Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exposure of aggregated murine P19 embryonal carcinoma cells to dimethylsulfoxide (DMSO) induces mesoderm and both embryonic cardiac and skeletal muscle differentiation, while retinoic acid (RA) is an inducer of neuroectodermal differentiation. P19 cells constitutively express the retinoic acid receptor alpha (RAR alpha) and RAR gamma mRNAs while RAR beta expression is induced by RA through a consensus RA-response element in the RAR beta promoter. In the present study we show that the RAR beta transcript is strongly expressed in both P19 cells and in a RA-nonresponsive derivative of P19 cells, called RAC65, during DMSO-induced mesoderm and muscle differentiation. Reverse transcriptase-polymerase chain reaction analysis indicated that RAR beta 2 is the predominant isoform expressed in DMSO-differentiated cells, providing the first evidence for RA-independent regulation of RAR beta 2 transcript levels. Immunoblot analysis showed a 3-fold increase in the RAR beta protein expression over basal levels in differentiated cells, and immunohistochemistry indicated that all cells in the culture including muscle reacted positively for the RAR beta protein. RAR beta 2 transcript expression was differentiation-dependent and occurred without transactivation of a transfected RARE beta 2 reporter gene. Little transcription of the RAR beta gene was detected in nuclear run-off assays of undifferentiated P19 cells and only a small increase in transcription was observed in nuclei from DMSO-treated cells. RA treatment of P19 cells stably transfected with the RA-responsive element from the RAR beta gene showed that RAR beta 2 mRNA expression during DMSO differentiation was associated with increased sensitivity to RA. Together these data show that RAR beta 2 is expressed spontaneously in an apparently RA-independent manner in differentiating mesoderm and mesoderm derivatives, resulting in increased sensitivity to RA in these cells.
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PMID:Spontaneous retinoic acid receptor beta 2 expression during mesoderm differentiation of P19 murine embryonal carcinoma cells. 1092 6

Transcription of TATA box-containing genes by RNA polymerase II is mediated by TBP-containing and TBP-free multisubunit complexes consisting of common and unique components. We have identified a highly stable TBP-TFIIA-containing complex, TAC, which is detectable in embryonal carcinoma (EC) cells but not in differentiated cells. TAC contains the TFIIAgamma subunit and the unprocessed form of TFIIAalphabeta, although the processed TFIIAalpha and TFIIAbeta subunits are present in EC cells. TAC mediates transcriptional activation by RNA polymerase II in vivo, even though it does not contain classical TAFs. Formaldehyde cross-linking revealed that in EC but not in differentiated cells, association of TBP with chromatin is strongly enhanced when complexed with TFIIA in vivo. Remarkably, the TFIIAalphabeta precursor is preferentially, if not exclusively, associated with chromatin as compared to the processed subunits present in "free" TFIIA in EC cells.
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PMID:TAC, a TBP-sans-TAFs complex containing the unprocessed TFIIAalphabeta precursor and the TFIIAgamma subunit. 1103 Mar 33

Differentiation in vitro of mouse F9 embryonal carcinoma (EC) cells to the parietal endoderm (PE) mimics processes of development of the early mouse embryo. This differentiation is accompanied by a dramatic down-regulation of all genes transcribed by RNA polymerase III (pol III). Complementation of extracts from cells, differentiated for various time periods with purified pol III transcription factors show for the first time that TFIIIC1 can substantially restore this impaired transcription, particularly in the early stages of differentiation. At later stages (day 7) the TBP (TATA-binding protein )-TAF complex, TFIIIBbeta, may also become limiting, which can contribute to but cannot account for the reduced transcription of type 2 promoters in PE cells. Because TFIIIBbeta is not required for the expression of type 3 promoters, other components must necessarily be involved, and our results show that U6 transcription can significantly be reactivated by TFIIIC1. By employing a variant type 3 promoter construct, which essentially requires a mutant form of TBP (TBP-DR2), we show that TBP is not limiting in PE extracts. The partial purification of pol III transcription factors from PE and EC cells revealed that TFIIIC2 activity could be purified from both cell types, whereas TFIIIC1 activity was dramatically reduced in extracts from PE cells.
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PMID:The activity of transcription factor IIIC1 is impaired during differentiation of F9 cells. 1174 93

The murine Nanog gene, a member of the homeobox family of DNA binding transcription factors, has been shown recently to maintain pluripotency of embryonic stem cells. We have used a sequence homology and expression screen to identify and clone the mouse and human Nanog genes and characterized their phylogenetic context and expression patterns. We report here the gene structure and expression patterns of the mouse Nanog gene, the human Nanog and Nanog2 genes, and six processed human Nanog pseudogenes. Mouse Nanog expression is high in undifferentiated embryonic stem cells and is down-regulated during embryonic stem cell differentiation, concomitant with loss of pluripotency. Murine embryonic Nanog expression is detected in the inner cell mass of the blastocyst. After implantation, Nanog is detectable at embryonic day (E) 6 in proximal epiblast in the region of the presumptive primitive streak. Expression extends distally as the streak elongates during gastrulation and remains restricted to epiblast. Nanog RNA is down-regulated in cells ingressing through the streak to form mesoderm and definitive endoderm. Nanog expression also marks the pluripotent germ cells of the nascent gonad at E11.5-E12.5 and is highly expressed in germ cell tumour and teratoma-derived cell lines. Reverse transcriptase-polymerase chain reaction analysis detected mouse Nanog expression at low levels in several adult tissues. The human Nanog genes are expressed in embryonic stem cells and down-regulated in all adult tissues and differentiated cell lines examined. High levels of human Nanog expression were detected by Northern analysis in the undifferentiated N-Tera embryonal carcinoma cell line. The conservation in gene sequence, structure, and expression of mouse and human Nanog and Nanog2 genes may reflect a common role in the maintenance of pluripotency in both species.
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PMID:Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human. 1510 23

Retinoic acid receptors (RARs) are the molecular relays of retinoid action on transcription, cellular differentiation and apoptosis. Transcriptional activation of retinoid-regulated promoters requires the dismissal of corepressors and the recruitment of coactivators to promoter-bound RAR. RARs recruit in vitro a plethora of coactivators whose actual contribution to retinoid-induced transcription is poorly characterized in vivo. Embryonal carcinoma P19 cells, which are highly sensitive to retinoids, were depleted from archetypical coactivators by RNAi. SRC1-deficient P19 cells showed severely compromised retinoid-induced responses, in agreement with the supposed role of SRC1 as a RAR coactivator. Unexpectedly, Med1/TRAP220/DRIP205-depleted cells exhibited an exacerbated response to retinoids, both in terms transcriptional responses and of cellular differentiation. Med1 depletion affected TFIIH and cdk9 detection at the prototypical retinoid-regulated RARbeta2 promoter, and favored a higher RNA polymerase II detection in transcribed regions of the RARbeta2 gene. Furthermore, the nature of the ligand impacted strongly on the ability of RARs to interact with a given coactivator and to activate transcription in intact cells. Thus RAR accomplishes transcriptional activation as a function of the ligand structure, by recruiting regulatory complexes which control distinct molecular events at retinoid-regulated promoters.
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PMID:Distinct roles of the steroid receptor coactivator 1 and of MED1 in retinoid-induced transcription and cellular differentiation. 1672 56

Hox gene expression is activated by all-trans retinoic acid (RA), through binding to retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimers bound at RA response elements (RAREs) of target genes. The RARs and RXRs each have three isotypes (alpha, beta, and gamma), which are encoded by distinct genes. Hox genes are also repressed by polycomb group proteins (PcG), though how these proteins are targeted is unclear. We used chromatin immunoprecipitation assays to investigate the association of RXRalpha, RARgamma, cofactors, and the PcG protein SUZ12 with the Hoxa1, RARbeta2, and Cyp26A1 RAREs in F9 embryonal carcinoma cells (teratocarcinoma stem cells) during RA treatment. We demonstrate that RARgamma and RXRalpha are associated with RAREs prior to and during RA treatment. pCIP, p300, and RNA polymerase II levels increased at target RAREs upon exposure to RA. Conversely, SUZ12 was found associated with all RAREs studied and these associations were attenuated by treatment with RA. Upon RA removal, SUZ12 re-associated with RAREs. H3ac, H3K4me2, and H3K27me3 marks were simultaneously detected at target loci, indicative of a bivalent domain chromatin structure. During RA mediated differentiation, H3K27me3 levels decreased at target RAREs whereas H3ac and H3K4me2 levels remained constant. These studies provide insight into the dynamics of association of co-regulators with RAREs and demonstrate a novel link between RA signaling and PcG repression.
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PMID:Retinoid regulated association of transcriptional co-regulators and the polycomb group protein SUZ12 with the retinoic acid response elements of Hoxa1, RARbeta(2), and Cyp26A1 in F9 embryonal carcinoma cells. 1766 92

Testicular germ cell tumors (TGCTs), comprised of seminomas and non-seminomas, are derived from premalignant and noninvasive intracellular germ cell neoplasias. Among TGCTs, seminomas are believed to resemble a transformed state of primordial germ cells (PGCs) and are known to exhibit a gene expression profile similar to that of embryonic stem (ES) cells, such as transcription factor OCT-4. OCT-4 has recently been recognized as a diagnostic marker for clinical aspects of seminomas. However, the role of the OCT-4 protein in seminomas has not been clarified. To determine a possible role of the OCT-4 protein in seminomas, in this paper, we studied a series of 41 testicular tumor tissues and four cell lines by immunohistochemistry, Western blotting, and reverse-transcriptase polymerase chain reaction (RT-PCR) to examine the expression and distribution of the OCT-4 transcription factor in seminomas. By utilizing immunohistochemical staining and Western blotting, we demonstrated that the OCT-4 transcription factor was aberrantly localized in the cytoplasm and nuclei of cells in the collected seminoma tissues. This observation was further confirmed using immunocytochemical staining of NCCIT (seminoma-embryonal carcinoma) and NT2 (embryonal carcinoma) cells. In addition, the RT-PCR results indicated that Oct-4 mRNA was relatively highly expressed in NCCIT, NT2 cells, and seminoma tissues when compared with human embryonic stem cells. The aberrant expression and distribution of the OCT-4 transcription factor in seminomas may provide some important clues concerning the cell transformation between germ line stem cells (like PGC) and testicular germ cell tumors.
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PMID:Aberrant expression and distribution of the OCT-4 transcription factor in seminomas. 1768 39

The gene of mouse kappa opioid receptor (KOR) utilizes two promoters, P1 and P2. P1 is active in various brain areas and constitutively in P19 mouse embryonal carcinoma cells. P2 is active in limited brain stem areas of adult animals and only in late differentiated cells of P19 induced for neuronal differentiation in the presence of nerve growth factor (NGF). NGF response of P2 was found to be mediated by a specific binding site for transcription factor activation protein 2 (AP2) located in P2. Electrophoretic gel shift assay showed specific binding of this AP2 site by AP2beta, but not AP2alpha. Knockdown of endogenous AP2beta with siRNA abolished the stimulating effect of NGF on the expression of transcripts driven by P2. Binding of endogenous AP2beta on the endogenous KOR P2 chromatin region was also confirmed by chromatin immunoprecipitation. The effect of NGF was inhibited by LY2942002 (phosphatidylinositol 3-kinase, PI3K inhibitor), suggesting that PI3K was involved in signaling pathway mediating the effect of NGF stimulation on KOR P2. The chromatin of P2 in P19 was found to be specifically modified following NGF stimulation, which included demethylation at Lys9 and dimethylation at Lys4 of histone H3 and was consistent with the increased recruitment of RNA polymerase II to this promoter. This study presents the first evidence for epigenetic changes occurred on a specific KOR promoter triggered by NGF in cells undergoing neuronal differentiation. This epigenetic change is mediated by recruited AP2beta to this promoter and involves the PI3K system.
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PMID:Epigenetic regulation of kappa opioid receptor gene in neuronal differentiation. 1820 39

Chromatin immunoprecipitation (ChIP) is a powerful technique for studying protein-DNA interactions. Drawbacks of current ChIP assays however are a requirement for large cell numbers, which limits applicability of ChIP to rare cell samples, and/or lengthy procedures with limited applications. There are to date no protocols for fast and parallel ChIPs of post-translationally modified histones from small cell numbers or biopsies, and importantly, no protocol allowing for investigations of transcription factor binding in small cell numbers. We report here the development of a micro (micro) ChIP assay suitable for up to nine parallel quantitative ChIPs of modified histones or RNA polymerase II from a single batch of 1000 cells. MicroChIP can also be downscaled to monitor the association of one protein with multiple genomic sites in as few as 100 cells. MicroChIP is applicable to small fresh tissue biopsies, and a cross-link-while-thawing procedure makes the assay suitable for frozen biopsies. Using MicroChIP, we characterize transcriptionally permissive and repressive histone H3 modifications on developmentally regulated promoters in human embryonal carcinoma cells and in osteosarcoma biopsies. muChIP creates possibilities for multiple parallel and rapid transcription factor binding and epigenetic analyses of rare cell and tissue samples.
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PMID:MicroChIP--a rapid micro chromatin immunoprecipitation assay for small cell samples and biopsies. 1820 78

Various compounds, including therapeutic drugs, can adversely impact the survival and development of embryos in the uterus. Identification of such development-interfering agents is a challenging task, although multi-angle approaches--including the use of in vitro toxicology studies involving embryonic stem cells--should alleviate some of the current difficulties. In the present study, we utilized the in vitro elongation of embryoid bodies (EBs) derived from mouse embryonal carcinoma stem cell line P19C5 as a model of early embryological events, specifically that of gastrulation and axial patterning. From our study, we identified donepezil, a medication indicated for the management of Alzheimer's disease, as a potential developmental toxicant. The extent of P19C5 EB axial elongation was diminished by donepezil in a dose-dependent manner. Although donepezil is a known inhibitor of acetylcholinesterase, interference of elongation was not mediated through this enzyme. Quantitative reverse-transcriptase PCR revealed that donepezil altered the expression pattern of a specific set of developmental regulator genes involved in patterning along the anterior-posterior body axis. When tested in mouse whole embryo culture, donepezil caused morphological abnormalities including impaired somitogenesis. Donepezil also diminished elongation morphogenesis of EBs generated from human embryonic stem cells. These results suggest that donepezil interferes with axial elongation morphogenesis of early embryos by altering the expression pattern of regulators of axial development.
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PMID:Morphology-based mammalian stem cell tests reveal potential developmental toxicity of donepezil. 2526 81


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