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In JoVE (1)
- Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells
Other Publications (7)
Articles by Shelley Force Aldred in JoVE
Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells
Shelley Force Aldred, Patrick Collins, Nathan Trinklein
MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.
Other articles by Shelley Force Aldred on PubMed
Genome Research. Jan, 2004 | Pubmed ID: 14707170
The alignment of full-length human cDNA sequences to the finished sequence of the human genome provides a unique opportunity to study the distribution of genes throughout the genome. By analyzing the distances between 23,752 genes, we identified a class of divergently transcribed gene pairs, representing more than 10% of the genes in the genome, whose transcription start sites are separated by less than 1000 base pairs. Although this bidirectional arrangement has been previously described in humans and other species, the prevalence of bidirectional gene pairs in the human genome is striking, and the mechanisms of regulation of all but a few bidirectional genes are unknown. Our work shows that the transcripts of many bidirectional pairs are coexpressed, but some are antiregulated. Further, we show that many of the promoter segments between two bidirectional genes initiate transcription in both directions and contain shared elements that regulate both genes. We also show that the bidirectional arrangement is often conserved among mouse orthologs. These findings demonstrate that a bidirectional arrangement provides a unique mechanism of regulation for a significant number of mammalian genes.
Polymorphisms Within the C-reactive Protein (CRP) Promoter Region Are Associated with Plasma CRP Levels
American Journal of Human Genetics. Jul, 2005 | Pubmed ID: 15897982
Elevated plasma levels of C-reactive protein (CRP), an inflammation-sensitive marker, have emerged as an important predictor of future cardiovascular disease and metabolic abnormalities in apparently healthy men and women. Here, we performed a systematic survey of common nucleotide variation across the genomic region encompassing the CRP gene locus. Of the common single-nucleotide polymorphisms (SNPs) identified, several in the CRP promoter region are strongly associated with CRP levels in a large cohort study of cardiovascular risk in European American and African American young adults. We also demonstrate the functional importance of these SNPs in vitro.
Integrated Analysis of Experimental Data Sets Reveals Many Novel Promoters in 1% of the Human Genome
Genome Research. Jun, 2007 | Pubmed ID: 17567992
The regulation of transcriptional initiation in the human genome is a critical component of global gene regulation, but a complete catalog of human promoters currently does not exist. In order to identify regulatory regions, we developed four computational methods to integrate 129 sets of ENCODE-wide chromatin immunoprecipitation data. They collectively predicted 1393 regions. Roughly 47% of the regions were unique to one method, as each method makes different assumptions about the data. Overall, predicted regions tend to localize to highly conserved, DNase I hypersensitive, and actively transcribed regions in the genome. Interestingly, a significant portion of the regions overlaps with annotated 3'-UTRs, suggesting that some of them might regulate anti-sense transcription. The majority of the predicted regions are >2 kb away from the 5'-ends of previously annotated human cDNAs and hence are novel. These novel regions may regulate unannotated transcripts or may represent new alternative transcription start sites of known genes. We tested 163 such regions for promoter activity in four cell lines using transient transfection assays, and 25% of them showed transcriptional activity above background in at least one cell line. We also performed 5'-RACE experiments on 62 novel regions, and 76% of the regions were associated with the 5'-ends of at least two RACE products. Our results suggest that there are at least 35% more functional promoters in the human genome than currently annotated.
Identification and Analysis of Functional Elements in 1% of the Human Genome by the ENCODE Pilot Project
Nature. Jun, 2007 | Pubmed ID: 17571346
We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view of chromatin structure has emerged, including its inter-relationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded new mechanistic and evolutionary insights concerning the functional landscape of the human genome. Together, these studies are defining a path for pursuit of a more comprehensive characterization of human genome function.
Functional Profiling Reveals Critical Role for MiRNA in Differentiation of Human Mesenchymal Stem Cells
PloS One. 2009 | Pubmed ID: 19440384
Mesenchymal stem (MS) cells are excellent candidates for cell-based therapeutic strategies to regenerate injured tissue. Although human MS cells can be isolated from bone marrow and directed to differentiate by means of an osteogenic pathway, the regulation of cell-fate determination is not well understood. Recent reports identify critical roles for microRNAs (miRNAs), regulators of gene expression either by inhibiting the translation or by stimulating the degradation of target mRNAs.
Toxicological Sciences : an Official Journal of the Society of Toxicology. Nov, 2009 | Pubmed ID: 19502547
Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1alpha and constitutively expressed HIF-1beta. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia-response elements (HREs) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based beta-lactamase HRE reporter gene assay in a quantitative high-throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, whereas two compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1alpha inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1alpha activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.
Two-tiered Approach Identifies a Network of Cancer and Liver Disease-related Genes Regulated by MiR-122
The Journal of Biological Chemistry. May, 2011 | Pubmed ID: 21402708
MicroRNAs function as important regulators of gene expression and are commonly linked to development, differentiation, and diseases such as cancer. To better understand their roles in various biological processes, identification of genes targeted by microRNAs is necessary. Although prediction tools have significantly helped with this task, experimental approaches are ultimately required for extensive target search and validation. We employed two independent yet complementary high throughput approaches to map a large set of mRNAs regulated by miR-122, a liver-specific microRNA implicated in regulation of fatty acid and cholesterol metabolism, hepatitis C infection, and hepatocellular carcinoma. The combination of luciferase reporter-based screening and shotgun proteomics resulted in the identification of 260 proteins significantly down-regulated in response to miR-122 in at least one method, 113 of which contain predicted miR-122 target sites. These proteins are enriched for functions associated with the cell cycle, differentiation, proliferation, and apoptosis. Among these miR-122-sensitive proteins, we identified a large group with strong connections to liver metabolism, diseases, and hepatocellular carcinoma. Additional analyses, including examination of consensus binding motifs for both miR-122 and target sequences, provide further insight into miR-122 function.