Genome-wide analyses in neuronal cells reveal that USF transcription factors regulate lysosomal gene expression
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Tomoyuki Yamanaka
PROVIDER: E-GEOD-76617 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
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.
2016-01-08 | E-GEOD-76615 | biostudies-arrayexpress
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.
2012-12-21 | E-GEOD-40988 | biostudies-arrayexpress
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.
2014-07-01 | E-GEOD-52831 | biostudies-arrayexpress
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.
2012-06-30 | E-GEOD-31180 | biostudies-arrayexpress
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.
2013-07-30 | E-GEOD-49308 | biostudies-arrayexpress
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. ChIP-chip analysis of USF1 and NF-Y in mouse brain cortex. To identify downstream targets of USF1 and NF-Y in neuronal cells, they were chromatin-immunoprecipitated from mouse brain cortical lysates. The obtained chromatin DNAs were labeled with biotin and hybridized on Affymetrix Mouse Promoter 1.0R Array.
2016-01-08 | E-GEOD-76616 | biostudies-arrayexpress
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
2011-12-10 | E-GEOD-25971 | biostudies-arrayexpress
Project description:A common experimental setup to investigate the function of a microRNA is a â??gain-of-functionâ?? approach, in which the microRNA of interest is overexpressed to analyze its impact on a given process. However, a major drawback of this technique is that microRNAs, when expressed significantly above endogenous levels, may target genes that are not affected under physiological conditions. To circumvent this problem, we have generated a retroviral microRNA-knockdown library encompassing the majority of the microRNA families expressed throughout lymphocyte development. MicroRNA knockdown is mediated by sponges, mRNAs that possess multiple binding sites for a certain microRNA family in their 3â??-UTR region. MiRNA sponges function as competitive inhibitors that sequester all specific microRNA/RISC complexes, thereby de-repressing the endogenous target genes. Using this library in pre-B cells as a model system, we have characterized all microRNAs expressed in early B cells development according to their impact on processes such as apoptosis and differentiation. In doing so, we show that functional knockdown of the known tumor-suppressive miR-15 family has only slight effects under cellular steady-state conditions. Upon withdrawal of the growth factor IL-7, however, loss of miR-15 function almost completely inhibits pre-B to immature B cell differentiation, partially protects from apoptosis and enables prolonged proliferation. In correspondence with that, knockdown of the miR-15 family confers a competitive advantage in suboptimal IL-7 concentrations, indicating that levels of miR-15 determine the sensitivity of cells towards growth factor receptor signaling. Moreover, IL-7 withdrawal seems to increase the activity of endogenous miR-15 family members in pre-B cells. Using microarray analysis, we have identified cyclin E1 as a direct and cyclin D3 as an indirect putative target gene of the miR-15 family in B cell precursors. Exogenous expression of cyclin E1 or cyclin D3 recapitulates the miR-15 loss-of-function phenotype in B cell precursors, validating their biological importance. Together, this suggests that the miR-15 family functions as a central element in a positive feedback loop that reinforces cell-cycle arrest at low growth factor concentrations, which is considered a prerequisite for light chain gene recombination and differentiation. Gene expression in cells expressing a scrambled sponge as a control vector or a sponge designed to sequester all members of the miR-15 family (miR-15a, miR-15b, miR-16, miR-497, miR-195, miR-322) was measured either in the presence or 36 hours after withdrawal of the growth factor IL-7. Two independent experiments were performed, using independently generated cell clones for each repetition.
2016-07-03 | E-GEOD-72767 | biostudies-arrayexpress
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 3âUTR-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.
2009-11-07 | E-GEOD-16962 | biostudies-arrayexpress
Project description:Gene expression was measured in human KPL-1 breast cancer cell lines, both with and without knock-down of RAI2 by shRNA. Measurements of each condition were done in triplicates.
2021-01-31 | E-MTAB-7071 | biostudies-arrayexpress