miR-212 decreases, whereas its inhibition increases cocaine intake in rats with extended but not restricted drug access, suggesting that miR-212 serves as a protective factor against the development of compulsive drug seeking. The transcription factor CREB (cAMP response element-binding protein) is considered a core regulator of cocaine reward. We show that miR-212 controls responsiveness to cocaine by dramatically amplifying striatal CREB signaling. This action occurs through miR-212-enhanced Raf-1 activity, resulting in adenylyl cyclase sensitization and increased expression of the essential CREB co-activator TORC (Transducer of Regulated CREB; also known as CRTC). Our findings suggest that striatal miR-212 signaling plays a key role in vulnerability to addiction, and that noncoding RNAs such as the miRNAs may serve as novel targets for the development of anti-addiction therapeutics. To identify potential targets for miR-212, we transfected cells with a vector to overexpress miR-212 or an empty vector. We then analyzed profiled gene expression using Affymetrix arrays. Human Affymetrix Study: Cells were transfected with a vector to overexpress miR-212 or an empty vector. We then analyzed profiled gene expression using Affymetrix arrays. The complete, unfiltered set of signal intensity data for Samples miR-212 and pMIF, including miR-212/pMIF ratios, are in the supplementary file at the foot of the record. Probes excluded from analysis (pMIF intensity <100) are indicated in the supplementary file by a no in the far-right column, 'Passed data filtering and included in analysis: no/yes'. Probes that were included in the analyses (pMIF intensity >100) are indicated in the supplementary file by a yes in the 'Passed data filtering and included in analysis: no/yes' column. Rat miRNA Study: Rats were permitted access to intravenous cocaine self-administration (0.5 mg/kg/infusion) for 7 consecutive days during restricted (1-h) or extended (6-h) daily sessions. Control rats remained coaine-naïve throughout. 24-h after the last session the dorsal striatum from each rats was removed, and RNA extracted for microarray profiling. There was a total of n=6 rats per access condition. RNA from 2 rats per access condition were pooled on to arrays, for a total of 3 arrays per access condition, and 9 arrays in total.
Project description:Argonautes, a family of highly evolutionarily conserved proteins, are the central platform for small RNA-mediated gene regulatory mechanisms which occur mainly in the cytoplasm. To understand a potential role of Argonaute 1 (Ago1) protein in the nucleus of mammalian cells in regulating gene transcription and epigenetics, we performed integrated analyses of Ago1 ChIP-sequencing (GSE40536) and gene expression profiling in cells depleted of Ago1. For gene expression profiling, we knocked down the expression of Ago1 by siRNA in PC-3 cells and compared gene expression profiles in the cells depleted of Ago1 and cells receiving control treatments. We found that Ago1 depletion resulted in more downregulated genes which were enriched in gene responsible for promoting cell cycle progression, DNA replication and repair, and response to endogenous stimuli. Integrated analyses of Ago1-chromosomal interactions and gene expression changes in response to Ago1 depletion reveal a significant correlation between Ago1-bound genes and genes altered by Ago1 depletion. These genes are significantly enriched in cancer-related pathways. Our data suggests a nuclear function of Ago1 in regulating gene transcription. PC-3 cells were mock transfected or transfected with an Ago1 specific siRNA or a control siRNA. Each treatment was done in duplicates.
Project description:Mononucleotide A and T repeats are abundant in human genome. Many of A repeats are bound by Argonaute proteins (AGOs). To evaluate the role of AGOs and A repeats in gene regulation, HEK293 cells were treated with 8-amino-3,6-dioxaoctanoic acid added peptide nucleic acid (PNA) AAAAAAAAAAAAAAA oligo (OO-A(15)). Matched two sets of two individual HEK293 cells were transfected with OO-A(15) and scramble OO-PNA oligo, and were analyzed as biological duplicates. Cells were grown in DMEM (Gibco-BRL) according to the manufacturer’s protocol. OO-A(15) and scramble oligo were dilute with dH2O to reach 10μM. Cells were transfected in 6-well plates, seed 6 x 105 cells per well with 50 nM OO-A(15) and scramble oligo using TransIT-siQUEST transfection reagent (Mirus). The transfected cell lines were cultured for 48 h post-transfection and harvested total RNA by Trizol reagent (Invitrogen). All RNA integrity assays were performed and hybridized on beadchip according to the protocol.
Project description:Transcription profiling of Neuropeptide S Receptor 1-A (NPSR1-A) and Neuropeptide S Receptor 1-B (NPSR1-B) transfected human epithelial kidney cells stimulated with neuropeptide S to identify downstream targets.
Project description:Toll-like receptor 4 (TLR-4) is a transmembrane protein. Its activation leads to NF-kB signaling pathway and proinflammatory cytokines’ production that are responsible for activating innate immunity system. Here, we design a co-immunoprecipitation based cross-linking proteomics approach to reveal the TLR-4 interactome upon treatment of lipopolysaccharides and statin drug in HA-TLR-4 transfected HEK293 cells. A total of 712 differentially expressed proteins were identified and quantified in this study. Selected candidate proteins, macrophage myristoylated alanine-rich C kinase substrate, and creatine kinase, exclusively identified in the treatment of statin along with cross-linkers, were further validated by immunoblotting.