Transcriptional Regulatory System Research Articles

Revealing System‐level Correlations between Aging and Calorie Restriction using a Mouse Transcriptome

The present study aimed to understand the most sensitive biological systems affected during aging and to reveal the systems underlying the crosstalk between aging and the ability of calorie restriction (CR), to effectively slowdown aging. We collected and analyzed 478 aging- and 586 CR-related mouse genes. For the given genes, the biological systems that are significantly related to aging and CR were examined according to three aspects. First, a global characterization by Gene Ontology (GO) was performed, where we found that the transcriptome (a set of genes) for both aging and CR were strongly related in the immune response, lipid metabolism, and cell adhesion functions. Second, the transcriptional modularity found in aging and CR was evaluated by identifying possible functional modules, sets of genes that show consistent expression patterns. Our analyses using the given functional modules, revealed systematic interactions among various biological processes as exemplified by the negative relation shown between lipid metabolism and the immune response at the system level. Third, transcriptional regulatory systems were predicted for both the aging and CR transcriptome. Here, we suggest a systems-biological framework to further understand the most important systems as they age. This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (NO. 2007-00376).

Calcineurin/NFAT-dependent regulation of 230-kDa bullous pemphigoid antigen (BPAG1) gene expression in normal human epidermal keratinocytes

Cyclosporin A (CsA) is utilized widely for treatment of inflammatory skin diseases, such as psoriasis vulgaris. The therapeutic effects of CsA are thought to be mediated by its immunosuppressive action on infiltrating lymphocytes in the lesional skin. CsA also inhibits epidermal keratinocyte proliferation, suggesting a direct biological action on keratinocytes. Here we tested the hypothesis that CsA can modulate the expression of the nuclear factor of activated T-cell (NFAT) in epidermal keratinocytes. We also investigated whether the keratinocyte-specific gene expression is modified by CsA through NFAT activity in association with differentiation induction. RT-PCR was performed using total RNAs extracted from cultured normal human epidermal keratinocytes (NHEK), normal human dermal fibroblasts (NHDF), and normal human epidermal melanocytes (NHEM) for detecting NFAT isomolecules. Transient transfections of NHEK with a 230-kDa bullous pemphigoid antigen (BPAG1) promoter/luciferase reporter gene and the luciferase assay were conducted for examining the effect of CsA on the promoter activity of the BPAG1 gene. Electrophoretic gel mobility shift assays (EMSA) with probes containing NFAT consensus sequences for analyzing the binding activities of the nuclear proteins extracted from NHEK. RT-PCR revealed expression of all of the five isoforms of NFAT in the cell lines examined. The mRNA expression levels of NFAT1, NFAT2, BPAG1, and involucrin were downregulated by CsA treatment in NHEK. The luciferase assay indicated suppression of the promoter activity by CsA. EMSA with NFAT consensus probes identified in the BPAG1 promoter region demonstrated specific binding activity in the nuclear proteins of epidermal keratinocytes. As reported previously, our results indicate that epidermal keratinocytes possess calcineurin/NFAT system, which is suppressed by CsA. In addition, the data suggest that CsA can downregulate the BPAG1 gene expression perhaps via the NFAT consensus cis-elements in the BPAG1 promoter region. Such transcriptional regulatory system might be involved in the regulation of keratinocyte differentiation and proliferation.

Effect of colored cross-correlated noise on the gene transcriptional regulatory system

The transient properties (e.g., the mean first passage time T) of the gene transcriptional regulatory system driven by colored cross-correlated noise are investigated. The approximate Fokker-Planck equation is obtained based on the Novikov theorem and the Fox approach and the explicit expression of the mean first passage time is derived. The numerical computation results show that for the case of strong correlation intensity and short correlation time，a successive switch process (i.e.，“on”→“off”→“on”) occurs; for tha case of weak correlation intensity and long correlation time，only one switch process occurs (i.e.，“on”→“off”). The reentrance phenomena appear in the gene transcriptional regulatory system.

Hypoxia and human disease—and the Journal of Molecular Medicine

This special issue of the Journal of Molecular Medicine is focused on the role of hypoxia in the pathogenesis of human disease. Oxygen homeostasis is a critical organizing principle of metazoan evolution, development, and physiology. The seven invited reviews in this issue highlight the rapid progress that is being made in delineating the molecular mechanisms that result in disruption of oxygen homeostasis in different disease states, the extent to which this disruption contributes to disease pathogenesis, and the prospects for novel therapies that target pathways involved in adaptive (or maladaptive) responses to hypoxia. In their review, entitled “Hypoxia and Gastrointestinal Disease,” Cormac Taylor and Sean Colgan summarize the results of published studies analyzing hypoxia-induced gene expression in intestinal epithelial cells and the role of hypoxia in the pathogenesis of inflammatory bowel disease. In “Hypoxia and Cancer,” Christiane Brahimi-Horn, Johanna Chiche, and Jacques Pouyssegur discuss the causes and consequences of intratumoral hypoxia. Joseph Garcia discusses ischemic heart disease with a specific focus on alterations in cardiac metabolism that promote the survival of ischemic cells. In “Hypoxia and Chronic Lung Disease,” Rubin Tuder and colleagues review the role of hypoxia in respiratory distress syndrome, high altitude pulmonary edema, pulmonary arterial hypertension, and chronic obstructive pulmonary disease. It is remarkable that despite receiving oxygen from the bronchial circulation and directly from the alveolar air sacs, hypoxia can develop within the lungs (even at sea level) and contribute to pathogenic tissue remodeling. Masaomi Nangaku and Kai-Uwe Eckardt discuss the role of the kidney in oxygen homeostasis, particularly in the production of erythropoietin, which is the glycoprotein hormone that controls red blood cell production. They also summarize recent data indicating that chronic hypoxia in the tubulointerstitium represents a final common pathway to end-stage kidney disease. Rajiv Ratan and his colleagues discuss novel strategies for promoting hypoxic adaptation in neurons as a means to prevent or treat brain injury after stroke. These strategies are based on recent advances in our understanding of the molecular physiology of oxygen sensing. Annelies Zinkernagel, Randall Johnson, and Victor Nizet review data indicating that the same transcriptional regulatory system that promotes adaptive responses to hypoxia, which are described in the other six papers in this issue, is also required for neutrophil inflammatory and innate immune functions. They also review data indicating that obligate intracellular bacteria and oncogenic viruses modulate hypoxia response pathways in host cells. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that plays an important role in cellular and systemic responses to reduced oxygen availability in all metazoan species. Purification of HIF-1 from human cells revealed that it is heterodimer composed of HIF-1α and HIF-1β subunits, which dimerize and bind to DNA containing the core sequence 5′-(A/G)CGTG-3′. Database searches for proteins that were homologous to HIF-1α led to the identification of HIF-2α, which can also dimerize with HIF-1β and bind to an overlapping but distinct set of target genes. HIF-1α and HIF-2α are each negatively regulated by O2-dependent hydroxylation of key proline and asparagine residues that dramatically reduce protein half-life and transcriptional activity, respectively, under aerobic conditions. Although I admit my bias in focusing on the role of J Mol Med (2007) 85:1293–1294 DOI 10.1007/s00109-007-0285-z

Function and Regulation of the Saccharomyces cerevisiae ENA Sodium ATPase System

Despite the fact that sodium ions are necessary to assist in the function of specific macromolecules (65), for most eukaryotic cells an elevated intracellular concentration of sodium ions is deleterious, probably because they interfere with the correct functioning of certain cellular targets (83). Because of the abundance of sodium ions in natural environments, yeasts and many other eukaryotic cells have developed mechanisms to maintain a relatively low intracellular concentration of this cation and/or to sequester it into organelles. The yeast Saccharomyces cerevisiae is a model organism used very often for cation tolerance studies. In this organism, maintenance of intracellular low sodium when sodium is present in excess in the external medium relies heavily on the capacity to extrude the cation. This is achieved by two main extrusion mechanisms. The first one is an H/Na antiporter encoded by the NHA1 gene, which is able to extrude Na ,L i, and even K cations by exchange with protons, gaining biological significance at rather acidic external pHs (8, 73). The second is based on a P-type ATPase pump encoded by the ENA system, which is the subject of this review. Although both mechanisms are complementary, comparison of the phenotypes of strains lacking functional antiporter or ATPase genes suggests that under standard growth conditions, the ENA system is the major determinant for sodium detoxification. The importance of this efflux system is highlighted by the observation that it seems to be present in all fungi as well as in certain nonflowering plants, such as bryophytes. In addition, active research with S. cerevisiae has revealed a complex and sophisticated system of transcriptional regulation in response to specific environmental changes, which is the major issue of this review.

Transcriptional and post-translational regulatory system for hypoxia specific gene expression using the erythropoietin enhancer and the oxygen-dependent degradation domain

Gene therapy with angiogenic factors is a promising strategy for the treatment of ischemic diseases. However, unregulated expression of an angiogenic factor may induce pathological angiogenesis. In this study, a hypoxia specific gene expression plasmid, pSV-Luc-ODD, was constructed with the oxygen-dependent degradation (ODD) domain for rapid degradation of a target protein under normoxia. In the transfection assay, luciferase activity in the pSV-Luc-ODD transfected cells was much lower under normoxia than that under hypoxia. However, the luciferase mRNA levels under hypoxia and normoxia were not significantly different. Therefore, decrease of luciferase activity under normoxia is not due to pre-translational events such as change of transcription rate or mRNA stability, but to post-translational degradation. For more hypoxia specific gene expression, pEpo-SV-Luc-ODD was constructed with the erythropoietin (Epo) enhancer and the ODD domain. pEpo-SV-Luc-ODD showed more than 1000 times increase of gene expression under hypoxia in Neuro2A cells, compared to normoxia. In addition, reoxygenation studies after hypoxia incubation showed that gene expression was decreased in response to increased oxygen concentration. This highly hypoxia specific gene expression system will be useful for development of targeting gene therapy for ischemic diseases.

Computational and Experimental Approaches for Modeling Gene Regulatory Networks

To understand most cellular processes, one must understand how genetic information is processed. A formidable challenge is the dissection of gene regulatory networks to delineate how eukaryotic cells coordinate and govern patterns of gene expression that ultimately lead to a phenotype. In this paper, we review several approaches for modeling eukaryotic gene regulatory networks and for reverse engineering such networks from experimental observations. Since we are interested in elucidating the transcriptional regulatory mechanisms of colon cancer progression, we use this important biological problem to illustrate various aspects of modeling gene regulation. We discuss four important models: gene networks, transcriptional regulatory systems, Boolean networks, and dynamical Bayesian networks. We review state-of-the-art functional genomics techniques, such as gene expression profiling, cis-regulatory element identification, TF target gene identification, and gene silencing by RNA interference, which can be used to extract information about gene regulation. We can employ this information, in conjunction with appropriately designed reverse engineering algorithms, to construct a computational model of gene regulation that sufficiently predicts experimental observations. In the last part of this review, we focus on the problem of reverse engineering transcriptional regulatory networks by gene perturbations. We mathematically formulate this problem and discuss the role of experimental resolution in our ability to reconstruct accurate models of gene regulation. We conclude, by discussing a promising approach for inferring a transcriptional regulatory system from microarray data obtained by gene perturbations.

268. Transcriptional and Post-Translational Regulation System for Hypoxia Specific Gene Therapy

268. Transcriptional and Post-Translational Regulation System for Hypoxia Specific Gene Therapy

The discoidin domain receptor 1 as a novel susceptibility gene for schizophrenia

Evidence suggests that myelin alterations could predispose to schizophrenia. Reduced expression of several myelin genes has been observed in schizophrenia patients. Recently, we identified the discoidin domain receptor 1 (DDR1; located at human chromosome 6p21.3) as a myelin gene in the mouse model and in a human oligodendroglial cell line. In the present study we screened for single nucleotide polymorphisms (SNPs) in the DNA from 100 schizophrenia patients. We identified a novel mutation within exon 10 that produces the amino-acid substitution N502S in the a-d isoforms, and M475V in the e isoform. However the frequency of the mutation (2%) was similar in schizophrenia patients and in control subjects. In a case-control assessment with 389 schizophrenic patients and 615 controls, we identified one SNP (SNP9, rs1049623) associated with schizophrenia (odds ratio=1.44, 95% confidence interval: 1.15-1.79, adjusted P=0.0016). This association was confirmed in haplotype analysis; the SNPs 9-10-11 (rs1049623, rs2267641 and rs2239518) haplotype remaining significant even after adjustment for multiple testing (adjusted P=0.0136). Of note was a strong gender dependence in the association, that is, statistical significance restricted to men (adjusted P-value=0.0002). Regression analysis of DDR1 mRNA expression in peripheral blood lymphocytes from schizophrenia patients showed that the presence of the G allele significantly decreased the relative number of mRNA copies in a dose-dependent manner (P=0.003). These data suggest that the risk haplotype tags a cis-acting variant involved in the transcription regulation system of the gene. In conclusion, we propose the DDR1 as a new susceptibility gene for schizophrenia.

Tetracycline-controlled transcriptional regulation systems: countermeasures to eliminate basal transgene leaks in Tet-based systems

Tetracycline-controlled transcriptional regulation systems: countermeasures to eliminate basal transgene leaks in Tet-based systems

Matrix Formalism to Describe Functional States of Transcriptional Regulatory Systems

Complex regulatory networks control the transcription state of a genome. These transcriptional regulatory networks (TRNs) have been mathematically described using a Boolean formalism, in which the state of a gene is represented as either transcribed or not transcribed in response to regulatory signals. The Boolean formalism results in a series of regulatory rules for the individual genes of a TRN that in turn can be used to link environmental cues to the transcription state of a genome, thereby forming a complete transcriptional regulatory system (TRS). Herein, we develop a formalism that represents such a set of regulatory rules in a matrix form. Matrix formalism allows for the systemic characterization of the properties of a TRS and facilitates the computation of the transcriptional state of the genome under any given set of environmental conditions. Additionally, it provides a means to incorporate mechanistic detail of a TRS as it becomes available. In this study, the regulatory network matrix, R, for a prototypic TRS is characterized and the fundamental subspaces of this matrix are described. We illustrate how the matrix representation of a TRS coupled with its environment (R*) allows for a sampling of all possible expression states of a given network, and furthermore, how the fundamental subspaces of the matrix provide a way to study key TRS features and may assist in experimental design.

Advances in the Discovery of cis-Regulatory Elements

Discovery of transcription regulatory elements has been an enormous challenge, both to biologists and computational scientists. Over the last three decades, significant progress has been achieved by various laboratories around the world. Earlier, laborious experimental methods were used to detect one or handful of elements at a time. With recent advances in DNA sequencing technology, many completed genomes became available. High throughput biological techniques and computational methods emerged. Comparative genomic approaches and their integration with microarray gene expression data provided promising results. In this review, we discuss the development of technology to decipher the complex transcription regulation system with a focus on the discovery of cis-regulatory elements in eukaryotes. Keywords: cis-regulatory elements, motifs, transcription factor binding sites, transcriptional regulation, gene expression, transcription factor

Stochastic Transcriptional Regulatory Systems with Time Delays: A Mean-Field Approximation

Modeling transcriptional regulation with time delays is an important problem of computational cell biology. In this paper, we propose a computational tool for studying transcriptional regulation in single cells based on a mean-field approximation method. The main idea is to replace the occurrence probabilities of the underlying transcriptional events by their mean values and use appropriately chosen additive noise terms to model statistical variations not accounted by this approximation. The proposed methodology allows us to characterize the transient and steady-state behavior of transcriptional regulation. Moreover, it provides a rather simple and computationally attractive tool for rapid statistical characterization of the dynamic behavior of a nonlinear transcriptional regulatory system with time delays.

Comprehensive Analysis of Combinatorial Regulation using the Transcriptional Regulatory Network of Yeast

Studies on various model systems have shown that a relatively small number of transcription factors can set up strikingly complex spatial and temporal patterns of gene expression. This is achieved mainly by means of combinatorial or differential gene regulation, i.e. regulation of a gene by two or more transcription factors simultaneously or under different conditions. While a number of specific molecular details of the mechanisms of combinatorial regulation have emerged, our understanding of the general principles of combinatorial regulation on a genomic scale is still limited. In this work, we approach this problem by using the largest assembled transcriptional regulatory network for yeast. A specific network transformation procedure was used to obtain the co-regulatory network describing the set of all significant associations among transcription factors in regulating common target genes. Analysis of the global properties of the co-regulatory network suggested the presence of two classes of regulatory hubs: (i) those that make many co-regulatory associations, thus serving as integrators of disparate cellular processes; and (ii) those that make few co-regulatory associations, and thereby specifically regulate one or a few major cellular processes. Investigation of the local structure of the co-regulatory network revealed a significantly higher than expected modular organization, which might have emerged as a result of selection by functional constraints. These constraints probably emerge from the need for extensive modular backup and the requirement to integrate transcriptional inputs of multiple distinct functional systems. We then explored the transcriptional control of three major regulatory systems (ubiquitin signaling, protein kinase and transcriptional regulation systems) to understand specific aspects of their upstream control. As a result, we observed that ubiquitin E3 ligases are regulated primarily by unique transcription factors, whereas E1 and E2 enzymes share common transcription factors to a much greater extent. This suggested that the deployment of E3s unique to specific functional contexts may be mediated significantly at the transcriptional level. Likewise, we were able to uncover evidence for much higher upstream transcription control of transcription factors themselves, in comparison to components of other regulatory systems. We believe that the results presented here might provide a framework for testing the role of co-regulatory associations in eukaryotic transcriptional control.

Ethanol Feeding Alters Death Signaling in the Pancreas

Alcohol abuse is a major cause of pancreatitis, which is associated with death of parenchymal cells. The goal of this study was to explore the effects of ethanol on cell death pathways in the pancreas. Adult male Wistar rats were fed with ethanol diets using the Lieber-DeCarli method. Caspase-8, caspase-3, and cathepsin B expression and activity in the pancreas of these animals as well as the signals that regulate their expression were studied using Western blot analysis and specific assays for biochemical enzyme activity. In the pancreas from rats fed with ethanol, the protein expression and activity of caspase-8 decreased by 48% and 45%, respectively, and caspase-3 activity decreased by 39%. In contrast, cathepsin B protein expression and activity increased with ethanol feeding by 189% and 143%, respectively. Evaluation of the transcriptional regulatory system for caspase-8 and cathepsin B showed that the ethanol effects on these pathways were largely transcriptional. Our findings show effects of ethanol on the expression of several signals involved in cell death in the pancreas through alteration of transcriptional regulators. The decrease in caspase expression and increase in cathepsin B expression indicate that ethanol feeding may prevent apoptosis and promote necrosis of pancreatic tissue with stresses that cause pancreatitis.

Statins attenuate sepsis

If (1) 3-hydroxy-3-methylglutaryl-coenzyme reductase inhibitors (statins) block the rate-limiting step in cholesterol synthesis and promote the expression of low-density lipoprotein receptors, (2) "Gram-negative sepsis" results from an abundant systemic response to bacterial lipopolysaccharide (endotoxin), (3) triglyceride-rich lipoproteins can bind endotoxin and low-density lipoprotein receptors enhance the uptake of both of these molecules, and (4) low-density lipoprotein receptor internalization of lipoproteins and endotoxin co-opts a common transcriptional regulatory system (NF-kappaB) that results in reduced cell vulnerability to inflammation, then statins, in addition to their lipid-lowering capacity, enhance endotoxin clearance from the circulation and attenuate the septic response.

Current status of transcriptional regulation systems

Many attempts have been undertaken to control transgene activity in mammalian cells. This is of importance for both applied biotechnology and basic research activities. State of the art regulatory systems use elements for transgene regulation which are unrelated to host regulatory networks and thus do not interfere with endogenous activities. Most of these regulation systems consist of transregulators and transregulator responding promoter elements that are derived from non mammalian origin. Apart from the tetracycline (Tet) regulated system which is most widely used for conditional gene expression at the moment, a number of new systems were created. These systems have been significantly refined and their performance makes them suitable for regulating transgenes not only in cellular systems but also in transgenic animals and for human therapeutic use.

Genome-Wide Survey of Transcription Factors in Prokaryotes Reveals Many Bacteria-Specific Families Not Found in Archaea

Assignment of all transcription factors (TFs) from genome sequence data is not a straightforward task due to the wide variation in TFs among different species. A DNA binding domain (DBD) and a contiguous non-DBD with a characteristic SCOP or Pfam domain combination are observed in most members of TF families. We found that most of the experimentally verified TFs in prokaryotes are detectable by a combination of SCOP or Pfam domains assigned to DBDs and non-DBDs. Based on this finding, we set up rules to detect TFs and classify them into 52 TF families. Application of the rules to 154 entirely sequenced prokaryotic genomes detected >18,000 TFs classified into families, which have been made publicly available from the 'GTOP_TF' database. Despite the rough proportionality of the number of TFs per genome with genome size, species with reduced genomes, i.e. obligatory parasites and symbionts, have only a few if any TFs, reflecting a nearly complete loss. Also the number of TFs is significantly lower in archaea than in bacteria. In addition, all but 1 of the 19 TF families present in archaea is present in bacteria, whereas 33 TF families are found exclusively in bacteria. This observation indicates that a number of new TF families have evolved in bacteria, making the transcription regulatory system more divergent in bacteria than in archaea.

A Hidden Markov Model for Transcriptional Regulation in Single Cells

We discuss several issues pertaining to the use of stochastic biochemical systems for modeling transcriptional regulation in single cells. By appropriately choosing the system state, we can model transcriptional regulation by a hidden Markov model (HMM). This opens the possibility of using well-known techniques for the statistical analysis and stochastic control of HMMs to mathematically and computationally study transcriptional regulation in single cells. Unfortunately, in all but a few simple cases, analytical characterization of the statistical behavior of the proposed HMM is not possible. Moreover, analysis by Monte Carlo simulation is computationally cumbersome. We discuss several techniques for approximating the HMM by one that is more tractable. We employ simulations, based on a biologically relevant transcriptional regulatory system, to show the relative merits and limitations of various approximation techniques and provide general guidelines for their use.

895. Separate rAAV1 Vectors Co-Infect Striatal Neurons Efficiently

Top of pageAbstract rAAV is a useful tool for gene therapy, however because of the limited size of the AAV genome large or multicistronic cDNAs are problematic. For example, the tetracycline responsive promoter-transactivator transcriptional regulation system is difficult to package in a single rAAV vector. In instances where more than one gene is required, co-infection by two viruses is necessary and often, both genes must be transferred to the same cell for the system to function properly. To date, the rate at which two mixed viral vectors infect the same cells has not been quantified. We hypothesized that a sequential surgical procedure separated by 24 hr would allow for receptor recycling and increase the number of double-hits. Six adult rats were injected unilaterally in the striatum with a mix of AAV1-GFP and AAV1 DS-Red2. The contralateral striatum was injected with AAV1 DS-Red2 then 24 hours later injected with AAV1-GFP at the same site. In both groups, approximately 75% of cells expressing GFP (direct fluorescence) also expressed dsred2 (indirect fluorescence). However, the number of GFP expressing cells far exceeded that of dsred2 despite very similar titers. In conclusion these results demonstrate that 2 vectors co-transduce striatal neurons at a very high rate even with simple mixing procedure.