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1
Ma, Le, Zhenyu Liu, and Zhimin Fan. "Potential Mechanisms of miR-143/Krupple Like Factor 5 Axis in Impeding the Proliferation of Michigan Cancer Foundation-7 Breast Cancer Cell Line." Journal of Biomaterials and Tissue Engineering 11, no. 2 (February 1, 2021): 326–32. http://dx.doi.org/10.1166/jbt.2021.2545.
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Breast cancer is one of the most prevailing cancers in females, while the cancerous heterogeneity hinders its early diagnosis and subsequent therapy. miR-143-3p is a critical mediator in malignancy development and tumorigenesis as a tumor suppressor. Its role in various tumor entities has been investigated, such as colon cancer and breast cancer. Using MCF-7 breast cancer cell model, we planned to explore the underlying mechanisms of miR-143/KLF-5 axis in retarding breast cancer cells growth. Bioinformatics analysis searched the target KLF5 of miR-143, and the miR-143-targeted mimic and inhibitor were employed to detect the changes of KLF5. After transfection of mimic miR-143, the CCK-8 reagent assessed cell proliferation. Based on optimal stimulation time, miR-143 stimulation model was established, followed by determining expression of KLF5, EGFR and PCNA via western blot and qPCR. Eventually, siRNA-KLF5 was applied to silencing KLF5 level to evaluate its role in MCF-7 cells. The transcription and translation levels of KLF5 were diminished in miR-143-mimic transfected MCF-7 cells, while enhanced in miR-143-inhibitor transfected MCF-7 cells. When MCF-7 cells were transfected with miR-143-mimic at different time points, 48 hours was found to be the optimal transfection time, with reduced transcription and translation levels of KLF5, EGFR and PCNA. The transcription and translation levels of PNCA and EGFR were declined after silencing KLF5 by siRNA. miR-143/KLF5 axis could retard the proliferation of MCF-7 breast cancer cells.
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Sampath, Deepak, Richard C. Winneker, and Zhiming Zhang. "Cyr61, a Member of the CCN Family, Is Required for MCF-7 Cell Proliferation: Regulation by 17β-Estradiol and Overexpression in Human Breast Cancer." Endocrinology 142, no. 6 (June 1, 2001): 2540–48. http://dx.doi.org/10.1210/endo.142.6.8186.
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Abstract Cyr61, a member of the CCN (CTGF/Cyr61/NOV) family of growth regulators, is a secreted cysteine-rich proangiogenic factor that has been implicated in tumorigenesis. Previous studies have also demonstrated that Cyr61 is regulated by 17β-estradiol (E2) in the uterus. Therefore, we hypothesized that hormonal regulation of Cyr61 may be important in estrogen-dependent pathogenic processes such as breast tumorigenesis. Our study demonstrates that both Cyr61 messenger RNA and protein are induced by E2 in MCF-7 mammary adenocarcinoma cells that primarily overexpress estrogen receptor α (ERα) in a dose-dependent and immediate early fashion. Cyr61 gene induction by E2 is transcriptionally regulated by ERα as the antiestrogen, ICI 182,780, and actinomycin D blocked induction completely. In addition, Cyr61 is up-regulated in MCF-7 cells by epidermal growth factor (EGF) in an immediate early fashion as well. The functional relevance of steroid induction of Cyr61 in breast cancer cell growth is demonstrated by anti-Cyr61 neutralizing antibodies, which diminished E2 and EGF-dependent DNA synthesis and dramatically reduced E2-driven cell proliferation by more than 70%. Most importantly, Cyr61 is overexpressed in 70% (28 of 40) of breast cancer patients with infiltrating ductal carcinoma and is localized exclusively to hyperplastic ductal epithelial cells. Moreover, the levels of Cyr61 protein are higher in breast tumors that are ER+/EGF receptor+ than those that are ER−/EGF receptor+, suggesting that estrogens may mediate Cyr61 expression in vivo. Collectively, our data suggest that Cyr61 may play a critical role in estrogen- as well as growth factor-dependent breast tumor growth.
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Abuarqoub, Abdelrahman. "D-FAP: Dual-Factor Authentication Protocol for Mobile Cloud Connected Devices." Journal of Sensor and Actuator Networks 9, no. 1 (December 20, 2019): 1. http://dx.doi.org/10.3390/jsan9010001.
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Emerging Mobile Cloud Computing (MCC) technologies offer a new world of promise by leveraging the quality of mobile services. With MCC, resource-constrained mobile devices could capitalize on the computation/storage resources of cloud servers via communication networks. While MCC adoption is growing significantly, several challenges need to be addressed to make MCC-based solutions scale and meet the ever-growing demand for more resource intensive applications. Security is a critical problem hindering the adoption of MCC. One of the most important aspects of MCC security is to establish authenticated communication sessions between mobile devices and cloud servers. The huge amount of data stored on mobile devices poses information security risks and privacy concerns for individuals, enterprises, and governments. The ability to establish authenticated communication sessions between mobile devices and cloud servers can resolve many security concerns. Limited computing and energy resources on mobile devices makes authentication and encryption a challenging task. In this paper, an overview of MCC authentication protocols is presented. Then, a Dual-Factor Authentication Protocol for MCC devices (D-FAP) is proposed. D-FAP aims at increasing authentication security by using multi-factors while offloading computation to the cloud to reduce battery consumption. The security of the protocol is formally verified and informal analysis is performed for various attacks. The results prove that the D-FAP is successful in mitigating various outsider and insider attacks.
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Vrana, K. E., M. E. Churchill, T. D. Tullius, and D. D. Brown. "Mapping functional regions of transcription factor TFIIIA." Molecular and Cellular Biology 8, no. 4 (April 1988): 1684–96. http://dx.doi.org/10.1128/mcb.8.4.1684.
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Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA 19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outside the DNA-binding domain. The elongated protein can be aligned over the internal control region (ICR) of the Xenopus 5S RNA gene with its carboxyl terminus oriented toward the 5' end of the gene and its amino terminus oriented toward the 3' end of the gene. The nine "zinc fingers" and the linkers that separate them comprise 80% of the protein mass and correspond to the DNA-binding domain of TFIIIA. The zinc fingers near the amino terminus of the protein contribute more to the overall binding energy of the protein to the ICR than do the zinc fingers near the carboxyl end. The most striking feature of TFIIIA is its modular structure. This is demonstrated by the fact that each zinc finger binds to just one of three short nucleotide sequences within the ICR.
5
Ni, Zhuoyu, Abbie Saunders, Nicholas J. Fuda, Jie Yao, Jose-Ramon Suarez, Watt W. Webb, and John T. Lis. "P-TEFb Is Critical for the Maturation of RNA Polymerase II into Productive Elongation In Vivo." Molecular and Cellular Biology 28, no. 3 (December 10, 2007): 1161–70. http://dx.doi.org/10.1128/mcb.01859-07.
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ABSTRACT Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. Here, we demonstrate that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.
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Elhady, Sameh S., Reda F. A. Abdelhameed, Salwa H. Zekry, Amany K. Ibrahim, Eman S. Habib, Khaled M. Darwish, Reem M. Hazem, Khadijah A. Mohammad, Hashim A. Hassanean, and Safwat A. Ahmed. "VEGFR-Mediated Cytotoxic Activity of Pulicaria undulata Isolated Metabolites: A Biological Evaluation and In Silico Study." Life 11, no. 8 (July 28, 2021): 759. http://dx.doi.org/10.3390/life11080759.
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Natural products play a remarkable role not only in the synthesis, design, and discovery of new drugs but also as the most prominent source of drugs and bioactive substances. Adding to the search for new sources of safe innovative antitumor drugs, here we reported a phytochemical study on Pulicaria undulata which revealed promising antiangiogenic agents. Six compounds were isolated and identified as xanthoxyline (1), stigmasterol (2), oleanolic acid (3), salvigenin (4), rhamnetin (5) and dihydroquercetin-4′-methyl ether (6) using nuclear magnetic resonance (NMR) spectroscopic techniques. Compound 3 and 4 are first reported in Pulicaria genus. Both the extract and isolated compounds were evaluated for in vitro antiproliferative activity against breast cancer cell line (MCF-7). In vivo antiproliferative activity against Ehrlich’s ascites carcinoma (EAC) were also assessed. The P. undulata extract and isolates showed significant reduction in tumor weight, decreased both serum vascular endothelial growth factor B (VEGF-B) levels and vascular endothelial growth factor receptor 2 (VEGFR-2) expression significantly compared to the control EAC group, suggesting an antiangiogenic activity through the inhibition of VEGF signaling. Besides, they displayed reduction in CD34 expression, confirming their antiangiogenic effect. Moreover, the potential affinity of isolated compounds to human estrogen nuclear receptor-alpha (hER-α), the most recognized modulator of VEGFR-2 expression, was virtually estimated through molecular modeling studies. The most promising activity profiles were assigned to the investigated flavonoids, compounds 4–6, as well as the alkyl-phenylketone, compound 1. Additionally, these four top active compounds showed respective high to intermediate docking scores while possessing preferential binding with hER-α critical pocket residues. Based on the provided data, these isolated compounds illustrated promising inhibitors of VEGF-stimulated angiogenesis, which could be a possible mechanism for their anticancer activity.
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Mohammad-Qureshi, Sarah S., Raphaël Haddad, Elizabeth J. Hemingway, Jonathan P. Richardson, and Graham D. Pavitt. "Critical Contacts between the Eukaryotic Initiation Factor 2B (eIF2B) Catalytic Domain and both eIF2β and -2γ Mediate Guanine Nucleotide Exchange." Molecular and Cellular Biology 27, no. 14 (May 25, 2007): 5225–34. http://dx.doi.org/10.1128/mcb.00495-07.
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ABSTRACT Diverse guanine nucleotide exchange factors (GEFs) regulate the activity of GTP binding proteins. One of the most complicated pairs is eukaryotic initiation factor 2B (eIF2B) and eIF2, which function during protein synthesis initiation in eukaryotes. We have mutated conserved surface residues within the eIF2B GEF domain, located at the eIF2Bε C terminus. Extensive genetic and biochemical characterization established how these residues contribute to GEF activity. We find that the universally conserved residue E569 is critical for activity and that even a conservative E569D substitution is lethal in vivo. Several mutations within residues close to E569 have no discernible effect on growth or GCN4 expression, but an alanine substitution at the adjacent L568 is cold sensitive and deregulates GCN4 activity at 15°C. The mutation of W699, found on a separate surface approximately 40 Å from E569, is also lethal. Binding studies show that W699 is critical for interaction with eIF2β, while L568 and E569 are not. In contrast, all three residues are critical for interaction with eIF2γ. These data show that multiple contacts between eIF2γ and eIF2Bε mediate nucleotide exchange.
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Lei, Lei, Delin Ren, and Zachary F. Burton. "The RAP74 Subunit of Human Transcription Factor IIF Has Similar Roles in Initiation and Elongation." Molecular and Cellular Biology 19, no. 12 (December 1, 1999): 8372–82. http://dx.doi.org/10.1128/mcb.19.12.8372.
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ABSTRACT Transcription factor IIF (TFIIF) is a protein allosteric effector for RNA polymerase II during the initiation and elongation phases of the transcription cycle. In initiation, TFIIF induces promoter DNA to wrap almost a full turn around RNA polymerase II in a complex that includes the general transcription factors TATA-binding protein, TFIIB, and TFIIE. During elongation, TFIIF also supports a more active conformation of RNA polymerase II. This conformational model for elongation is supported by three lines of experimental evidence. First, a region within the RNA polymerase II-associating protein 74 (RAP74) subunit of TFIIF (amino acids T154 to M177), a region that is critical for isomerization of the preinitiation complex, is also critical for elongation stimulation. Amino acid substitutions within this region are shown to have very similar effects on initiation and elongation, and mutagenic analysis indicates that L155, W164, N172, I176, and M177 are the most important residues in this region for transcription. Second, TFIIF is shown to have a higher affinity for rapidly elongating RNA polymerase II than for the stalled elongation complex, indicating that RNA polymerase II alternates between active and inactive states during elongation and that TFIIF stimulates elongation by supporting the active conformational state of RNA polymerase II. The deleterious I176A substitution in the critical region of RAP74 decreases the affinity of TFIIF for the active form of the elongation complex. Third, TFIIF is shown by Arrhenius analysis to stimulate elongation by populating an activated state of RNA polymerase II.
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DEUTSCH, CLAUDE, and NAEEM A. TAHIR. "Fusion reactions and matter–antimatter annihilation for space propulsion." Laser and Particle Beams 24, no. 4 (October 2006): 605–16. http://dx.doi.org/10.1017/s0263034606060691.
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Magnetic confinement fusion (MCF) and inertial confinement fusion (ICF) are critically contrasted in the context of far-distant travels throughout solar system. Both are shown to potentially display superior capabilities for vessel maneuvering at high speed, which are unmatched by standard cryogenic propulsion (SCP). Costs constraints seem less demanding than for ground-based power plants. Main issue is the highly problematic takeoff from earth, in view of safety hazards concomitant to radioactive spills in case of emergency. So, it is recommended to assemble the given powered vessel at high earth altitude ∼ 700 km, above upper atmosphere. Fusion propulsion is also compared to fission powered one, which secures a factor of two improvement over SCP. As far a specific impulse (s) is considered, one expects 500–3000 from fission and as much as 104–105 from fusion through deuterium–tritium (D-T). Next, we turn attention to the most performing fusion reaction, i.e., proton–antiproton annihilation with specific impulse ∼ 103–106 and thrust–to–weight ratio ∼ 10−3–1. Production and costs are timely reviewed. The latter could drop by four orders of magnitude, which is possible with successful MCF or ICF. Appropriate vessel designs will be presented for fusion as well as for antimatter propulsion. In particular, ion compressed antimatter nuclear II (ICAN-II) project to Mars in 30 days with fusion catalyzed by 140 ng of antiprotons will be detailed (specific impulse ∼ 13500 s).
10
Vrana, K. E., M. E. Churchill, T. D. Tullius, and D. D. Brown. "Mapping functional regions of transcription factor TFIIIA." Molecular and Cellular Biology 8, no. 4 (April 1988): 1684–96. http://dx.doi.org/10.1128/mcb.8.4.1684-1696.1988.
Full textAbstract:
Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA 19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outside the DNA-binding domain. The elongated protein can be aligned over the internal control region (ICR) of the Xenopus 5S RNA gene with its carboxyl terminus oriented toward the 5' end of the gene and its amino terminus oriented toward the 3' end of the gene. The nine "zinc fingers" and the linkers that separate them comprise 80% of the protein mass and correspond to the DNA-binding domain of TFIIIA. The zinc fingers near the amino terminus of the protein contribute more to the overall binding energy of the protein to the ICR than do the zinc fingers near the carboxyl end. The most striking feature of TFIIIA is its modular structure. This is demonstrated by the fact that each zinc finger binds to just one of three short nucleotide sequences within the ICR.
11
Du, Kevin L., Hon S. Ip, Jian Li, Mary Chen, Frederic Dandre, William Yu, Min Min Lu, Gary K. Owens, and Michael S. Parmacek. "Myocardin Is a Critical Serum Response Factor Cofactor in the Transcriptional Program Regulating Smooth Muscle Cell Differentiation." Molecular and Cellular Biology 23, no. 7 (April 1, 2003): 2425–37. http://dx.doi.org/10.1128/mcb.23.7.2425-2437.2003.
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ABSTRACT The SAP family transcription factor myocardin functionally synergizes with serum response factor (SRF) and plays an important role in cardiac development. To determine the function of myocardin in the smooth muscle cell (SMC) lineage, we mapped the pattern of myocardin gene expression and examined the molecular mechanisms underlying transcriptional activity of myocardin in SMCs and embryonic stem (ES) cells. The human and murine myocardin genes were expressed in vascular and visceral SMCs at levels equivalent to or exceeding those observed in the heart. During embryonic development, the myocardin gene was expressed abundantly in a precise, developmentally regulated pattern in SMCs. Forced expression of myocardin transactivated multiple SMC-specific transcriptional regulatory elements in non-SMCs. By contrast, myocardin-induced transactivation was not observed in SRF−/− ES cells but could be rescued by forced expression of SRF or the SRF DNA-binding domain. Furthermore, expression of a dominant-negative myocardin mutant protein or small-interfering-RNA-induced myocardin knockdown significantly reduced SM22α promoter activity in SMCs. Most importantly, forced expression of myocardin activated expression of the SM22α, smooth muscle α-actin, and calponin-h1 genes in undifferentiated mouse ES cells. Taken together, these data demonstrate that myocardin plays an important role in the SRF-dependent transcriptional program that regulates SMC development and differentiation.
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Gozdecka, Malgorzata, and Wolfgang Breitwieser. "The roles of ATF2 (activating transcription factor 2) in tumorigenesis." Biochemical Society Transactions 40, no. 1 (January 19, 2012): 230–34. http://dx.doi.org/10.1042/bst20110630.
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MAPK (mitogen-activated protein kinase) pathways are among the most frequently deregulated signalling events in cancer. Among the critical targets of MAPK activities are members of the AP-1 (activator protein 1) transcription factor, a dimeric complex consisting of Jun, Fos, Maf and ATF (activating transcription factor) family DNA-binding proteins. Depending on the cellular context, the composition of the dimeric complexes determines the regulation of growth, survival or apoptosis. JNK (c-Jun N-terminal kinase), p38 and a number of Jun and Fos family proteins have been analysed for their involvement in oncogenic transformation and tumour formation. These data are also emerging for the ATF components of the AP-1 factor. The aim of the present review is to provide an overview of the functions of two ATF family proteins, ATF2 and ATF7, in mammalian development and their potential functions in tumour formation.
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Bayly, Richard, Takayuki Murase, Brandy D. Hyndman, Rachel Savage, Salima Nurmohamed, Kim Munro, Richard Casselman, Steven P. Smith, and David P. LeBrun. "Critical Role for a Single Leucine Residue in Leukemia Induction by E2A-PBX1." Molecular and Cellular Biology 26, no. 17 (September 1, 2006): 6442–52. http://dx.doi.org/10.1128/mcb.02025-05.
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ABSTRACT In roughly 5% of cases of acute lymphoblastic leukemia, a chromosomal translocation leads to expression of the oncogenic protein E2A-PBX1. The N-terminal portion of E2A-PBX1, encoded by the E2A gene, is identical in sequence to the corresponding portion of the E proteins E12/E47 and includes transcriptional activation domains. The C terminus consists of most of the HOX interacting transcription factor PBX1, including its DNA-binding homeodomain. Structure-function correlative experiments have suggested that oncogenesis by E2A-PBX1 requires an activation domain, called AD1, at the extreme N terminus. We recently demonstrated that a potentially helical portion of AD1 interacts directly with the transcriptional coactivator protein cyclic AMP response element-binding protein (CBP) and that this interaction is essential in the immortalization of primary bone marrow cells in tissue culture. Here we show that a conserved LXXLL motif within AD1 is required in the interaction between E2A-PBX1 and the KIX domain of CBP. We show by circular dichroism spectroscopy that the LXXLL-containing portion of AD1 undergoes a helical transition upon interacting with the KIX domain and that amino acid substitutions that prevent helix formation prevent both the KIX interaction and cell immortalization by E2A-PBX1. Perhaps most strikingly, substitution of a single, conserved leucine residue (L20) within the LXXLL motif impairs leukemia induction in mice after transplantation with E2A-PBX1-expressing bone marrow. The KIX domain of CBP mediates well-characterized interactions with several transcription factors of relevance to leukemia induction. Circumstantial evidence suggests that the side chain of L20 might interact with a deep hydrophobic pocket in the KIX domain. Therefore, our results serve to identify a potential new drug target.
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Chen, Linyun, Mandy Kwong, Ronghua Lu, David Ginzinger, Candy Lee, Laura Leung, and Jefferson Y. Chan. "Nrf1 Is Critical for Redox Balance and Survival of Liver Cells during Development." Molecular and Cellular Biology 23, no. 13 (July 1, 2003): 4673–86. http://dx.doi.org/10.1128/mcb.23.13.4673-4686.2003.
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ABSTRACT The Nrf1 transcription factor belongs to the CNC subfamily of basic leucine zipper proteins. Knockout of Nrf1 is lethal in mouse embryos, but nothing is known about the cell types that absolutely require its function during development. We show by chimera analysis that Nrf1 is essential for the hepatocyte lineage. Mouse embryonic stem cells lacking Nrf1 developed normally and contributed to most tissues in adult chimeras where Nrf1 is normally expressed. Nrf1-deficient cells contributed to fetal, but not adult, liver cells. Loss of Nrf1 function resulted in liver cell apoptosis in late-gestation chimeric fetuses. Fetal livers from mutant embryos exhibited increased oxidative stress and impaired expression of antioxidant genes, and primary cultures of nrf1−/− fetal hepatocytes were sensitive to tert-butyl hydroperoxide-induced cell death, suggesting that impaired antioxidant defense may be responsible for the apoptosis observed in the livers of chimeric mice. In addition, cells deficient in Nrf1 were sensitized to the cytotoxic effects of tumor necrosis factor (TNF). Our results provide in vivo evidence demonstrating an essential role of Nrf1 in the survival of hepatocytes during development. Our results also suggest that Nrf1 may promote cell survival by maintaining redox balance and protecting embryonic hepatocytes from TNF-mediated apoptosis during development.
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Chen, Xiaoyong, and James J. Bieker. "Stage-Specific Repression by the EKLF Transcriptional Activator." Molecular and Cellular Biology 24, no. 23 (December 1, 2004): 10416–24. http://dx.doi.org/10.1128/mcb.24.23.10416-10424.2004.
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ABSTRACT Dynamic changes in transcription factor function can be mediated by switching its interaction with coactivators and corepressors. Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific transcription factor that plays a critical role in β-globin gene activation via its interactions with CBP/p300 and SWI/SNF proteins. Unexpectedly, it also interacts with Sin3A and histone deacetylase 1 (HDAC1) corepressors via its zinc finger domain. We now find that selected point mutants can uncouple activation and repression and that an intact finger structure is not required for interactions with Sin3A/HDAC1 or for transrepression. Most intriguingly, EKLF repression exhibits stage specificity, with reversible EKLF-Sin3A interactions playing a key role in this process. Finally, we have located a key lysine residue that is both a substrate for CBP acetylation and required for Sin3A interaction. These data suggest a model whereby the stage of the erythroid cell alters the acetylation status of EKLF and plays a critical role in directing its coactivator-corepressor interactions and downstream transcriptional effects.
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Schlaeger, Thorsten M., Anna Schuh, Simon Flitter, Andreas Fisher, Hanna Mikkola, Stuart H. Orkin, Paresh Vyas, and Catherine Porcher. "Decoding Hematopoietic Specificity in the Helix-Loop-Helix Domain of the Transcription Factor SCL/Tal-1." Molecular and Cellular Biology 24, no. 17 (September 1, 2004): 7491–502. http://dx.doi.org/10.1128/mcb.24.17.7491-7502.2004.
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ABSTRACT The helix-loop-helix (HLH) domain is employed by many transcription factors that control cell fate choice in multiple developmental settings. Previously, we demonstrated that the HLH domain of the class II basic HLH (bHLH) protein SCL/Tal-1 is critical for hematopoietic specification. We have now identified residues in this domain that are essential for restoring hematopoietic development to SCL−/− embryonic stem cells and sufficient to convert a muscle-specific HLH domain to one able to rescue hematopoiesis. Most of these critical residues are distributed in the loop of SCL, with one in helix 2. This is in contrast to the case for MyoD, the prototype of class II bHLH proteins, where the loop seems to serve mainly as a linker between the two helices. Among the identified residues, some promote heterodimerization with the bHLH partners of SCL (E12/E47), while others, unimportant for this property, are still crucial for the biological function of SCL. Importantly, the residue in helix 2 specifically promotes interaction with a known partner of SCL, the LIM-only protein LMO2, a finding that strengthens genetic evidence that these proteins interact. Our data highlight the functional complexity of bHLH proteins, provide mechanistic insight into SCL function, and strongly support the existence of an active SCL/LMO2-containing multiprotein complex in early hematopoietic cells.
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Chang, Yuh-Long, Balas King, Shu-Chun Lin, James A. Kennison, and Der-Hwa Huang. "A Double-Bromodomain Protein, FSH-S, Activates the Homeotic Gene Ultrabithorax through a Critical Promoter-Proximal Region." Molecular and Cellular Biology 27, no. 15 (May 25, 2007): 5486–98. http://dx.doi.org/10.1128/mcb.00692-07.
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ABSTRACT More than a dozen trithorax group (trxG) proteins are involved in activation of Drosophila HOX genes. How they act coordinately to integrate signals from distantly located enhancers is not fully understood. The female sterile (1) homeotic (fs(1)h) gene is one of the trxG genes that is most critical for Ultrabithorax (Ubx) activation. We show that one of the two double-bromodomain proteins encoded by fs(1)h acts as an essential factor in the Ubx proximal promoter. First, overexpression of the small isoform FSH-S, but not the larger one, can induce ectopic expression of HOX genes and cause body malformation. Second, FSH-S can stimulate Ubx promoter in cultured cells through a critical proximal region in a bromodomain-dependent manner. Third, purified FSH-S can bind specifically to a motif within this region that was previously known as the ZESTE site. The physiological relevance of FSH-S is ascertained using transgenic embryos containing a modified Ubx proximal promoter and chromatin immunoprecipitation. In addition, we show that FSH-S is involved in phosphorylation of itself and other regulatory factors. We suggest that FSH-S acts as a critical component of a regulatory circuitry mediating long-range effects of distant enhancers.
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Schwartz, David C., and Roy Parker. "Mutations in Translation Initiation Factors Lead to Increased Rates of Deadenylation and Decapping of mRNAs inSaccharomyces cerevisiae." Molecular and Cellular Biology 19, no. 8 (August 1, 1999): 5247–56. http://dx.doi.org/10.1128/mcb.19.8.5247.
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ABSTRACT The turnover of most mRNAs in Saccharomyces cerevisiaebegins with deadenylation followed by decapping and 5′→3′ exonucleolytic digestion. An important question involves the mechanisms that allow particular mRNAs to exhibit different rates of both deadenylation and decapping. Since the cap structure plays a critical role in the assembly of translation initiation factors, we hypothesized that the status of the cytoplasmic cap binding complex would affect the rate of decapping. To test this hypothesis, we examined mRNA decay rates in yeast strains that were defective in several translation initiation factors that are part of the cap binding complex. These experiments yielded three significant observations. First, any mutation known to inhibit translation initiation also increased the rate of decapping. Second, decapping still occurred only after deadenylation, suggesting that the ability of the poly(A) tail to inhibit decapping does not require efficient translation of the transcript. Third, mutants with defects in translation initiation factors also showed an increase in the rate of deadenylation, suggesting that the rate of deadenylation may be controlled primarily by the translation status of the transcript. These results argue that the nature of the translation initiation complex is a critical factor in determining the mRNA half-life. This view also implies that some cis-acting sequences that modulate mRNA decay rate do so by affecting the translation status of the transcript.
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Cao, Qing-Fu, Junichi Yamamoto, Tomoyasu Isobe, Shumpei Tateno, Yuki Murase, Yexi Chen, Hiroshi Handa, and Yuki Yamaguchi. "Characterization of the Human Transcription Elongation Factor Rtf1: Evidence for Nonoverlapping Functions of Rtf1 and the Paf1 Complex." Molecular and Cellular Biology 35, no. 20 (July 27, 2015): 3459–70. http://dx.doi.org/10.1128/mcb.00601-15.
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Restores TBP function 1 (Rtf1) is generally considered to be a subunit of the Paf1 complex (PAF1C), a multifunctional protein complex involved in histone modification and transcriptional or posttranscriptional regulation. Rtf1, however, is not stably associated with the PAF1C in most species exceptSaccharomyces cerevisiae, and its biochemical functions are not well understood. Here, we show that human Rtf1 is a transcription elongation factor that may function independently of the PAF1C. Rtf1 requires “Rtf1 coactivator” activity, which is most likely unrelated to the PAF1C or DSIF, for transcriptional activationin vitro. A mutational study revealed that the Plus3 domain of human Rtf1 is critical for its coactivator-dependent function. Transcriptome sequencing (RNA-seq) and chromatin immunoprecipitation studies in HeLa cells showed that Rtf1 and the PAF1C play distinct roles in regulating the expression of a subset of genes. Moreover, contrary to the finding inS. cerevisiae, the PAF1C was apparently recruited to the genes examined in an Rtf1-independent manner. The present study establishes a role for human Rtf1 as a transcription elongation factor and highlights the similarities and differences between theS. cerevisiaeand human Rtf1 proteins.
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Singh, Chingakham Ranjit, Cynthia Curtis, Yasufumi Yamamoto, Nathan S. Hall, Dustin S. Kruse, Hui He, Ernest M. Hannig, and Katsura Asano. "Eukaryotic Translation Initiation Factor 5 Is Critical for Integrity of the Scanning Preinitiation Complex and Accurate Control of GCN4 Translation." Molecular and Cellular Biology 25, no. 13 (July 1, 2005): 5480–91. http://dx.doi.org/10.1128/mcb.25.13.5480-5491.2005.
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ABSTRACT The integrity of eukaryotic translation initiation factor (eIF) interactions in ribosomal preinitiation complexes is critical for the proper regulation of GCN4 mRNA translation in response to amino acid availability. Increased phosphorylation of eIF2 under amino acid starvation conditions leads to a corresponding increase in GCN4 mRNA translation. The carboxyl-terminal domain (CTD) of eIF5 (eIF5-CTD) has been identified as a potential nucleation site for preinitiation complex assembly. To further characterize eIF5 and delineate its role in GCN4 translational control, we isolated mutations leading to temperature sensitivity (Ts− phenotype) targeted at TIF5, the structural gene encoding eIF5 in yeast (Saccharomyces cerevisiae). Nine single point mutations were isolated, in addition to an allele in which the last 15 amino acids were deleted. The nine point mutations clustered in the eIF5-CTD, which contains two conserved aromatic/acidic boxes. Six of the point mutations derepressed GCN4 translation independent of eIF2 phosphorylation (Gcd− phenotype) at a permissive temperature, directly implicating eIF5-CTD in the eIF2/GTP/Met-tRNAi Met ternary complex binding process required for GCN4 translational control. In addition, stronger restriction of eIF5-CTD function at an elevated temperature led to failure to derepress GCN4 translation (Gcn− phenotype) in all of the mutants, most likely due to leaky scanning of the first upstream open reading frame of GCN4 mRNA. This latter result directly implicates eIF5-CTD in the process of accurate scanning for, or recognition of, AUG codons. Taken together, our results indicate that eIF5-CTD plays a critical role in both the assembly of the 43S complex and the postassembly process in the 48S complex, likely during the scanning process.
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Meredith, David M., Mark D. Borromeo, Tye G. Deering, Bradford H. Casey, Trisha K. Savage, Paul R. Mayer, Chinh Hoang, et al. "Program Specificity for Ptf1a in Pancreas versus Neural Tube Development Correlates with Distinct Collaborating Cofactors and Chromatin Accessibility." Molecular and Cellular Biology 33, no. 16 (June 10, 2013): 3166–79. http://dx.doi.org/10.1128/mcb.00364-13.
Full textAbstract:
The lineage-specific basic helix-loop-helix transcription factor Ptf1a is a critical driver for development of both the pancreas and nervous system. How one transcription factor controls diverse programs of gene expression is a fundamental question in developmental biology. To uncover molecular strategies for the program-specific functions of Ptf1a, we identified bound genomic regionsin vivoduring development of both tissues. Most regions bound by Ptf1a are specific to each tissue, lie near genes needed for proper formation of each tissue, and coincide with regions of open chromatin. The specificity of Ptf1a binding is encoded in the DNA surrounding the Ptf1a-bound sites, because these regions are sufficient to direct tissue-restricted reporter expression in transgenic mice. Fox and Sox factors were identified as potential lineage-specific modifiers of Ptf1a binding, since binding motifs for these factors are enriched in Ptf1a-bound regions in pancreas and neural tube, respectively. Of the Fox factors expressed during pancreatic development, Foxa2 plays a major role. Indeed, Ptf1a and Foxa2 colocalize in embryonic pancreatic chromatin and can act synergistically in cell transfection assays. Together, these findings indicate that lineage-specific chromatin landscapes likely constrain the DNA binding of Ptf1a, and they identify Fox and Sox gene families as part of this process.
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Pavitt, G. D., W. Yang, and A. G. Hinnebusch. "Homologous segments in three subunits of the guanine nucleotide exchange factor eIF2B mediate translational regulation by phosphorylation of eIF2." Molecular and Cellular Biology 17, no. 3 (March 1997): 1298–313. http://dx.doi.org/10.1128/mcb.17.3.1298.
Full textAbstract:
eIF2B is a five-subunit guanine nucleotide exchange factor that is negatively regulated by phosphorylation of the alpha subunit of its substrate, eIF2, leading to inhibition of translation initiation. To analyze this regulatory mechanism, we have characterized 29 novel mutations in the homologous eIF2B subunits encoded by GCD2, GCD7, and GCN3 that reduce or abolish inhibition of eIF2B activity by eIF2 phosphorylated on its alpha subunit [eIF2(alphaP)]. Most, if not all, of the mutations decrease sensitivity to eIF2(alphaP) without excluding GCN3, the nonessential subunit, from eIF2B; thus, all three proteins are critical for regulation of eIF2B by eIF2(alphaP). The mutations are clustered at both ends of the homologous region of each subunit, within two segments each of approximately 70 amino acids in length. Several mutations alter residues at equivalent positions in two or all three subunits. These results imply that structurally similar segments in GCD2, GCD7, and GCN3 perform related functions in eIF2B regulation. We propose that these segments form a single domain in eIF2B that makes multiple contacts with the alpha subunit of eIF2, around the phosphorylation site, allowing eIF2B to detect and respond to phosphoserine at residue 51. Most of the eIF2 is phosphorylated in certain mutants, suggesting that these substitutions allow eIF2B to accept phosphorylated eIF2 as a substrate for nucleotide exchange.
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Moriguchi, Takashi, Lei Yu, Akihito Otsuki, Keiko Ainoya, Kim-Chew Lim, Masayuki Yamamoto, and James Douglas Engel. "Gata3Hypomorphic Mutant Mice Rescued with a Yeast Artificial Chromosome Transgene Suffer a Glomerular Mesangial Cell Defect." Molecular and Cellular Biology 36, no. 17 (June 13, 2016): 2272–81. http://dx.doi.org/10.1128/mcb.00173-16.
Full textAbstract:
GATA3 is a zinc finger transcription factor that plays a crucial role in embryonic kidney development, while its precise functions in the adult kidney remain largely unexplored. Here, we demonstrate that GATA3 is specifically expressed in glomerular mesangial cells and plays a critical role in the maintenance of renal glomerular function. Newly generatedGata3hypomorphic mutant mice exhibited neonatal lethality associated with severe renal hypoplasia. Normal kidney size was restored by breeding the hypomorphic mutant with a rescuing transgenic mouse line bearing a 662-kbGata3yeast artificial chromosome (YAC), and these animals (termed G3YR mice) survived to adulthood. However, most of the G3YR mice showed degenerative changes in glomerular mesangial cells, which deteriorated progressively during postnatal development. Consequently, the G3YR adult mice suffered severe renal failure. We found that the 662-kbGata3YAC transgene recapitulatedGata3expression in the renal tubules but failed to direct sufficient GATA3 activity to mesangial cells. Renal glomeruli of the G3YR mice had significantly reduced amounts of platelet-derived growth factor receptor (PDGFR), which is known to participate in the development and maintenance of glomerular mesangial cells. These results demonstrate a critical role for GATA3 in the maintenance of mesangial cells and its absolute requirement for prevention of glomerular disease.
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Ravid, K., T. Doi, D. L. Beeler, D. J. Kuter, and R. D. Rosenberg. "Transcriptional regulation of the rat platelet factor 4 gene: interaction between an enhancer/silencer domain and the GATA site." Molecular and Cellular Biology 11, no. 12 (December 1991): 6116–27. http://dx.doi.org/10.1128/mcb.11.12.6116.
Full textAbstract:
We used various segments of the 5' upstream region of the rat platelet factor 4 (PF4) gene coupled to the human growth hormone gene and heterologous promoters to identify domains which are critical for tissue-specific expression. Transient expression experiments with rat bone marrow cells and other cell lines revealed a complex interplay between a core promoter domain from -97 to the transcriptional start site and an enhancer/silencer domain from -448 to -112. The core promoter contains a GATA site at -31 to -28 whose mutation to TATA or AATA decreases tissue specificity and moderately affects expression in megakaryocytes as well as a positively acting subdomain from -97 to -83 whose removal decreases overall transcription without affecting tissue specificity. The enhancer/silencer domain possesses three positively acting subdomains from -380 to -362, -270 to -257, and -137 to -120 as well as a negatively acting subdomain at -184 to -151 which is able to reduce overall transcription but has no effect on tissue specificity. The subdomain from -380 to -362 is most critical in restricting gene expression driven either by the PF4 promoter or by a heterologous promoter to the megakaryocytic lineage. The subdomains from -270 to -257 and -137 to -120 function together with the subdomain from -380 to -362 to somewhat increase tissue specificity. Simultaneous mutation of the GATA site and deletion of either the whole enhancer/silencer domain or the subdomain from -380 to -362 or -137 to -120 reduce transcription in megakaryocytes by 10- to 30-fold. On the basis of the above-described results, we propose that the megakaryocyte-specific enhancer/silencer domain and the GATA site are responsible for high-level expression of the PF4 gene in a lineage-specific manner.
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Lahlil, Rachid, Eric Lécuyer, Sabine Herblot, and Trang Hoang. "SCL Assembles a Multifactorial Complex That Determines Glycophorin A Expression." Molecular and Cellular Biology 24, no. 4 (February 15, 2004): 1439–52. http://dx.doi.org/10.1128/mcb.24.4.1439-1452.2004.
Full textAbstract:
ABSTRACT SCL/TAL1 is a hematopoietic-specific transcription factor of the basic helix-loop-helix (bHLH) family that is essential for erythropoiesis. Here we identify the erythroid cell-specific glycophorin A gene (GPA) as a target of SCL in primary hematopoietic cells and show that SCL occupies the GPA locus in vivo. GPA promoter activation is dependent on the assembly of a multifactorial complex containing SCL as well as ubiquitous (E47, Sp1, and Ldb1) and tissue-specific (LMO2 and GATA-1) transcription factors. In addition, our observations suggest functional specialization within this complex, as SCL provides its HLH protein interaction motif, GATA-1 exerts a DNA-tethering function through its binding to a critical GATA element in the GPA promoter, and E47 requires its N-terminal moiety (most likely entailing a transactivation function). Finally, endogenous GPA expression is disrupted in hematopoietic cells through the dominant-inhibitory effect of a truncated form of E47 (E47-bHLH) on E-protein activity or of FOG (Friend of GATA) on GATA activity or when LMO2 or Ldb-1 protein levels are decreased. Together, these observations reveal the functional complementarities of transcription factors within the SCL complex and the essential role of SCL as a nucleation factor within a higher-order complex required to activate gene GPA expression.
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Smoak, Kathleen, and John A. Cidlowski. "Glucocorticoids Regulate Tristetraprolin Synthesis and Posttranscriptionally Regulate Tumor Necrosis Factor Alpha Inflammatory Signaling." Molecular and Cellular Biology 26, no. 23 (September 18, 2006): 9126–35. http://dx.doi.org/10.1128/mcb.00679-06.
Full textAbstract:
ABSTRACT Glucocorticoids are used to treat various inflammatory disorders, but the mechanisms underlying these actions are incompletely understood. The zinc finger protein tristetraprolin (TTP) destabilizes several proinflammatory cytokine mRNAs by binding to AU-rich elements within their 3′ untranslated regions, targeting them for degradation. Here we report that glucocorticoids induce the synthesis of TTP mRNA and protein in A549 lung epithelial cells and in rat tissues. Dexamethasone treatment leads to a sustained induction of TTP mRNA expression that is abrogated by RU486. Glucocorticoid induction of TTP mRNA is also blocked by actinomycin D but not by cycloheximide, suggesting a transcriptional mechanism which has been confirmed by transcription run-on experiments. The most widely characterized TTP-regulated gene is the AU-rich tumor necrosis factor alpha (TNF-α) gene. Dexamethasone represses TNF-α mRNA in A549 cells and decreases luciferase expression of a TNF-α 3′ untranslated region reporter plasmid in an orientation-dependent manner. Small interfering RNAs to TTP significantly prevent this effect, and a cell line stably expressing a short-hairpin RNA to TTP conclusively establishes that TTP is critical for dexamethasone inhibition of TNF-α mRNA expression. These studies provide the molecular evidence for glucocorticoid regulation of human TTP and reflect a novel inductive anti-inflammatory signaling pathway for glucocorticoids that acts via posttranscriptional mechanisms.
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Han, Yu, and Véronique Lefebvre. "L-Sox5 and Sox6 Drive Expression of the Aggrecan Gene in Cartilage by Securing Binding of Sox9 to a Far-Upstream Enhancer." Molecular and Cellular Biology 28, no. 16 (June 16, 2008): 4999–5013. http://dx.doi.org/10.1128/mcb.00695-08.
Full textAbstract:
ABSTRACT The Sry-related high-mobility-group box transcription factor Sox9 recruits the redundant L-Sox5 and Sox6 proteins to effect chondrogenesis, but the mode of action of the trio remains unclear. We identify here a highly conserved 359-bp sequence 10 kb upstream of the Agc1 gene for aggrecan, a most essential cartilage proteoglycan and key marker of chondrocyte differentiation. This sequence directs expression of a minimal promoter in both embryonic and adult cartilage in transgenic mice, in a manner that matches Agc1 expression. The chondrogenic trio is required and sufficient to mediate the activity of this enhancer. It acts directly, Sox9 binding to a critical cis-acting element and L-Sox5/Sox6 binding to three additional elements, which are cooperatively needed. Upon binding to their specific sites, L-Sox5/Sox6 increases the efficiency of Sox9 binding to its own recognition site and thereby robustly potentiates the ability of Sox9 to activate the enhancer. L-Sox5/Sox6 similarly secures Sox9 binding to Col2a1 (encoding collagen-2) and other cartilage-specific enhancers. This study thus uncovers critical cis-acting elements and transcription factors driving Agc1 expression in cartilage and increases understanding of the mode of action of the chondrogenic Sox trio.
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Ravid, K., T. Doi, D. L. Beeler, D. J. Kuter, and R. D. Rosenberg. "Transcriptional regulation of the rat platelet factor 4 gene: interaction between an enhancer/silencer domain and the GATA site." Molecular and Cellular Biology 11, no. 12 (December 1991): 6116–27. http://dx.doi.org/10.1128/mcb.11.12.6116-6127.1991.
Full textAbstract:
We used various segments of the 5' upstream region of the rat platelet factor 4 (PF4) gene coupled to the human growth hormone gene and heterologous promoters to identify domains which are critical for tissue-specific expression. Transient expression experiments with rat bone marrow cells and other cell lines revealed a complex interplay between a core promoter domain from -97 to the transcriptional start site and an enhancer/silencer domain from -448 to -112. The core promoter contains a GATA site at -31 to -28 whose mutation to TATA or AATA decreases tissue specificity and moderately affects expression in megakaryocytes as well as a positively acting subdomain from -97 to -83 whose removal decreases overall transcription without affecting tissue specificity. The enhancer/silencer domain possesses three positively acting subdomains from -380 to -362, -270 to -257, and -137 to -120 as well as a negatively acting subdomain at -184 to -151 which is able to reduce overall transcription but has no effect on tissue specificity. The subdomain from -380 to -362 is most critical in restricting gene expression driven either by the PF4 promoter or by a heterologous promoter to the megakaryocytic lineage. The subdomains from -270 to -257 and -137 to -120 function together with the subdomain from -380 to -362 to somewhat increase tissue specificity. Simultaneous mutation of the GATA site and deletion of either the whole enhancer/silencer domain or the subdomain from -380 to -362 or -137 to -120 reduce transcription in megakaryocytes by 10- to 30-fold. On the basis of the above-described results, we propose that the megakaryocyte-specific enhancer/silencer domain and the GATA site are responsible for high-level expression of the PF4 gene in a lineage-specific manner.
29
DuRose, Jenny B., Donalyn Scheuner, Randal J. Kaufman, Lawrence I. Rothblum, and Maho Niwa. "Phosphorylation of Eukaryotic Translation Initiation Factor 2α Coordinates rRNA Transcription and Translation Inhibition during Endoplasmic Reticulum Stress." Molecular and Cellular Biology 29, no. 15 (May 26, 2009): 4295–307. http://dx.doi.org/10.1128/mcb.00260-09.
Full textAbstract:
ABSTRACT The endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed the capacity of the ER, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Here, we report that the UPR downregulates the synthesis of rRNA by inactivation of the RNA polymerase I basal transcription factor RRN3/TIF-IA. Inhibition of rRNA synthesis does not appear to involve the well-characterized mTOR (mammalian target of rapamycin) pathway; instead, PERK-dependent phosphorylation of eIF2α plays a critical role in the inactivation of RRN3/TIF-IA. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2α phosphorylation-induced translation repression. Since rRNA is the most abundant RNA species, constituting ∼90% of total cellular RNA, its downregulation exerts a significant impact on cell physiology. Our study demonstrates the first link between regulation of translation and rRNA synthesis with phosphorylation of eIF2α, suggesting that this pathway may be broadly utilized by stresses that activate eIF2α kinases in order to coordinately regulate translation and ribosome biogenesis during cellular stress.
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Chen, H. E., S. Chang, T. Trub, and B. G. Neel. "Regulation of colony-stimulating factor 1 receptor signaling by the SH2 domain-containing tyrosine phosphatase SHPTP1." Molecular and Cellular Biology 16, no. 7 (July 1996): 3685–97. http://dx.doi.org/10.1128/mcb.16.7.3685.
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SHPTP1 (PTP1C, HCP, SHP) is an SH2 domain-containing tyrosine phosphatase expressed predominantly in hematopoietic cells. A frameshift mutation in the SHPTP1 gene causes the motheaten (me/me) mouse. These mice are essentially SHPTP1 null and display multiple hematopoietic abnormalities, most prominently hyperproliferation and inappropriate activation of granulocytes and macrophages. The me/me phenotype suggests that SHPTP1 negatively regulates macrophage proliferative pathways. Using primary bone marrow-derived macrophages from me/me mice and normal littermates, we examined the role of SHPTP1 in regulating signaling by the major macrophage mitogen colony-stimulating factor 1 (CSF-1) (also known as macrophage colony-stimulating factor). Macrophages from me/me mice hyperproliferate in response to CSF-1. In the absence of SHPTP1, the CSF-1 receptor (CSF-1R) is hyperphosphorylated upon CSF-1 stimulation, suggesting that SHPTP1 dephosphorylates the CSF-1R. At least some CSF-1R-associated proteins also are hyperactivated. SHPTP1 is associated constitutively, via its SH2 domains, with an unidentified 130-kDa phosphotyrosyl protein (P130). P130 and SHPTP1 are further tyrosyl phosphorylated upon CSF-1 stimulation. Tyrosyl-phosphorylated SHPTP1 binds to Grb2 via the Grb2 SH2 domain. Moreover, in me/me macrophages, Grb2 is associated, via its SH3 domains, with several tyrosyl phosphoproteins. These proteins are hyperphosphorylated on tyrosyl residues in me/me macrophages, suggesting that Grb2 may recruit substrates for SHPTP1. Our results indicate that SHPTP1 is a critical negative regulator of CSF-1 signaling in vivo and suggest a potential new function for Grb2.
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Wang, Y., V. Buggia-Prévot, M. E. Zavorka, R. C. Bleackley, R. G. MacDonald, G. Thinakaran, and S. Kar. "Overexpression of the Insulin-Like Growth Factor II Receptor Increases β-Amyloid Production and Affects Cell Viability." Molecular and Cellular Biology 35, no. 14 (May 4, 2015): 2368–84. http://dx.doi.org/10.1128/mcb.01338-14.
Full textAbstract:
Amyloid β (Aβ) peptides originating from amyloid precursor protein (APP) in the endosomal-lysosomal compartments play a critical role in the development of Alzheimer's disease (AD), the most common type of senile dementia affecting the elderly. Since insulin-like growth factor II (IGF-II) receptors facilitate the delivery of nascent lysosomal enzymes from thetrans-Golgi network to endosomes, we evaluated their role in APP metabolism and cell viability using mouse fibroblast MS cells deficient in the murine IGF-II receptor and corresponding MS9II cells overexpressing the human IGF-II receptors. Our results show that IGF-II receptor overexpression increases the protein levels of APP. This is accompanied by an increase of β-site APP-cleaving enzyme 1 levels and an increase of β- and γ-secretase enzyme activities, leading to enhanced Aβ production. At the cellular level, IGF-II receptor overexpression causes localization of APP in perinuclear tubular structures, an increase of lipid raft components, and increased lipid raft partitioning of APP. Finally, MS9II cells are more susceptible to staurosporine-induced cytotoxicity, which can be attenuated by β-secretase inhibitor. Together, these results highlight the potential contribution of IGF-II receptor to AD pathology not only by regulating expression/processing of APP but also by its role in cellular vulnerability.
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Ueki, Kohjiro, David A. Fruman, Saskia M. Brachmann, Yu-Hua Tseng, Lewis C. Cantley, and C. Ronald Kahn. "Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival." Molecular and Cellular Biology 22, no. 3 (February 1, 2002): 965–77. http://dx.doi.org/10.1128/mcb.22.3.965-977.2002.
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ABSTRACT Class Ia phosphoinositide (PI) 3-kinase is a central component in growth factor signaling and is comprised of a p110 catalytic subunit and a regulatory subunit, the most common family of which is derived from the p85α gene (Pik3r1). Optimal signaling through the PI 3-kinase pathway depends on a critical molecular balance between the regulatory and catalytic subunits. In wild-type cells, the p85 subunit is more abundant than p110, leading to competition between the p85 monomer and the p85-p110 dimer and ineffective signaling. Heterozygous disruption of Pik3r1 results in increased Akt activity and decreased apoptosis by insulin-like growth factor 1 (IGF-1) through up-regulated phosphatidylinositol (3,4,5)-triphosphate production. Complete depletion of p85α, on the other hand, results in significantly increased apoptosis due to reduced PI 3-kinase-dependent signaling. Thus, a reduction in p85α represents a novel therapeutic target for enhancing IGF-1/insulin signaling, prolongation of cell survival, and protection against apoptosis.
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Andrisani, O. M., D. A. Pot, Z. Zhu, and J. E. Dixon. "Three sequence-specific DNA-protein complexes are formed with the same promoter element essential for expression of the rat somatostatin gene." Molecular and Cellular Biology 8, no. 5 (May 1988): 1947–56. http://dx.doi.org/10.1128/mcb.8.5.1947.
Full textAbstract:
We identified three sequence-specific DNA-protein complexes which are formed after in vitro binding of nuclear extracts, derived from neuronal (CA-77, rat brain) or non-neuronal (HeLa) cells, to positions -70 to -29 of the rat somatostatin promoter. The protein(s) responsible for the formation of the three sequence-specific complexes was fractionated from rat brain whole cell extracts by DEAE-Sepharose chromatography. The critical contact residues of the factor(s) in each complex, as determined by methylation interference analyses, are located within positions -59 to -35, which is protected from DNase I digestion; these include the G residues of a TGACGTCA consensus also found in the cAMP-responsive human enkephalin (positions -105 to -76) and E1A-inducible adenovirus type 5 E3 (positions -72 to -42) promoters. Competition assays with these heterologous promoters reveal that the factor(s) of each complex displays approximately 50-fold greater affinity for the somatostatin promoter-binding site. Synthetic oligonucleotides spanning positions -70 to -29 of the somatostatin promoter and containing single-base substitutions of the G residues in the TGACGTCA consensus were utilized in competition assays. The G residues located in the center of the module are the most critical determinants in the formation of the three sequence-specific complexes. Deletions disrupting the TGACGTCA consensus abolish not only formation of the three complexes in vitro but also expression of the somatostatin promoter in vivo, suggesting that formation of one or more of these complexes is essential for transcription of the rat somatostatin gene.
34
Carreira, S., T. J. Dexter, U. Yavuzer, D. J. Easty, and C. R. Goding. "Brachyury-Related Transcription Factor Tbx2 and Repression of the Melanocyte-Specific TRP-1 Promoter." Molecular and Cellular Biology 18, no. 9 (September 1, 1998): 5099–108. http://dx.doi.org/10.1128/mcb.18.9.5099.
Full textAbstract:
ABSTRACT Previous work has demonstrated that two key melanocyte-specific elements termed the MSEu and MSEi play critical roles in the expression of the melanocyte-specific tyrosinase-related protein 1 (TRP-1) promoter. Both the MSEu and MSEi, located at position −237 and at the initiator, respectively, bind a melanocyte-specific factor termed MSF but are also recognized by a previously uncharacterized repressor, since mutations affecting either of these elements result in strong up-regulation of TRP-1 promoter activity in melanoma cells. Here we demonstrate that repression mediated by the MSEu and MSEi also operates in melanocytes. We also report that both the MSEu and MSEi are recognized by the brachyury-related transcription factor Tbx2, a member of the recently described T-box family, and that Tbx2 is expressed in melanocyte and melanoblast cell lines but not in melanoblast precursor cells. Although Tbx2 and MSF each recognize the TRP-1 MSEu and MSEi motifs, it is binding by Tbx-2, not binding by MSF, that correlates with repression. Several lines of evidence tend to point to the brachyury-related transcription factor Tbx2 as being the repressor of TRP-1 expression: both the MSEu and MSEi bind Tbx2, and mutations in either element that result in derepression of the TRP-1 promoter diminish binding by Tbx2; the TRP-1 promoter, but not the tyrosinase, microphthalmia, or glyceraldehyde-3-phosphate dehydrogenase (G3PDH) promoter, is repressed by Tbx2 in cotransfection assays; a high-affinity consensus brachyury/Tbx2-binding site is able to constitutively repress expression of the heterologous IE110 promoter; and a low-affinity brachyury/Tbx2 binding site is able to mediate Tbx2-dependent repression of the G3PDH promoter. Although we cannot rule out the presence of an additional, as yet unidentified factor playing a role in the negative regulation of TRP-1 in vivo, the evidence presented here suggests that Tbx2 most likely is the previously unidentified repressor of TRP-1 expression and as such is likely to represent the first example of transcriptional repression by a T-box family member.
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Barnard, Daron C., Quiping Cao, and Joel D. Richter. "Differential Phosphorylation Controls Maskin Association with Eukaryotic Translation Initiation Factor 4E and Localization on the Mitotic Apparatus." Molecular and Cellular Biology 25, no. 17 (September 1, 2005): 7605–15. http://dx.doi.org/10.1128/mcb.25.17.7605-7615.2005.
Full textAbstract:
ABSTRACT Several cytoplasmic polyadenylation element (CPE)-containing mRNAs that are repressed in Xenopus oocytes become active during meiotic maturation. A group of factors that are anchored to the CPE are responsible for this repression and activation. Two of the most important are CPEB, which binds directly to the CPE, and Maskin, which associates with CPEB. In oocytes, Maskin also binds eukaryotic translation initiation factor 4E (eIF4E), an interaction that excludes eIF4G and prevents formation of the eIF4F initiation complex. When the oocytes are stimulated to reenter the meiotic divisions (maturation), CPEB promotes cytoplasmic polyadenylation. The newly elongated poly(A) tail becomes bound by poly(A) binding protein (PABP), which in turn binds eIF4G and helps it displace Maskin from eIF4E, thereby inducing translation. Here we show that Maskin undergoes several phosphorylation events during oocyte maturation, some of which are important for its dissociation from eIF4E and translational activation of CPE-containing mRNA. These sites are T58, S152, S311, S343, S453, and S638 and are phosphorylated by cdk1. Mutation of these sites to alanine alleviates the cdk1-induced dissociation of Maskin from eIF4E. Prior to maturation, Maskin is phosphorylated on S626 by protein kinase A. While this modification has no detectable effect on translation during oocyte maturation, it is critical for this protein to localize on the mitotic apparatus in somatic cells. These results show that Maskin activity and localization is controlled by differential phosphorylation.
36
Andrisani, O. M., D. A. Pot, Z. Zhu, and J. E. Dixon. "Three sequence-specific DNA-protein complexes are formed with the same promoter element essential for expression of the rat somatostatin gene." Molecular and Cellular Biology 8, no. 5 (May 1988): 1947–56. http://dx.doi.org/10.1128/mcb.8.5.1947-1956.1988.
Full textAbstract:
We identified three sequence-specific DNA-protein complexes which are formed after in vitro binding of nuclear extracts, derived from neuronal (CA-77, rat brain) or non-neuronal (HeLa) cells, to positions -70 to -29 of the rat somatostatin promoter. The protein(s) responsible for the formation of the three sequence-specific complexes was fractionated from rat brain whole cell extracts by DEAE-Sepharose chromatography. The critical contact residues of the factor(s) in each complex, as determined by methylation interference analyses, are located within positions -59 to -35, which is protected from DNase I digestion; these include the G residues of a TGACGTCA consensus also found in the cAMP-responsive human enkephalin (positions -105 to -76) and E1A-inducible adenovirus type 5 E3 (positions -72 to -42) promoters. Competition assays with these heterologous promoters reveal that the factor(s) of each complex displays approximately 50-fold greater affinity for the somatostatin promoter-binding site. Synthetic oligonucleotides spanning positions -70 to -29 of the somatostatin promoter and containing single-base substitutions of the G residues in the TGACGTCA consensus were utilized in competition assays. The G residues located in the center of the module are the most critical determinants in the formation of the three sequence-specific complexes. Deletions disrupting the TGACGTCA consensus abolish not only formation of the three complexes in vitro but also expression of the somatostatin promoter in vivo, suggesting that formation of one or more of these complexes is essential for transcription of the rat somatostatin gene.
37
Itoh, K., K. Igarashi, N. Hayashi, M. Nishizawa, and M. Yamamoto. "Cloning and characterization of a novel erythroid cell-derived CNC family transcription factor heterodimerizing with the small Maf family proteins." Molecular and Cellular Biology 15, no. 8 (August 1995): 4184–93. http://dx.doi.org/10.1128/mcb.15.8.4184.
Full textAbstract:
The chicken beta-globin enhancer is critical for the tissue- and developmental stage-specific expression of the beta-globin genes. This enhancer contains two indispensable cis elements, one containing two GATA sites and the other containing an NF-E2 site. To identify the putative transcription factor acting through the NF-E2 motif in the chicken beta-globin enhancer, we screened chicken cDNA libraries with a mouse p45 NF-E2 cDNA probe and isolated cDNA clones which encode a protein of 582 amino acid residues. This protein contains a region that includes the basic region-leucine zipper domain which is well conserved among members of the CNC family proteins (Cap 'n' collar, p45 NF-E2, LCR-F1, Nrf1, and Nrf2). Hence, we named this protein ECH (erythroid cell-derived protein with CNC homology). ECH is expressed abundantly in cultured erythroid cells undergoing terminal differentiation, peripheral erythrocytes, and some nonhematopoietic tissues. Since most of the cDNA clones obtained from the chicken erythrocyte cDNA library encoded ECH, ECH is likely the predominant CNC family protein present in avian peripheral erythrocytes. Like p45 NF-E2, ECH can heterodimerize with any of the small Maf family proteins and bind the NF-E2 site as a heterodimer in vitro. In a transfection assay, ECH transactivates transcription depending on the presence of NF-E2 sites on the reporter gene plasmid. These results indicate that ECH is likely a key regulator of avian erythropoiesis.
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Pan, Shi, Ping An, Rong Zhang, Xiangrong He, Guoyong Yin, and Wang Min. "Etk/Bmx as a Tumor Necrosis Factor Receptor Type 2-Specific Kinase: Role in Endothelial Cell Migration and Angiogenesis." Molecular and Cellular Biology 22, no. 21 (November 1, 2002): 7512–23. http://dx.doi.org/10.1128/mcb.22.21.7512-7523.2002.
Full textAbstract:
ABSTRACT Tumor necrosis factor (TNF) is a cytokine that mediates many pathophysiologial processes, including angiogenesis. However, the molecular signaling involved in TNF-induced angiogenesis has not been determined. In this study, we examined the role of Etk/Bmx, an endothelial/epithelial tyrosine kinase involved in cell adhesion, migration, and survival in TNF-induced angiogenesis. We show that TNF activates Etk specifically through TNF receptor type 2 (TNFR2) as demonstrated by studies using a specific agonist to TNFR2 and TNFR2-deficient cells. Etk forms a preexisting complex with TNFR2 in a ligand-independent manner, and the association is through multiple domains (pleckstrin homology domain, TEC homology domain, and SH2 domain) of Etk and the C-terminal domain of TNFR2. The C-terminal 16-amino-acid residues of TNFR2 are critical for Etk association and activation, and this Etk-binding and activating motif in TNFR2 is not overlapped with the TNFR-associated factor type 2 (TRAF2)-binding sequence. Thus, TRAF2 is not involved in TNF-induced Etk activation, suggesting a novel mechanism for Etk activation by cytokine receptors. Moreover, a constitutively active form of Etk enhanced, whereas a dominant-negative Etk blocked, TNF-induced endothelial cell migration and tube formation. While most TNF actions have been attributed to TNFR1, our studies demonstrate that Etk is a TNFR2-specific kinase involved in TNF-induced angiogenic events.
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Dent, C. L., K. A. Lillycrop, J. K. Estridge, N. S. Thomas, and D. S. Latchman. "The B-cell and neuronal forms of the octamer-binding protein Oct-2 differ in DNA-binding specificity and functional activity." Molecular and Cellular Biology 11, no. 8 (August 1991): 3925–30. http://dx.doi.org/10.1128/mcb.11.8.3925.
Full textAbstract:
B lymphocytes contain an octamer-binding transcription factor, Oct-2, that is absent in most other cell types and plays a critical role in the B-cell-specific transcription of the immunoglobulin genes. A neuronal form of this protein has also been detected in brain and neuronal cell lines by using a DNA mobility shift assay, and an Oct-2 mRNA is observed in these cells by Northern (RNA) blotting and in situ hybridization. We show that the neuronal form of Oct-2 differs from that found in B cells with respect to both DNA-binding specificity and functional activity. In particular, whereas the B-cell protein activates octamer-containing promoters, the neuronal protein inhibits octamer-mediated gene expression. The possible role of the neuronal form of Oct-2 in the regulation of neuronal gene expression and its relationship to B-cell Oct-2 are discussed.
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Tao, Ye, Jianbo Wang, Tsuyoshi Tokusumi, Kathleen Gajewski, and Robert A. Schulz. "Requirement of the LIM Homeodomain Transcription Factor Tailup for Normal Heart and Hematopoietic Organ Formation in Drosophila melanogaster." Molecular and Cellular Biology 27, no. 11 (March 19, 2007): 3962–69. http://dx.doi.org/10.1128/mcb.00093-07.
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ABSTRACT Dorsal vessel morphogenesis in Drosophila melanogaster serves as a superb system with which to study the cellular and genetic bases of heart tube formation. We used a cardioblast-expressed Toll-GFP transgene to screen for additional genes involved in heart development and identified tailup as a locus essential for normal dorsal vessel formation. tailup, related to vertebrate islet1, encodes a LIM homeodomain transcription factor expressed in all cardioblasts and pericardial cells of the heart tube as well as in associated lymph gland hematopoietic organs and alary muscles that attach the dorsal vessel to the epidermis. A transcriptional enhancer regulating expression in these four cell types was identified and used as a tailup-GFP transgene with additional markers to characterize dorsal vessel defects resulting from gene mutations. Two reproducible phenotypes were observed in mutant embryos: hypoplastic heart tubes with misaligned cardioblasts and the absence of most lymph gland and pericardial cells. Conversely, a significant expansion of the lymph glands and abnormal morphology of the heart were observed when tailup was overexpressed in the mesoderm. Tailup was shown to bind to two DNA recognition sequences in the dorsal vessel enhancer of the Hand basic helix-loop-helix transcription factor gene, with one site proven to be essential for the lymph gland, pericardial cell, and Svp/Doc cardioblast expression of Hand. Together, these results establish Tailup as being a critical new transcription factor in dorsal vessel morphogenesis and lymph gland formation and place this regulator directly upstream of Hand in these developmental processes.
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Fiorillo, Marco, Fanni Tóth, Matteo Brindisi, Federica Sotgia, and Michael P. Lisanti. "Deferiprone (DFP) Targets Cancer Stem Cell (CSC) Propagation by Inhibiting Mitochondrial Metabolism and Inducing ROS Production." Cells 9, no. 6 (June 23, 2020): 1529. http://dx.doi.org/10.3390/cells9061529.
Full textAbstract:
Deferiprone (DFP), also known as Ferriprox, is an FDA-approved, orally active, iron chelator that is currently used clinically for the treatment of iron-overload, especially in thalassaemia major. As iron is a critical factor in Fe-S cluster assembly that is absolutely required for the metabolic function of mitochondria, we hypothesized that DFP treatment could be used to selectively target mitochondria in cancer stem cells (CSCs). For this purpose, we used two ER(+) human breast cancer cell lines, namely MCF7 and T47D cells, as model systems. More specifically, a 3D tumorsphere assay was employed as a functional readout of CSC activity which measures anchorage-independent growth under low attachment conditions. Here, we show that DFP dose dependently inhibited the propagation of CSCs, with an IC-50 of ~100 nM for MCF7 and an IC-50 of ~0.5 to 1 μM for T47D cells, making DFP one the most potent FDA-approved drugs that we and others have thus far identified for targeting CSCs. Mechanistically, we show that high concentrations of DFP metabolically targeted both mitochondrial oxygen consumption (OCR) and glycolysis (extracellular acidification rates (ECAR)) in MCF7 and T47D cell monolayers. Most importantly, we demonstrate that DFP also induced a generalized increase in reactive oxygen species (ROS) and mitochondrial superoxide production, and its effects reverted in the presence of N-acetyl-cysteine (NAC). Therefore, we propose that DFP is a new candidate therapeutic for drug repurposing and for Phase II clinical trials aimed at eradicating CSCs.
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Salton, S. R., D. J. Fischberg, and K. W. Dong. "Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells." Molecular and Cellular Biology 11, no. 5 (May 1991): 2335–49. http://dx.doi.org/10.1128/mcb.11.5.2335.
Full textAbstract:
Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.
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Reeves, Wendy M., and Steven Hahn. "Targets of the Gal4 Transcription Activator in Functional Transcription Complexes." Molecular and Cellular Biology 25, no. 20 (October 15, 2005): 9092–102. http://dx.doi.org/10.1128/mcb.25.20.9092-9102.2005.
Full textAbstract:
ABSTRACT Although biochemical and genetic methods have detected many activator-transcription factor interactions, the direct functional targets of most activators remain undetermined. For this study, photo-cross-linkers positioned within the Gal4 C-terminal acidic activating region were used to identify polypeptides in close physical proximity to Gal4 during transcription activation in vitro. Of six specifically cross-linked polypeptides, three (Tra1, Taf12, and Gal11) are subunits of four complexes (SAGA, Mediator, NuA4, and TFIID) known to play a role in gene regulation. These cross-linking targets had differential effects on activation. SAGA was critical for activation by Gal4, Gal11 contributed modestly to activation, and TFIID and NuA4 were not important for activation under our conditions. Tra1, Taf12, and Gal11 have also been identified as cross-linking targets of the Gcn4 acidic central activating region. Our results demonstrate that two unrelated acidic activators converge on the same set of functional targets.
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Dent, C. L., K. A. Lillycrop, J. K. Estridge, N. S. Thomas, and D. S. Latchman. "The B-cell and neuronal forms of the octamer-binding protein Oct-2 differ in DNA-binding specificity and functional activity." Molecular and Cellular Biology 11, no. 8 (August 1991): 3925–30. http://dx.doi.org/10.1128/mcb.11.8.3925-3930.1991.
Full textAbstract:
B lymphocytes contain an octamer-binding transcription factor, Oct-2, that is absent in most other cell types and plays a critical role in the B-cell-specific transcription of the immunoglobulin genes. A neuronal form of this protein has also been detected in brain and neuronal cell lines by using a DNA mobility shift assay, and an Oct-2 mRNA is observed in these cells by Northern (RNA) blotting and in situ hybridization. We show that the neuronal form of Oct-2 differs from that found in B cells with respect to both DNA-binding specificity and functional activity. In particular, whereas the B-cell protein activates octamer-containing promoters, the neuronal protein inhibits octamer-mediated gene expression. The possible role of the neuronal form of Oct-2 in the regulation of neuronal gene expression and its relationship to B-cell Oct-2 are discussed.
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Samarsky, Dmitry A., and Maurille J. Fournier. "Functional Mapping of the U3 Small Nucleolar RNA from the Yeast Saccharomyces cerevisiae." Molecular and Cellular Biology 18, no. 6 (June 1, 1998): 3431–44. http://dx.doi.org/10.1128/mcb.18.6.3431.
Full textAbstract:
ABSTRACT The U3 small nucleolar RNA participates in early events of eukaryotic pre-rRNA cleavage and is essential for formation of 18S rRNA. Using an in vivo system, we have developed a functional map of the U3 small nucleolar RNA from Saccharomyces cerevisiae. The test strain features a galactose-dependent U3 gene in the chromosome and a plasmid-encoded allele with a unique hybridization tag. Effects of mutations on U3 production were analyzed by evaluating RNA levels in cells grown on galactose medium, and effects on U3 function were assessed by growing cells on glucose medium. The major findings are as follows: (i) boxes C′ and D and flanking helices are critical for U3 accumulation; (ii) boxes B and C are not essential for U3 production but are important for function, most likely due to binding of a trans-acting factor(s); (iii) the 5′ portion of U3 is required for function but not stability; and, (iv) strikingly, the nonconserved hairpins 2, 3, and 4, which account for 50% of the molecule, are not required for accumulation or function.
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Holt, S. E., W. E. Wright, and J. W. Shay. "Regulation of telomerase activity in immortal cell lines." Molecular and Cellular Biology 16, no. 6 (June 1996): 2932–39. http://dx.doi.org/10.1128/mcb.16.6.2932.
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Telomerase is a ribonucleoprotein whose activity has been detected in germ line cells, immortal cells, and most cancer cells. Except in stem cells, which have a low level of telomerase activity, its activity is absent from normal somatic tissues. Understanding the regulation of telomerase activity is critical for the development of potential tools for the diagnosis and treatment of cancer. Using the telomeric repeat amplification protocol, we found that immortal, telomerase-positive, pseudodiploid human cells (HT1080 and HL60 cells) sorted by flow repressed in quiescent cells. This was true whether quiescence was induced by contact inhibition (NIH 3T3 mouse cells), growth factor removal (bromodeoxyuridine-blocked mouse myoblasts), reexpression of cellular senescence (the reversibly immortalized IDH4 cells), or irreversible cell differentiation (HL60 promyelocytic leukemia cells and C2C12 mouse myoblasts). Taken together, these results indicate that telomerase is active throughout the cell in dividing, immortal cells but that its activity is repressed in cells that exit the cell cycle. This suggests that quiescent stem cells that have the potential to express telomerase may remain unaffected by potential antitelomerase cancer therapies.
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Hatanaka, Fumiyuki, Chiaki Matsubara, Jihwan Myung, Takashi Yoritaka, Naoko Kamimura, Shuichi Tsutsumi, Akinori Kanai, et al. "Genome-Wide Profiling of the Core Clock Protein BMAL1 Targets Reveals a Strict Relationship with Metabolism." Molecular and Cellular Biology 30, no. 24 (October 11, 2010): 5636–48. http://dx.doi.org/10.1128/mcb.00781-10.
Full textAbstract:
ABSTRACT Circadian rhythms are common to most organisms and govern much of homeostasis and physiology. Since a significant fraction of the mammalian genome is controlled by the clock machinery, understanding the genome-wide signaling and epigenetic basis of circadian gene expression is essential. BMAL1 is a critical circadian transcription factor that regulates genes via E-box elements in their promoters. We used multiple high-throughput approaches, including chromatin immunoprecipitation-based systematic analyses and DNA microarrays combined with bioinformatics, to generate genome-wide profiles of BMAL1 target genes. We reveal that, in addition to E-boxes, the CCAATG element contributes to elicit robust circadian expression. BMAL1 occupancy is found in more than 150 sites, including all known clock genes. Importantly, a significant proportion of BMAL1 targets include genes that encode central regulators of metabolic processes. The database generated in this study constitutes a useful resource to decipher the network of circadian gene control and its intimate links with several fundamental physiological functions.
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Salton, S. R., D. J. Fischberg, and K. W. Dong. "Structure of the gene encoding VGF, a nervous system-specific mRNA that is rapidly and selectively induced by nerve growth factor in PC12 cells." Molecular and Cellular Biology 11, no. 5 (May 1991): 2335–49. http://dx.doi.org/10.1128/mcb.11.5.2335-2349.1991.
Full textAbstract:
Nerve growth factor (NGF) plays a critical role in the development and survival of neurons in the peripheral nervous system. Following treatment with NGF but not epidermal growth factor, rat pheochromocytoma (PC12) cells undergo neural differentiation. We have cloned a nervous system-specific mRNA, NGF33.1, that is rapidly and relatively selectively induced by treatment of PC12 cells with NGF and basic fibroblast growth factor in comparison with epidermal growth factor. Analysis of the nucleic acid and predicted amino acid sequences of the NGF33.1 cDNA clone suggested that this clone corresponded to the NGF-inducible mRNA called VGF (A. Levi, J. D. Eldridge, and B. M. Paterson, Science 229:393-395, 1985; R. Possenti, J. D. Eldridge, B. M. Paterson, A. Grasso, and A. Levi, EMBO J. 8:2217-2223, 1989). We have used the NGF33.1 cDNA clone to isolate and characterize the VGF gene, and in this paper we report the complete sequence of the VGF gene, including 853 bases of 5' flank revealed TATAA and CCAAT elements, several GC boxes, and a consensus cyclic AMP response element-binding protein binding site. The VGF promoter contains sequences homologous to other NGF-inducible, neuronal promoters. We further show that VGF mRNA is induced in PC12 cells to a greater extent by depolarization and by phorbol-12-myristate-13-acetate treatment than by 8-bromo-cyclic AMP treatment. By Northern (RNA) and RNase protection analysis, VGF mRNA is detectable in embryonic and postnatal central and peripheral nervous tissues but not in a number of nonneural tissues. In the cascade of events which ultimately leads to the neural differentiation of NGF-treated PC12 cells, the VGF gene encodes the most rapidly and selectively regulated, nervous-system specific mRNA yet identified.
49
Barrett, David M., Karen S. Gustafson, Jing Wang, Shou Zhen Wang, and Gordon D. Ginder. "A GATA Factor Mediates Cell Type-Restricted Induction of HLA-E Gene Transcription by Gamma Interferon." Molecular and Cellular Biology 24, no. 14 (July 15, 2004): 6194–204. http://dx.doi.org/10.1128/mcb.24.14.6194-6204.2004.
Full textAbstract:
ABSTRACT The human major histocompatibility complex (MHC) class Ib gene, HLA-E, codes for the major ligand of the inhibitory receptor NK-G-2A, which is present on most natural killer (NK) cells and some CD8+ cytotoxic T lymphocytes. We have previously shown that gamma interferon (IFN-γ) induction of HLA-E gene transcription is mediated through a distinct IFN-γ-responsive element, the IFN response region (IRR), in all cell types studied. We have now identified and characterized a cell type-restricted enhancer of IFN-γ-mediated induction of HLA-E gene transcription, designated the upstream interferon response region (UIRR), which is located immediately upstream of the IRR. The UIRR mediates a three- to eightfold enhancement of IFN-γ induction of HLA-E transcription in some cell lines but not in others, and it functions only in the presence of an adjacent IRR. The UIRR contains a variant GATA binding site (AGATAC) that is critical to both IFN-γ responsiveness and to the formation of a specific binding complex containing GATA-1 in K562 cell nuclear extracts. The binding of GATA-1 to this site in response to IFN-γ was confirmed in vivo in a chromatin immunoprecipitation assay. Forced expression of GATA-1 in nonexpressing U937 cells resulted in a four- to fivefold enhancement of the IFN-γ response from HLA-E promoter constructs containing a wild-type but not a GATA-1 mutant UIRR sequence and increased the IFN-γ response of the endogenous HLA-E gene. Knockdown of GATA-1 expression in K562 cells resulted in a ∼4-fold decrease in the IFN-γ response of the endogenous HLA-E gene, consistent with loss of the increase in IFN-γ response of HLA-E promoter-driven constructs containing the UIRR in wild-type K562 cells. Coexpression of wild-type and mutant adenovirus E1a proteins that sequester p300/CBP eliminated IFN-γ-mediated enhancement through the UIRR, but only partially reduced induction through the IRR, implicating p300/CBP binding to Stat-1α at the IRR in the recruitment of GATA-1 to mediate the cooperation between the UIRR and IRR. We propose that the GATA-1 transcription factor represents a cell type-restricted mediator of IFN-γ induction of the HLA-E gene.
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Trasanidis, Nikolaos, Jaime Alvarez-Benayas, Alexia Katsarou, Aristeidis Chaidos, Philippa C. May, Kanagaraju Ponnusamy, Xiaolin Xiao, et al. "Distinct Chromatin Accessibility Changes, Aberrant Transcription Factor Networks Combined with Novel Oncogenic Enhancers Characterise Myeloma-Initiating Genetic Events." Blood 134, Supplement_1 (November 13, 2019): 1769. http://dx.doi.org/10.1182/blood-2019-124761.
Full textAbstract:
In multiple myeloma (MM), a malignancy of the bone marrow plasma cells (BMPC), hyperdiploidy (HY) and oncogene over-expression via chromosomal translocation [including CCND1- t(11;14), MAF- t(14;16), MMSET-t(4;14)] are the primary myeloma initiating events (MIE) that drive distinct transcriptional programs. These are further shaped by secondary SNV and CNV events. This genetic heterogeneity converges, in most cases, to a functionally dichotomous state of CCND1 or CCND2 overexpression. The molecular mechanisms underlying each of the distinct myelomagenic transcriptomes and the CCND1 vs CCND2 dichotomy have not been defined. To address these questions, we obtained highly purified BMPC from 3 healthy donors and 30 MM patients (HY: 15; CCND1: 4; MMSET: 5; MAF: 2; other: 4), either at diagnosis or relapse, and mapped their chromatin accessibility and transcriptome profiles by ATAC-seq and RNA-seq, respectively. In total, we obtained ~300K regions with accessible chromatin in either MM or normal PC. Overall chromatin accessibility increased in myeloma compared to normal PC, particularly in MAF- and MMSET-translocated subtypes. Analysis of combined ATAC-seq/RNA-seq by Multi-Omics Factor Analysis (MOFA) resulted in a clearer samples distinction than either ATAC-seq or RNA-seq alone, with altered chromatin accessibility accounting for more of the variance than expression. Of the top five identified factors, the top two (one transcriptome driven, one accessibility driven) distinguished normal from MM samples, whilst two more separated MMSET, MAF and CCND1 subgroups. Ninety seven, 157, 256 and 348 overexpressed genes in the CCND1, HY, MMSET and MAF subgroups, respectively, were predicted to be regulated by differentially accessible enhancers. Twenty percent (165/858) of these genes were overexpressed in >1 subgroup suggesting a process of chromatin accessibility-based convergence evolution. Enrichment analysis suggested direct or indirect involvement of Polycomb and chromatin remodellers; significant enrichment was also found for genes involved in neurogenesis. ATAC-seq footprinting predicted binding sites for 250 expressed transcription factors (TFs), 116 of which displayed higher binding frequency in myeloma than in normal PC and included both known (e.g., XBP1, RELA, IRF4, PRDM1) and potentially novel regulators of myeloma biology (e.g., CXXC1 and NFE2L1). The remaining 134 TF were predicted to be present in at least one MM subgroup, but absent in normal PC. Amongst them, as expected, MAF was active in the MMSET- and more so in the MAF-translocated subgroups. DepMap database analysis suggested myeloma cell dependency on 181/250 TF (CRISPR/Cas9 CERES score < -0.1 in >3/14 MMCL analysed). In dissecting the regulatory basis of CCND2 vs CCND1 dichotomy, one MOFA factor completely separated MAF from CCND1 samples, placing extreme opposite weights on the expression of CCND2 and CCND1 respectively. Interestingly, the same factor identified open-chromatin clusters upstream of CCND2 and linked them to its over-expression. These clusters were also open in the MMSET group and in CCND2-expressing HY samples. Conversely, no accessibility was detected in the CCND1 group, the CCND1-expressing HY samples or in normal PCs. Further, super-enhancer calling using the H3K27ac histone mark in MAF-translocated JJN3 cells identified the region of interest as a bona fide super-enhancer. Chromatin long range interactions, as assessed by Capture-HiC, demonstrated high frequency interactions of the CCND2 promoter with the constituent elements of the putative super-enhancer. Experimental validation using a CRISPR/Cas9i system confirmed the functional role of all 4 super-enhancer constituents tested in the regulation of CCND2 expression, while TF footprinting predicted MAF binding to the super-enhancer in MAF-translocated PC. In conclusion, we show that distinct oncogenic transcriptomes in MM are underpinned by extensive chromatin changes, accompanied by TF activity 're-wiring' that does not necessarily require transcriptional deregulation of the TF themselves. We identify novel, non-oncogene TF dependencies that suggest therapeutic opportunities in MM and we discover and characterise the critical super-enhancer that drives overexpression of the CCND2 oncogene in MM. Disclosures Auner: Amgen: Other: Consultancy and Research Funding; Takeda: Consultancy; Karyopharm: Consultancy. Hatjiharissi:Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.