Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin

The TSC2 gene, mutated in patients with tuberous sclerosis complex (TSC), encodes a 200 kDa protein TSC2 (tuberin). The importance of TSC2 in the regulation of cell growth and proliferation is irrefutable. TSC2 in complex with TSC1 negatively regulates the mTOR complex 1 (mTORC1) via RHEB in the PI3K-AKT-mTOR pathway and in turn regulates cell proliferation. It shows nuclear as well as cytoplasmic localization. However, its nuclear function remains elusive. In order to identify the nuclear function of TSC2, a whole-genome expression profiling of TSC2 overexpressing cells was performed, and the results showed differential regulation of 266 genes. Interestingly, transcription was found to be the most populated functional category. EREG (Epiregulin), a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in TSC lesions, making its regulatory aspects intriguing. Using the luciferase reporter, ChIP and EMSA techniques, we show that TSC2 binds to the EREG promoter between -352 bp and -303 bp and negatively regulates its expression. This is the first evidence for the role of TSC2 as a transcription factor and of TSC2 binding to the promoter of any gene.

Trm1p, a Zn(II)(2)Cys(6)-type transcription factor, is essential for the transcriptional activation of genes of methanol utilization pathway, in Pichia pastoris

The zinc finger transcription factors Mxr1p and Rop are key regulators of methanol metabolism in the methylotrophic yeast, Pichia pastoris, while Trm1p and Trm2p regulate methanol metabolism in Candida boidinii. Here, we demonstrate that Trm1p is essential for the expression of genes of methanol utilization (mut) pathway in P. pastoris as well. Expression of AOXI and other genes of mut pathway is severely compromised in P. pastoris Delta Trm1 strain resulting in impaired growth on media containing methanol as the sole source of carbon. Trm1p localizes to the nucleus of cells cultured on glucose or methanol. The zinc finger domain of Mxr1p but not Trm1p binds to AOXI promoter sequences in vitro, indicating that these two positive regulators act by different mechanisms. We conclude that both Trm1p and Mxr1p are essential for the expression of genes of mut pathway in P. pastoris and the mechanism of transcriptional regulation of mut pathway may be similar in P. pastoris and C. boidinii. (C) 2014 Elsevier Inc. All rights reserved.

Genomic mapping of cAMP receptor protein (CRPMt) in Mycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMt as a transcription factor

Chromatin immunoprecipitation identified 191 binding sites of Mycobacterium tuberculosis cAMP receptor protein (CRPMt) at endogenous expression levels using a specific alpha-CRPMt antibody. Under these native conditions an equal distribution between intragenic and intergenic locations was observed. CRPMt binding overlapped a palindromic consensus sequence. Analysis by RNA sequencing revealed widespread changes in transcriptional profile in a mutant strain lacking CRPMt during exponential growth, and in response to nutrient starvation. Differential expression of genes with a CRPMt-binding site represented only a minor portion of this transcriptional reprogramming with similar to 19% of those representing transcriptional regulators potentially controlled by CRPMt. The subset of genes that are differentially expressed in the deletion mutant under both culture conditions conformed to a pattern resembling canonical CRP regulation in Escherichia coli, with binding close to the transcriptional start site associated with repression and upstream binding with activation. CRPMt can function as a classical transcription factor in M. tuberculosis, though this occurs at only a subset of CRPMt-binding sites.

Dual role of MsRbpA: transcription activation and rescue of transcription from the inhibitory effect of rifampicin

MsRbpA is an RNA polymerase (RNAP) binding protein from Mycobacterium smegmatis. According to previous studies, MsRbpA rescues rifampicin-induced transcription inhibition upon binding to the RNAP. Others have shown that RbpA from Mycobacterium tuberculosis (MtbRbpA) is a transcription activator. In this study, we report that both MsRbpA and MtbRbpA activate transcription as well as rescue rifampicin-induced transcription inhibition. Transcription activation is achieved through the increased formation of closed RNAP-promoter complex as well as enhanced rate of conversion of this complex to a stable transcriptionally competent RNAP promoter complex. When a 16 aa peptide fragment (Asp 58 to Lys 73) was deleted from MsRbpA, the resulting protein showed 1000-fold reduced binding with core RNAP. The deletion results in abolition of transcription activation and rescue of transcription from the inhibitory effect of rifampicin. Through alanine scanning of this essential region of MsRbpA, Gly 67, Val 69, Pro 70 and Pro 72 residues are identified to be important for MsRbpA function. Furthermore, we report here that the protein is indispensable for M. smegmatis, and it appears to help the organism grow in the presence of the antibiotic rifampicin.

Estimation of intrinsic diffusion coefficients in a pseudo-binary diffusion couple

Major drawback of studying diffusion in multi-component systems is the lack of suitable techniques to estimate the diffusion parameters. In this study, a generalized treatment to determine the intrinsic diffusion coefficients in multi-component systems is developed utilizing the concept of a pseudo-binary approach. This is explained with the help of experimentally developed diffusion profiles in the Cu(Sn, Ga) and Cu(Sn, Si) solid solutions. (C) 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Different Modes of Transactivation of Bacteriophage Mu Late Promoters by Transcription Factor C

Transactivator protein C is required for the expression of bacteriophage Mu late genes from lys, I, P and mom promoters during lytic life cycle of the phage. The mechanism of transcription activation of mom gene by C protein is well understood. C activates transcription at Pmom by initial unwinding of the promoter DNA, thereby facilitating RNA polymerase (RNAP) recruitment. Subsequently, C interacts with the (sic) subunit of RNAP to enhance promoter clearance. The mechanism by which C activates other late genes of the phage is not known. We carried out promoter-polymerase interaction studies with all the late gene promoters to determine the individual step of C mediated activation. Unlike at P-mom, at the other three promoters, RNAP recruitment and closed complex formation are not C dependent. Instead, the action of C at P-lys, P-I, and P-P is during the isomerization from closed complex to open complex with no apparent effect at other steps of initiation pathway. The mechanism of transcription activation of mom and other late promoters by their common activator is different. This distinction in the mode of activation (promoter recruitment and escape versus isomerization) by the same activator at different promoters appears to be important for optimized expression of each of the late genes.

Evolution of bacterial transcription factors: how proteins take on new tasks, but do not always stop doing the old ones

Many bacterial transcription factors do not behave as per the textbook operon model. We draw on whole genome work, as well as reported diversity across different bacteria, to argue that transcription factors may have evolved from nucleoid-associated proteins. This view would explain a large amount of recent data gleaned from high-throughput sequencing and bioinformatic analyses.

Electronic structure origin of conductivity and oxygen reduction activity changes in low-level Cr-substituted (La, Sr)MnO3

The electronic structure of the (La0.8Sr0.2)(0.98)Mn1-xCrxO3 model series (x = 0, 0.05, or 0.1) was measured using soft X-ray synchrotron radiation at room and elevated temperature. O K-edge near-edge X-ray absorption fine structure (NEXAFS) spectra showed that low-level chromium substitution of (La, Sr)MnO3 resulted in lowered hybridisation between O 2p orbitals and M 3d and M 4sp valance orbitals. Mn L-3-edge resonant photoemission spectroscopy measurements indicated lowered Mn 3d-O 2p hybridisation with chromium substitution. Deconvolution of O K-edge NEXAFS spectra took into account the effects of exchange and crystal field splitting and included a novel approach whereby the pre-peak region was described using the nominally filled t(2g) up arrow state. 10% chromium substitution resulted in a 0.17 eV lowering in the energy of the t(2g) up arrow state, which appears to provide an explanation for the 0.15 eV rise in activation energy for the oxygen reduction reaction, while decreased overlap between hybrid O 2p-Mn 3d states was in qualitative agreement with lowered electronic conductivity. An orbital-level understanding of the thermodynamically predicted solid oxide fuel cell cathode poisoning mechanism involving low-level chromium substitution on the B-site of (La, Sr)MnO3 is presented. (C) 2015 AIP Publishing LLC.

Comparative study of Eu3+ -activated LnOCl (Ln=La and Gd) phosphors and their Judd-Ofelt analysis

Eu3+-activated layered LnOCl (Ln=La and Gd) phosphors were synthesized by the conventional solid-state method at relatively low temperature (700 degrees C) and shorter duration of 2 h. The structural parameters were refined by the Rietveld refinement analysis and confirmed by the high resolution transmission electron microscopy (HRTEM). Both the compounds were crystallized in the tetragonal structure with space group P4/nmm (No. 129). The homogeneity of the elements were analyzed by TEM mapping and found to be uniformly distributed. The photoluminescence spectra revealed that the intensity of D-5(0)-> F-7(2) transition (619 nm) was more intense in Eu3+-activated GdOCl compared to LaOCl. This was due to the property of Gd3+ ions to act as an intermediate sublattice to facilitate the energy transfer to Eu3+ ions. Intensity parameters and radiative properties such as transition probabilities, radiative lifetime and branching ratio were calculated using the Judd-Ofelt theory. The CIE color coordinates result revealed that the Eu3+-activated GdOCl (0.641, 0.354) phosphor was close to the commercial red phosphors like, Y2O3:Eu3+ (0.645, 0.347), (Y2OS)-S-2:Eu3+ (0.647, 0.343) and National Television System Committee (NTSC) (0.67, 0.33). The results suggest that the present GdOCl:Eu3+ compound acts as a potential candidate for red phosphor materials.

Common effect of chemical and external pressures on the magnetic properties of RCoPO (R = La, Pr, Nd, Sm). II.

We investigate the direct correspondence between Co band ferromagnetism and structural parameters in the pnictide oxides RCoPO for different rare-earth ions (R = La, Pr, Nd, Sm) by means of muon-spin spectroscopy and ab initio calculations, complementing our results published previously G. Prando et al., Common effect of chemical and external pressures on the magnetic properties of RCoPO (R = La, Pr), Phys. Rev. B 87, 064401 (2013)]. We find that both the transition temperature to the ferromagnetic phase T-C and the volume of the crystallographic unit cell V are conveniently tuned by the R ionic radius and/or external pressure. We report a linear correlation between T-C and V and our ab initio calculations unambiguously demonstrate a full equivalence of chemical and external pressures. As such, we show that R ions influence the ferromagnetic phase only via the induced structural shrinkage without involving any active role from the electronic f degrees of freedom, which are only giving a sizable magnetic contribution at much lower temperatures.

HuR Displaces Polypyrimidine Tract Binding Protein To Facilitate La Binding to the 3 ` Untranslated Region and Enhances Hepatitis C Virus Replication

HuR is a ubiquitous, RNA binding protein that influences the stability and translation of several cellular mRNAs. Here, we report a novel role for HuR, as a regulator of proteins assembling at the 3' untranslated region (UTR) of viral RNA in the context of hepatitis C virus (HCV) infection. HuR relocalizes from the nucleus to the cytoplasm upon HCV infection, interacts with the viral polymerase (NS5B), and gets redistributed into compartments of viral RNA synthesis. Depletion in HuR levels leads to a significant reduction in viral RNA synthesis. We further demonstrate that the interaction of HuR with the 3' UTR of the viral RNA affects the interaction of two host proteins, La and polypyrimidine tract binding protein (PTB), at this site. HuR interacts with La and facilitates La binding to the 3' UTR, enhancing La-mediated circularization of the HCV genome and thus viral replication. In addition, it competes with PTB for association with the 3' UTR, which might stimulate viral replication. Results suggest that HuR influences the formation of a cellular/viral ribonucleoprotein complex, which is important for efficient initiation of viral RNA replication. Our study unravels a novel strategy of regulation of HCV replication through an interplay of host and viral proteins, orchestrated by HuR. IMPORTANCE Hepatitis C virus (HCV) is highly dependent on various host factors for efficient replication of the viral RNA. Here, we have shown how a host factor (HuR) migrates from the nucleus to the cytoplasm and gets recruited in the protein complex assembling at the 3' untranslated region (UTR) of HCV RNA. At the 3' UTR, it facilitates circularization of the viral genome through interaction with another host factor, La, which is critical for replication. Also, it competes with the host protein PTB, which is a negative regulator of viral replication. Results demonstrate a unique strategy of regulation of HCV replication by a host protein through alteration of its subcellular localization and interacting partners. The study has advanced our knowledge of the molecular mechanism of HCV replication and unraveled the complex interplay between the host factors and viral RNA that could be targeted for therapeutic interventions.

Roles for Transcription Factors Sp1, NF-kappa B, IRF3, and IRF7 in Expression of the Human IFNL4 Gene

The expression of a biologically active human IFN4 depends on the presence of a frameshift deletion polymorphism within the first exon of the interferon lambda 4 (IFNL4) gene. In this report, we use the lung carcinoma-derived cell line, A549, which is genetically viable to express a functional IFN4, to address transcriptional requirements of the IFNL4 gene. We show that the GC-rich DNA-binding transcription factor (TF) specificity protein 1 (Sp1) is recruited to the IFNL4 promoter and has a role in induction of gene expression upon stimulation with viral RNA mimic poly(I:C). By using RNAi and overexpression strategies, we also show key roles in IFNL4 gene expression for the virus-inducible TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B), IFN regulatory factor 3 (IRF3), and IRF7. Interestingly, we also observe that overexpression of IFN4 influences IFNL4 promoter activity, which may further be dependent on the retinoic acid-inducible gene-I (RIG-I)-like receptor pathway. Together, our work for the first time reports on the functional characterization of the human IFNL4 promoter.

Role of (La, Gd) co-doping on the enhanced dielectric and magnetic properties of BiFeO3 ceramics

The effect of the La3+ and Gd3+ co-doping on the structure, electric and magnetic properties of BiFeO3 (BFO) ceramics are investigated. For the compositions (x=0 and 0 <= y <= 0.15) in the perovskite structured LaxGdyBi1-xFeO3 system, a tiny residual phase of Bi2Fe4O9 is noticed. Such a secondary phase is suppressed with the incorporation of `La' content (x). The magnitude of dielectric constant (epsilon(r) increases progressively by increasing the `La' content from x=0 to 0.15 with a remarkable decrease of dielectric loss. For x=0.15, the system LaxGdyBi1-x(x+y)FeO3 exhibits highest remanent magnetization (M-r) of 0.18 emu/g and coercive magnetic field (H-c) of similar to 1 Tin the presence of external magnetic field of 9 T at 300 K. The origin of enhanced dielectric and magnetic properties of LaxGdyBil (x+y)Fe03 and the role of doping elements, La3+, Gd3+ has been discussed. (C) 2015 Elsevier Ltd and Techna Group S.r.l. All rights reserved.

Autoregulation of topoisomerase I expression by supercoiling sensitive transcription

The opposing catalytic activities of topoisomerase I (TopoI/relaxase) and DNA gyrase (supercoiling enzyme) ensure homeostatic maintenance of bacterial chromosome supercoiling. Earlier studies in Es-cherichia coli suggested that the alteration in DNA supercoiling affects the DNA gyrase and TopoI expression. Although, the role of DNA elements around the promoters were proposed in regulation of gyrase, the molecular mechanism of supercoiling mediated control of TopoI expression is not yet understood. Here, we describe the regulation of TopoI expression from Mycobacterium tuberculosis and Mycobac-terium smegmatis by a mechanism termed Supercoiling Sensitive Transcription (SST). In both the organisms, topoI promoter(s) exhibited reduced activity in response to chromosome relaxation suggesting that SST is intrinsic to topoI promoter(s). We elucidate the role of promoter architecture and high transcriptional activity of upstream genes in topoI regulation. Analysis of the promoter(s) revealed the presence of suboptimal spacing between the -35 and -10 elements, rendering them supercoiling sensitive. Accordingly, upon chromosome relaxation, RNA polymerase occupancy was decreased on the topoI promoter region implicating the role of DNA topology in SST of topoI. We propose that negative supercoiling induced DNA twisting/writhing align the -35 and -10 elements to facilitate the optimal transcription of topoI.