Absence Of Transcription Factors Research Articles

Entropy-driven electrochemiluminescence ultra-sensitive detection strategy of NF-κB p50 as the regulator of cytokine storm

2019 novel coronavirus (2019-nCoV) with strong contagion in the crowd, has ravaged worldwide and severely impacts the human health and epidemic prevention system, by producing a series of significant stress reactions in the body to induce further cytokine storm. Transcription factors (TFs) served as essential DNA binding proteins play an integral role in regulating cytokine storm, and the detection of it in the human coronavirus environment provides especially valuable approaches to diagnosis and treatment of 2019-nCoV and development of antiviral drugs. In this work, an entropy-driven electrochemiluminescence (ECL) biosensor was constructed for ultra-sensitive bioassay of NF-κB p50. The strategy primarily capitalizing the splendid double-stranded DNA (dsDNA) binding properties of transcription factors, employing GOAu-Ru composite material as ECL emitter, utilizing entropy-driven reactions for signal amplification method, offered a repeatable proposal for TFs detection. In the absence of TFs, the released DNA1 further went in the entropy-driven reaction, contributing to an “ECL off” state. However, in the presence of TFs, the dsDNA avoided being digested, which blocked DNA1 for participating in the entropy-driven reaction, and the system exhibited an “ECL on” state. Most importantly, the ECL bioanalytical method denoted broad application prospects for NF-κB p50 detection with a lower detection limit (9.1 pM).

Assessment of transcriptional importance of cell line-specific features based on GTRD and FANTOM5 data.

Creating a complete picture of the regulation of transcription seems to be an urgent task of modern biology. Regulation of transcription is a complex process carried out by transcription factors (TFs) and auxiliary proteins. Over the past decade, ChIP-Seq has become the most common experimental technology studying genome-wide interactions between TFs and DNA. We assessed the transcriptional significance of cell line-specific features using regression analysis of ChIP-Seq datasets from the GTRD database and transcriptional start site (TSS) activities from the FANTOM5 expression atlas. For this purpose, we initially generated a large number of features that were defined as the presence or absence of TFs in different promoter regions around TSSs. Using feature selection and regression analysis, we identified sets of the most important TFs that affect expression activity of TSSs in human cell lines such as HepG2, K562 and HEK293. We demonstrated that some TFs can be classified as repressors and activators depending on their location relative to TSS.

The Role of Some Transcription Factors in Expression of GyrA and GyrB Following Exposure to Ciprofloxacin

Background: GyrA and gyrB genes encode DNA gyrase subunits. This enzyme regulates DNA supercoiling. Inhibitors of this enzyme, such as ciprofloxacin, may change the level of supercoiling and the expression level of genes, including gyrA and gyrB. Objectives: The aims of this research were first to select some transcription factors, which regulate the expression of gyrA and gyrB. Secondly, the effect of these transcription factors was investigated on the expression of these genes in Escherichia coli mutants with different levels of resistance to ciprofloxacin in the presence and absence of these transcription factors. Methods: For this purpose, the online software called Promoter Analyzer in Virtual Footprint version 3 was used to find and select some transcription factors. The relative expression of genes was determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results: Theoretical results showed that CspA, FhlA, and SoxS transcription factors (with a score of match higher than 6), could be selected for further analysis. The expression of gyrA and gyrB genes remained unchanged in the presence and absence of CspA and FhlA transcription factors following exposure to the low amount of ciprofloxacin. However, SoxS transcription activator might have indirect effects on the expression of these genes, as soxS gene was overexpressed following treatment with a higher amount of ciprofloxacin. Conclusions: It is concluded that overexpression of gyrA and gyrB genes is not dependent on CspA and FhlA transcription factors, but may be dependent indirectly on regulatory proteins involved in oxidative stress following exposure to ciprofloxacin.

DNA-silver nanoclusters/polypyrrole nanoparticles: A label-free and enzyme-free platform for multiplexed transcription factors detection

Transcription factors (TFs) regulate information flow from gene to protein, and they are recognized as key indicators to reflect cellular processes. Facile monitoring of TFs may aid diagnostics and treatment, but we still lack of techniques for highly efficient detection of them. In this research, a hybrid material consisting of DNA-silver nanoclusters (DNA-AgNCs) and polypyrrole nanoparticles (PPyNPs) was built for TFs detection. The designed DNA-AgNCs have a hairpin-shaped nucleic acid architecture with a double-stranded stem for recognizing TFs and a single-stranded loop for interacting with PPyNPs. In the absence of TFs, DNA-AgNCs are absorbed to PPyNPs, resulting in fluorescent quenching. While in the presence of TFs, the binding between TFs and the DNA-AgNCs caused desorption of DNA-AgNCs via steric hindrance mechanism. Accordingly, the increase of fluorescence derived from desorption is used for quantifications. Derived from low non-specific protein absorption features of PPyNPs, detection limit of 70 pM for NF-κB p50 and 110 pM for p53 were obtained. Then, by absorbing two kinds of DNA-AgNCs to PPyNPs, label-free multiplexed detection of TFs was first realized. Additionally, we suggested that this platform can be developed for drug screening by evaluating inhibitory effect of a pair of optical isomers towards TFs.

Sensitive and Label-Free Fluorescent Detection of Transcription Factors Based on DNA-Ag Nanoclusters Molecular Beacons and Exonuclease III-Assisted Signal Amplification.

Transcription factors (TFs) regulate gene expression by binding to regulatory regions, and their dysregulation is involved in numerous diseases. Thus, they are therapeutic targets and potential diagnostic markers. However, widely used methods for TFs detection are either cumbersome or costly. Herein, we first applied DNA-Ag nanoclusters molecular beacons (AgMBs) in TFs analysis and designed an assay based on the switchable fluorescence of AgMBs. In the absence of TFs, a single-stranded DNA functioned as a reporter is released from a double-stranded DNA probe (referred as dsTFs probe) under exonuclease III (Exo III) digestion. Then, the reporter triggers downstream Exo III-assisted signal amplification by continuously consuming the guanine-rich enhancer sequences in AgMBs, resulting in significant fluorescent decrease eventually. Conversely, the presence of TFs protects the dsTFs probe from digestion and blocks the downstream reaction to keep a highly fluorescent state. To testify this rationale, we utilized nuclear factor-kappa B p50 (NF-κB p50) as a model TFs. Owing to the amplification strategy, this method exhibited high sensitivity toward NF-κB p50 with a limit of detection of 10 pM, and a broad linear range from 30 pM to 1.5 nM. Furthermore, this method could detect multiple TFs in human colon cancer DLD-1 cells and reflect the variation in their cellular levels after stimulation. Finally, by conducting an inhibition assay we revealed the potential of this method for screening TFs-targeted drugs and calculating the IC50 of corresponding inhibitors.

Phenotypic and Molecular Characterization of Domestic Cat (Felis catus) Spermatogonial Stem Cells.

In many mammalian species, surface markers have been used to obtain enriched populations of spermatogonial stem cells (SSCs) for assisted reproduction and other applications; however, little is known about the expression patterns of feline SSCs. In this study, we assessed expression of the SSC surface markers commonly used in other species, KIT, ITGA6, CD9, GFRalpha1, ADGRA3, and THY1, in addition to the less frequently used pluripotent markers TRA-1-60, TRA-1-81, SSEA-1, and SSEA-4 in SSCs of both prepubertal and adult domestic cats (Felis catus). To further characterize cat SSCs, we sorted cells using SSC-specific markers and evaluated the expression of the pluripotent transcription factors NANOG, POU5F1, and SOX2 and the proto-oncogene MYC within these populations. We concluded that SSC surface markers used in other mammalian species were not specific for identifying cat SSCs. However, the pluripotent markers we evaluated were more specific to cat spermatogonia, and the presence of SSEA-1 and SSEA-4 in fewer and primarily individual cells suggests that these two markers may be used for enrichment of cat SSCs. The expression of pluripotent transcription factors at mRNA level by single-stained cells positive for SSEA-4 and by dual-stained cells positive for both GFRalpha1 and SSEA-4 reflects the undifferentiated stage of cat SSCs. The absence of transcription factors in double-stained cells positive for only one marker implies the loss of the stem cell-like identity with the loss of either GFRalpha1 or SSEA-4. Further investigation is warranted to elucidate the biological characteristics of these spermatogonial subpopulations.

Inflammatory Cytokines and Thrombosis

Abstract SCI-44Platelets circulate in abundance in a quiescent state, yet are readily activated in physiologic control of blood loss and support for vascular integrity, with excessive or prolonged responsiveness underlying a range of cardiovascular events. Platelet activation is initiated by soluble agonists, including thrombin, platelet-activating factor, and ADP, and is accompanied by adhesion to injured vascular walls or to other circulating inflammatory cells. Activated platelets produce thromboxane A2, ATP and ADP, growth factors, and cytokines such as CD40 ligand, and they shed highly thrombotic microparticles. Anucleate platelets cannot generate new RNA to alter their proteome, but they store factors for prompt release after stimulation. Despite the handicap of lacking a nucleus to direct RNA transcript formation, platelets do, in fact, produce a number of new proteins after appropriate stimulation. The questions, then, are: how do platelets respond to inflammatory signaling in the absence of transcription factors that integrate so much of inflammatory signaling? How are new proteins produced in the absence of stored mRNA? To what events do new platelet proteins contribute? The answers to these questions derive both from stimulated translation of stored mRNA in unactivated platelets and from the presence of unspliced heteronuclear RNA transcripts of select genes, accompanied by stimulated posttranscriptional splicing that removes introns from stored heteronuclear RNA to generate functional mRNA, followed by stimulated translation to produce new protein. These mechanisms are retained in murine platelets, suggesting a continued role for the unique process of posttranscriptional protein production by activated platelets. Thrombin stimulates mTOR and S6 kinase signaling to stimulate spliceosome activity, while platelets respond to lipopolysaccharide through their TLR4 receptor, ultimately to stimulate the AKT and JNK kinases and then spliceosome activation. Human and murine platelets store interleukin-1β heteronuclear RNA, and, upon activation, both produce new pro-IL-1β, but also process it via caspase-1 activity to active cytokine. Activated platelets release functional IL-1β, in association with shed microparticles, to active endothelial cells and naïve platelets themselves. Platelets express signaling type 1 receptors for IL-1, and recombinant IL-1α and IL-1β each activate IL-1β RNA processing and cytokine production. Accordingly, specific blockade of IL-1 signaling with IL-1Ra, in clinical use as Anakinra, suppressed platelet stimulation by soluble IL-1, and suppressed microparticles shed from activated platelets. IL-1 signaling additionally affects platelets’ structure and their interaction with immobile surfaces. Remarkably, IL-1Ra also abolished platelet stimulation by lipopolysaccharide, showing that platelets amplify TLR4 signaling by the IL-1 signaling axis. IL-1β accumulates in association with platelets within thrombi formed after FeCl3 damage to murine carotid arteries, not by captured or infiltrating mononuclear cells. The process of posttranscriptional RNA splicing and translation uniquely generates the paradigmatic inflammatory cytokine IL-1β in thrombi, and because this process can be tightly targeted by inhibition of posttranslational splicing, the contribution of platelets to vascular remodeling during and after thrombosis may be specifically addressed. Disclosures:No relevant conflicts of interest to declare.

The transcription reinitiation properties of RNA polymerase III in the absence of transcription factors.

Transcription reinitiation by RNA polymerase (Pol) III proceeds through facilitated recycling, a process by which the terminating Pol III, assisted by the transcription factors TFIIIB and TFIIIC, rapidly reloads onto the same transcription unit. To get further insight into the Pol III transcription mechanism, we analyzed the kinetics of transcription initiation and reinitiation of a simplified in vitro transcription system consisting only of Pol III and template DNA. The data indicates that, in the absence of transcription factors, first-round transcription initiation by Pol III proceeds at a normal rate, while facilitated reinitiation during subsequent cycles is compromised.

The Proteasome Regulates HIV-1 Transcription by Both Proteolytic and Nonproteolytic Mechanisms

Although the proteasome facilitates transcription from several yeast promoters, it is unclear if its role is proteolytic or which subunits are involved. We show that the proteasome regulates the HIV-1 promoter in both proteolytic and nonproteolytic modes. In the absence of transcription factor, Tat, proteasome was associated with promoter and coding regions, and its proteolytic activity regulated the level of basal transcription emanating from the promoter. Tat switched the proteasome to a nonproteolytic mode by recruiting a proteasome-associated protein, PAAF1, which favors proteasome dissociation into 19S and 20S particles. Gel filtration chromatography showed that expression of both Tat and PAAF1 enhanced the abundance of a 19S-like complex in nuclear extracts. 19S, but not 20S, subunits were strongly recruited to the promoter in the presence of Tat and PAAF1 and coactivated Tat-dependent transcription. 19S components facilitated transcriptional elongation and may be involved in clearance of paused transcriptional elongation complexes from the promoter.

Identification of interventricular septum precursor cells in the mouse embryo

Little is known about the formation of the interventricular septum (IVS), a central event during cardiogenesis. Here, we describe a novel population of myocardial progenitor cells in the primitive ventricle of the mouse embryo, which is characterized by expression of lysozyme M (lysM). Using LysM-Cre mice we show that lysozyme expressing cells give rise to the IVS and to a part of the left ventricular free wall, demonstrating that these heart regions are developmentally related. LysM+ precursors are not of hematopoietic origin and develop in the absence of transcription factors that regulate lysozyme expression in macrophages. LysM-deficient mice lack an overt cardiac phenotype, perhaps due to compensation by the related lysozyme P, which we also found to be expressed in the developing heart. Direct visualization of lysM expression, using LysM-EGFP knock-in mice, showed that ventricular septation is initiated at embryonic day 9 by the movement of myocardial trabeculae from the primitive ventricle towards the bulbo-ventricular groove and revealed the dynamics of IVS formation at later stages. Our studies predict that LysM-Cre mice will be useful to inactivate genes in the developing IVS.

Analysis of transcription factor OCT.1, OCT.2 and BOB.1 expression using tissue arrays in classical Hodgkin's lymphoma

Hodgkin's lymphoma can be considered in most cases a B-cell lymphoma due to the presence of potentially functional immunoglobulin (Ig) gene rearrangements in the neoplastic cells. In contrast to lymphocyte-predominant Hodgkin's lymphoma, Hodgkin/Reed–Sternberg (HRS) cells from classical Hodgkin's lymphoma have low frequency of B-cell marker expression and lack Ig light and Ig heavy messenger RNA. Recent studies have shown transcription machinery deficiency in Hodgkin's lymphoma caused by an absence of the transcription factors OCT.1, OCT.2 and/or BOB.1. By using the tissue microarray technique, we have performed an immunohistochemical study of OCT.1, OCT.2 and BOB.1 in 325 classical Hodgkin's lymphoma cases. The results have been correlated with the expression of the B-cell markers CD20, CD79a, B-cell-specific activator protein (BSAP) and MUM.1, the presence of Epstein–Barr virus and the histological subtype. The percentage of CD20 and CD79a positivity was low (18 and 18%, respectively), whereas MUM.1 and BSAP were positive in the majority of cases. Considering the positive cases with independence of the intensity of staining, 62% of them expressed OCT.2, 59% OCT.1 and 37% BOB.1. Nevertheless, when we considered only the strongly positive cases, the results were similar to those previously described by others. No statistical association was found between the transcription factor expression, histological subtype and Epstein–Barr virus presence. To our knowledge, this is the largest series of classical Hodgkin's lymphoma cases in which the expression of transcription factors has been studied. We have found a notorious percentage of cases displaying weak positivity for OCT.2 and BOB.1 factors in HRS cells. We propose that other mechanisms different from the absence of transcription factors OCT.2 and BOB.1 might be involved in the control of Ig transcription and B lineage in classical Hodgkin's lymphoma.

Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity cross-linking.

The subunits of Saccharomyces cerevisiae RNA polymerase II (RNAP II) in proximity to the DNA during transcription elongation have been identified by photoaffinity cross-linking. In the absence of transcription factors, RNAP II will transcribe a double-stranded DNA fragment containing a 3'-extension of deoxycytidines, a "tailed template". We designed a DNA template allowing the RNAP to transcribe 76 bases before it was stalled by omission of CTP in the transcription reaction. This stall site oriented the RNAP on the DNA template and allowed us to map the RNAP subunits along the DNA. The DNA analogue 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUTP (N(3)RdUTP) [Bartholomew, B., Kassavetis, G. A., Braun, B. R., and Geiduschek, E. P. (1990) EMBO J. 9, 2197-205] was synthesized and enzymatically incorporated into the DNA at specified positions upstream or downstream of the stall site, in either the template or nontemplate strand of the DNA. Radioactive nucleotides were positioned beside the photoactivatable nucleotides, and cross-linking by brief ultraviolet irradiation transferred the radioactive tag from the DNA onto the RNAP subunits. In addition to N(3)RdUTP, which has a photoreactive azido group 9 A from the uridine base, we used the photoaffinity cross-linker 5N(3)dUTP with an azido group directly on the uridine ring to identify the RNAP II subunits closest to the DNA at positions where multiple subunits cross-linked. In cross-linking reactions dependent on transcription, RPB1, RPB2, and RPB5 were cross-linked with N(3)RdUTP. With 5N(3)dUTP, only RPB1 and RPB2 were cross-linked. Under certain circumstances, RPB3, RPB4, and RPB7 were cross-linked. From the information obtained in this topological study, we developed a model of yeast RNAP II in a transcription elongation complex.

Altered Development and Cytokine Responses of Myeloid Progenitors in the Absence of Transcription Factor, Interferon Consensus Sequence Binding Protein

AbstractMice deficient for the transcription factor, interferon consensus sequence binding protein (ICSBP), are immunodeficient and develop disease symptoms similar to human chronic myeloid leukemia (CML). To elucidate the hematopoietic disorder of ICSBP−/− mice, we investigated the growth, differentiation, and leukemogenic potential of ICSBP−/−myeloid progenitor cells in vitro, as well as by cell-transfers in vivo. We report that adult bone marrow, as well as fetal liver of ICSBP-deficient mice harbor increased numbers of progenitor cells, which are hyperresponsive to both granulocyte macrophage colony-stimulating factor (GM-CSF) and G-CSF in vitro. In contrast, their response to M-CSF is strongly reduced and, surprisingly, ICSBP−/− colonies formed in the presence of M-CSF are mostly of granulocytic morphology. This disproportional differentiation toward cells of the granulocytic lineage in vitro parallels the expansion of granulocytes in ICSBP−/− mice and correlates with a 4-fold reduction of M-CSF receptor expressing cells in bone marrow. Cell transfer studies showed an intrinsic leukemogenic potential and long-term reconstitution capability of ICSBP−/− progenitors. Further experiments demonstrated strongly reduced adhesion of colony-forming cells from ICSBP−/− bone marrow to fibronectin. In summary, ICSBP−/− myeloid progenitor cells share several abnormal features with CML progenitors, suggesting that the distal parts of signaling pathways of these two disorders are overlapping.

Altered Development and Cytokine Responses of Myeloid Progenitors in the Absence of Transcription Factor, Interferon Consensus Sequence Binding Protein

Mice deficient for the transcription factor, interferon consensus sequence binding protein (ICSBP), are immunodeficient and develop disease symptoms similar to human chronic myeloid leukemia (CML). To elucidate the hematopoietic disorder of ICSBP−/− mice, we investigated the growth, differentiation, and leukemogenic potential of ICSBP−/−myeloid progenitor cells in vitro, as well as by cell-transfers in vivo. We report that adult bone marrow, as well as fetal liver of ICSBP-deficient mice harbor increased numbers of progenitor cells, which are hyperresponsive to both granulocyte macrophage colony-stimulating factor (GM-CSF) and G-CSF in vitro. In contrast, their response to M-CSF is strongly reduced and, surprisingly, ICSBP−/− colonies formed in the presence of M-CSF are mostly of granulocytic morphology. This disproportional differentiation toward cells of the granulocytic lineage in vitro parallels the expansion of granulocytes in ICSBP−/− mice and correlates with a 4-fold reduction of M-CSF receptor expressing cells in bone marrow. Cell transfer studies showed an intrinsic leukemogenic potential and long-term reconstitution capability of ICSBP−/− progenitors. Further experiments demonstrated strongly reduced adhesion of colony-forming cells from ICSBP−/− bone marrow to fibronectin. In summary, ICSBP−/− myeloid progenitor cells share several abnormal features with CML progenitors, suggesting that the distal parts of signaling pathways of these two disorders are overlapping.

Localization and functional analysis of DNase-I-hypersensitive sites in the human c-sis/PDGF-B gene transcription unit and its flanking regions.

We studied the regulation of the expression of the human c-sis/PDGF-B gene in the following panel of cell lines: K562 cells, in which expression is inducible by phorbol esters; cytotrophoblast-derived cell lines JEG-3 and JAR; carcinoma-derived cell lines PC3, T24 and HeLa, which show extensive differences in c-sis mRNA content; dermal fibroblasts, which do not express the gene. We demonstrate that the wide variety of levels of c-sis mRNA in these cells is mainly determined at the transcription level. Extensive gene rearrangements or amplifications, or significant differences in the stability of the c-sis transcript could not be found. In fibroblasts and placenta cell lines, inaccessibility of the c-sis promoter, rather than the absence of transcription factors that activate it, inhibits expression of the endogenous gene. Examination of the chromatin structure of the transcription unit and immediate flanking regions revealed several cell-type-specific DNase-I-hypersensitivity (DH) sites. Functional analysis of genomic fragments harbouring one or more DH sites showed the presence of negative regulatory elements within intron 1, and of an activating element downstream of the gene. A DH site, located immediately downstream of the promoter in dermal fibroblasts, may regulate accessibility of the promoter by means of specific nucleosome phasing.