167 research outputs found

    Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection

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    Background: Respiratory syncytial virus (RSV) is the major viral driver of a global pediatric respiratory disease burden disproportionately borne by the poor1. Thus, RSV, like SARS-CoV-2, combines with congenital and environmental and host-history-dependent factors to create a spectrum of disease with greatest severity most frequently occurring in those least able to procure treatment. Methods: Here we apply whole genome sequencing and a suite of other molecular biological techniques to survey host-virus dynamics in infections of two distinct cell lines (HEp2 and A549) with four strains representative of known RSV genetic diversity. Results: We observed non-gradient patterns of RSV gene expression and a single major difference in transcriptional readthrough correlating with a deep split in the RSV phylogenetic tree. We also observed increased viral replication in HEp2 cells along with a pro-inflammatory host-response; and decreased viral replication in A549 cells with a more potent antiviral response in host gene expression and levels of secreted cytokines. Conclusions: Our findings suggest HEp2 and A549 cell lines can be used as complementary models of host response leading to more or less severe RSV disease. In vitro perturbations inspired by actual environmental and host-history-dependent factors associated with greater disease can be tested for their ability to shift the antiviral response of A549 cells to the more pro-inflammatory response of HEp2 cells. Such studies would help illuminate the tragic costs of poverty and suggest public health-level interventions to reduce the global disease burden from RSV and other respiratory viruses

    DNMT3A-coordinated splicing governs the stem state switch towards differentiation in embryonic and haematopoietic stem cells

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    Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3 alpha (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation.Ramabadran et al. identify a DNA methylation-independent role for DNMT3A in stem cell activation, mediated through recruitment of SF3B1 and splicing regulation

    Imaging-Based Screening of Deubiquitinating Proteases Identifies Otubain-1 as a Stabilizer of c-MYC

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    The ubiquitin–proteasome pathway precisely controls the turnover of transcription factors in the nucleus, playing an important role in maintaining appropriate quantities of these regulatory proteins. The transcription factor c-MYC is essential for normal development and is a critical cancer driver. Despite being highly expressed in several tissues and malignancies, the c-MYC protein is also continuously targeted by the ubiquitin–proteasome pathway, which can either facilitate or inhibit c-MYC degradation. Deubiquitinating proteases can remove ubiquitin chains from target proteins and rescue them from proteasomal digestion. This study sought to determine novel elements of the ubiquitin–proteasome pathway that regulate c-MYC levels. We performed an overexpression screen with 41 human proteases to identify which deubiquitinases stabilize c-MYC. We discovered that the highly expressed Otubain-1 (OTUB1) protease increases c-MYC protein levels. Confirming its role in enhancing c-MYC activity, we found that elevated OTUB1 correlates with inferior clinical outcomes in the c-MYC-dependent cancer multiple myeloma, and overexpression of OTUB1 accelerates the growth of myeloma cells. In summary, our study identifies OTUB1 as a novel amplifier of the proto-oncogene c-MYC

    Enhancer RNA m6A methylation facilitates transcriptional condensate formation and gene activation

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    The mechanistic understanding of nascent RNAs in transcriptional control remains limited. Here, by a high sensitivity method methylation-inscribed nascent transcripts sequencing (MINT-seq), we characterized the landscapes of N6-methyladenosine (m6A) on nascent RNAs. We uncover heavy but selective m6A deposition on nascent RNAs produced by transcription regulatory elements, including promoter upstream antisense RNAs and enhancer RNAs (eRNAs), which positively correlates with their length, inclusion of m6A motif, and RNA abundances. m6A-eRNAs mark highly active enhancers, where they recruit nuclear m6A reader YTHDC1 to phase separate into liquid-like condensates, in a manner dependent on its C terminus intrinsically disordered region and arginine residues. The m6A-eRNA/YTHDC1 condensate co-mixes with and facilitates the formation of BRD4 coactivator condensate. Consequently, YTHDC1 depletion diminished BRD4 condensate and its recruitment to enhancers, resulting in inhibited enhancer and gene activation. We propose that chemical modifications of eRNAs together with reader proteins play broad roles in enhancer activation and gene transcriptional control

    Targeted brachyury degradation disrupts a highly specific autoregulatory program controlling chordoma cell identity

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    © 2020 The Authors Sheppard et al. map the brachyury regulatory landscape in chordoma and explore its targeting using transcriptional CDK inhibition and targeted brachyury degradation. Brachyury is a highly selective transcriptional regulator of chordoma identity, and they confirm that brachyury targeting is a promising therapeutic strategy

    Classification of estrogenic compounds by coupling high content analysis and machine learning algorithms.

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    Environmental toxicants affect human health in various ways. Of the thousands of chemicals present in the environment, those with adverse effects on the endocrine system are referred to as endocrine-disrupting chemicals (EDCs). Here, we focused on a subclass of EDCs that impacts the estrogen receptor (ER), a pivotal transcriptional regulator in health and disease. Estrogenic activity of compounds can be measured by many in vitro or cell-based high throughput assays that record various endpoints from large pools of cells, and increasingly at the single-cell level. To simultaneously capture multiple mechanistic ER endpoints in individual cells that are affected by EDCs, we previously developed a sensitive high throughput/high content imaging assay that is based upon a stable cell line harboring a visible multicopy ER responsive transcription unit and expressing a green fluorescent protein (GFP) fusion of ER. High content analysis generates voluminous multiplex data comprised of minable features that describe numerous mechanistic endpoints. In this study, we present a machine learning pipeline for rapid, accurate, and sensitive assessment of the endocrine-disrupting potential of benchmark chemicals based on data generated from high content analysis. The multidimensional imaging data was used to train a classification model to ultimately predict the impact of unknown compounds on the ER, either as agonists or antagonists. To this end, both linear logistic regression and nonlinear Random Forest classifiers were benchmarked and evaluated for predicting the estrogenic activity of unknown compounds. Furthermore, through feature selection, data visualization, and model discrimination, the most informative features were identified for the classification of ER agonists/antagonists. The results of this data-driven study showed that highly accurate and generalized classification models with a minimum number of features can be constructed without loss of generality, where these machine learning models serve as a means for rapid mechanistic/phenotypic evaluation of the estrogenic potential of many chemicals

    Tributyltin chloride (TBT) induces RXRA down-regulation and lipid accumulation in human liver cells.

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    A subset of environmental chemicals acts as "obesogens" as they increase adipose mass and lipid content in livers of treated rodents. One of the most studied class of obesogens are the tin-containing chemicals that have as a central moiety tributyltin (TBT), which bind and activate two nuclear hormone receptors, Peroxisome Proliferator Activated Receptor Gamma (PPARG) and Retinoid X Receptor Alpha (RXRA), at nanomolar concentrations. Here, we have tested whether TBT chloride at such concentrations may affect the neutral lipid level in two cell line models of human liver. Indeed, using high content image analysis (HCA), TBT significantly increased neutral lipid content in a time- and concentration-dependent manner. Consistent with the observed increased lipid accumulation, RNA fluorescence in situ hybridization (RNA FISH) and RT-qPCR experiments revealed that TBT enhanced the steady-state mRNA levels of two key genes for de novo lipogenesis, the transcription factor SREBF1 and its downstream enzymatic target, FASN. Importantly, pre-treatment of cells with 2-deoxy-D-glucose reduced TBT-mediated lipid accumulation, thereby suggesting a role for active glycolysis during the process of lipid accumulation. As other RXRA binding ligands can promote RXRA protein turnover via the 26S proteasome, TBT was tested for such an effect in the two liver cell lines. We found that TBT, in a time- and dose-dependent manner, significantly reduced steady-state RXRA levels in a proteasome-dependent manner. While TBT promotes both RXRA protein turnover and lipid accumulation, we found no correlation between these two events at the single cell level, thereby suggesting an additional mechanism may be involved in TBT promotion of lipid accumulation, such as glycolysis

    HDAC 阻害剤は Diethylstilbestrol による性腺刺激ホルモン細胞からのプロラクチン細胞への分化転換を抑制する

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    Diethylstilbestrol (DES), an estrogen agonist, increases prolactin (PRL) cells through transdifferentiation of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) cells to PRL cells as well as proliferation of PRL cells in adult male mouse pituitary. Since hyperacetylation of histone H3 is implicated in the regulation of activation of various genes, we examined the effect of DES on the state of histone H3 acetylation. DES significantly reduced the immunohistochemical signal for acetylated histone H3 at lysine 9 (H3K9ac) in PRL, LH and FSH cells, but not for H3K18ac or H3K23ac. DES-treated mice were injected intraperitoneally with HDAC inhibitors (HDACi), sodium phenylbutyrate (NaPB) or valproic acid (VPA), to mimic the acetylation level of histone H3. As expected, HDACi treatment restored the level of H3K9ac expression in these cells, and also inhibited DES-induced increase in PRL cells. Furthermore, NaPB and VPA also abrogated the effects of DES on the population density of both LH and FSH cells. Similarly, the numbers of proliferating and apoptotic cells in the pituitary in NaPB- or VPA-treated mice were comparable to those of the control mice. Considered together, these results indicated that the acetylation level of histone H3 plays an important role in DES-induced transdifferentiation of LH to PRL cells as well as proliferation of PRL cells.長崎大学学位論文 学位記番号:博(医歯薬)甲第1128号 学位授与年月日:平成31年3月20日Author: Nandar Tun, Yasuaki Shibata, Myat Thu Soe, Myo Win Htun, Takehiko KojiCitation: Histochemistry and Cell Biology, 151(4), pp.291-303; 2018Nagasaki University (長崎大学)課程博

    Steroid Receptor Coactivator-2 Controls the Pentose Phosphate Pathway through RPIA in Human Endometrial Cancer Cells

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    Abstract Steroid receptor coactivator-2 (SRC-2) is a transcriptional coregulator that modulates the activity of many transcription factors. Levels of SRC-2 are elevated in endometrial biopsies from polycystic ovary syndrome patients, a population predisposed to endometrial cancer (EC). Increased expression of SRC-2 is also detected in neoplastic endometrium suggesting a causal link between elevated SRC-2 expression and the emergence of endometrial disorders that can lead to cancer. Here, we reveal that SRC-2 knockdown reduces EC cell proliferation and anchorage-independence. Additionally, SRC-2 is required to maintain cellular glycolytic capacity and oxidative phosphorylation, processes essential for EC cell proliferation. Importantly, SRC-2 is critical for the normal performance of the pentose phosphate pathway (PPP). Perturbation of the PPP due to loss of SRC-2 expression may result from the depletion of ribose-5-P isomerase (RPIA), a key enzyme of the PPP. As with SRC-2, RPIA knockdown reduces EC cell proliferation, which is accompanied by a decrease in glycolytic capacity and oxidative phosphorylation. Glucose metabolite tracking experiments confirmed that knockdown of SRC-2 and RPIA downregulates the metabolic rate of both glycolysis and the PPP, highlighting a novel regulatory cross-talk between glycolysis and the PPP modulated by SRC-2