Project description:The upstream stimulating factors (USFs) USF1 and USF2 are ubiquitously expressed transcription factors characterized by a conserved basic helix-loop-helix leucine zipper DNA-binding domain. They form homo- or heterodimers and recognize E-box motifs to modulate gene expression. They are known to regulate diverse cellular functions including the cell cycle, immune response and glucose-lipid metabolism, but their roles in neuronal cells remain to be clarified. Here, we performed chromatin immunoprecipitation of USF1 from mouse brain cortex preparations. Subsequent promoter array analysis (ChIP-chip) indicated that USF1 exclusively bound to the CACGTG E-box motifs in the proximal promoter regions. Importantly, functional annotation of the USF1-binding targets revealed an enrichment of genes related to lysosomal functions. Gene expression arrays using a neuronal cell line subsequently revealed that knockdown of USFs deregulated lysosomal gene expression. Altered expression was validated by quantitative RT-PCR, supporting the conclusion that USFs regulate lysosomal gene expression. Furthermore, USFs knockdown slightly increased LysoTracker staining, implying a role for USFs in modulating lysosomal homeostasis. Together, our comprehensive, genome-scale analyses identified lysosomal genes as targets of USFs in neuronal cells, suggesting a potential additional pathway of lysosomal regulation. Gene expression profiling of neuro2a cells knocking down USFs. To screen USFs-downstream genes, mouse neuro2a cells were transfected with vectors for USFs miR RNAi or an empty vector, and then subjected to microarray analysis using Affymetrix Mouse Gene 1.0 ST Array.
Project description:By using high-density DNA microarrays, we analyzed the gene-expression profile of Hodgkin's lymphoma cell line L-428 after knock-down of the tumor antigen PRAME (preferentially expressed antigen in melanoma) Kewitz and Staege, submitted RNA was extracted from established Hodgkin's lymphoma cell line L-540 and hybridized with Affymetrix HG_U133A microarrays.
Project description:By using high-density DNA microarrays, we analyzed the gene-expression profile of Hodgkin's lymphoma cell line L-428 after knock-down of DUSP5 (dual specificity phosphatase 5) RNA was extracted from established Hodgkin's lymphoma cell line L-428 and hybridized with Affymetrix HG_U133A microarrays.
Project description:For this study we selected a gene, α-synuclein (SNCA), that is consistently under-expressed in MCF7 cells and breast tumors. Following transfection with an SNCA expression construct, two stable MCF7 clones (named MCF7-SNCA #1 and 2) were selected and examined for expression differences relative to the parental MCF7 cells. MCF7 cells were transfected with an expression vector containing the full-length SNCA sequence. After selection with blasticidin, two different clones were isolated and examined for expression differences relative to the parental MCF7 cells.
Project description:We examined the function of miR-150 in T-cell lymphomagenesis. We transfected GFP-control or GFP-miR-150 into several T-cell lymphoma lines and sought which genes were regulated by miR-150. To examine the function of miR-150 in T-cell lymphomagenesis, we first transfected GFP-control (Mock) or GFP-miR-150 into several T-cell lymphoma lines (ATN-1, HUT78, My-La, and HH). To detect genes commonly downregulated among these cells, we used the CodeLinkTM Human Whole Genome Bioarray platform with the criterion that the miR-150/GFP-control ratio must be <0.75 in all 3 CTCL lines.
Project description:MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. Previously, we demonstrated that miR-210 is a key player of endothelial cell (EC) response to hypoxia, modulating EC survival, migration and ability to form capillary like-structures. Moreover, the receptor tyrosine kinase ligand Ephrin-A3 was identified as one functionally relevant target. Since each miRNA regulates hundreds of mRNAs, different approaches were combined to identify new miR-210 targets: a Using target prediction software, 32 new miR-210 potential targets were identified. b The proteomic profiling of miR-210 over-expressing ECs identified 11 proteins that were specifically inhibited by miR-210, either directly or indirectly. c Affymetrix based gene expression profiles identified 51 genes that were both down-modulated by miR-210 over-expression and de-repressed when miR-210 was blocked. Surprisingly, only few genes identified either by proteomics or transcriptomics were recognized as miR-210 targets by target prediction algorithms. However, a low-stringency pairing research revealed enrichment for miR-210 putative binding sites, raising the possibility that these genes were targeted via non-canonical recognition sequences. To clarify this issue, miR-210-loaded RISC was purified by immuno-precipitation along with its mRNA targets. The presence of Ephrin-A3 mRNA in the complex validated this approach. We found that 32 potential targets were indeed enriched in miR-210-loaded RISC, and thus can be considered as genuine miR-210 targets. In keeping with this conclusion, we were able to further validate a sub-set of them by 3’UTR-reporter assays. Gene ontology analysis of the targets confirmed the known miR-210 activity in differentiation and cell cycle regulation, highlighting new functions such as involvement in RNA processing, DNA binding, development, membrane trafficking and amino acid catabolism. In conclusion, we validated a multidisciplinary approach for miRNAs target identification and indicated novel molecular mechanisms underpinning miR-210 role in EC response to hypoxia. Experiment Overall Design: Gene expression modifications induced by both miR-210 over-expression and blockade were evaluated. In order to identify new direct and indirect miR-210 targets, transcripts repressed by miR-210 over-expression and up-regulated by miR-210 inhibition (and vice versa) were selected.
Project description:B-cell acute lymphoblastic leukemia (B-ALL) is often associated with chromosomal translocations leading to the deregulation of proto-oncogenes. MicroRNAs can also be affected by chromosomal alterations and thus contribute to carcinogenesis. The microRNA miR-125b-1 is over-expressed in B-ALL cases with the t(11;14)(q24;q34) translocation, therefore we sought to determine the role of this microRNA in B-cell fate. We used murine pre-BI cells alongside murine and human leukemic B-cell lines to show that miR-125b expression enhances proliferation by targeting Bright/ARID3a, an activator of immunoglobulin heavy-chain transcription. Accordingly, this target gene was down-regulated in B-ALL patients with the t(11;14)(q24;q34) translocation. Repression of Bright/ARID3A blocked differentiation and conferred a survival advantage to Ba/F3 cells under IL3 starvation. In addition, over-expression of miR-125b protected pre-BI and leukemic B-cell lines from apoptosis through blockade of caspase activation via a mechanism that was independent of p53 and BAK1. In summary, miR-125b can act as an oncogene in B-ALL by targeting ARID3a and mediating its repression, thus leading to a blockage in differentiation, increased proliferation and inhibition of apoptosis. To identify specific targets of mir-125b, we overexpressed miR-125b as well as a control miRNA in 70Z/3 and 18-81 pre-B leukaemia cells. Chemically synthesized miRNA duplexes, called pre-miR-125b and miR-Neg, were purchased from Ambion. The cells were transfected with pre-miRNA at a final concentration of 10 nM using Lipofectamin RNAi MAX (Invitrogen) according to the manufacturer’s instructions. The medium was replaced 8 hours after transfection. RNA samples were harvested at 48 hours post-transfection. 2 independent experiments were carried out for a total of 8 samples labeled with Cy3 dye (one color design).
Project description:MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. miR-210 can be considered a master miRNA of hypoxic response and is currently regarded as a promising novel non-invasive tumor hypoxia marker. The targets identified to date indicate that miR-210 plays a role in cell cycle regulation, differentiation, mitochondrial metabolism repression, DNA repair and apoptosis. In order to identify miRNAs sub-sequentely modulated by miR-210, miRNA expression profiles of human umbilical vein endothelial cells (HUVEC) over-expressing miR-210 were generated, allowing the identification of miRNAs modulated upon miR-210 up-regulation. HUVEC over-expressing pre-miR-210 or a scramble sequence were generated by retroviral infection, yielding a selected population that expressed mature miR-210 levels comparable with those observed in hypoxic cells. miRNA expression profiles were then measured and miRNAs modulated upon miR-210 up-regulation were identified. This Sample represents four hybridizations - one of which was a dye-swap.
Project description:In this study, we analyzed the DNA methylation levels of 4799 IAP LTRs in three murine cell types: AB2.2 ES cells, somatic cells and a neuroblastoma cell line Neuro2A. According to the results, half of the IAP LTR retrotransposons show constant methylation patterns between the three cell types whereas the remaining half display variable levels of methylation. About half of the variably methylated IAP LTRs tend to be hypomethylated in ES cells, and nearly all of this group are hypomethylated in Neuro2A cells. Interestingly, the observed hypomethylation in both cell types occur in a non-uniform, locus-specific manner and to various degrees of severity, with some of them being easily detectible by COBRA. Overall, this study demonstrates the feasibility of HT-TREBS to study alterations in DNA methylation at retrotransposons in a locus-specific manner in multiple cell types and further suggests the potential utility of this technique in developing epigenetic biomarkers for tracking disease progression. HT-TREBS has been used with the Ion Torrent PGM platform to analyze the DNA methylation of 4799 IAP LTRs in a locus-specific manner in 3 cell types: somatic cells (previously submitted under GEO Accession GSE49222), AB2.2 ES cells and Neuro2A cells
Project description:Understanding the function of individual miRNA species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA ‘Tough Decoys’ (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuD depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122—but not anti-let-7—TuD reduced serum cholesterol by 40% for 18 weeks in wild-type mice and reduced serum LDL by 50% in LDL receptor-deficient mice. High throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNA, but no other miRNAs, were depleted and revealed that TuD RNAs induce miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation. Examining the effect of Tough Decoy miRNA inhibitors on miRNA stability and integrity