62 research outputs found

    Enhanced immunoprecipitation techniques for the identification of RNA-binding protein partners: IGF2BP1 interactions in mammary epithelial cells

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    RNA-binding proteins (RBPs) regulate the expression of large cohorts of RNA species to produce programmatic changes in cellular phenotypes. To describe the function of RBPs within a cell, it is key to identify their mRNA-binding partners. This is often done by crosslinking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, which involves complex processing with attendant sample losses, thus large amounts of starting materials and prone to artifacts. To evaluate potential alternative technologies, we tested exclusion-based purification of immunoprecipitates (IFAST or SLIDE) and report here that these methods can efficiently, rapidly, and specifically isolate RBP-RNA complexes. The analysis requires less than 1% of the starting material required for techniques that include crosslinking. Depending on the antibody used, 50% to 100% starting protein can be retrieved, facilitating the assay of endogenous levels of RBPs; the isolated ribonucleoproteins are subsequently analyzed using standard techniques, to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA-binding partners for RBP IGF2BP1 in cultured mammary epithelial cells are enriched in mRNAs important for detoxifying superoxides (specifically glutathione peroxidase [GPX]-1 and GPX-2) and mRNAs encoding mitochondrial proteins. We show that these interactions are functionally significant, as loss of function of IGF2BP1 leads to destabilization of GPX mRNAs and reduces mitochondrial membrane potential and oxygen consumption. We speculate that this underlies a consistent requirement for IGF2BP1 for the expression of clonogenic activity in vitro

    A systems approach identifies Enhancer of Zeste Homolog 2 (EZH2) as a protective factor in epilepsy.

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    Complex neurological conditions can give rise to large scale transcriptomic changes that drive disease progression. It is likely that alterations in one or a few transcription factors or cofactors underlie these transcriptomic alterations. Identifying the driving transcription factors/cofactors is a non-trivial problem and a limiting step in the understanding of neurological disorders. Epilepsy has a prevalence of 1% and is the fourth most common neurological disorder. While a number of anti-seizure drugs exist to treat seizures symptomatically, none is curative or preventive. This reflects a lack of understanding of disease progression. We used a novel systems approach to mine transcriptome profiles of rodent and human epileptic brain samples to identify regulators of transcriptional networks in the epileptic brain. We find that Enhancer of Zeste Homolog 2 (EZH2) regulates differentially expressed genes in epilepsy across multiple rodent models of acquired epilepsy. EZH2 undergoes a prolonged upregulation in the epileptic brain. A transient inhibition of EZH2 immediately after status epilepticus (SE) robustly increases spontaneous seizure burden weeks later. This suggests that EZH2 upregulation is a protective. These findings are the first to characterize a role for EZH2 in opposing epileptogenesis and debut a bioinformatic approach to identify nuclear drivers of complex transcriptional changes in disease

    Syndecan-1 Is Required to Maintain Intradermal Fat and Prevent Cold Stress

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    <div><p>Homeostatic temperature regulation is fundamental to mammalian physiology and is controlled by acute and chronic responses of local, endocrine and nervous regulators. Here, we report that loss of the heparan sulfate proteoglycan, syndecan-1, causes a profoundly depleted intradermal fat layer, which provides crucial thermogenic insulation for mammals. Mice without syndecan-1 enter torpor upon fasting and show multiple indicators of cold stress, including activation of the stress checkpoint p38őĪ in brown adipose tissue, liver and lung. The metabolic phenotype in mutant mice, including reduced liver glycogen, is rescued by housing at thermoneutrality, suggesting that reduced insulation in cool temperatures underlies the observed phenotypes. We find that syndecan-1, which functions as a facultative lipoprotein uptake receptor, is required for adipocyte differentiation <i>in vitro</i>. Intradermal fat shows highly dynamic differentiation, continuously expanding and involuting in response to hair cycle and ambient temperature. This physiology probably confers a unique role for Sdc1 in this adipocyte sub-type. The PPARő≥ agonist rosiglitazone rescues <i>Sdc1‚ąí/‚ąí</i> intradermal adipose tissue, placing PPARő≥ downstream of Sdc1 in triggering adipocyte differentiation. Our study indicates that disruption of intradermal adipose tissue development results in cold stress and complex metabolic pathology.</p></div

    Tumor Suppressive Function of mir-205 in Breast Cancer Is Linked to HMGB3 Regulation

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    <div><p>Identifying targets of dysregulated microRNAs (miRNAs) will enhance our understanding of how altered miRNA expression contributes to the malignant phenotype of breast cancer. The expression of miR-205 was reduced in four breast cancer cell lines compared to the normal-like epithelial cell line MCF10A and in tumor and metastatic tissues compared to adjacent benign breast tissue. Two predicted binding sites for miR-205 were identified in the 3’ untranslated region of the high mobility group box 3 gene, HMGB3. Both dual-luciferase reporter assay and Western blotting confirmed that miR-205 binds to and regulates HMGB3. To further explore miR-205 targeting of HMGB3, WST-1 proliferation and in vitro invasion assays were performed in MDA-MB-231 and BT549 cells transiently transfected with precursor miR-205 oligonucleotide or HMGB3 small interfering RNA (siRNA). Both treatments reduced the proliferation and invasion of the cancer cells. The mRNA and protein levels of HMGB3 were higher in the tumor compared to adjacent benign specimens and there was an indirect correlation between the expression of HMGB3 mRNA and patient survival. Treatment of breast cancer cells with 5-Aza/TSA derepressed miR-205 and reduced HMGB3 mRNA while knockdown of the transcriptional repressor NRSF/REST, reduced miR-205 and increased HMGB3. In conclusion, regulation of HMGB3 by miR-205 reduced both proliferation and invasion of breast cancer cells. Our findings suggest that modulating miR-205 and/or targeting HMGB3 are potential therapies for advanced breast cancer.</p> </div

    Reduced Juvenile Long-Term Depression in Tuberous Sclerosis Complex Is Mitigated in Adults by Compensatory Recruitment of mGluR5 and Erk Signaling

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    <div><p>Tuberous sclerosis complex (TSC) is a multisystem genetic disease that manifests with mental retardation, tumor formation, autism, and epilepsy. Heightened signaling through the mammalian target of rapamycin (mTOR) pathway is involved in TSC pathology, however it remains unclear how other signaling pathways are perturbed and contribute to disease symptoms. Reduced long-term depression (LTD) was recently reported in TSC mutant mice. We find that although reduced LTD is a feature of the juvenile mutant hippocampus, heightened expression of metabotropic glutamate receptor 5 and constitutively activated Erk signaling in the adult hippocampus drives wild-type levels of LTD. Increased mGluR5 and Erk results in a novel mTOR-independent LTD in CA1 hippocampus of adult mice, and contributes to the development of epileptiform bursting activity in the TSC2<sup>+/‚ąí</sup> CA3 region of the hippocampus. Inhibition of mGluR5 or Erk signaling restores appropriate mTOR-dependence to LTD, and significantly reduces epileptiform bursting in TSC2<sup>+/‚ąí</sup> hippocampal slices. We also report that adult TSC2<sup>+/‚ąí</sup> mice exhibit a subtle perseverative behavioral phenotype that is eliminated by mGluR5 antagonism. These findings highlight the potential of modulating the mGluR5-Erk pathway in a developmental stage-specific manner to treat TSC.</p></div

    Endogenous Fluorescence Signatures in Living Pluripotent Stem Cells Change with Loss of Potency

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    <div><p>The therapeutic potential of stem cells is limited by the non-uniformity of their phenotypic state. Thus it would be advantageous to noninvasively monitor stem cell status. Driven by this challenge, we employed multidimensional multiphoton microscopy to quantify changes in endogenous fluorescence occurring with pluripotent stem cell differentiation. We found that global and cellular-scale fluorescence lifetime of human embryonic stem cells (hESC) and murine embryonic stem cells (mESC) consistently decreased with differentiation. Less consistent were trends in endogenous fluorescence intensity with differentiation, suggesting intensity is more readily impacted by nuances of species and scale of analysis. What emerges is a practical and accessible approach to evaluate, and ultimately enrich, living stem cell populations based on changes in metabolism that could be exploited for both research and clinical applications.</p> </div

    A REST derived gene signature stratifies glioblastomas into chemotherapy resistant and responsive disease

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    <p>Abstract</p> <p>Background</p> <p>Glioblastomas are the most common central nervous system neoplasia in adults, with 9,000 cases in the US annually. Glioblastoma multiformae, the most aggressive glioma subtype, has an 18% one-year survival rate, and 3% two year survival rate. Recent work has highlighted the role of the transcription factor RE1 Silencing Transcription Factor, REST in glioblastoma but how REST function correlates with disease outcome has not been described.</p> <p>Method</p> <p>Using a bioinformatic approach and mining of publicly available microarray datasets, we describe an aggressive subtype of gliomas defined by a gene signature derived from REST. Using this REST gene signature we predict that REST function is enhanced in advanced glioblastoma. We compare disease outcomes between tumors based on REST status and treatment regimen, and describe downstream targets of REST that may contribute to the decreased benefits observed with high dose chemotherapy in REM tumors.</p> <p>Results</p> <p>We present human data showing that patients with ‚ÄúREST Enhanced Malignancies‚ÄĚ (REM) tumors present with a shorter disease free survival compared to non-REM gliomas. Importantly, REM tumors are refractory to multiple rounds of chemotherapy and patients fail to respond to this line of treatment.</p> <p>Conclusions</p> <p>This report is the first to describe a REST gene signature that predicts response to multiple rounds of chemotherapy, the mainline therapy for this disease. The REST gene signature may have important clinical implications for the treatment of glioblastoma.</p
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