ResearchPad - dna-transcription https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Scedar: A scalable Python package for single-cell RNA-seq exploratory data analysis]]> https://www.researchpad.co/article/elastic_article_13837 In single-cell RNA-seq (scRNA-seq) experiments, the number of individual cells has increased exponentially, and the sequencing depth of each cell has decreased significantly. As a result, analyzing scRNA-seq data requires extensive considerations of program efficiency and method selection. In order to reduce the complexity of scRNA-seq data analysis, we present scedar, a scalable Python package for scRNA-seq exploratory data analysis. The package provides a convenient and reliable interface for performing visualization, imputation of gene dropouts, detection of rare transcriptomic profiles, and clustering on large-scale scRNA-seq datasets. The analytical methods are efficient, and they also do not assume that the data follow certain statistical distributions. The package is extensible and modular, which would facilitate the further development of functionalities for future requirements with the open-source development community. The scedar package is distributed under the terms of the MIT license at https://pypi.org/project/scedar.

]]>
<![CDATA[HSPA6 augments garlic extract-induced inhibition of proliferation, migration, and invasion of bladder cancer EJ cells; Implication for cell cycle dysregulation, signaling pathway alteration, and transcription factor-associated MMP-9 regulation]]> https://www.researchpad.co/article/5989db4fab0ee8fa60bdb9fe

Although recent studies have demonstrated the anti-tumor effects of garlic extract (GE), the exact molecular mechanism is still unclear. In this study, we investigated the molecular mechanism associated with the inhibitory action of GE against bladder cancer EJ cell responses. Treatment with GE significantly inhibited proliferation of EJ cells dose-dependently through G2/M-phase cell cycle arrest. This G2/M-phase cell cycle arrest by GE was due to the activation of ATM and CHK2, which appears to inhibit phosphorylation of Cdc25C (Ser216) and Cdc2 (Thr14/Tyr15), this in turn was accompanied by down-regulation of cyclin B1 and up-regulation of p21WAF1. Furthermore, GE treatment was also found to induce phosphorylation of MAPK (ERK1/2, p38MAPK, and JNK) and AKT. In addition, GE impeded the migration and invasion of EJ cells via inhibition of MMP-9 expression followed by decreased binding activities of AP-1, Sp-1, and NF-κB motifs. Based on microarray datasets, we selected Heat shock protein A6 (HSPA6) as the most up-regulated gene responsible for the inhibitory effects of GE. Interestingly, overexpression of HSPA6 gene resulted in an augmentation effect with GE inhibiting proliferation, migration, and invasion of EJ cells. The augmentation effect of HSPA6 was verified by enhancing the induction of G2/M-phase-mediated ATM-CHK2-Cdc25C-p21WAF1-Cdc2 cascade, phosphorylation of MAPK and AKT signaling, and suppression of transcription factor-associated MMP-9 regulation in response to GE in EJ cells. Overall, our novel results indicate that HSPA6 reinforces the GE-mediated inhibitory effects of proliferation, migration, and invasion of EJ cells and may provide a new approach for therapeutic treatment of malignancies.

]]>
<![CDATA[Age-related transcriptional modules and TF-miRNA-mRNA interactions in neonatal and infant human thymus]]> https://www.researchpad.co/article/Ne5173bb6-5611-4e9c-b8d8-f6fe9062bcd6

The human thymus suffers a transient neonatal involution, recovers and then starts a process of decline between the 1st and 2nd years of life. Age-related morphological changes in thymus were extensively investigated, but the genomic mechanisms underlying this process remain largely unknown. Through Weighted Gene Co-expression Network Analysis (WGCNA) and TF-miRNA-mRNA integrative analysis we studied the transcriptome of neonate and infant thymic tissues grouped by age: 0–30 days (A); 31days-6 months (B); 7–12 months (C); 13–18 months (D); 19-31months (E). Age-related transcriptional modules, hubs and high gene significance (HGS) genes were identified, as well as TF-miRNA-hub/HGS co-expression correlations. Three transcriptional modules were correlated with A and/or E groups. Hubs were mostly related to cellular/metabolic processes; few were differentially expressed (DE) or related to T-cell development. Inversely, HGS genes in groups A and E were mostly DE. In A (neonate) one third of the hyper-expressed HGS genes were related to T-cell development, against one-twentieth in E, what may correlate with the early neonatal depletion and recovery of thymic T-cell populations. This genomic mechanism is tightly regulated by TF-miRNA-hub/HGS interactions that differentially govern cellular and molecular processes involved in the functioning of the neonate thymus and in the beginning of thymic decline.

]]>
<![CDATA[Global transcriptional regulation of innate immunity by ATF-7 in C. elegans]]> https://www.researchpad.co/article/5c784faad5eed0c4840072b2

The nematode Caenorhabditis elegans has emerged as a genetically tractable animal host in which to study evolutionarily conserved mechanisms of innate immune signaling. We previously showed that the PMK-1 p38 mitogen-activated protein kinase (MAPK) pathway regulates innate immunity of C. elegans through phosphorylation of the CREB/ATF bZIP transcription factor, ATF-7. Here, we have undertaken a genomic analysis of the transcriptional response of C. elegans to infection by Pseudomonas aeruginosa, combining genome-wide expression analysis by RNA-seq with ATF-7 chromatin immunoprecipitation followed by sequencing (ChIP-Seq). We observe that PMK-1-ATF-7 activity regulates a majority of all genes induced by pathogen infection, and observe ATF-7 occupancy in regulatory regions of pathogen-induced genes in a PMK-1-dependent manner. Moreover, functional analysis of a subset of these ATF-7-regulated pathogen-induced target genes supports a direct role for this transcriptional response in host defense. The genome-wide regulation through PMK-1– ATF-7 signaling reveals a striking level of control over the innate immune response to infection through a single transcriptional regulator.

]]>
<![CDATA[Mapping DNA sequence to transcription factor binding energy in vivo]]> https://www.researchpad.co/article/5c61e8e5d5eed0c48496f361

Despite the central importance of transcriptional regulation in biology, it has proven difficult to determine the regulatory mechanisms of individual genes, let alone entire gene networks. It is particularly difficult to decipher the biophysical mechanisms of transcriptional regulation in living cells and determine the energetic properties of binding sites for transcription factors and RNA polymerase. In this work, we present a strategy for dissecting transcriptional regulatory sequences using in vivo methods (massively parallel reporter assays) to formulate quantitative models that map a transcription factor binding site’s DNA sequence to transcription factor-DNA binding energy. We use these models to predict the binding energies of transcription factor binding sites to within 1 kBT of their measured values. We further explore how such a sequence-energy mapping relates to the mechanisms of trancriptional regulation in various promoter contexts. Specifically, we show that our models can be used to design specific induction responses, analyze the effects of amino acid mutations on DNA sequence preference, and determine how regulatory context affects a transcription factor’s sequence specificity.

]]>
<![CDATA[Transcription-driven chromatin repression of Intragenic transcription start sites]]> https://www.researchpad.co/article/5c5df308d5eed0c484580b95

Progression of RNA polymerase II (RNAPII) transcription relies on the appropriately positioned activities of elongation factors. The resulting profile of factors and chromatin signatures along transcription units provides a “positional information system” for transcribing RNAPII. Here, we investigate a chromatin-based mechanism that suppresses intragenic initiation of RNAPII transcription. We demonstrate that RNAPII transcription across gene promoters represses their function in plants. This repression is characterized by reduced promoter-specific molecular signatures and increased molecular signatures associated with RNAPII elongation. The conserved FACT histone chaperone complex is required for this repression mechanism. Genome-wide Transcription Start Site (TSS) mapping reveals thousands of discrete intragenic TSS positions in fact mutants, including downstream promoters that initiate alternative transcript isoforms. We find that histone H3 lysine 4 mono-methylation (H3K4me1), an Arabidopsis RNAPII elongation signature, is enriched at FACT-repressed intragenic TSSs. Our analyses suggest that FACT is required to repress intragenic TSSs at positions that are in part characterized by elevated H3K4me1 levels. In sum, conserved and plant-specific chromatin features correlate with the co-transcriptional repression of intragenic TSSs. Our insights into TSS repression by RNAPII transcription promise to inform the regulation of alternative transcript isoforms and the characterization of gene regulation through the act of pervasive transcription across eukaryotic genomes.

]]>
<![CDATA[Transvection-like interchromosomal interaction is not observed at the transcriptional level when tested in the Rosa26 locus in mouse]]> https://www.researchpad.co/article/5c6f1494d5eed0c48467a353

Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a comprehensive view of intra- and interchromosomal contacts between specific genomic sites. The locus control region of the β-globin genes (β-LCR) is a super-enhancer that is capable of activating all of the β-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3’HS1 in the human β-globin locus, in this way thought to contribute to the formation of a “chromatin hub”. The β-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared “transcription factory” for co-transcription. Theoretically, enhancers could also activate target gene promoters at the identical loci, yet on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the β-LCR and the β-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because of a lack of suitable regulatory elements that might facilitate such trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and β-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans, their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the β-globin gene in trans even in this idealized experimental context.

]]>
<![CDATA[Epidermal growth factor receptor inhibition attenuates non-alcoholic fatty liver disease in diet-induced obese mice]]> https://www.researchpad.co/article/5c673077d5eed0c484f37b8e

Non-alcoholic fatty liver disease (NAFLD) is one of the main causes of chronic liver disease. NAFLD begins with excessive lipid accumulation in the liver and progresses to nonalcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is closely linked to dysregulated hepatic lipid metabolism. Although recent studies have reported that epidermal growth factor receptor (EGFR) signaling regulates lipid metabolism, the roles of EGFR and EGFR inhibitors as modulators of lipid metabolism are largely unknown. Here, we investigated whether inhibiting EGFR using the EGFR tyrosine kinase inhibitor (TKI) PD153035 improves NAFLD. Our results demonstrate that EGFR was activated in liver tissues from high fat diet (HFD)-induced NAFLD mice. Inhibiting EGFR using PD153035 significantly reduced phosphatidylinositol-3-kinase/protein kinase B signaling and sterol responsive elementary binding protein 1 and 2 expression, which prevented HFD-induced hepatic steatosis and hypercholesterolemia by reducing de novo lipogenesis and cholesterol synthesis and enhancing fatty acid oxidation. Additionally, inhibiting EGFR improved HFD-induced glucose intolerance. In conclusion, these results indicate that EGFR plays an important role in NAFLD and is a potential therapeutic target.

]]>
<![CDATA[The inhibitor apoptosis protein antagonist Debio 1143 Is an attractive HIV-1 latency reversal candidate]]> https://www.researchpad.co/article/5c61e8bbd5eed0c48496f07d

Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can resupply active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. In this study, we tested efficacy of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 at reversing HIV latency and investigated its mechanisms of action. Debio 1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. Debio 1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. Debio 1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that Debio 1143 activates the non-canonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, Debio 1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that Debio 1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. Debio 1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. Interestingly, daily oral administration of Debio 1143 in cancer patients at well-tolerated doses elicited BIRC2 target engagement in PBMCs and induced a moderate increase in cytokines and chemokines mechanistically related to NF-kB signaling. In conclusion, we provide strong evidences that the IAPa Debio 1143, by initially activating the non-canonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new attractive viral latency reversal agent (LRA).

]]>
<![CDATA[Single-molecule dynamics and genome-wide transcriptomics reveal that NF-kB (p65)-DNA binding times can be decoupled from transcriptional activation]]> https://www.researchpad.co/article/5c4a3091d5eed0c4844c0568

Transcription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an “enhanceosome,” nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.

]]>
<![CDATA[Orf virus (ORFV) infection in a three-dimensional human skin model: Characteristic cellular alterations and interference with keratinocyte differentiation]]> https://www.researchpad.co/article/5c5b523fd5eed0c4842bc547

ORF virus (ORFV) is the causative agent of contagious ecthyma, a pustular dermatitis of small ruminants and humans. Even though the development of lesions caused by ORFV was extensively studied in animals, only limited knowledge exists about the lesion development in human skin. The aim of the present study was to evaluate a three-dimensional (3D) organotypic culture (OTC) as a human skin model for ORFV infection considering lesion development, replication of the virus, viral gene transcription and modulation of differentiation of human keratinocytes by ORFV. ORFV infection of OTC was performed using the ORFV isolate B029 derived from a human patient. The OTC sections showed a similar structure of stratified epidermal keratinocytes as human foreskin and a similar expression profile of the differentiation markers keratin 1 (K1), K10, and loricrin. Upon ORFV infection, OTCs exhibited histological cytopathic changes including hyperkeratosis and ballooning degeneration of the keratinocytes. ORFV persisted for 10 days and was located in keratinocytes of the outer epidermal layers. ORFV-specific early, intermediate and late genes were transcribed, but limited viral spread and restricted cell infection were noticed. ORFV infection resulted in downregulation of K1, K10, and loricrin at the transcriptional level without affecting proliferation as shown by PCNA or Ki-67 expression. In conclusion, OTC provides a suitable model to study the interaction of virus with human keratinocytes in a similar structural setting as human skin and reveals that ORFV infection downregulates several differentiation markers in the epidermis of the human skin, a hitherto unknown feature of dermal ORFV infection in man.

]]>
<![CDATA[The nuclear hormone receptor NHR-86 controls anti-pathogen responses in C. elegans]]> https://www.researchpad.co/article/5c50c466d5eed0c4845e86e5

Nuclear hormone receptors (NHRs) are ligand-gated transcription factors that control adaptive host responses following recognition of specific endogenous or exogenous ligands. Although NHRs have expanded dramatically in C. elegans compared to other metazoans, the biological function of only a few of these genes has been characterized in detail. Here, we demonstrate that an NHR can activate an anti-pathogen transcriptional program. Using genetic epistasis experiments, transcriptome profiling analyses and chromatin immunoprecipitation-sequencing, we show that, in the presence of an immunostimulatory small molecule, NHR-86 binds to the promoters of immune effectors to activate their transcription. NHR-86 is not required for resistance to the bacterial pathogen Pseudomonas aeruginosa at baseline, but activation of NHR-86 by this compound drives a transcriptional program that provides protection against this pathogen. Interestingly, NHR-86 targets immune effectors whose basal regulation requires the canonical p38 MAPK PMK-1 immune pathway. However, NHR-86 functions independently of PMK-1 and modulates the transcription of these infection response genes directly. These findings characterize a new transcriptional regulator in C. elegans that can induce a protective host response towards a bacterial pathogen.

]]>
<![CDATA[Unique and overlapping GLI1 and GLI2 transcriptional targets in neoplastic chondrocytes]]> https://www.researchpad.co/article/5c59ff07d5eed0c48413599d

Excessive Hedgehog (Hh) signaling in chondrocytes is sufficient to cause formation of enchondroma-like lesions which can progress to chondrosarcoma. To elucidate potential underlying mechanisms, we identified GLI1 and GLI2 target genes in human chondrosarcoma. Using chromatin immunoprecipitation (ChIP) sequencing and microarray data, in silico analyses were conducted to identify and characterize unique and overlapping GLI1 and GLI2 binding regions in neoplastic chondrocytes. After overlaying microarray data from human chondrosarcoma, 204 upregulated and 106 downregulated genes were identified as Hh-responsive Gli binding targets. After overlaying published Gli ChIP-on-chip data from mouse, 48 genes were identified as potential direct downstream targets of Hedgehog signaling with shared GLI binding regions in evolutionarily conserved DNA elements. Among these was BMP2, pointing to potential cross-talk between TGF beta signaling and Hh signaling. Our identification of potential target genes that are unique and common to GLI1 and GLI2 in neoplastic chondrocytes contributes to elucidating potential pathways through which Hh signaling impacts cartilage tumor biology.

]]>
<![CDATA[INDETERMINATE-DOMAIN 4 (IDD4) coordinates immune responses with plant-growth in Arabidopsis thaliana]]> https://www.researchpad.co/article/5c536a50d5eed0c484a471ee

INDETERMINATE DOMAIN (IDD)/ BIRD proteins are a highly conserved plant-specific family of transcription factors which play multiple roles in plant development and physiology. Here, we show that mutation in IDD4/IMPERIAL EAGLE increases resistance to the hemi-biotrophic pathogen Pseudomonas syringae, indicating that IDD4 may act as a repressor of basal immune response and PAMP-triggered immunity. Furthermore, the idd4 mutant exhibits enhanced plant-growth indicating IDD4 as suppressor of growth and development. Transcriptome comparison of idd4 mutants and IDD4ox lines aligned to genome-wide IDD4 DNA-binding studies revealed major target genes related to defense and developmental-biological processes. IDD4 is a phospho-protein that interacts and becomes phosphorylated on two conserved sites by the MAP kinase MPK6. DNA-binding studies of IDD4 after flg22 treatment and with IDD4 phosphosite mutants show enhanced binding affinity to ID1 motif-containing promoters and its function as a transcriptional regulator. In contrast to the IDD4-phospho-dead mutant, the IDD4 phospho-mimicking mutant shows altered susceptibility to PstDC3000, salicylic acid levels and transcriptome reprogramming. In summary, we found that IDD4 regulates various hormonal pathways thereby coordinating growth and development with basal immunity.

]]>
<![CDATA[A variant of the Escherichia coli anaerobic transcription factor FNR exhibiting diminished promoter activation function enhances ionizing radiation resistance]]> https://www.researchpad.co/article/5c5217d1d5eed0c484794619

We have previously generated four replicate populations of ionizing radiation (IR)-resistant Escherichia coli though directed evolution. Sequencing of isolates from these populations revealed that mutations affecting DNA repair (through DNA double-strand break repair and replication restart), ROS amelioration, and cell wall metabolism were prominent. Three mutations involved in DNA repair explained the IR resistance phenotype in one population, and similar DNA repair mutations were prominent in two others. The remaining population, IR-3-20, had no mutations in the key DNA repair proteins, suggesting that it had taken a different evolutionary path to IR resistance. Here, we present evidence that a variant of the anaerobic metabolism transcription factor FNR, unique to and isolated from population IR-3-20, plays a role in IR resistance. The F186I allele of FNR exhibits a diminished ability to activate transcription from FNR-activatable promoters, and furthermore reduces levels of intracellular ROS. The FNR F186I variant is apparently capable of enhancing resistance to IR under chronic irradiation conditions, but does not increase cell survival when exposed to acute irradiation. Our results underline the importance of dose rate on cell survival of IR exposure.

]]>
<![CDATA[Allele-specific RNA imaging shows that allelic imbalances can arise in tissues through transcriptional bursting]]> https://www.researchpad.co/article/5c3fa5f1d5eed0c484caa55b

Extensive cell-to-cell variation exists even among putatively identical cells, and there is great interest in understanding how the properties of transcription relate to this heterogeneity. Differential expression from the two gene copies in diploid cells could potentially contribute, yet our ability to measure from which gene copy individual RNAs originated remains limited, particularly in the context of tissues. Here, we demonstrate quantitative, single molecule allele-specific RNA FISH adapted for use on tissue sections, allowing us to determine the chromosome of origin of individual RNA molecules in formaldehyde-fixed tissues. We used this method to visualize the allele-specific expression of Xist and multiple autosomal genes in mouse kidney. By combining these data with mathematical modeling, we evaluated models for allele-specific heterogeneity, in particular demonstrating that apparent expression from only one of the alleles in single cells can arise as a consequence of low-level mRNA abundance and transcriptional bursting.

]]>
<![CDATA[KDM2B regulates choline kinase expression and neuronal differentiation of neuroblastoma cells]]> https://www.researchpad.co/article/5c40f7add5eed0c4843865c6

The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells.

]]>
<![CDATA[Biological evaluation of isoflavonoids from Genista halacsyi using estrogen-target cells: Activities of glucosides compared to aglycones]]> https://www.researchpad.co/article/5c3e4fb8d5eed0c484d7862d

The purpose of this study was to evaluate the response of estrogen target cells to a series of isoflavone glucosides and aglycones from Genista halacsyi Heldr. The methanolic extract of aerial parts of this plant was processed using fast centrifugal partition chromatography, resulting in isolation of four archetypal isoflavones (genistein, daidzein, isoprunetin, 8-C-β-D-glucopyranosyl-genistein) and ten derivatives thereof. 7-O-β-D-glucopyranosyl-genistein and 7,4΄-di-O-β-D-glucopyranosyl-genistein were among the most abundant constituents of the isolate. All fourteen, except genistein, displayed low binding affinity for estrogen receptors (ER). Models of binding to ERα could account for the low binding affinity of monoglucosides. Genistein and its glucosides displayed full efficacy in inducing alkaline phosphatase (AlkP) in Ishikawa cells, proliferation of MCF-7 cells and ER-dependent gene expression in reporter cells at low concentrations (around 0.3 μM). ICI182,780 fully antagonized these effects. The AlkP-inducing efficacy of the fourteen isoflavonoids was more strongly correlated with their transcriptional efficacy through ERα. O-monoglucosides displayed higher area under the dose-response curve (AUC) of AlkP response relative to the AUC of ERα-transcriptional response compared to the respective aglycones. In addition, 7-O-β-D-glucopyranosyl-genistein and 7,4΄-di-O-β-D-glucopyranosyl-genistein displayed estradiol-like efficacy in promoting differentiation of MC3T3-E1 cells to osteoblasts, while genistein was not convincingly effective in this respect. Moreover, 7,4΄-di-O-β-D-glucopyranosyl-genistein suppressed lipopolysaccharide-induced tumor necrosis factor mRNA expression in RAW 264.7 cells, while 7-O-β-D-glucopyranosyl-genistein was not convincingly effective and genistein was ineffective. However, genistein and its O-glucosides were ineffective in inhibiting differentiation of RAW 264.7 cells to osteoclasts and in protecting glutamate-challenged HT22 hippocampal neurons from oxidative stress-induced cell death. These findings suggest that 7-O-β-D-glucopyranosyl-genistein and 7,4΄-di-O-β-D-glucopyranosyl-genistein display higher estrogen-like and/or anti-inflammatory activity compared to the aglycone. The possibility of using preparations rich in O-β-D-glucopyranosides of genistein to substitute for low-dose estrogen in formulations for menopausal symptoms is discussed.

]]>
<![CDATA[Different duration of parathyroid hormone exposure distinctively regulates primary response genes Nurr1 and RANKL in osteoblasts]]> https://www.researchpad.co/article/5c269783d5eed0c48470fc21

Parathyroid hormone (PTH) exerts dual effects, anabolic or catabolic, on bone when administrated intermittently or continuously, via mechanisms that remain largely unknown. PTH binding to cells induces PTH-responsive genes including primary response genes (PRGs). PRGs are rapidly induced without the need for de novo protein synthesis, thereby playing pivotal roles in directing subsequent molecular responses. In this study, to understand the role of PRGs in mediating osteoblastic cellular responses to PTH, we investigated whether various durations of PTH differentially induce PRGs in primary osteoblasts and MC3T3-E1. Nurr1 and RANKL, PRGs known for their anabolic and catabolic roles in bone metabolism respectively, presented distinctive transient vs. sustained induction kinetics. Corroborating their roles, maximum induction of Nurr1 was sufficiently achieved by brief PTH in as little as 30 minutes and continued beyond that, while maximum induction of RANKL was achieved only by prolonged PTH over 4 hours. Our data suggested distinctive regulatory mechanisms for Nurr1 and RANKL: PKA-mediated chromatin rearrangement for transcriptional regulation of both PRGs and ERK-mediated transcriptional regulation for RANKL but not Nurr1. Lastly, we classified PRGs into two groups based on the induction kinetics: The group that required brief PTH for maximum induction included Nur77, cox-2, and Nurr1, all of which are reported to play roles in bone formation. The other group that required prolonged PTH for maximum induction included IL-6 and RANKL, which play roles in bone resorption. Together, our data suggested the crucial role of PRG groups in mediating differential osteoblastic cellular responses to intermittent vs. continuous PTH. Continued research into the regulatory mechanisms of PKA and ERK for PRGs will help us better understand the molecular mechanisms underlying the dual effects of PTH, thereby optimizing the current therapeutic use of PTH for osteoporosis.

]]>
<![CDATA[Activation of E2F-dependent transcription by the mouse cytomegalovirus M117 protein affects the viral host range]]> https://www.researchpad.co/article/5c1813ccd5eed0c484775d94

Cytomegaloviruses (CMVs) have a highly restricted host range as they replicate only in cells of their own or closely related species. To date, the molecular mechanisms underlying the CMV host restriction remain poorly understood. However, it has been shown that mouse cytomegalovirus (MCMV) can be adapted to human cells and that adaptation goes along with adaptive mutations in several viral genes. In this study, we identify MCMV M117 as a novel host range determinant. Mutations in this gene enable the virus to cross the species barrier and replicate in human RPE-1 cells. We show that the M117 protein is expressed with early kinetics, localizes to viral replication compartments, and contributes to the inhibition of cellular DNA synthesis. Mechanistically, M117 interacts with members of the E2F transcription factor family and induces E2F target gene expression in murine and human cells. While the N-terminal part of M117 mediates E2F interaction, the C-terminal part mediates self-interaction. Both parts are required for the activation of E2F-dependent transcription. We further show that M117 is dispensable for viral replication in cultured mouse fibroblasts and endothelial cells, but is required for colonization of mouse salivary glands in vivo. Conversely, inactivation of M117 or pharmacological inhibition of E2F facilitates MCMV replication in human RPE-1 cells, whereas replacement of M117 by adenovirus E4orf6/7, a known E2F activator, prevents it. These results indicate that E2F activation is detrimental for MCMV replication in human cells. In summary, this study identifies MCMV M117 as a novel E2F activator that functions as a host range determinant by precluding MCMV replication in human cells.

]]>