rSNPBase 3.0: an updated database of SNP-related regulatory elements, element-gene pairs and SNP-based gene regulatory networks

PubMed Central

2018-01-01

Abstract Here, we present the updated rSNPBase 3.0 database (http://rsnp3.psych.ac.cn), which provides human SNP-related regulatory elements, element-gene pairs and SNP-based regulatory networks. This database is the updated version of the SNP regulatory annotation database rSNPBase and rVarBase. In comparison to the last two versions, there are both structural and data adjustments in rSNPBase 3.0: (i) The most significant new feature is the expansion of analysis scope from SNP-related regulatory elements to include regulatory element–target gene pairs (E–G pairs), therefore it can provide SNP-based gene regulatory networks. (ii) Web function was modified according to data content and a new network search module is provided in the rSNPBase 3.0 in addition to the previous regulatory SNP (rSNP) search module. The two search modules support data query for detailed information (related-elements, element-gene pairs, and other extended annotations) on specific SNPs and SNP-related graphic networks constructed by interacting transcription factors (TFs), miRNAs and genes. (3) The type of regulatory elements was modified and enriched. To our best knowledge, the updated rSNPBase 3.0 is the first data tool supports SNP functional analysis from a regulatory network prospective, it will provide both a comprehensive understanding and concrete guidance for SNP-related regulatory studies. PMID:29140525

Repression of enhancer II activity by a negative regulatory element in the hepatitis B virus genome.

PubMed Central

Lo, W Y; Ting, L P

1994-01-01

Enhancer II of human hepatitis B virus has dual functions in vivo. Located at nucleotides (nt) 1646 to 1741, it can stimulate the surface and X promoters from a downstream position. Moreover, the same sequence can also function as upstream regulatory element that activates the core promoter in a position- and orientation-dependent manner. In this study, we report the identification and characterization of a negative regulatory element (NRE) upstream of enhancer II (nt 1613 to 1636) which can repress both the enhancer and upstream stimulatory function of the enhancer II sequence in differentiated liver cells. This NRE has marginal inhibitory effect by itself but a strong repressive function in the presence of a functional enhancer II. Mutational analysis reveals that sequence from nt 1616 to 1621 is required for repression of enhancer activity by the NRE. Gel shift analysis reveals that this negative regulatory region can be recognized by a specific protein factor(s) present at the 0.4 M NaCl fraction of HepG2 nuclear extracts. The discovery of the NRE indicates that HBV gene transcription is controlled by combined effects of both positive and negative regulation. It also provides a unique system with which to study the mechanism of negative regulation of gene expression. Images PMID:8107237

Transcription factor trapping by RNA in gene regulatory elements.

PubMed

Sigova, Alla A; Abraham, Brian J; Ji, Xiong; Molinie, Benoit; Hannett, Nancy M; Guo, Yang Eric; Jangi, Mohini; Giallourakis, Cosmas C; Sharp, Phillip A; Young, Richard A

2015-11-20

Transcription factors (TFs) bind specific sequences in promoter-proximal and -distal DNA elements to regulate gene transcription. RNA is transcribed from both of these DNA elements, and some DNA binding TFs bind RNA. Hence, RNA transcribed from regulatory elements may contribute to stable TF occupancy at these sites. We show that the ubiquitously expressed TF Yin-Yang 1 (YY1) binds to both gene regulatory elements and their associated RNA species across the entire genome. Reduced transcription of regulatory elements diminishes YY1 occupancy, whereas artificial tethering of RNA enhances YY1 occupancy at these elements. We propose that RNA makes a modest but important contribution to the maintenance of certain TFs at gene regulatory elements and suggest that transcription of regulatory elements produces a positive-feedback loop that contributes to the stability of gene expression programs. Copyright © 2015, American Association for the Advancement of Science.

Decoding the role of regulatory element polymorphisms in complex disease.

PubMed

Vockley, Christopher M; Barrera, Alejandro; Reddy, Timothy E

2017-04-01

Genetic variation in gene regulatory elements contributes to diverse human diseases, ranging from rare and severe developmental defects to common and complex diseases such as obesity and diabetes. Early examples of regulatory mechanisms of human diseases involve large chromosomal rearrangements that change the regulatory connections within the genome. Single nucleotide variants in regulatory elements can also contribute to disease, potentially via demonstrated associations with changes in transcription factor binding, enhancer activity, post-translational histone modifications, long-range enhancer-promoter interactions, or RNA polymerase recruitment. Establishing causality between non-coding genetic variants, gene regulation, and disease has recently become more feasible with advances in genome-editing and epigenome-editing technologies. As establishing causal regulatory mechanisms of diseases becomes routine, functional annotation of target genes is likely to emerge as a major bottleneck for translation into patient benefits. In this review, we discuss the history and recent advances in understanding the regulatory mechanisms of human disease, and new challenges likely to be encountered once establishing those mechanisms becomes rote. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genome-Wide Discovery of Drug-Dependent Human Liver Regulatory Elements

PubMed Central

Morrissey, Kari M.; Luizon, Marcelo R.; Hoffmann, Thomas J.; Sun, Xuefeng; Jones, Stacy L.; Force Aldred, Shelley; Ramamoorthy, Anuradha; Desta, Zeruesenay; Liu, Yunlong; Skaar, Todd C.; Trinklein, Nathan D.; Giacomini, Kathleen M.; Ahituv, Nadav

2014-01-01

Inter-individual variation in gene regulatory elements is hypothesized to play a causative role in adverse drug reactions and reduced drug activity. However, relatively little is known about the location and function of drug-dependent elements. To uncover drug-associated elements in a genome-wide manner, we performed RNA-seq and ChIP-seq using antibodies against the pregnane X receptor (PXR) and three active regulatory marks (p300, H3K4me1, H3K27ac) on primary human hepatocytes treated with rifampin or vehicle control. Rifampin and PXR were chosen since they are part of the CYP3A4 pathway, which is known to account for the metabolism of more than 50% of all prescribed drugs. We selected 227 proximal promoters for genes with rifampin-dependent expression or nearby PXR/p300 occupancy sites and assayed their ability to induce luciferase in rifampin-treated HepG2 cells, finding only 10 (4.4%) that exhibited drug-dependent activity. As this result suggested a role for distal enhancer modules, we searched more broadly to identify 1,297 genomic regions bearing a conditional PXR occupancy as well as all three active regulatory marks. These regions are enriched near genes that function in the metabolism of xenobiotics, specifically members of the cytochrome P450 family. We performed enhancer assays in rifampin-treated HepG2 cells for 42 of these sequences as well as 7 sequences that overlap linkage-disequilibrium blocks defined by lead SNPs from pharmacogenomic GWAS studies, revealing 15/42 and 4/7 to be functional enhancers, respectively. A common African haplotype in one of these enhancers in the GSTA locus was found to exhibit potential rifampin hypersensitivity. Combined, our results further suggest that enhancers are the predominant targets of rifampin-induced PXR activation, provide a genome-wide catalog of PXR targets and serve as a model for the identification of drug-responsive regulatory elements. PMID:25275310

Dynamics and function of distal regulatory elements during neurogenesis and neuroplasticity

PubMed Central

Thakurela, Sudhir; Sahu, Sanjeeb Kumar; Garding, Angela; Tiwari, Vijay K.

2015-01-01

Gene regulation in mammals involves a complex interplay between promoters and distal regulatory elements that function in concert to drive precise spatiotemporal gene expression programs. However, the dynamics of the distal gene regulatory landscape and its function in the transcriptional reprogramming that underlies neurogenesis and neuronal activity remain largely unknown. Here, we performed a combinatorial analysis of genome-wide data sets for chromatin accessibility (FAIRE-seq) and the enhancer mark H3K27ac, revealing the highly dynamic nature of distal gene regulation during neurogenesis, which gets progressively restricted to distinct genomic regions as neurons acquire a post-mitotic, terminally differentiated state. We further find that the distal accessible and active regions serve as target sites for distinct transcription factors that function in a stage-specific manner to contribute to the transcriptional program underlying neuronal commitment and maturation. Mature neurons respond to a sustained activity of NMDA receptors by epigenetic reprogramming at a large number of distal regulatory regions as well as dramatic reorganization of super-enhancers. Such massive remodeling of the distal regulatory landscape in turn results in a transcriptome that confers a transient loss of neuronal identity and gain of cellular plasticity. Furthermore, NMDA receptor activity also induces many novel prosurvival genes that function in neuroprotective pathways. Taken together, these findings reveal the dynamics of the distal regulatory landscape during neurogenesis and uncover novel regulatory elements that function in concert with epigenetic mechanisms and transcription factors to generate the transcriptome underlying neuronal development and activity. PMID:26170447

Diverse activities of viral cis-acting RNA regulatory elements revealed using multicolor, long-term, single-cell imaging

PubMed Central

Pocock, Ginger M.; Zimdars, Laraine L.; Yuan, Ming; Eliceiri, Kevin W.; Ahlquist, Paul; Sherer, Nathan M.

2017-01-01

Cis-acting RNA structural elements govern crucial aspects of viral gene expression. How these structures and other posttranscriptional signals affect RNA trafficking and translation in the context of single cells is poorly understood. Herein we describe a multicolor, long-term (>24 h) imaging strategy for measuring integrated aspects of viral RNA regulatory control in individual cells. We apply this strategy to demonstrate differential mRNA trafficking behaviors governed by RNA elements derived from three retroviruses (HIV-1, murine leukemia virus, and Mason-Pfizer monkey virus), two hepadnaviruses (hepatitis B virus and woodchuck hepatitis virus), and an intron-retaining transcript encoded by the cellular NXF1 gene. Striking behaviors include “burst” RNA nuclear export dynamics regulated by HIV-1’s Rev response element and the viral Rev protein; transient aggregations of RNAs into discrete foci at or near the nuclear membrane triggered by multiple elements; and a novel, pulsiform RNA export activity regulated by the hepadnaviral posttranscriptional regulatory element. We incorporate single-cell tracking and a data-mining algorithm into our approach to obtain RNA element–specific, high-resolution gene expression signatures. Together these imaging assays constitute a tractable, systems-based platform for studying otherwise difficult to access spatiotemporal features of viral and cellular gene regulation. PMID:27903772

Prediction of transcriptional regulatory elements for plant hormone responses based on microarray data

PubMed Central

2011-01-01

Background Phytohormones organize plant development and environmental adaptation through cell-to-cell signal transduction, and their action involves transcriptional activation. Recent international efforts to establish and maintain public databases of Arabidopsis microarray data have enabled the utilization of this data in the analysis of various phytohormone responses, providing genome-wide identification of promoters targeted by phytohormones. Results We utilized such microarray data for prediction of cis-regulatory elements with an octamer-based approach. Our test prediction of a drought-responsive RD29A promoter with the aid of microarray data for response to drought, ABA and overexpression of DREB1A, a key regulator of cold and drought response, provided reasonable results that fit with the experimentally identified regulatory elements. With this succession, we expanded the prediction to various phytohormone responses, including those for abscisic acid, auxin, cytokinin, ethylene, brassinosteroid, jasmonic acid, and salicylic acid, as well as for hydrogen peroxide, drought and DREB1A overexpression. Totally 622 promoters that are activated by phytohormones were subjected to the prediction. In addition, we have assigned putative functions to 53 octamers of the Regulatory Element Group (REG) that have been extracted as position-dependent cis-regulatory elements with the aid of their feature of preferential appearance in the promoter region. Conclusions Our prediction of Arabidopsis cis-regulatory elements for phytohormone responses provides guidance for experimental analysis of promoters to reveal the basis of the transcriptional network of phytohormone responses. PMID:21349196

Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, R. L., Hamaguchi, L., Busch, M. A., and Weigel, D.

2003-06-01

OAK-B135 In Arabidopsis thaliana, cis-regulatory sequences of the floral homeotic gene AGAMOUS (AG) are located in the second intron. This 3 kb intron contains binding sites for two direct activators of AG, LEAFY (LFY) and WUSCHEL (WUS), along with other putative regulatory elements. We have used phylogenetic footprinting and the related technique of phylogenetic shadowing to identify putative cis-regulatory elements in this intron. Among 29 Brassicaceae, several other motifs, but not the LFY and WUS binding sites previously identified, are largely invariant. Using reporter gene analyses, we tested six of these motifs and found that they are all functionally importantmore » for activity of AG regulatory sequences in A. thaliana. Although there is little obvious sequence similarity outside the Brassicaceae, the intron from cucumber AG has at least partial activity in A. thaliana. Our studies underscore the value of the comparative approach as a tool that complements gene-by-gene promoter dissection, but also highlight that sequence-based studies alone are insufficient for a complete identification of cis-regulatory sites.« less

Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters.

PubMed

Yamaguchi-Shinozaki, Kazuko; Shinozaki, Kazuo

2005-02-01

cis-Acting regulatory elements are important molecular switches involved in the transcriptional regulation of a dynamic network of gene activities controlling various biological processes, including abiotic stress responses, hormone responses and developmental processes. In particular, understanding regulatory gene networks in stress response cascades depends on successful functional analyses of cis-acting elements. The ever-improving accuracy of transcriptome expression profiling has led to the identification of various combinations of cis-acting elements in the promoter regions of stress-inducible genes involved in stress and hormone responses. Here we discuss major cis-acting elements, such as the ABA-responsive element (ABRE) and the dehydration-responsive element/C-repeat (DRE/CRT), that are a vital part of ABA-dependent and ABA-independent gene expression in osmotic and cold stress responses.

Diverse activities of viral cis-acting RNA regulatory elements revealed using multicolor, long-term, single-cell imaging.

PubMed

Pocock, Ginger M; Zimdars, Laraine L; Yuan, Ming; Eliceiri, Kevin W; Ahlquist, Paul; Sherer, Nathan M

2017-02-01

Cis-acting RNA structural elements govern crucial aspects of viral gene expression. How these structures and other posttranscriptional signals affect RNA trafficking and translation in the context of single cells is poorly understood. Herein we describe a multicolor, long-term (>24 h) imaging strategy for measuring integrated aspects of viral RNA regulatory control in individual cells. We apply this strategy to demonstrate differential mRNA trafficking behaviors governed by RNA elements derived from three retroviruses (HIV-1, murine leukemia virus, and Mason-Pfizer monkey virus), two hepadnaviruses (hepatitis B virus and woodchuck hepatitis virus), and an intron-retaining transcript encoded by the cellular NXF1 gene. Striking behaviors include "burst" RNA nuclear export dynamics regulated by HIV-1's Rev response element and the viral Rev protein; transient aggregations of RNAs into discrete foci at or near the nuclear membrane triggered by multiple elements; and a novel, pulsiform RNA export activity regulated by the hepadnaviral posttranscriptional regulatory element. We incorporate single-cell tracking and a data-mining algorithm into our approach to obtain RNA element-specific, high-resolution gene expression signatures. Together these imaging assays constitute a tractable, systems-based platform for studying otherwise difficult to access spatiotemporal features of viral and cellular gene regulation. © 2017 Pocock et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Comparing anterior and posterior Hox complex formation reveals guidelines for predicting cis-regulatory elements

PubMed Central

Uhl, Juli D.; Cook, Tiffany A.; Gebelein, Brian

2010-01-01

Hox transcription factors specify numerous cell fates along the anterior-posterior axis by regulating the expression of downstream target genes. While expression analysis has uncovered large numbers of de-regulated genes in cells with altered Hox activity, determining which are direct versus indirect targets has remained a significant challenge. Here, we characterize the DNA binding activity of Hox transcription factor complexes on eight experimentally verified cis-regulatory elements. Hox factors regulate the activity of each element by forming protein complexes with two cofactor proteins, Extradenticle (Exd) and Homothorax (Hth). Using comparative DNA binding assays, we found that a number of flexible arrangements of Hox, Exd, and Hth binding sites mediate cooperative transcription factor complexes. Moreover, analysis of a Distal-less regulatory element (DMXR) that is repressed by abdominal Hox factors revealed that suboptimal binding sites can be combined to form high affinity transcription complexes. Lastly, we determined that the anterior Hox factors are more dependent upon Exd and Hth for complex formation than posterior Hox factors. Based upon these findings, we suggest a general set of guidelines to serve as a basis for designing bioinformatics algorithms aimed at identifying Hox regulatory elements using the wealth of recently sequenced genomes. PMID:20398649

Multiple cis-regulatory elements are involved in the complex regulation of the sieve element-specific MtSEO-F1 promoter from Medicago truncatula.

PubMed

Bucsenez, M; Rüping, B; Behrens, S; Twyman, R M; Noll, G A; Prüfer, D

2012-09-01

The sieve element occlusion (SEO) gene family includes several members that are expressed specifically in immature sieve elements (SEs) in the developing phloem of dicotyledonous plants. To determine how this restricted expression profile is achieved, we analysed the SE-specific Medicago truncatula SEO-F1 promoter (PMtSEO-F1) by constructing deletion, substitution and hybrid constructs and testing them in transgenic tobacco plants using green fluorescent protein as a reporter. This revealed four promoter regions, each containing cis-regulatory elements that activate transcription in SEs. One of these segments also contained sufficient information to suppress PMtSEO-F1 transcription in the phloem companion cells (CCs). Subsequent in silico analysis revealed several candidate cis-regulatory elements that PMtSEO-F1 shares with other SEO promoters. These putative sieve element boxes (PSE boxes) are promising candidates for cis-regulatory elements controlling the SE-specific expression of PMtSEO-F1. © 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.

Open chromatin defined by DNaseI and FAIRE identifies regulatory elements that shape cell-type identity

PubMed Central

Song, Lingyun; Zhang, Zhancheng; Grasfeder, Linda L.; Boyle, Alan P.; Giresi, Paul G.; Lee, Bum-Kyu; Sheffield, Nathan C.; Gräf, Stefan; Huss, Mikael; Keefe, Damian; Liu, Zheng; London, Darin; McDaniell, Ryan M.; Shibata, Yoichiro; Showers, Kimberly A.; Simon, Jeremy M.; Vales, Teresa; Wang, Tianyuan; Winter, Deborah; Zhang, Zhuzhu; Clarke, Neil D.; Birney, Ewan; Iyer, Vishwanath R.; Crawford, Gregory E.; Lieb, Jason D.; Furey, Terrence S.

2011-01-01

The human body contains thousands of unique cell types, each with specialized functions. Cell identity is governed in large part by gene transcription programs, which are determined by regulatory elements encoded in DNA. To identify regulatory elements active in seven cell lines representative of diverse human cell types, we used DNase-seq and FAIRE-seq (Formaldehyde Assisted Isolation of Regulatory Elements) to map “open chromatin.” Over 870,000 DNaseI or FAIRE sites, which correspond tightly to nucleosome-depleted regions, were identified across the seven cell lines, covering nearly 9% of the genome. The combination of DNaseI and FAIRE is more effective than either assay alone in identifying likely regulatory elements, as judged by coincidence with transcription factor binding locations determined in the same cells. Open chromatin common to all seven cell types tended to be at or near transcription start sites and to be coincident with CTCF binding sites, while open chromatin sites found in only one cell type were typically located away from transcription start sites and contained DNA motifs recognized by regulators of cell-type identity. We show that open chromatin regions bound by CTCF are potent insulators. We identified clusters of open regulatory elements (COREs) that were physically near each other and whose appearance was coordinated among one or more cell types. Gene expression and RNA Pol II binding data support the hypothesis that COREs control gene activity required for the maintenance of cell-type identity. This publicly available atlas of regulatory elements may prove valuable in identifying noncoding DNA sequence variants that are causally linked to human disease. PMID:21750106

Rapid evolution of regulatory element libraries for tunable transcriptional and translational control of gene expression.

PubMed

Jin, Erqing; Wong, Lynn; Jiao, Yun; Engel, Jake; Holdridge, Benjamin; Xu, Peng

2017-12-01

Engineering cell factories for producing biofuels and pharmaceuticals has spurred great interests to develop rapid and efficient synthetic biology tools customized for modular pathway engineering. Along the way, combinatorial gene expression control through modification of regulatory element offered tremendous opportunity for fine-tuning gene expression and generating digital-like genetic circuits. In this report, we present an efficient evolutionary approach to build a range of regulatory control elements. The reported method allows for rapid construction of promoter, 5'UTR, terminator and trans -activating RNA libraries. Synthetic overlapping oligos with high portion of degenerate nucleotides flanking the regulatory element could be efficiently assembled to a vector expressing fluorescence reporter. This approach combines high mutation rate of the synthetic DNA with the high assembly efficiency of Gibson Mix. Our constructed library demonstrates broad range of transcriptional or translational gene expression dynamics. Specifically, both the promoter library and 5'UTR library exhibits gene expression dynamics spanning across three order of magnitude. The terminator library and trans -activating RNA library displays relatively narrowed gene expression pattern. The reported study provides a versatile toolbox for rapidly constructing a large family of prokaryotic regulatory elements. These libraries also facilitate the implementation of combinatorial pathway engineering principles and the engineering of more efficient microbial cell factory for various biomanufacturing applications.

Cell Type-Specific Chromatin Signatures Underline Regulatory DNA Elements in Human Induced Pluripotent Stem Cells and Somatic Cells.

PubMed

Zhao, Ming-Tao; Shao, Ning-Yi; Hu, Shijun; Ma, Ning; Srinivasan, Rajini; Jahanbani, Fereshteh; Lee, Jaecheol; Zhang, Sophia L; Snyder, Michael P; Wu, Joseph C

2017-11-10

Regulatory DNA elements in the human genome play important roles in determining the transcriptional abundance and spatiotemporal gene expression during embryonic heart development and somatic cell reprogramming. It is not well known how chromatin marks in regulatory DNA elements are modulated to establish cell type-specific gene expression in the human heart. We aimed to decipher the cell type-specific epigenetic signatures in regulatory DNA elements and how they modulate heart-specific gene expression. We profiled genome-wide transcriptional activity and a variety of epigenetic marks in the regulatory DNA elements using massive RNA-seq (n=12) and ChIP-seq (chromatin immunoprecipitation combined with high-throughput sequencing; n=84) in human endothelial cells (CD31 + CD144 + ), cardiac progenitor cells (Sca-1 + ), fibroblasts (DDR2 + ), and their respective induced pluripotent stem cells. We uncovered 2 classes of regulatory DNA elements: class I was identified with ubiquitous enhancer (H3K4me1) and promoter (H3K4me3) marks in all cell types, whereas class II was enriched with H3K4me1 and H3K4me3 in a cell type-specific manner. Both class I and class II regulatory elements exhibited stimulatory roles in nearby gene expression in a given cell type. However, class I promoters displayed more dominant regulatory effects on transcriptional abundance regardless of distal enhancers. Transcription factor network analysis indicated that human induced pluripotent stem cells and somatic cells from the heart selected their preferential regulatory elements to maintain cell type-specific gene expression. In addition, we validated the function of these enhancer elements in transgenic mouse embryos and human cells and identified a few enhancers that could possibly regulate the cardiac-specific gene expression. Given that a large number of genetic variants associated with human diseases are located in regulatory DNA elements, our study provides valuable resources for deciphering

Regulatory element-based prediction identifies new susceptibility regulatory variants for osteoporosis.

PubMed

Yao, Shi; Guo, Yan; Dong, Shan-Shan; Hao, Ruo-Han; Chen, Xiao-Feng; Chen, Yi-Xiao; Chen, Jia-Bin; Tian, Qing; Deng, Hong-Wen; Yang, Tie-Lin

2017-08-01

Despite genome-wide association studies (GWASs) have identified many susceptibility genes for osteoporosis, it still leaves a large part of missing heritability to be discovered. Integrating regulatory information and GWASs could offer new insights into the biological link between the susceptibility SNPs and osteoporosis. We generated five machine learning classifiers with osteoporosis-associated variants and regulatory features data. We gained the optimal classifier and predicted genome-wide SNPs to discover susceptibility regulatory variants. We further utilized Genetic Factors for Osteoporosis Consortium (GEFOS) and three in-house GWASs samples to validate the associations for predicted positive SNPs. The random forest classifier performed best among all machine learning methods with the F1 score of 0.8871. Using the optimized model, we predicted 37,584 candidate SNPs for osteoporosis. According to the meta-analysis results, a list of regulatory variants was significantly associated with osteoporosis after multiple testing corrections and contributed to the expression of known osteoporosis-associated protein-coding genes. In summary, combining GWASs and regulatory elements through machine learning could provide additional information for understanding the mechanism of osteoporosis. The regulatory variants we predicted will provide novel targets for etiology research and treatment of osteoporosis.

Evolutionary conservation of regulatory elements in vertebrate HOX gene clusters

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santini, Simona; Boore, Jeffrey L.; Meyer, Axel

2003-12-31

Due to their high degree of conservation, comparisons of DNA sequences among evolutionarily distantly-related genomes permit to identify functional regions in noncoding DNA. Hox genes are optimal candidate sequences for comparative genome analyses, because they are extremely conserved in vertebrates and occur in clusters. We aligned (Pipmaker) the nucleotide sequences of HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human and mouse (over 500 million years of evolutionary distance). We identified several highly conserved intergenic sequences, likely to be important in gene regulation. Only a few of these putative regulatory elements have been previously described as being involvedmore » in the regulation of Hox genes, while several others are new elements that might have regulatory functions. The majority of these newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac). The conserved intergenic regions located between the most rostrally expressed genes in the developing embryo are longer and better retained through evolution. We document that presumed regulatory sequences are retained differentially in either A or A clusters resulting from a genome duplication in the fish lineage. This observation supports both the hypothesis that the conserved elements are involved in gene regulation and the Duplication-Deletion-Complementation model.« less

DiRE: identifying distant regulatory elements of co-expressed genes

PubMed Central

Gotea, Valer; Ovcharenko, Ivan

2008-01-01

Regulation of gene expression in eukaryotic genomes is established through a complex cooperative activity of proximal promoters and distant regulatory elements (REs) such as enhancers, repressors and silencers. We have developed a web server named DiRE, based on the Enhancer Identification (EI) method, for predicting distant regulatory elements in higher eukaryotic genomes, namely for determining their chromosomal location and functional characteristics. The server uses gene co-expression data, comparative genomics and profiles of transcription factor binding sites (TFBSs) to determine TFBS-association signatures that can be used for discriminating specific regulatory functions. DiRE's unique feature is its ability to detect REs outside of proximal promoter regions, as it takes advantage of the full gene locus to conduct the search. DiRE can predict common REs for any set of input genes for which the user has prior knowledge of co-expression, co-function or other biologically meaningful grouping. The server predicts function-specific REs consisting of clusters of specifically-associated TFBSs and it also scores the association of individual transcription factors (TFs) with the biological function shared by the group of input genes. Its integration with the Array2BIO server allows users to start their analysis with raw microarray expression data. The DiRE web server is freely available at http://dire.dcode.org. PMID:18487623

Deciphering RNA Regulatory Elements Involved in the Developmental and Environmental Gene Regulation of Trypanosoma brucei.

PubMed

Gazestani, Vahid H; Salavati, Reza

2015-01-01

Trypanosoma brucei is a vector-borne parasite with intricate life cycle that can cause serious diseases in humans and animals. This pathogen relies on fine regulation of gene expression to respond and adapt to variable environments, with implications in transmission and infectivity. However, the involved regulatory elements and their mechanisms of actions are largely unknown. Here, benefiting from a new graph-based approach for finding functional regulatory elements in RNA (GRAFFER), we have predicted 88 new RNA regulatory elements that are potentially involved in the gene regulatory network of T. brucei. We show that many of these newly predicted elements are responsive to both transcriptomic and proteomic changes during the life cycle of the parasite. Moreover, we found that 11 of predicted elements strikingly resemble previously identified regulatory elements for the parasite. Additionally, comparison with previously predicted motifs on T. brucei suggested the superior performance of our approach based on the current limited knowledge of regulatory elements in T. brucei.

In silico analysis of cis-acting regulatory elements in 5' regulatory regions of sucrose transporter gene families in rice (Oryza sativa Japonica) and Arabidopsis thaliana.

PubMed

Ibraheem, Omodele; Botha, Christiaan E J; Bradley, Graeme

2010-12-01

The regulation of gene expression involves a multifarious regulatory system. Each gene contains a unique combination of cis-acting regulatory sequence elements in the 5' regulatory region that determines its temporal and spatial expression. Cis-acting regulatory elements are essential transcriptional gene regulatory units; they control many biological processes and stress responses. Thus a full understanding of the transcriptional gene regulation system will depend on successful functional analyses of cis-acting elements. Cis-acting regulatory elements present within the 5' regulatory region of the sucrose transporter gene families in rice (Oryza sativa Japonica cultivar-group) and Arabidopsis thaliana, were identified using a bioinformatics approach. The possible cis-acting regulatory elements were predicted by scanning 1.5kbp of 5' regulatory regions of the sucrose transporter genes translational start sites, using Plant CARE, PLACE and Genomatix Matinspector professional databases. Several cis-acting regulatory elements that are associated with plant development, plant hormonal regulation and stress response were identified, and were present in varying frequencies within the 1.5kbp of 5' regulatory region, among which are; A-box, RY, CAT, Pyrimidine-box, Sucrose-box, ABRE, ARF, ERE, GARE, Me-JA, ARE, DRE, GA-motif, GATA, GT-1, MYC, MYB, W-box, and I-box. This result reveals the probable cis-acting regulatory elements that possibly are involved in the expression and regulation of sucrose transporter gene families in rice and Arabidopsis thaliana during cellular development or environmental stress conditions. Copyright © 2010 Elsevier Ltd. All rights reserved.

Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene.

PubMed

Dale, Rodney M; Topczewski, Jacek

2011-09-15

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5' of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue

Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene

PubMed Central

Dale, Rodney M.; Topczewski, Jacek

2011-01-01

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a. We focused our analysis on col2a1a, as it is expressed in both the stacked chondrocytes and the perichondrium. By comparing the genomic sequence surrounding the predicted transcriptional start site of col2a1a among several species of teleosts we identified a small highly conserved sequence (R2) located 1.7 kb upstream of the presumptive transcriptional initiation site. Interestingly, neither the sequence nor location of this element is conserved between teleost and mammalian Col2a1. We generated transient and stable transgenic lines with just the R2 element or the entire 1.7 kb fragment 5’ of the transcriptional initiation site. The identified regulatory elements enable the tracking of cellular development in various tissues by driving robust reporter expression in craniofacial cartilage, ear, notochord, floor plate, hypochord and fins in a pattern similar to the expression of endogenous col2a1a. Using a reporter gene driven by the R2 regulatory element, we analyzed the morphogenesis of the notochord sheath cells as they withdraw from the stack of initially uniform cells and encase the inflating vacuolated notochord cells. Finally, we show that like endogenous col2a1a, craniofacial expression of these reporter constructs depends on Sox9a transcription factor activity. At the same time, notochord expression is maintained after Sox9a knockdown, suggesting that other factors can activate expression through the identified regulatory element in this tissue

A conserved RNA structural element within the hepatitis B virus post-transcriptional regulatory element enhance nuclear export of intronless transcripts and repress the splicing mechanism.

PubMed

Visootsat, Akasit; Payungporn, Sunchai; T-Thienprasert, Nattanan P

2015-12-01

Hepatitis B virus (HBV) infection is a primary cause of hepatocellular carcinoma and liver cirrhosis worldwide. To develop novel antiviral drugs, a better understanding of HBV gene expression regulation is vital. One important aspect is to understand how HBV hijacks the cellular machinery to export unspliced RNA from the nucleus. The HBV post-transcriptional regulatory element (HBV PRE) has been proposed to be the HBV RNA nuclear export element. However, the function remains controversial, and the core element is unclear. This study, therefore, aimed to identify functional regulatory elements within the HBV PRE and investigate their functions. Using bioinformatics programs based on sequence conservation and conserved RNA secondary structures, three regulatory elements were predicted, namely PRE 1151-1410, PRE 1520-1620 and PRE 1650-1684. PRE 1151-1410 significantly increased intronless and unspliced luciferase activity in both HepG2 and COS-7 cells. Likewise, PRE 1151-1410 significantly elevated intronless and unspliced HBV surface transcripts in liver cancer cells. Moreover, motif analysis predicted that PRE 1151-1410 contains several regulatory motifs. This study reported the roles of PRE 1151-1410 in intronless transcript nuclear export and the splicing mechanism. Additionally, these results provide knowledge in the field of HBV RNA regulation. Moreover, PRE 1151-1410 may be used to enhance the expression of other mRNAs in intronless reporter plasmids.

Cis-regulatory Elements and Human Evolution

PubMed Central

Siepel, Adam

2014-01-01

Modification of gene regulation has long been considered an important force in human evolution, particularly through changes to cis-regulatory elements (CREs) that function in transcriptional regulation. For decades, however, the study of cis-regulatory evolution was severely limited by the available data. New data sets describing the locations of CREs and genetic variation within and between species have now made it possible to study CRE evolution much more directly on a genome-wide scale. Here, we review recent research on the evolution of CREs in humans based on large-scale genomic data sets. We consider inferences based on primate divergence, human polymorphism, and combinations of divergence and polymorphism. We then consider “new frontiers” in this field stemming from recent research on transcriptional regulation. PMID:25218861

Genomic identification of regulatory elements by evolutionary sequence comparison and functional analysis.

PubMed

Loots, Gabriela G

2008-01-01

Despite remarkable recent advances in genomics that have enabled us to identify most of the genes in the human genome, comparable efforts to define transcriptional cis-regulatory elements that control gene expression are lagging behind. The difficulty of this task stems from two equally important problems: our knowledge of how regulatory elements are encoded in genomes remains elementary, and there is a vast genomic search space for regulatory elements, since most of mammalian genomes are noncoding. Comparative genomic approaches are having a remarkable impact on the study of transcriptional regulation in eukaryotes and currently represent the most efficient and reliable methods of predicting noncoding sequences likely to control the patterns of gene expression. By subjecting eukaryotic genomic sequences to computational comparisons and subsequent experimentation, we are inching our way toward a more comprehensive catalog of common regulatory motifs that lie behind fundamental biological processes. We are still far from comprehending how the transcriptional regulatory code is encrypted in the human genome and providing an initial global view of regulatory gene networks, but collectively, the continued development of comparative and experimental approaches will rapidly expand our knowledge of the transcriptional regulome.

Regulatory elements involved in tax-mediated transactivation of the HTLV-I LTR.

PubMed

Seeler, J S; Muchardt, C; Podar, M; Gaynor, R B

1993-10-01

HTLV-I is the etiologic agent of adult T-cell leukemia. In this study, we investigated the regulatory elements and cellular transcription factors which function in modulating HTLV-I gene expression in response to the viral transactivator protein, tax. Transfection experiments into Jurkat cells of a variety of site-directed mutants in the HTLV-1 LTR indicated that each of the three motifs A, B, and C within the 21-bp repeats, the binding sites for the Ets family of proteins, and the TATA box all influenced the degree of tax-mediated activation. Tax is also able to activate gene expression of other viral and cellular promoters. Tax activation of the IL-2 receptor and the HIV-1 LTR is mediated through NF-kappa B motifs. Interestingly, sequences in the 21-bp repeat B and C motifs contain significant homology with NF-kappa B regulatory elements. We demonstrated that an NF-kappa B binding protein, PRDII-BF1, but not the rel protein, bound to the B and C motifs in the 21-bp repeat. PRDII-BF1 was also able to stimulate activation of HTLV-I gene expression by tax. The role of the Ets proteins on modulating tax activation was also studied. Ets 1 but not Ets 2 was capable of increasing the degree of tax activation of the HTLV-I LTR. These results suggest that tax activates gene expression by either direct or indirect interaction with several cellular transcription factors that bind to the HTLV-I LTR.

Identification of germline transcriptional regulatory elements in Aedes aegypti.

PubMed

Akbari, Omar S; Papathanos, Philippos A; Sandler, Jeremy E; Kennedy, Katie; Hay, Bruce A

2014-02-04

The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UD(MEL), and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. Here we report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference.

Identification of germline transcriptional regulatory elements in Aedes aegypti

NASA Astrophysics Data System (ADS)

Akbari, Omar S.; Papathanos, Philippos A.; Sandler, Jeremy E.; Kennedy, Katie; Hay, Bruce A.

2014-02-01

The mosquito Aedes aegypti is the principal vector for the yellow fever and dengue viruses, and is also responsible for recent outbreaks of the alphavirus chikungunya. Vector control strategies utilizing engineered gene drive systems are being developed as a means of replacing wild, pathogen transmitting mosquitoes with individuals refractory to disease transmission, or bringing about population suppression. Several of these systems, including Medea, UDMEL, and site-specific nucleases, which can be used to drive genes into populations or bring about population suppression, utilize transcriptional regulatory elements that drive germline-specific expression. Here we report the identification of multiple regulatory elements able to drive gene expression specifically in the female germline, or in the male and female germline, in the mosquito Aedes aegypti. These elements can also be used as tools with which to probe the roles of specific genes in germline function and in the early embryo, through overexpression or RNA interference.

Genome-wide colonization of gene regulatory elements by G4 DNA motifs

PubMed Central

Du, Zhuo; Zhao, Yiqiang; Li, Ning

2009-01-01

G-quadruplex (or G4 DNA), a stable four-stranded structure found in guanine-rich regions, is implicated in the transcriptional regulation of genes involved in growth and development. Previous studies on the role of G4 DNA in gene regulation mostly focused on genomic regions proximal to transcription start sites (TSSs). To gain a more comprehensive understanding of the regulatory role of G4 DNA, we examined the landscape of potential G4 DNA (PG4Ms) motifs in the human genome and found that G4 motifs, not restricted to those found in the TSS-proximal regions, are bias toward gene-associated regions. Significantly, analyses of G4 motifs in seven types of well-known gene regulatory elements revealed a constitutive enrichment pattern and the clusters of G4 motifs tend to be colocalized with regulatory elements. Considering our analysis from a genome evolutionary perspective, we found evidence that the occurrence and accumulation of certain progenitors and canonical G4 DNA motifs within regulatory regions were progressively favored by natural selection. Our results suggest that G4 DNA motifs are ‘colonized’ in regulatory regions, supporting a likely genome-wide role of G4 DNA in gene regulation. We hypothesize that G4 DNA is a regulatory apparatus situated in regulatory elements, acting as a molecular switch that can modulate the role of the host functional regions, by transition in DNA structure. PMID:19759215

Genome-Wide Identification of Regulatory Elements and Reconstruction of Gene Regulatory Networks of the Green Alga Chlamydomonas reinhardtii under Carbon Deprivation

PubMed Central

Vischi Winck, Flavia; Arvidsson, Samuel; Riaño-Pachón, Diego Mauricio; Hempel, Sabrina; Koseska, Aneta; Nikoloski, Zoran; Urbina Gomez, David Alejandro; Rupprecht, Jens; Mueller-Roeber, Bernd

2013-01-01

The unicellular green alga Chlamydomonas reinhardtii is a long-established model organism for studies on photosynthesis and carbon metabolism-related physiology. Under conditions of air-level carbon dioxide concentration [CO2], a carbon concentrating mechanism (CCM) is induced to facilitate cellular carbon uptake. CCM increases the availability of carbon dioxide at the site of cellular carbon fixation. To improve our understanding of the transcriptional control of the CCM, we employed FAIRE-seq (formaldehyde-assisted Isolation of Regulatory Elements, followed by deep sequencing) to determine nucleosome-depleted chromatin regions of algal cells subjected to carbon deprivation. Our FAIRE data recapitulated the positions of known regulatory elements in the promoter of the periplasmic carbonic anhydrase (Cah1) gene, which is upregulated during CCM induction, and revealed new candidate regulatory elements at a genome-wide scale. In addition, time series expression patterns of 130 transcription factor (TF) and transcription regulator (TR) genes were obtained for cells cultured under photoautotrophic condition and subjected to a shift from high to low [CO2]. Groups of co-expressed genes were identified and a putative directed gene-regulatory network underlying the CCM was reconstructed from the gene expression data using the recently developed IOTA (inner composition alignment) method. Among the candidate regulatory genes, two members of the MYB-related TF family, Lcr1 (Low-CO 2 response regulator 1) and Lcr2 (Low-CO 2 response regulator 2), may play an important role in down-regulating the expression of a particular set of TF and TR genes in response to low [CO2]. The results obtained provide new insights into the transcriptional control of the CCM and revealed more than 60 new candidate regulatory genes. Deep sequencing of nucleosome-depleted genomic regions indicated the presence of new, previously unknown regulatory elements in the C. reinhardtii genome. Our work can

Evolution of UCP1 Transcriptional Regulatory Elements Across the Mammalian Phylogeny

PubMed Central

Gaudry, Michael J.; Campbell, Kevin L.

2017-01-01

Uncoupling protein 1 (UCP1) permits non-shivering thermogenesis (NST) when highly expressed in brown adipose tissue (BAT) mitochondria. Exclusive to placental mammals, BAT has commonly been regarded to be advantageous for thermoregulation in hibernators, small-bodied species, and the neonates of larger species. While numerous regulatory control motifs associated with UCP1 transcription have been proposed for murid rodents, it remains unclear whether these are conserved across the eutherian mammal phylogeny and hence essential for UCP1 expression. To address this shortcoming, we conducted a broad comparative survey of putative UCP1 transcriptional regulatory elements in 139 mammals (135 eutherians). We find no evidence for presence of a UCP1 enhancer in monotremes and marsupials, supporting the hypothesis that this control region evolved in a stem eutherian ancestor. We additionally reveal that several putative promoter elements (e.g., CRE-4, CCAAT) identified in murid rodents are not conserved among BAT-expressing eutherians, and together with the putative regulatory region (PRR) and CpG island do not appear to be crucial for UCP1 expression. The specificity and importance of the upTRE, dnTRE, URE1, CRE-2, RARE-2, NBRE, BRE-1, and BRE-2 enhancer elements first described from rats and mice are moreover uncertain as these motifs differ substantially—but generally remain highly conserved—in other BAT-expressing eutherians. Other UCP1 enhancer motifs (CRE-3, PPRE, and RARE-3) as well as the TATA box are also highly conserved in nearly all eutherian lineages with an intact UCP1. While these transcriptional regulatory motifs are generally also maintained in species where this gene is pseudogenized, the loss or degeneration of key basal promoter (e.g., TATA box) and enhancer elements in other UCP1-lacking lineages make it unlikely that the enhancer region is pleiotropic (i.e., co-regulates additional genes). Importantly, differential losses of (or mutations within

Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells.

PubMed

Laurette, Patrick; Strub, Thomas; Koludrovic, Dana; Keime, Céline; Le Gras, Stéphanie; Seberg, Hannah; Van Otterloo, Eric; Imrichova, Hana; Siddaway, Robert; Aerts, Stein; Cornell, Robert A; Mengus, Gabrielle; Davidson, Irwin

2015-03-24

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.

Deep conservation of cis-regulatory elements in metazoans

PubMed Central

Maeso, Ignacio; Irimia, Manuel; Tena, Juan J.; Casares, Fernando; Gómez-Skarmeta, José Luis

2013-01-01

Despite the vast morphological variation observed across phyla, animals share multiple basic developmental processes orchestrated by a common ancestral gene toolkit. These genes interact with each other building complex gene regulatory networks (GRNs), which are encoded in the genome by cis-regulatory elements (CREs) that serve as computational units of the network. Although GRN subcircuits involved in ancient developmental processes are expected to be at least partially conserved, identification of CREs that are conserved across phyla has remained elusive. Here, we review recent studies that revealed such deeply conserved CREs do exist, discuss the difficulties associated with their identification and describe new approaches that will facilitate this search. PMID:24218633

The 3’-Jα Region of the TCRα Locus Bears Gene Regulatory Activity in Thymic and Peripheral T Cells

PubMed Central

Kučerová-Levisohn, Martina; Knirr, Stefan; Mejia, Rosa I.; Ortiz, Benjamin D.

2015-01-01

Much progress has been made in understanding the important cis-mediated controls on mouse TCRα gene function, including identification of the Eα enhancer and TCRα locus control region (LCR). Nevertheless, previous data have suggested that other cis-regulatory elements may reside in the locus outside of the Eα/LCR. Based on prior findings, we hypothesized the existence of gene regulatory elements in a 3.9-kb region 5’ of the Cα exons. Using DNase hypersensitivity assays and TCRα BAC reporter transgenes in mice, we detected gene regulatory activity within this 3.9-kb region. This region is active in both thymic and peripheral T cells, and selectively affects upstream, but not downstream, gene expression. Together, these data indicate the existence of a novel cis-acting regulatory complex that contributes to TCRα transgene expression in vivo. The active chromatin sites we discovered within this region would remain in the locus after TCRα gene rearrangement, and thus may contribute to endogenous TCRα gene activity, particularly in peripheral T cells, where the Eα element has been found to be inactive. PMID:26177549

Potential Novel Mechanism for Axenfeld-Rieger Syndrome: Deletion of a Distant Region Containing Regulatory Elements of PITX2

PubMed Central

Volkmann, Bethany A.; Zinkevich, Natalya S.; Mustonen, Aki; Schilter, Kala F.; Bosenko, Dmitry V.; Reis, Linda M.; Broeckel, Ulrich; Link, Brian A.

2011-01-01

Purpose. Mutations in PITX2 are associated with Axenfeld-Rieger syndrome (ARS), which involves ocular, dental, and umbilical abnormalities. Identification of cis-regulatory elements of PITX2 is important to better understand the mechanisms of disease. Methods. Conserved noncoding elements surrounding PITX2/pitx2 were identified and examined through transgenic analysis in zebrafish; expression pattern was studied by in situ hybridization. Patient samples were screened for deletion/duplication of the PITX2 upstream region using arrays and probes. Results. Zebrafish pitx2 demonstrates conserved expression during ocular and craniofacial development. Thirteen conserved noncoding sequences positioned within a gene desert as far as 1.1 Mb upstream of the human PITX2 gene were identified; 11 have enhancer activities consistent with pitx2 expression. Ten elements mediated expression in the developing brain, four regions were active during eye formation, and two sequences were associated with craniofacial expression. One region, CE4, located approximately 111 kb upstream of PITX2, directed a complex pattern including expression in the developing eye and craniofacial region, the classic sites affected in ARS. Screening of ARS patients identified an approximately 7600-kb deletion that began 106 to 108 kb upstream of the PITX2 gene, leaving PITX2 intact while removing regulatory elements CE4 to CE13. Conclusions. These data suggest the presence of a complex distant regulatory matrix within the gene desert located upstream of PITX2 with an essential role in its activity and provides a possible mechanism for the previous reports of ARS in patients with balanced translocations involving the 4q25 region upstream of PITX2 and the current patient with an upstream deletion. PMID:20881290

Tissue-Specific Enrichment of Lymphoma Risk Loci in Regulatory Elements

PubMed Central

Hayes, James E.; Trynka, Gosia; Vijai, Joseph; Offit, Kenneth; Raychaudhuri, Soumya; Klein, Robert J.

2015-01-01

Though numerous polymorphisms have been associated with risk of developing lymphoma, how these variants function to promote tumorigenesis is poorly understood. Here, we report that lymphoma risk SNPs, especially in the non-Hodgkin’s lymphoma subtype chronic lymphocytic leukemia, are significantly enriched for co-localization with epigenetic marks of active gene regulation. These enrichments were seen in a lymphoid-specific manner for numerous ENCODE datasets, including DNase-hypersensitivity as well as multiple segmentation-defined enhancer regions. Furthermore, we identify putatively functional SNPs that are both in regulatory elements in lymphocytes and are associated with gene expression changes in blood. We developed an algorithm, UES, that uses a Monte Carlo simulation approach to calculate the enrichment of previously identified risk SNPs in various functional elements. This multiscale approach integrating multiple datasets helps disentangle the underlying biology of lymphoma, and more broadly, is generally applicable to GWAS results from other diseases as well. PMID:26422229

Disease-associated variants in different categories of disease located in distinct regulatory elements.

PubMed

Ma, Meng; Ru, Ying; Chuang, Ling-Shiang; Hsu, Nai-Yun; Shi, Li-Song; Hakenberg, Jörg; Cheng, Wei-Yi; Uzilov, Andrew; Ding, Wei; Glicksberg, Benjamin S; Chen, Rong

2015-01-01

The invention of high throughput sequencing technologies has led to the discoveries of hundreds of thousands of genetic variants associated with thousands of human diseases. Many of these genetic variants are located outside the protein coding regions, and as such, it is challenging to interpret the function of these genetic variants by traditional genetic approaches. Recent genome-wide functional genomics studies, such as FANTOM5 and ENCODE have uncovered a large number of regulatory elements across hundreds of different tissues or cell lines in the human genome. These findings provide an opportunity to study the interaction between regulatory elements and disease-associated genetic variants. Identifying these diseased-related regulatory elements will shed light on understanding the mechanisms of how these variants regulate gene expression and ultimately result in disease formation and progression. In this study, we curated and categorized 27,558 Mendelian disease variants, 20,964 complex disease variants, 5,809 cancer predisposing germline variants, and 43,364 recurrent cancer somatic mutations. Compared against nine different types of regulatory regions from FANTOM5 and ENCODE projects, we found that different types of disease variants show distinctive propensity for particular regulatory elements. Mendelian disease variants and recurrent cancer somatic mutations are 22-fold and 10- fold significantly enriched in promoter regions respectively (q<0.001), compared with allele-frequency-matched genomic background. Separate from these two categories, cancer predisposing germline variants are 27-fold enriched in histone modification regions (q<0.001), 10-fold enriched in chromatin physical interaction regions (q<0.001), and 6-fold enriched in transcription promoters (q<0.001). Furthermore, Mendelian disease variants and recurrent cancer somatic mutations share very similar distribution across types of functional effects. We further found that regulatory regions are

Disease-associated variants in different categories of disease located in distinct regulatory elements

PubMed Central

2015-01-01

Background The invention of high throughput sequencing technologies has led to the discoveries of hundreds of thousands of genetic variants associated with thousands of human diseases. Many of these genetic variants are located outside the protein coding regions, and as such, it is challenging to interpret the function of these genetic variants by traditional genetic approaches. Recent genome-wide functional genomics studies, such as FANTOM5 and ENCODE have uncovered a large number of regulatory elements across hundreds of different tissues or cell lines in the human genome. These findings provide an opportunity to study the interaction between regulatory elements and disease-associated genetic variants. Identifying these diseased-related regulatory elements will shed light on understanding the mechanisms of how these variants regulate gene expression and ultimately result in disease formation and progression. Results In this study, we curated and categorized 27,558 Mendelian disease variants, 20,964 complex disease variants, 5,809 cancer predisposing germline variants, and 43,364 recurrent cancer somatic mutations. Compared against nine different types of regulatory regions from FANTOM5 and ENCODE projects, we found that different types of disease variants show distinctive propensity for particular regulatory elements. Mendelian disease variants and recurrent cancer somatic mutations are 22-fold and 10- fold significantly enriched in promoter regions respectively (q<0.001), compared with allele-frequency-matched genomic background. Separate from these two categories, cancer predisposing germline variants are 27-fold enriched in histone modification regions (q<0.001), 10-fold enriched in chromatin physical interaction regions (q<0.001), and 6-fold enriched in transcription promoters (q<0.001). Furthermore, Mendelian disease variants and recurrent cancer somatic mutations share very similar distribution across types of functional effects. We further found that

Regulatory elements driving the expression of skeletal lineage reporters differ during bone development and adulthood.

PubMed

Stiers, Pieter-Jan; van Gastel, Nick; Moermans, Karen; Stockmans, Ingrid; Carmeliet, Geert

2017-12-01

To improve bone healing or regeneration more insight in the fate and role of the different skeletal cell types is required. Mouse models for fate mapping and lineage tracing of skeletal cells, using stage-specific promoters, have advanced our understanding of bone development, a process that is largely recapitulated during bone repair. However, validation of these models is often only performed during development, whereas proof of the activity and specificity of the used promoters during the bone regenerative process is limited. Here, we show that the regulatory elements of the 6kb collagen type II promoter are not adequate to drive gene expression during bone repair. Similarly, the 2.3kb promoter of collagen type I lacks activity in adult mice, but the 3.2kb promoter is suitable. Furthermore, Cre-mediated fate mapping allows the visualization of progeny, but this label retention may hinder to distinguish these cells from ones with active expression of the marker at later time points. Together, our results show that the lineage-specific regulatory elements driving gene expression during bone development differ from those required later in life and during bone repair, and justify validation of lineage-specific cell tracing and gene silencing strategies during fracture healing and bone regenerative applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements.

PubMed

Kyrchanova, Olga; Chetverina, Darya; Maksimenko, Oksana; Kullyev, Andrey; Georgiev, Pavel

2008-12-01

Insulators are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. According to previous data, an interaction (pairing) between some Drosophila insulators can support distant activation of a promoter by an enhancer. Here, we have demonstrated that pairs of well-studied insulators such as scs-scs, scs'-scs', 1A2-1A2 and Wari-Wari support distant activation of the white promoter by the yeast GAL4 activator in an orientation-dependent manner. The same is true for the efficiency of the enhancer that stimulates white expression in the eyes. In all insulator pairs tested, stimulation of the white gene was stronger when insulators were inserted between the eye enhancer or GAL4 and the white promoter in opposite orientations relative to each other. As shown previously, Zw5, Su(Hw) and dCTCF proteins are required for the functioning of different insulators that do not interact with each other. Here, strong functional interactions have been revealed between DNA fragments containing binding sites for either Zw5 or Su(Hw) or dCTCF protein but not between heterologous binding sites [Zw5-Su(Hw), dCTCF-Su(Hw), or dCTCF-Zw5]. These results suggest that insulator proteins can support selective interactions between distant regulatory elements.

An Autonomous BMP2 Regulatory Element in Mesenchymal Cells

PubMed Central

Kruithof, Boudewijn P.T.; Fritz, David T.; Liu, Yijun; Garsetti, Diane E.; Frank, David B.; Pregizer, Steven K.; Gaussin, Vinciane; Mortlock, Douglas P.; Rogers, Melissa B.

2014-01-01

BMP2 is a morphogen that controls mesenchymal cell differentiation and behavior. For example, BMP2 concentration controls the differentiation of mesenchymal precursors into myocytes, adipocytes, chondrocytes, and osteoblasts. Sequences within the 3′untranslated region (UTR) of the Bmp2 mRNA mediate a post-transcriptional block of protein synthesis. Interaction of cell and developmental stage-specific trans-regulatory factors with the 3′UTR is a nimble and versatile mechanism for modulating this potent morphogen in different cell types. We show here, that an ultra-conserved sequence in the 3′UTR functions independently of promoter, coding region, and 3′UTR context in primary and immortalized tissue culture cells and in transgenic mice. Our findings indicate that the ultra-conserved sequence is an autonomously functioning post-transcriptional element that may be used to modulate the level of BMP2 and other proteins while retaining tissue specific regulatory elements. PMID:21268088

Two regulatory RNA elements affect TisB-dependent depolarization and persister formation.

PubMed

Berghoff, Bork A; Hoekzema, Mirthe; Aulbach, Lena; Wagner, E Gerhart H

2017-03-01

Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR-1 toxin-antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR-1 in TisB-dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug-tolerant by arresting growth. The RNA antitoxin IstR-1 sets a threshold for TisB-dependent depolarization under DNA-damaging conditions, resulting in two sub-populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5' UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub-population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity. © 2016 John Wiley & Sons Ltd.

The identification of cis-regulatory elements: A review from a machine learning perspective.

PubMed

Li, Yifeng; Chen, Chih-Yu; Kaye, Alice M; Wasserman, Wyeth W

2015-12-01

The majority of the human genome consists of non-coding regions that have been called junk DNA. However, recent studies have unveiled that these regions contain cis-regulatory elements, such as promoters, enhancers, silencers, insulators, etc. These regulatory elements can play crucial roles in controlling gene expressions in specific cell types, conditions, and developmental stages. Disruption to these regions could contribute to phenotype changes. Precisely identifying regulatory elements is key to deciphering the mechanisms underlying transcriptional regulation. Cis-regulatory events are complex processes that involve chromatin accessibility, transcription factor binding, DNA methylation, histone modifications, and the interactions between them. The development of next-generation sequencing techniques has allowed us to capture these genomic features in depth. Applied analysis of genome sequences for clinical genetics has increased the urgency for detecting these regions. However, the complexity of cis-regulatory events and the deluge of sequencing data require accurate and efficient computational approaches, in particular, machine learning techniques. In this review, we describe machine learning approaches for predicting transcription factor binding sites, enhancers, and promoters, primarily driven by next-generation sequencing data. Data sources are provided in order to facilitate testing of novel methods. The purpose of this review is to attract computational experts and data scientists to advance this field. Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.

Extracellular Acidic pH Activates the Sterol Regulatory Element-Binding Protein 2 to Promote Tumor Progression.

PubMed

Kondo, Ayano; Yamamoto, Shogo; Nakaki, Ryo; Shimamura, Teppei; Hamakubo, Takao; Sakai, Juro; Kodama, Tatsuhiko; Yoshida, Tetsuo; Aburatani, Hiroyuki; Osawa, Tsuyoshi

2017-02-28

Conditions of the tumor microenvironment, such as hypoxia and nutrient starvation, play critical roles in cancer progression. However, the role of acidic extracellular pH in cancer progression is not studied as extensively as that of hypoxia. Here, we show that extracellular acidic pH (pH 6.8) triggered activation of sterol regulatory element-binding protein 2 (SREBP2) by stimulating nuclear translocation and promoter binding to its targets, along with intracellular acidification. Interestingly, inhibition of SREBP2, but not SREBP1, suppressed the upregulation of low pH-induced cholesterol biosynthesis-related genes. Moreover, acyl-CoA synthetase short-chain family member 2 (ACSS2), a direct SREBP2 target, provided a growth advantage to cancer cells under acidic pH. Furthermore, acidic pH-responsive SREBP2 target genes were associated with reduced overall survival of cancer patients. Thus, our findings show that SREBP2 is a key transcriptional regulator of metabolic genes and progression of cancer cells, partly in response to extracellular acidification. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Wnt-mediated activation of NeuroD1 and retro-elements during adult neurogenesis.

PubMed

Kuwabara, Tomoko; Hsieh, Jenny; Muotri, Alysson; Yeo, Gene; Warashina, Masaki; Lie, Dieter Chichung; Moore, Lynne; Nakashima, Kinichi; Asashima, Makoto; Gage, Fred H

2009-09-01

In adult hippocampus, new neurons are continuously generated from neural stem cells (NSCs), but the molecular mechanisms regulating adult neurogenesis remain elusive. We found that Wnt signaling, together with the removal of Sox2, triggered the expression of NeuroD1 in mice. This transcriptional regulatory mechanism was dependent on a DNA element containing overlapping Sox2 and T-cell factor/lymphoid enhancer factor (TCF/LEF)-binding sites (Sox/LEF) in the promoter. Notably, Sox/LEF sites were also found in long interspersed nuclear element 1 (LINE-1) elements, consistent with their critical roles in the transition of NSCs to proliferating neuronal progenitors. Our results describe a previously unknown Wnt-mediated regulatory mechanism that simultaneously coordinates activation of NeuroD1 and LINE-1, which is important for adult neurogenesis and survival of neuronal progenitors. Moreover, the discovery that LINE-1 retro-elements embedded in the mammalian genome can function as bi-directional promoters suggests that Sox/LEF regulatory sites may represent a general mechanism, at least in part, for relaying environmental signals to other nearby loci to promote adult hippocampal neurogenesis.

A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design.

PubMed

Smith, Robin P; Riesenfeld, Samantha J; Holloway, Alisha K; Li, Qiang; Murphy, Karl K; Feliciano, Natalie M; Orecchia, Lorenzo; Oksenberg, Nir; Pollard, Katherine S; Ahituv, Nadav

2013-07-18

Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences. We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly. This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.

Orientation-dependent interaction between Drosophila insulators is a property of this class of regulatory elements

PubMed Central

Kyrchanova, Olga; Chetverina, Darya; Maksimenko, Oksana; Kullyev, Andrey; Georgiev, Pavel

2008-01-01

Insulators are defined as a class of regulatory elements that delimit independent transcriptional domains within eukaryotic genomes. According to previous data, an interaction (pairing) between some Drosophila insulators can support distant activation of a promoter by an enhancer. Here, we have demonstrated that pairs of well-studied insulators such as scs–scs, scs’–scs’, 1A2–1A2 and Wari–Wari support distant activation of the white promoter by the yeast GAL4 activator in an orientation-dependent manner. The same is true for the efficiency of the enhancer that stimulates white expression in the eyes. In all insulator pairs tested, stimulation of the white gene was stronger when insulators were inserted between the eye enhancer or GAL4 and the white promoter in opposite orientations relative to each other. As shown previously, Zw5, Su(Hw) and dCTCF proteins are required for the functioning of different insulators that do not interact with each other. Here, strong functional interactions have been revealed between DNA fragments containing binding sites for either Zw5 or Su(Hw) or dCTCF protein but not between heterologous binding sites [Zw5–Su(Hw), dCTCF–Su(Hw), or dCTCF–Zw5]. These results suggest that insulator proteins can support selective interactions between distant regulatory elements. PMID:18987002

RNA-Seq of Bacillus licheniformis: active regulatory RNA features expressed within a productive fermentation.

PubMed

Wiegand, Sandra; Dietrich, Sascha; Hertel, Robert; Bongaerts, Johannes; Evers, Stefan; Volland, Sonja; Daniel, Rolf; Liesegang, Heiko

2013-10-01

The production of enzymes by an industrial strain requires a complex adaption of the bacterial metabolism to the conditions within the fermenter. Regulatory events within the process result in a dynamic change of the transcriptional activity of the genome. This complex network of genes is orchestrated by proteins as well as regulatory RNA elements. Here we present an RNA-Seq based study considering selected phases of an industry-oriented fermentation of Bacillus licheniformis. A detailed analysis of 20 strand-specific RNA-Seq datasets revealed a multitude of transcriptionally active genomic regions. 3314 RNA features encoded by such active loci have been identified and sorted into ten functional classes. The identified sequences include the expected RNA features like housekeeping sRNAs, metabolic riboswitches and RNA switches well known from studies on Bacillus subtilis as well as a multitude of completely new candidates for regulatory RNAs. An unexpectedly high number of 855 RNA features are encoded antisense to annotated protein and RNA genes, in addition to 461 independently transcribed small RNAs. These antisense transcripts contain molecules with a remarkable size range variation from 38 to 6348 base pairs in length. The genome of the type strain B. licheniformis DSM13 was completely reannotated using data obtained from RNA-Seq analyses and from public databases. The hereby generated data-sets represent a solid amount of knowledge on the dynamic transcriptional activities during the investigated fermentation stages. The identified regulatory elements enable research on the understanding and the optimization of crucial metabolic activities during a productive fermentation of Bacillus licheniformis strains.

Sheep genome functional annotation reveals proximal regulatory elements contributed to the evolution of modern breeds.

PubMed

Naval-Sanchez, Marina; Nguyen, Quan; McWilliam, Sean; Porto-Neto, Laercio R; Tellam, Ross; Vuocolo, Tony; Reverter, Antonio; Perez-Enciso, Miguel; Brauning, Rudiger; Clarke, Shannon; McCulloch, Alan; Zamani, Wahid; Naderi, Saeid; Rezaei, Hamid Reza; Pompanon, Francois; Taberlet, Pierre; Worley, Kim C; Gibbs, Richard A; Muzny, Donna M; Jhangiani, Shalini N; Cockett, Noelle; Daetwyler, Hans; Kijas, James

2018-02-28

Domestication fundamentally reshaped animal morphology, physiology and behaviour, offering the opportunity to investigate the molecular processes driving evolutionary change. Here we assess sheep domestication and artificial selection by comparing genome sequence from 43 modern breeds (Ovis aries) and their Asian mouflon ancestor (O. orientalis) to identify selection sweeps. Next, we provide a comparative functional annotation of the sheep genome, validated using experimental ChIP-Seq of sheep tissue. Using these annotations, we evaluate the impact of selection and domestication on regulatory sequences and find that sweeps are significantly enriched for protein coding genes, proximal regulatory elements of genes and genome features associated with active transcription. Finally, we find individual sites displaying strong allele frequency divergence are enriched for the same regulatory features. Our data demonstrate that remodelling of gene expression is likely to have been one of the evolutionary forces that drove phenotypic diversification of this common livestock species.

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

PubMed Central

Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

1992-01-01

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

Design and testing of regulatory cassettes for optimal activity in skeletal and cardiac muscles.

PubMed

Himeda, Charis L; Chen, Xiaolan; Hauschka, Stephen D

2011-01-01

Gene therapy for muscular dystrophies requires efficient gene delivery to the striated musculature and specific, high-level expression of the therapeutic gene in a physiologically diverse array of muscles. This can be achieved by the use of recombinant adeno-associated virus vectors in conjunction with muscle-specific regulatory cassettes. We have constructed several generations of regulatory cassettes based on the enhancer and promoter of the muscle creatine kinase gene, some of which include heterologous enhancers and individual elements from other muscle genes. Since the relative importance of many control elements varies among different anatomical muscles, we are aiming to tailor these cassettes for high-level expression in cardiac muscle, and in fast and slow skeletal muscles. With the achievement of efficient intravascular gene delivery to isolated limbs, selected muscle groups, and heart in large animal models, the design of cassettes optimized for activity in different muscle types is now a practical goal. In this protocol, we outline the key steps involved in the design of regulatory cassettes for optimal activity in skeletal and cardiac muscle, and testing in mature muscle fiber cultures. The basic principles described here can also be applied to engineering tissue-specific regulatory cassettes for other cell types.

In silico modeling of epigenetic-induced changes in photoreceptor cis-regulatory elements.

PubMed

Hossain, Reafa A; Dunham, Nicholas R; Enke, Raymond A; Berndsen, Christopher E

2018-01-01

DNA methylation is a well-characterized epigenetic repressor of mRNA transcription in many plant and vertebrate systems. However, the mechanism of this repression is not fully understood. The process of transcription is controlled by proteins that regulate recruitment and activity of RNA polymerase by binding to specific cis-regulatory sequences. Cone-rod homeobox (CRX) is a well-characterized mammalian transcription factor that controls photoreceptor cell-specific gene expression. Although much is known about the functions and DNA binding specificity of CRX, little is known about how DNA methylation modulates CRX binding affinity to genomic cis-regulatory elements. We used bisulfite pyrosequencing of human ocular tissues to measure DNA methylation levels of the regulatory regions of RHO , PDE6B, PAX6 , and LINE1 retrotransposon repeats. To describe the molecular mechanism of repression, we used molecular modeling to illustrate the effect of DNA methylation on human RHO regulatory sequences. In this study, we demonstrate an inverse correlation between DNA methylation in regulatory regions adjacent to the human RHO and PDE6B genes and their subsequent transcription in human ocular tissues. Docking of CRX to the DNA models shows that CRX interacts with the grooves of these sequences, suggesting changes in groove structure could regulate binding. Molecular dynamics simulations of the RHO promoter and enhancer regions show changes in the flexibility and groove width upon epigenetic modification. Models also demonstrate changes in the local dynamics of CRX binding sites within RHO regulatory sequences which may account for the repression of CRX-dependent transcription. Collectively, these data demonstrate epigenetic regulation of CRX binding sites in human retinal tissue and provide insight into the mechanism of this mode of epigenetic regulation to be tested in future experiments.

An ant colony optimization based algorithm for identifying gene regulatory elements.

PubMed

Liu, Wei; Chen, Hanwu; Chen, Ling

2013-08-01

It is one of the most important tasks in bioinformatics to identify the regulatory elements in gene sequences. Most of the existing algorithms for identifying regulatory elements are inclined to converge into a local optimum, and have high time complexity. Ant Colony Optimization (ACO) is a meta-heuristic method based on swarm intelligence and is derived from a model inspired by the collective foraging behavior of real ants. Taking advantage of the ACO in traits such as self-organization and robustness, this paper designs and implements an ACO based algorithm named ACRI (ant-colony-regulatory-identification) for identifying all possible binding sites of transcription factor from the upstream of co-expressed genes. To accelerate the ants' searching process, a strategy of local optimization is presented to adjust the ants' start positions on the searched sequences. By exploiting the powerful optimization ability of ACO, the algorithm ACRI can not only improve precision of the results, but also achieve a very high speed. Experimental results on real world datasets show that ACRI can outperform other traditional algorithms in the respects of speed and quality of solutions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia.

PubMed Central

Castresana, C; Garcia-Luque, I; Alonso, E; Malik, V S; Cashmore, A R

1988-01-01

We have analyzed promoter regulatory elements from a photoregulated CAB gene (Cab-E) isolated from Nicotiana plumbaginifolia. These studies have been performed by introducing chimeric gene constructs into tobacco cells via Agrobacterium tumefaciens-mediated transformation. Expression studies on the regenerated transgenic plants have allowed us to characterize three positive and one negative cis-acting elements that influence photoregulated expression of the Cab-E gene. Within the upstream sequences we have identified two positive regulatory elements (PRE1 and PRE2) which confer maximum levels of photoregulated expression. These sequences contain multiple repeated elements related to the sequence-ACCGGCCCACTT-. We have also identified within the upstream region a negative regulatory element (NRE) extremely rich in AT sequences, which reduces the level of gene expression in the light. We have defined a light regulatory element (LRE) within the promoter region extending from -396 to -186 bp which confers photoregulated expression when fused to a constitutive nopaline synthase ('nos') promoter. Within this region there is a 132-bp element, extending from -368 to -234 bp, which on deletion from the Cab-E promoter reduces gene expression from high levels to undetectable levels. Finally, we have demonstrated for a full length Cab-E promoter conferring high levels of photoregulated expression, that sequences proximal to the Cab-E TATA box are not replaceable by corresponding sequences from a 'nos' promoter. This contrasts with the apparent equivalence of these Cab-E and 'nos' TATA box-proximal sequences in truncated promoters conferring low levels of photoregulated expression. Images PMID:2901343

BET Bromodomain Inhibition Releases the Mediator Complex from Select cis-Regulatory Elements.

PubMed

Bhagwat, Anand S; Roe, Jae-Seok; Mok, Beverly Y L; Hohmann, Anja F; Shi, Junwei; Vakoc, Christopher R

2016-04-19

The bromodomain and extraterminal (BET) protein BRD4 can physically interact with the Mediator complex, but the relevance of this association to the therapeutic effects of BET inhibitors in cancer is unclear. Here, we show that BET inhibition causes a rapid release of Mediator from a subset of cis-regulatory elements in the genome of acute myeloid leukemia (AML) cells. These sites of Mediator eviction were highly correlated with transcriptional suppression of neighboring genes, which are enriched for targets of the transcription factor MYB and for functions related to leukemogenesis. A shRNA screen of Mediator in AML cells identified the MED12, MED13, MED23, and MED24 subunits as performing a similar regulatory function to BRD4 in this context, including a shared role in sustaining a block in myeloid maturation. These findings suggest that the interaction between BRD4 and Mediator has functional importance for gene-specific transcriptional activation and for AML maintenance. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences

NASA Technical Reports Server (NTRS)

Li, X.; Eastman, E. M.; Schwartz, R. J.; Draghia-Akli, R.

1999-01-01

Relatively low levels of expression from naturally occurring promoters have limited the use of muscle as a gene therapy target. Myogenic restricted gene promoters display complex organization usually involving combinations of several myogenic regulatory elements. By random assembly of E-box, MEF-2, TEF-1, and SRE sites into synthetic promoter recombinant libraries, and screening of hundreds of individual clones for transcriptional activity in vitro and in vivo, several artificial promoters were isolated whose transcriptional potencies greatly exceed those of natural myogenic and viral gene promoters.

Decoding a Signature-Based Model of Transcription Cofactor Recruitment Dictated by Cardinal Cis-Regulatory Elements in Proximal Promoter Regions

PubMed Central

Benner, Christopher; Hutt, Kasey R.; Stunnenberg, Rieka; Garcia-Bassets, Ivan

2013-01-01

Genome-wide maps of DNase I hypersensitive sites (DHSs) reveal that most human promoters contain perpetually active cis-regulatory elements between −150 bp and +50 bp (−150/+50 bp) relative to the transcription start site (TSS). Transcription factors (TFs) recruit cofactors (chromatin remodelers, histone/protein-modifying enzymes, and scaffold proteins) to these elements in order to organize the local chromatin structure and coordinate the balance of post-translational modifications nearby, contributing to the overall regulation of transcription. However, the rules of TF-mediated cofactor recruitment to the −150/+50 bp promoter regions remain poorly understood. Here, we provide evidence for a general model in which a series of cis-regulatory elements (here termed ‘cardinal’ motifs) prefer acting individually, rather than in fixed combinations, within the −150/+50 bp regions to recruit TFs that dictate cofactor signatures distinctive of specific promoter subsets. Subsequently, human promoters can be subclassified based on the presence of cardinal elements and their associated cofactor signatures. In this study, furthermore, we have focused on promoters containing the nuclear respiratory factor 1 (NRF1) motif as the cardinal cis-regulatory element and have identified the pervasive association of NRF1 with the cofactor lysine-specific demethylase 1 (LSD1/KDM1A). This signature might be distinctive of promoters regulating nuclear-encoded mitochondrial and other particular genes in at least some cells. Together, we propose that decoding a signature-based, expanded model of control at proximal promoter regions should lead to a better understanding of coordinated regulation of gene transcription. PMID:24244184

Identification of functional elements and regulatory circuits by Drosophila modENCODE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roy, Sushmita; Ernst, Jason; Kharchenko, Peter V.

2010-12-22

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- andmore » tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation. Several years after the complete genetic sequencing of many species, it is still unclear how to translate genomic information into a functional map of cellular and developmental programs. The Encyclopedia of DNA Elements (ENCODE) (1) and model organism ENCODE (modENCODE) (2) projects use diverse genomic assays to comprehensively annotate the Homo sapiens (human), Drosophila melanogaster (fruit fly), and Caenorhabditis elegans (worm) genomes, through systematic generation and computational integration of functional genomic data sets. Previous genomic studies in flies have made seminal contributions to our understanding of basic biological mechanisms and genome functions, facilitated by genetic, experimental, computational, and manual annotation of the euchromatic and heterochromatic genome (3), small genome size, short life cycle, and a deep knowledge of development, gene function, and chromosome biology. The

Regulation of acrosomal exocytosis. II. The zona pellucida-induced acrosome reaction of bovine spermatozoa is controlled by extrinsic positive regulatory elements.

PubMed

Florman, H M; First, N L

1988-08-01

The effects of accessory sex gland secretions on the zona pellucida-induced acrosome reaction of bovine spermatozoa were investigated. Soluble extracts of zonae pellucidae initiated exocytosis in ejaculated spermatozoa. This process had an ED50 of 20 ng/microliter zona pellucida protein and saturated at 50 ng/microliter (Florman and First, 1988. Dev. Biol. 128, 453-463). In epididymal sperm this dose-response relationship was shifted toward greater agonist concentrations by at least a factor of 10(3). Reconstitution of high potency agonist response was achieved in vitro by incubation of epididymal sperm with bovine seminal plasma. Reconstitution was dependent on the seminal plasma protein concentration. The ED50 of this process was 62 micrograms protein/10(8) sperm and saturation was observed with 124 micrograms protein/10(8) sperm. Agonist responses in reconstituted epididymal sperm and in ejaculated sperm were indistinguishable with regard to dependence on the zona pellucida protein concentration and the kinetics of induced acrosome reactions. Kinetic studies suggest that reconstitution is due to adsorption of regulatory factors from seminal plasma. In addition to the positive regulatory elements responsible for reconstituting activity, seminal plasma also contains negative regulatory elements which inhibit agonist response. These negative factors are inactivated during sperm capacitation, permitting the expression of positive regulators. Acting together, these regulatory elements could coordinate high affinity agonist response with the availability of eggs in vivo.

[Main regulatory element (MRE) of the Danio rerio α/β-globin gene domain exerts enhancer activity toward the promoters of the embryonic-larval and adult globin genes].

PubMed

Kovina, A P; Petrova, N V; Razin, S V; Yarovaia, O V

2016-01-01

In warm-blooded vertebrates, the α- and β-globin genes are organized in domains of different types and are regulated in different fashion. In cold-blooded vertebrates and, in particular, the tropical fish Danio rerio, the α- and β-globin genes form two gene clusters. A major D. rerio globin gene cluster is in chromosome 3 and includes the α- and β-globin genes of embryonic-larval and adult types. The region upstream of the cluster contains c16orf35, harbors the main regulatory element (MRE) of the α-globin gene domain in warm-blooded vertebrates. In this study, transient transfection of erythroid cells with genetic constructs containing a reporter gene under the control of potential regulatory elements of the domain was performed to characterize the promoters of the embryonic-larval and adult α- and β-globin genes of the major cluster. Also, in the 5th intron of c16orf35 in Danio reriowas detected a functional analog of the warm-blooded vertebrate MRE. This enhancer stimulated activity of the promoters of both adult and embryonic-larval α- and β-globin genes.

The Regulatory Properties of Autonomous Subtelomeric P Elements Are Sensitive to a Suppressor of Variegation in Drosophila Melanogaster

PubMed Central

Ronsseray, S.; Lehmann, M.; Nouaud, D.; Anxolabehere, D.

1996-01-01

Genetic recombination was used in Drosophila melanogaster to isolate P elements, inserted at the telomeres of X chromosomes (cytological site 1A) from natural populations, in a genetic background devoid of other P elements. We show that complete maternally inherited P repression in the germline (P cytotype) can be elicited by only two autonomous P elements at 1A and that a single element at this site has partial regulatory properties. The analysis of the surrounding chromosomal regions of the P elements at 1A shows that in all cases these elements are flanked by Telomeric Associated Sequences, tandemly repetitive noncoding sequences that have properties of heterochromatin. In addition, we show that the regulatory properties of P elements at 1A can be inhibited by some of the mutant alleles of the Su(var)205 gene and by a deficiency of this gene. However, the regulatory properties of reference P strains (Harwich and Texas 007) are not impaired by Su(var)205 mutations. Su(var)205 encodes Heterochromatin Protein 1 (HP1). These results suggest that the HP1 dosage effect on the P element properties is site-dependent and could involve the structure of the chromatin. PMID:8844154

Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

PubMed Central

Chappell, James; Jensen, Kirsten; Freemont, Paul S.

2013-01-01

A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology. PMID:23371936

Computational Approaches to Identify Promoters and cis-Regulatory Elements in Plant Genomes1

PubMed Central

Rombauts, Stephane; Florquin, Kobe; Lescot, Magali; Marchal, Kathleen; Rouzé, Pierre; Van de Peer, Yves

2003-01-01

The identification of promoters and their regulatory elements is one of the major challenges in bioinformatics and integrates comparative, structural, and functional genomics. Many different approaches have been developed to detect conserved motifs in a set of genes that are either coregulated or orthologous. However, although recent approaches seem promising, in general, unambiguous identification of regulatory elements is not straightforward. The delineation of promoters is even harder, due to its complex nature, and in silico promoter prediction is still in its infancy. Here, we review the different approaches that have been developed for identifying promoters and their regulatory elements. We discuss the detection of cis-acting regulatory elements using word-counting or probabilistic methods (so-called “search by signal” methods) and the delineation of promoters by considering both sequence content and structural features (“search by content” methods). As an example of search by content, we explored in greater detail the association of promoters with CpG islands. However, due to differences in sequence content, the parameters used to detect CpG islands in humans and other vertebrates cannot be used for plants. Therefore, a preliminary attempt was made to define parameters that could possibly define CpG and CpNpG islands in Arabidopsis, by exploring the compositional landscape around the transcriptional start site. To this end, a data set of more than 5,000 gene sequences was built, including the promoter region, the 5′-untranslated region, and the first introns and coding exons. Preliminary analysis shows that promoter location based on the detection of potential CpG/CpNpG islands in the Arabidopsis genome is not straightforward. Nevertheless, because the landscape of CpG/CpNpG islands differs considerably between promoters and introns on the one side and exons (whether coding or not) on the other, more sophisticated approaches can probably be

Sterol regulatory element binding protein-1 (SREBP1) gene expression is similarly increased in polycystic ovary syndrome and endometrial cancer.

PubMed

Shafiee, Mohamad N; Mongan, Nigel; Seedhouse, Claire; Chapman, Caroline; Deen, Suha; Abu, Jafaru; Atiomo, William

2017-05-01

Women with polycystic ovary syndrome have a three-fold higher risk of endometrial cancer. Insulin resistance and hyperlipidemia may be pertinent factors in the pathogenesis of both conditions. The aim of this study was to investigate endometrial sterol regulatory element binding protein-1 gene expression in polycystic ovary syndrome and endometrial cancer endometrium, and to correlate endometrial sterol regulatory element binding protein-1 gene expression with serum lipid profiles. A cross-sectional study was performed at Nottingham University Hospital, UK. A total of 102 women (polycystic ovary syndrome, endometrial cancer and controls; 34 participants in each group) were recruited. Clinical and biochemical assessments were performed before endometrial biopsies were obtained from all participants. Taqman real-time polymerase chain reaction for endometrial sterol regulatory element binding protein-1 gene and its systemic protein expression were analyzed. The body mass indices of women with polycystic ovary syndrome (29.28 ± 2.91 kg/m 2 ) and controls (28.58 ± 2.62 kg/m 2 ) were not significantly different. Women with endometrial cancer had a higher mean body mass index (32.22 ± 5.70 kg/m 2 ). Sterol regulatory element binding protein-1 gene expression was significantly increased in polycystic ovary syndrome and endometrial cancer endometrium compared with controls (p < 0.0001). Sterol regulatory element binding protein-1 gene expression was positively correlated with body mass index (r = 0.017, p = 0.921) and waist-hip ratio (r = 0.023, p = 0.544) in polycystic ovary syndrome, but this was not statistically significant. Similarly, statistically insignificant positive correlations were found between endometrial sterol regulatory element binding protein-1 gene expression and body mass index in endometrial cancer (r = 0.643, p = 0.06) and waist-hip ratio (r = 0.096, p = 0.073). Sterol regulatory element binding protein-1 gene expression

Identification of Regulatory DNA Elements Using Genome-wide Mapping of DNase I Hypersensitive Sites during Tomato Fruit Development.

PubMed

Qiu, Zhengkun; Li, Ren; Zhang, Shuaibin; Wang, Ketao; Xu, Meng; Li, Jiayang; Du, Yongchen; Yu, Hong; Cui, Xia

2016-08-01

Development and ripening of tomato fruit are precisely controlled by transcriptional regulation, which depends on the orchestrated accessibility of regulatory proteins to promoters and other cis-regulatory DNA elements. This accessibility and its effect on gene expression play a major role in defining the developmental process. To understand the regulatory mechanism and functional elements modulating morphological and anatomical changes during fruit development, we generated genome-wide high-resolution maps of DNase I hypersensitive sites (DHSs) from the fruit tissues of the tomato cultivar "Moneymaker" at 20 days post anthesis as well as break stage. By exploring variation of DHSs across fruit development stages, we pinpointed the most likely hypersensitive sites related to development-specific genes. By detecting binding motifs on DHSs of these development-specific genes or genes in the ascorbic acid biosynthetic pathway, we revealed the common regulatory elements contributing to coordinating gene transcription of plant ripening and specialized metabolic pathways. Our results contribute to a better understanding of the regulatory dynamics of genes involved in tomato fruit development and ripening. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Rationales for regulatory activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perhac, R.M.

1997-02-01

The author provides an outline which touches on the types of concerns about risk evaluation which are addressed in the process of establishing regulatory guides. Broadly he says regulatory activity serves three broad constituents: (1) Paternalism (private risk); (2) Promotion of social welfare (public risks); (3) Protection of individual rights (public risks). He then discusses some of the major issues encountered in reaching a decision on what is an acceptable level of risk within each of these areas, and how one establishes such a level.

Identification of high-confidence RNA regulatory elements by combinatorial classification of RNA-protein binding sites.

PubMed

Li, Yang Eric; Xiao, Mu; Shi, Binbin; Yang, Yu-Cheng T; Wang, Dong; Wang, Fei; Marcia, Marco; Lu, Zhi John

2017-09-08

Crosslinking immunoprecipitation sequencing (CLIP-seq) technologies have enabled researchers to characterize transcriptome-wide binding sites of RNA-binding protein (RBP) with high resolution. We apply a soft-clustering method, RBPgroup, to various CLIP-seq datasets to group together RBPs that specifically bind the same RNA sites. Such combinatorial clustering of RBPs helps interpret CLIP-seq data and suggests functional RNA regulatory elements. Furthermore, we validate two RBP-RBP interactions in cell lines. Our approach links proteins and RNA motifs known to possess similar biochemical and cellular properties and can, when used in conjunction with additional experimental data, identify high-confidence RBP groups and their associated RNA regulatory elements.

Binding of TFIIIC to sine elements controls the relocation of activity-dependent neuronal genes to transcription factories.

PubMed

Crepaldi, Luca; Policarpi, Cristina; Coatti, Alessandro; Sherlock, William T; Jongbloets, Bart C; Down, Thomas A; Riccio, Antonella

2013-01-01

In neurons, the timely and accurate expression of genes in response to synaptic activity relies on the interplay between epigenetic modifications of histones, recruitment of regulatory proteins to chromatin and changes to nuclear structure. To identify genes and regulatory elements responsive to synaptic activation in vivo, we performed a genome-wide ChIPseq analysis of acetylated histone H3 using somatosensory cortex of mice exposed to novel enriched environmental (NEE) conditions. We discovered that Short Interspersed Elements (SINEs) located distal to promoters of activity-dependent genes became acetylated following exposure to NEE and were bound by the general transcription factor TFIIIC. Importantly, under depolarizing conditions, inducible genes relocated to transcription factories (TFs), and this event was controlled by TFIIIC. Silencing of the TFIIIC subunit Gtf3c5 in non-stimulated neurons induced uncontrolled relocation to TFs and transcription of activity-dependent genes. Remarkably, in cortical neurons, silencing of Gtf3c5 mimicked the effects of chronic depolarization, inducing a dramatic increase of both dendritic length and branching. These findings reveal a novel and essential regulatory function of both SINEs and TFIIIC in mediating gene relocation and transcription. They also suggest that TFIIIC may regulate the rearrangement of nuclear architecture, allowing the coordinated expression of activity-dependent neuronal genes.

Binding of TFIIIC to SINE Elements Controls the Relocation of Activity-Dependent Neuronal Genes to Transcription Factories

PubMed Central

Crepaldi, Luca; Policarpi, Cristina; Coatti, Alessandro; Sherlock, William T.; Jongbloets, Bart C.; Down, Thomas A.; Riccio, Antonella

2013-01-01

In neurons, the timely and accurate expression of genes in response to synaptic activity relies on the interplay between epigenetic modifications of histones, recruitment of regulatory proteins to chromatin and changes to nuclear structure. To identify genes and regulatory elements responsive to synaptic activation in vivo, we performed a genome-wide ChIPseq analysis of acetylated histone H3 using somatosensory cortex of mice exposed to novel enriched environmental (NEE) conditions. We discovered that Short Interspersed Elements (SINEs) located distal to promoters of activity-dependent genes became acetylated following exposure to NEE and were bound by the general transcription factor TFIIIC. Importantly, under depolarizing conditions, inducible genes relocated to transcription factories (TFs), and this event was controlled by TFIIIC. Silencing of the TFIIIC subunit Gtf3c5 in non-stimulated neurons induced uncontrolled relocation to TFs and transcription of activity-dependent genes. Remarkably, in cortical neurons, silencing of Gtf3c5 mimicked the effects of chronic depolarization, inducing a dramatic increase of both dendritic length and branching. These findings reveal a novel and essential regulatory function of both SINEs and TFIIIC in mediating gene relocation and transcription. They also suggest that TFIIIC may regulate the rearrangement of nuclear architecture, allowing the coordinated expression of activity-dependent neuronal genes. PMID:23966877

Structural characterization and regulatory element analysis of the heart isoform of cytochrome c oxidase VIa

NASA Technical Reports Server (NTRS)

Wan, B.; Moreadith, R. W.; Blomqvist, C. G. (Principal Investigator)

1995-01-01

In order to investigate the mechanism(s) governing the striated muscle-specific expression of cytochrome c oxidase VIaH we have characterized the murine gene and analyzed its transcriptional regulatory elements in skeletal myogenic cell lines. The gene is single copy, spans 689 base pairs (bp), and is comprised of three exons. The 5'-ends of transcripts from the gene are heterogeneous, but the most abundant transcript includes a 5'-untranslated region of 30 nucleotides. When fused to the luciferase reporter gene, the 3.5-kilobase 5'-flanking region of the gene directed the expression of the heterologous protein selectively in differentiated Sol8 cells and transgenic mice, recapitulating the pattern of expression of the endogenous gene. Deletion analysis identified a 300-bp fragment sufficient to direct the myotube-specific expression of luciferase in Sol8 cells. The region lacks an apparent TATA element, and sequence motifs predicted to bind NRF-1, NRF-2, ox-box, or PPAR factors known to regulate other nuclear genes encoding mitochondrial proteins are not evident. Mutational analysis, however, identified two cis-elements necessary for the high level expression of the reporter protein: a MEF2 consensus element at -90 to -81 bp and an E-box element at -147 to -142 bp. Additional E-box motifs at closely located positions were mutated without loss of transcriptional activity. The dependence of transcriptional activation of cytochrome c oxidase VIaH on cis-elements similar to those found in contractile protein genes suggests that the striated muscle-specific expression is coregulated by mechanisms that control the lineage-specific expression of several contractile and cytosolic proteins.

Identification of N-Terminal Lobe Motifs that Determine the Kinase Activity of the Catalytic Domains and Regulatory Strategies of Src and Csk Protein Tyrosine Kinases†

PubMed Central

Huang, Kezhen; Wang, Yue-Hao; Brown, Alex; Sun, Gongqin

2009-01-01

Csk and Src protein tyrosine kinases are structurally homologous, but use opposite regulatory strategies. The isolated catalytic domain of Csk is intrinsically inactive and is activated by interactions with the regulatory SH3 and SH2 domains, while the isolated catalytic domain of Src is intrinsically active and is suppressed by interactions with the regulatory SH3 and SH2 domains. The structural basis for why one isolated catalytic domain is intrinsically active while the other is inactive is not clear. In this current study, we identify the structural elements in the N-terminal lobe of the catalytic domain that render the Src catalytic domain active. These structural elements include the α-helix C region, a β-turn between the β-4 and β-5 strands, and an Arg residue at the beginning of the catalytic domain. These three motifs interact with each other to activate the Src catalytic domain, but the equivalent motifs in Csk directly interact with the regulatory domains that are important for Csk activation. The Src motifs can be grafted to the Csk catalytic domain to obtain an active Csk catalytic domain. These results, together with available Src and Csk tertiary structures, reveal an important structural switch that determines the kinase activity of a catalytic domain and dictates the regulatory strategy of a kinase. PMID:19244618

BLSSpeller: exhaustive comparative discovery of conserved cis-regulatory elements.

PubMed

De Witte, Dieter; Van de Velde, Jan; Decap, Dries; Van Bel, Michiel; Audenaert, Pieter; Demeester, Piet; Dhoedt, Bart; Vandepoele, Klaas; Fostier, Jan

2015-12-01

The accurate discovery and annotation of regulatory elements remains a challenging problem. The growing number of sequenced genomes creates new opportunities for comparative approaches to motif discovery. Putative binding sites are then considered to be functional if they are conserved in orthologous promoter sequences of multiple related species. Existing methods for comparative motif discovery usually rely on pregenerated multiple sequence alignments, which are difficult to obtain for more diverged species such as plants. As a consequence, misaligned regulatory elements often remain undetected. We present a novel algorithm that supports both alignment-free and alignment-based motif discovery in the promoter sequences of related species. Putative motifs are exhaustively enumerated as words over the IUPAC alphabet and screened for conservation using the branch length score. Additionally, a confidence score is established in a genome-wide fashion. In order to take advantage of a cloud computing infrastructure, the MapReduce programming model is adopted. The method is applied to four monocotyledon plant species and it is shown that high-scoring motifs are significantly enriched for open chromatin regions in Oryza sativa and for transcription factor binding sites inferred through protein-binding microarrays in O.sativa and Zea mays. Furthermore, the method is shown to recover experimentally profiled ga2ox1-like KN1 binding sites in Z.mays. BLSSpeller was written in Java. Source code and manual are available at http://bioinformatics.intec.ugent.be/blsspeller Klaas.Vandepoele@psb.vib-ugent.be or jan.fostier@intec.ugent.be. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press.

Massive contribution of transposable elements to mammalian regulatory sequences.

PubMed

Rayan, Nirmala Arul; Del Rosario, Ricardo C H; Prabhakar, Shyam

2016-09-01

Barbara McClintock discovered the existence of transposable elements (TEs) in the late 1940s and initially proposed that they contributed to the gene regulatory program of higher organisms. This controversial idea gained acceptance only much later in the 1990s, when the first examples of TE-derived promoter sequences were uncovered. It is now known that half of the human genome is recognizably derived from TEs. It is thus important to understand the scope and nature of their contribution to gene regulation. Here, we provide a timeline of major discoveries in this area and discuss how transposons have revolutionized our understanding of mammalian genomes, with a special emphasis on the massive contribution of TEs to primate evolution. Our analysis of primate-specific functional elements supports a simple model for the rate at which new functional elements arise in unique and TE-derived DNA. Finally, we discuss some of the challenges and unresolved questions in the field, which need to be addressed in order to fully characterize the impact of TEs on gene regulation, evolution and disease processes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Improved regulatory element prediction based on tissue-specific local epigenomic signatures

PubMed Central

He, Yupeng; Gorkin, David U.; Dickel, Diane E.; Nery, Joseph R.; Castanon, Rosa G.; Lee, Ah Young; Shen, Yin; Visel, Axel; Pennacchio, Len A.; Ren, Bing; Ecker, Joseph R.

2017-01-01

Accurate enhancer identification is critical for understanding the spatiotemporal transcriptional regulation during development as well as the functional impact of disease-related noncoding genetic variants. Computational methods have been developed to predict the genomic locations of active enhancers based on histone modifications, but the accuracy and resolution of these methods remain limited. Here, we present an algorithm, regulatory element prediction based on tissue-specific local epigenetic marks (REPTILE), which integrates histone modification and whole-genome cytosine DNA methylation profiles to identify the precise location of enhancers. We tested the ability of REPTILE to identify enhancers previously validated in reporter assays. Compared with existing methods, REPTILE shows consistently superior performance across diverse cell and tissue types, and the enhancer locations are significantly more refined. We show that, by incorporating base-resolution methylation data, REPTILE greatly improves upon current methods for annotation of enhancers across a variety of cell and tissue types. REPTILE is available at https://github.com/yupenghe/REPTILE/. PMID:28193886

A novel regulatory element (E77) isolated from CHO-K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells.

PubMed

Kang, Shin-Young; Kim, Yeon-Gu; Kang, Seunghee; Lee, Hong Weon; Lee, Eun Gyo

2016-05-01

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO-K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO-K1 genomic DNA fragments with a CMV promoter-driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study. © 2016 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Who owns Australia's water--elements of an effective regulatory model.

PubMed

McKay, J

2003-01-01

This paper identifies and describes a number of global trends in regulatory theory and legal scholarship. It points out the huge level of complexity demanded by globalisation and the unfortunate complication of this is that there is legal indeterminacy. The legal indeterminacy springs from the desire to amend and alter existing models. That has been the thrust of the Council of Australian Governments changes to adapt and add huge amounts of complexity to a flawed system. This paper argues that an effective water regulatory model requires a fundamental re-examination of the concept of water ownership and a capturing by the State of the right to allocate rainfall. This foundation is effective and the way forward to deal with the key issues in this transition phase. The second key element to an effective regulatory model is the concept of performance-based assessment. This requires information and schemes to be set up to work out ways to monitor and evaluate the performance of the utility on selected criteria. For Australia at present there is a dire lack of agreed criteria on these key issues and these have the potential to pull apart the whole process. The key issues are indigenous rights, governance issues, public participation, alteration of pre-existing rights and incorporation of environmental requirements.

p75 Neurotrophin Receptor Signaling Activates Sterol Regulatory Element-binding Protein-2 in Hepatocyte Cells via p38 Mitogen-activated Protein Kinase and Caspase-3.

PubMed

Pham, Dan Duc; Do, Hai Thi; Bruelle, Céline; Kukkonen, Jyrki P; Eriksson, Ove; Mogollón, Isabel; Korhonen, Laura T; Arumäe, Urmas; Lindholm, Dan

2016-05-13

Nerve growth factor (NGF) influences the survival and differentiation of a specific population of neurons during development, but its role in non-neuronal cells has been less studied. We observed here that NGF and its pro-form, pro-NGF, are elevated in fatty livers from leptin-deficient mice compared with controls, concomitant with an increase in low density lipoprotein receptors (LDLRs). Stimulation of mouse primary hepatocytes with NGF or pro-NGF increased LDLR expression through the p75 neurotrophin receptor (p75NTR). Studies using Huh7 human hepatocyte cells showed that the neurotrophins activate the sterol regulatory element-binding protein-2 (SREBP2) that regulates genes involved in lipid metabolism. The mechanisms for this were related to stimulation of p38 mitogen-activated protein kinase (p38 MAPK) and activation of caspase-3 and SREBP2 cleavage following NGF and pro-NGF stimulations. Cell fractionation experiments showed that caspase-3 activity was increased particularly in the membrane fraction that harbors SREBP2 and caspase-2. Experiments showed further that caspase-2 interacts with pro-caspase-3 and that p38 MAPK reduced this interaction and caused caspase-3 activation. Because of the increased caspase-3 activity, the cells did not undergo cell death following p75NTR stimulation, possibly due to concomitant activation of nuclear factor-κB (NF-κB) pathway by the neurotrophins. These results identify a novel signaling pathway triggered by ligand-activated p75NTR that via p38 MAPK and caspase-3 mediate the activation of SREBP2. This pathway may regulate LDLRs and lipid uptake particularly after injury or during tissue inflammation accompanied by an increased production of growth factors, including NGF and pro-NGF. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

BLSSpeller: exhaustive comparative discovery of conserved cis-regulatory elements

PubMed Central

De Witte, Dieter; Van de Velde, Jan; Decap, Dries; Van Bel, Michiel; Audenaert, Pieter; Demeester, Piet; Dhoedt, Bart; Vandepoele, Klaas; Fostier, Jan

2015-01-01

Motivation: The accurate discovery and annotation of regulatory elements remains a challenging problem. The growing number of sequenced genomes creates new opportunities for comparative approaches to motif discovery. Putative binding sites are then considered to be functional if they are conserved in orthologous promoter sequences of multiple related species. Existing methods for comparative motif discovery usually rely on pregenerated multiple sequence alignments, which are difficult to obtain for more diverged species such as plants. As a consequence, misaligned regulatory elements often remain undetected. Results: We present a novel algorithm that supports both alignment-free and alignment-based motif discovery in the promoter sequences of related species. Putative motifs are exhaustively enumerated as words over the IUPAC alphabet and screened for conservation using the branch length score. Additionally, a confidence score is established in a genome-wide fashion. In order to take advantage of a cloud computing infrastructure, the MapReduce programming model is adopted. The method is applied to four monocotyledon plant species and it is shown that high-scoring motifs are significantly enriched for open chromatin regions in Oryza sativa and for transcription factor binding sites inferred through protein-binding microarrays in O.sativa and Zea mays. Furthermore, the method is shown to recover experimentally profiled ga2ox1-like KN1 binding sites in Z.mays. Availability and implementation: BLSSpeller was written in Java. Source code and manual are available at http://bioinformatics.intec.ugent.be/blsspeller Contact: Klaas.Vandepoele@psb.vib-ugent.be or jan.fostier@intec.ugent.be Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26254488

Scan for Motifs: a webserver for the analysis of post-transcriptional regulatory elements in the 3' untranslated regions (3' UTRs) of mRNAs.

PubMed

Biswas, Ambarish; Brown, Chris M

2014-06-08

Gene expression in vertebrate cells may be controlled post-transcriptionally through regulatory elements in mRNAs. These are usually located in the untranslated regions (UTRs) of mRNA sequences, particularly the 3'UTRs. Scan for Motifs (SFM) simplifies the process of identifying a wide range of regulatory elements on alignments of vertebrate 3'UTRs. SFM includes identification of both RNA Binding Protein (RBP) sites and targets of miRNAs. In addition to searching pre-computed alignments, the tool provides users the flexibility to search their own sequences or alignments. The regulatory elements may be filtered by expected value cutoffs and are cross-referenced back to their respective sources and literature. The output is an interactive graphical representation, highlighting potential regulatory elements and overlaps between them. The output also provides simple statistics and links to related resources for complementary analyses. The overall process is intuitive and fast. As SFM is a free web-application, the user does not need to install any software or databases. Visualisation of the binding sites of different classes of effectors that bind to 3'UTRs will facilitate the study of regulatory elements in 3' UTRs.

Reconstructing genome-wide regulatory network of E. coli using transcriptome data and predicted transcription factor activities

PubMed Central

2011-01-01

Background Gene regulatory networks play essential roles in living organisms to control growth, keep internal metabolism running and respond to external environmental changes. Understanding the connections and the activity levels of regulators is important for the research of gene regulatory networks. While relevance score based algorithms that reconstruct gene regulatory networks from transcriptome data can infer genome-wide gene regulatory networks, they are unfortunately prone to false positive results. Transcription factor activities (TFAs) quantitatively reflect the ability of the transcription factor to regulate target genes. However, classic relevance score based gene regulatory network reconstruction algorithms use models do not include the TFA layer, thus missing a key regulatory element. Results This work integrates TFA prediction algorithms with relevance score based network reconstruction algorithms to reconstruct gene regulatory networks with improved accuracy over classic relevance score based algorithms. This method is called Gene expression and Transcription factor activity based Relevance Network (GTRNetwork). Different combinations of TFA prediction algorithms and relevance score functions have been applied to find the most efficient combination. When the integrated GTRNetwork method was applied to E. coli data, the reconstructed genome-wide gene regulatory network predicted 381 new regulatory links. This reconstructed gene regulatory network including the predicted new regulatory links show promising biological significances. Many of the new links are verified by known TF binding site information, and many other links can be verified from the literature and databases such as EcoCyc. The reconstructed gene regulatory network is applied to a recent transcriptome analysis of E. coli during isobutanol stress. In addition to the 16 significantly changed TFAs detected in the original paper, another 7 significantly changed TFAs have been detected by

Sterols regulate 3β-hydroxysterol Δ24-reductase (DHCR24) via dual sterol regulatory elements: cooperative induction of key enzymes in lipid synthesis by Sterol Regulatory Element Binding Proteins.

PubMed

Zerenturk, Eser J; Sharpe, Laura J; Brown, Andrew J

2012-10-01

3β-Hydroxysterol Δ24-reductase (DHCR24) catalyzes a final step in cholesterol synthesis, and has been ascribed diverse functions, such as being anti-apoptotic and anti-inflammatory. How this enzyme is regulated transcriptionally by sterols is currently unclear. Some studies have suggested that its expression is regulated by Sterol Regulatory Element Binding Proteins (SREBPs) while another suggests it is through the Liver X Receptor (LXR). However, these transcription factors have opposing effects on cellular sterol levels, so it is likely that one predominates. Here we establish that sterol regulation of DHCR24 occurs predominantly through SREBP-2, and identify the particular region of the DHCR24 promoter to which SREBP-2 binds. We demonstrate that sterol regulation is mediated by two sterol regulatory elements (SREs) in the promoter of the gene, assisted by two nearby NF-Y binding sites. Moreover, we present evidence that the dual SREs work cooperatively to regulate DHCR24 expression by comparison to two known SREBP target genes, the LDL receptor with one SRE, and farnesyl-diphosphate farnesyltransferase 1, with two SREs. Copyright © 2012 Elsevier B.V. All rights reserved.

Deciphering RNA regulatory elements in trypanosomatids: one piece at a time or genome-wide?

PubMed

Gazestani, Vahid H; Lu, Zhiquan; Salavati, Reza

2014-05-01

Morphological and metabolic changes in the life cycle of Trypanosoma brucei are accomplished by precise regulation of hundreds of genes. In the absence of transcriptional control, RNA-binding proteins (RBPs) shape the structure of gene regulatory maps in this organism, but our knowledge about their target RNAs, binding sites, and mechanisms of action is far from complete. Although recent technological advances have revolutionized the RBP-based approaches, the main framework for the RNA regulatory element (RRE)-based approaches has not changed over the last two decades in T. brucei. In this Opinion, after highlighting the current challenges in RRE inference, we explain some genome-wide solutions that can significantly boost our current understanding about gene regulatory networks in T. brucei. Copyright © 2014 Elsevier Ltd. All rights reserved.

PROSPECT improves cis-acting regulatory element prediction by integrating expression profile data with consensus pattern searches

PubMed Central

Fujibuchi, Wataru; Anderson, John S. J.; Landsman, David

2001-01-01

Consensus pattern and matrix-based searches designed to predict cis-acting transcriptional regulatory sequences have historically been subject to large numbers of false positives. We sought to decrease false positives by incorporating expression profile data into a consensus pattern-based search method. We have systematically analyzed the expression phenotypes of over 6000 yeast genes, across 121 expression profile experiments, and correlated them with the distribution of 14 known regulatory elements over sequences upstream of the genes. Our method is based on a metric we term probabilistic element assessment (PEA), which is a ranking of potential sites based on sequence similarity in the upstream regions of genes with similar expression phenotypes. For eight of the 14 known elements that we examined, our method had a much higher selectivity than a naïve consensus pattern search. Based on our analysis, we have developed a web-based tool called PROSPECT, which allows consensus pattern-based searching of gene clusters obtained from microarray data. PMID:11574681

Exaptation of Transposable Elements into Novel Cis-Regulatory Elements: Is the Evidence Always Strong?

PubMed Central

de Souza, Flávio S.J.; Franchini, Lucía F.; Rubinstein, Marcelo

2013-01-01

Transposable elements (TEs) are mobile genetic sequences that can jump around the genome from one location to another, behaving as genomic parasites. TEs have been particularly effective in colonizing mammalian genomes, and such heavy TE load is expected to have conditioned genome evolution. Indeed, studies conducted both at the gene and genome levels have uncovered TE insertions that seem to have been co-opted—or exapted—by providing transcription factor binding sites (TFBSs) that serve as promoters and enhancers, leading to the hypothesis that TE exaptation is a major factor in the evolution of gene regulation. Here, we critically review the evidence for exaptation of TE-derived sequences as TFBSs, promoters, enhancers, and silencers/insulators both at the gene and genome levels. We classify the functional impact attributed to TE insertions into four categories of increasing complexity and argue that so far very few studies have conclusively demonstrated exaptation of TEs as transcriptional regulatory regions. We also contend that many genome-wide studies dealing with TE exaptation in recent lineages of mammals are still inconclusive and that the hypothesis of rapid transcriptional regulatory rewiring mediated by TE mobilization must be taken with caution. Finally, we suggest experimental approaches that may help attributing higher-order functions to candidate exapted TEs. PMID:23486611

Defective distal regulatory element at the 5' upstream of rat prolactin gene of steroid-nonresponsive GH-subclone.

PubMed

Kumar, V; Wong, D T; Pasion, S G; Biswas, D K

1987-12-08

The prolactin-nonproducing (PRL-) GH cell strains (rat pituitary tumor cells in culture). GH12C1 and F1BGH12C1, do not respond to steroid hormones estradiol or hydrocortisone (HC). However, the stimulatory effect of estradiol and the inhibitory effect of hydrocortisone on prolactin synthesis can be demonstrated in the prolactin-producing GH cell strain, GH4C1. In this investigation we have examined the 5' end flanking region of rat prolactin (rat PRL) gene of steroid-responsive, GH4C1 cells to identify the positive and negative regulatory elements and to verify the status of these elements in steroid-nonresponsive F1BGH12C1 cells. Results presented in this report demonstrate that the basel level expression of the co-transferred Neo gene (neomycin phosphoribosyl transferase) is modulated by the distal upstream regulatory elements of rat PRL gene in response to steroid hormones. The expression of adjacent Neo gene is inhibited by dexamethasone and is stimulated by estradiol in transfectants carrying distal regulatory elements (SRE) of steroid-responsive cells. These responses are not observed in transfectants with the rat PRL upstream sequences derived from steroid-nonresponsive cells. The basal level expression of the host cell alpha-2 tubulin gene is not affected by dexamethasone. We report here the identification of the distal steroid regulatory element (SRE) located between 3.8 and 7.8 kb upstream of the transcription initiation site of rat PRL gene. Both the positive and the negative effects of steroid hormones can be identified within this upstream sequence. This distal SRE appears to be nonfunctional in steroid-nonresponsive cells. Though the proximal SRE is functional, the defect in the distal SRE makes the GH substrain nonresponsive to steroid hormones. These results suggest that both the proximal and the distal SREs are essential for the mediation of action of steroid hormones in GH cells.

Oxidative stress activates endothelial innate immunity via sterol regulatory element binding protein 2 (SREBP2) transactivation of microRNA-92a.

PubMed

Chen, Zhen; Wen, Liang; Martin, Marcy; Hsu, Chien-Yi; Fang, Longhou; Lin, Feng-Mao; Lin, Ting-Yang; Geary, McKenna J; Geary, Greg G; Zhao, Yongli; Johnson, David A; Chen, Jaw-Wen; Lin, Shing-Jong; Chien, Shu; Huang, Hsien-Da; Miller, Yury I; Huang, Po-Hsun; Shyy, John Y-J

2015-03-03

Oxidative stress activates endothelial innate immunity and disrupts endothelial functions, including endothelial nitric oxide synthase-derived nitric oxide bioavailability. Here, we postulated that oxidative stress induces sterol regulatory element-binding protein 2 (SREBP2) and microRNA-92a (miR-92a), which in turn activate endothelial innate immune response, leading to dysfunctional endothelium. Using cultured endothelial cells challenged by diverse oxidative stresses, hypercholesterolemic zebrafish, and angiotensin II-infused or aged mice, we demonstrated that SREBP2 transactivation of microRNA-92a (miR-92a) is oxidative stress inducible. The SREBP2-induced miR-92a targets key molecules in endothelial homeostasis, including sirtuin 1, Krüppel-like factor 2, and Krüppel-like factor 4, leading to NOD-like receptor family pyrin domain-containing 3 inflammasome activation and endothelial nitric oxide synthase inhibition. In endothelial cell-specific SREBP2 transgenic mice, locked nucleic acid-modified antisense miR-92a attenuates inflammasome, improves vasodilation, and ameliorates angiotensin II-induced and aging-related atherogenesis. In patients with coronary artery disease, the level of circulating miR-92a is inversely correlated with endothelial cell-dependent, flow-mediated vasodilation and is positively correlated with serum level of interleukin-1β. Our findings suggest that SREBP2-miR-92a-inflammasome exacerbates endothelial dysfunction during oxidative stress. Identification of this mechanism may help in the diagnosis or treatment of disorders associated with oxidative stress, innate immune activation, and endothelial dysfunction. © 2014 American Heart Association, Inc.

Identification of Regulatory Elements That Control PPARγ Expression in Adipocyte Progenitors

PubMed Central

Chou, Wen-Ling; Galmozzi, Andrea; Partida, David; Kwan, Kevin; Yeung, Hui; Su, Andrew I.; Saez, Enrique

2013-01-01

Adipose tissue renewal and obesity-driven expansion of fat cell number are dependent on proliferation and differentiation of adipose progenitors that reside in the vasculature that develops in coordination with adipose depots. The transcriptional events that regulate commitment of progenitors to the adipose lineage are poorly understood. Because expression of the nuclear receptor PPARγ defines the adipose lineage, isolation of elements that control PPARγ expression in adipose precursors may lead to discovery of transcriptional regulators of early adipocyte determination. Here, we describe the identification and validation in transgenic mice of 5 highly conserved non-coding sequences from the PPARγ locus that can drive expression of a reporter gene in a manner that recapitulates the tissue-specific pattern of PPARγ expression. Surprisingly, these 5 elements appear to control PPARγ expression in adipocyte precursors that are associated with the vasculature of adipose depots, but not in mature adipocytes. Characterization of these five PPARγ regulatory sequences may enable isolation of the transcription factors that bind these cis elements and provide insight into the molecular regulation of adipose tissue expansion in normal and pathological states. PMID:24009687

Novel green tissue-specific synthetic promoters and cis-regulatory elements in rice.

PubMed

Wang, Rui; Zhu, Menglin; Ye, Rongjian; Liu, Zuoxiong; Zhou, Fei; Chen, Hao; Lin, Yongjun

2015-12-11

As an important part of synthetic biology, synthetic promoter has gradually become a hotspot in current biology. The purposes of the present study were to synthesize green tissue-specific promoters and to discover green tissue-specific cis-elements. We first assembled several regulatory sequences related to tissue-specific expression in different combinations, aiming to obtain novel green tissue-specific synthetic promoters. GUS assays of the transgenic plants indicated 5 synthetic promoters showed green tissue-specific expression patterns and different expression efficiencies in various tissues. Subsequently, we scanned and counted the cis-elements in different tissue-specific promoters based on the plant cis-elements database PLACE and the rice cDNA microarray database CREP for green tissue-specific cis-element discovery, resulting in 10 potential cis-elements. The flanking sequence of one potential core element (GEAT) was predicted by bioinformatics. Then, the combination of GEAT and its flanking sequence was functionally identified with synthetic promoter. GUS assays of the transgenic plants proved its green tissue-specificity. Furthermore, the function of GEAT flanking sequence was analyzed in detail with site-directed mutagenesis. Our study provides an example for the synthesis of rice tissue-specific promoters and develops a feasible method for screening and functional identification of tissue-specific cis-elements with their flanking sequences at the genome-wide level in rice.

FK506 biosynthesis is regulated by two positive regulatory elements in Streptomyces tsukubaensis

PubMed Central

2012-01-01

Background FK506 (Tacrolimus) is an important immunosuppressant, produced by industrial biosynthetic processes using various Streptomyces species. Considering the complex structure of FK506, it is reasonable to expect complex regulatory networks controlling its biosynthesis. Regulatory elements, present in gene clusters can have a profound influence on the final yield of target product and can play an important role in development of industrial bioprocesses. Results Three putative regulatory elements, namely fkbR, belonging to the LysR-type family, fkbN, a large ATP-binding regulator of the LuxR family (LAL-type) and allN, a homologue of AsnC family regulatory proteins, were identified in the FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488, a progenitor of industrial strains used for production of FK506. Inactivation of fkbN caused a complete disruption of FK506 biosynthesis, while inactivation of fkbR resulted in about 80% reduction of FK506 yield. No functional role in the regulation of the FK506 gene cluster has been observed for the allN gene. Using RT-PCR and a reporter system based on a chalcone synthase rppA, we demonstrated, that in the wild type as well as in fkbN- and fkbR-inactivated strains, fkbR is transcribed in all stages of cultivation, even before the onset of FK506 production, whereas fkbN expression is initiated approximately with the initiation of FK506 production. Surprisingly, inactivation of fkbN (or fkbR) does not abolish the transcription of the genes in the FK506 gene cluster in general, but may reduce expression of some of the tested biosynthetic genes. Finally, introduction of a second copy of the fkbR or fkbN genes under the control of the strong ermE* promoter into the wild type strain resulted in 30% and 55% of yield improvement, respectively. Conclusions Our results clearly demonstrate the positive regulatory role of fkbR and fkbN genes in FK506 biosynthesis in S. tsukubaensis NRRL 18488. We have shown that regulatory

Global reorganisation of cis-regulatory units upon lineage commitment of human embryonic stem cells

PubMed Central

Freire-Pritchett, Paula; Schoenfelder, Stefan; Várnai, Csilla; Wingett, Steven W; Cairns, Jonathan; Collier, Amanda J; García-Vílchez, Raquel; Furlan-Magaril, Mayra; Osborne, Cameron S; Fraser, Peter; Rugg-Gunn, Peter J; Spivakov, Mikhail

2017-01-01

Long-range cis-regulatory elements such as enhancers coordinate cell-specific transcriptional programmes by engaging in DNA looping interactions with target promoters. Deciphering the interplay between the promoter connectivity and activity of cis-regulatory elements during lineage commitment is crucial for understanding developmental transcriptional control. Here, we use Promoter Capture Hi-C to generate a high-resolution atlas of chromosomal interactions involving ~22,000 gene promoters in human pluripotent and lineage-committed cells, identifying putative target genes for known and predicted enhancer elements. We reveal extensive dynamics of cis-regulatory contacts upon lineage commitment, including the acquisition and loss of promoter interactions. This spatial rewiring occurs preferentially with predicted changes in the activity of cis-regulatory elements and is associated with changes in target gene expression. Our results provide a global and integrated view of promoter interactome dynamics during lineage commitment of human pluripotent cells. DOI: http://dx.doi.org/10.7554/eLife.21926.001 PMID:28332981

Stress-Induced Transcriptional Regulation in the Developing Rat Brain Involves Increased Cyclic Adenosine 3′,5′-Monophosphate-Regulatory Element Binding Activity

PubMed Central

Hatalski, Carolyn G.; Baram, Tallie Z.

2012-01-01

The cAMP-regulatory element (CRE) binding protein (CREB) functions as a trans-acting regulator of genes containing the CRE sequence in their promoter. These include a number of critical genes, such as CRF, involved in the hypothalamic response to stressful stimuli in the adult. The ability of the developing rat (during the first 2 postnatal weeks) to mount the full complement of this stress response has been questioned. We have previously demonstrated the stress-induced up-regulation of the transcription of hypothalamic CRF during the second postnatal week in the rat. The focus of the current study was to explore the mechanism of transcriptional regulation in response to stress through the physiological induction of transcriptional trans-activators that bind to the CRE in the developing rat brain. CRE-binding activity was detected via gel shift analysis in extracts from both the hypothalamus and the cerebral cortex of the developing rat. CREB was identified in these extracts by Western blot analysis and was shown to be the major contributor to the CRE-binding activity by gel shift analysis with two specific antibodies directed against CREB. After acute hypothermic stress, the abundance of CRE-binding activity (but not of total immunoreactive CREB), increased in hypothalamic extracts. This enhanced CRE-binding activity was blocked by an antiserum directed against CREB and was accompanied by an apparent increase in CREB phosphorylation. These results indicate that posttranslational enhancement of CRE-binding activity is likely to constitute an important mechanism for up-regulation of genes possessing the CRE sequence in the developing rat hypothalamus by adverse external signals. PMID:9415405

Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements.

PubMed

Kelwick, Richard; Webb, Alexander J; MacDonald, James T; Freemont, Paul S

2016-11-01

Cell-free transcription-translation systems were originally applied towards in vitro protein production. More recently, synthetic biology is enabling these systems to be used within a systematic design context for prototyping DNA regulatory elements, genetic logic circuits and biosynthetic pathways. The Gram-positive soil bacterium, Bacillus subtilis, is an established model organism of industrial importance. To this end, we developed several B. subtilis-based cell-free systems. Our improved B. subtilis WB800N-based system was capable of producing 0.8µM GFP, which gave a ~72x fold-improvement when compared with a B. subtilis 168 cell-free system. Our improved system was applied towards the prototyping of a B. subtilis promoter library in which we engineered several promoters, derived from the wild-type P grac (σA) promoter, that display a range of comparable in vitro and in vivo transcriptional activities. Additionally, we demonstrate the cell-free characterisation of an inducible expression system, and the activity of a model enzyme - renilla luciferase. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

45 CFR 73a.735-502 - Employees in regulatory activities.

Code of Federal Regulations, 2014 CFR

2014-10-01

... regulatory activities. (a) An employee in regulatory activities (“control activity” employee) may hold... control of the employee that resulted in the interest becoming prohibited; (iii) No direct relationship... provisions within this part, the following interpretations apply: (1) A “control activity” employee (“control...

45 CFR 73a.735-502 - Employees in regulatory activities.

Code of Federal Regulations, 2010 CFR

2010-10-01

... regulatory activities. (a) An employee in regulatory activities (“control activity” employee) may hold... control of the employee that resulted in the interest becoming prohibited; (iii) No direct relationship... provisions within this part, the following interpretations apply: (1) A “control activity” employee (“control...

45 CFR 73a.735-502 - Employees in regulatory activities.

Code of Federal Regulations, 2012 CFR

2012-10-01

... regulatory activities. (a) An employee in regulatory activities (“control activity” employee) may hold... control of the employee that resulted in the interest becoming prohibited; (iii) No direct relationship... provisions within this part, the following interpretations apply: (1) A “control activity” employee (“control...

45 CFR 73a.735-502 - Employees in regulatory activities.

Code of Federal Regulations, 2011 CFR

2011-10-01

... regulatory activities. (a) An employee in regulatory activities (“control activity” employee) may hold... control of the employee that resulted in the interest becoming prohibited; (iii) No direct relationship... provisions within this part, the following interpretations apply: (1) A “control activity” employee (“control...

45 CFR 73a.735-502 - Employees in regulatory activities.

Code of Federal Regulations, 2013 CFR

2013-10-01

... regulatory activities. (a) An employee in regulatory activities (“control activity” employee) may hold... control of the employee that resulted in the interest becoming prohibited; (iii) No direct relationship... provisions within this part, the following interpretations apply: (1) A “control activity” employee (“control...

Functional analysis of two sterol regulatory element binding proteins in Penicillium digitatum

PubMed Central

Ruan, Ruoxin; Wang, Mingshuang; Liu, Xin; Sun, Xuepeng; Chung, Kuang-Ren

2017-01-01

The sterol regulatory element binding proteins (SREBPs) are key regulators for sterol homeostasis in most fungi. In the citrus postharvest pathogen Penicillium digitatum, the SREBP homolog is required for fungicide resistance and regulation of CYP51 expression. In this study, we identified another SREBP transcription factor PdSreB in P. digitatum, and the biological functions of both SREBPs were characterized and compared. Inactivation of PdsreA, PdsreB or both genes in P. digitatum reduced ergosterol contents and increased sensitivities to sterol 14-α-demethylation inhibitors (DMIs) and cobalt chloride. Fungal strains impaired at PdsreA but not PdsreB increased sensitivity to tridemorph and an iron chelator 2,2’-dipyridyl. Virulence assays on citrus fruit revealed that fungal strains impaired at PdsreA, PdsreB or both induce maceration lesions similar to those induced by wild-type. However, ΔPdsreA, ΔPdsreB or the double mutant strain rarely produce aerial mycelia on infected citrus fruit peels. RNA-Seq analysis showed the broad regulatory functions of both SREBPs in biosynthesis, transmembrane transportation and stress responses. Our results provide new insights into the conserved and differentiated regulatory functions of SREBP homologs in plant pathogenic fungi. PMID:28467453

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

PubMed

Zhang, Jun; Li, Jing; Craig, Theodore A; Kumar, Rajiv; Gross, Michael L

2017-07-18

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca 2+ -binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca 2+ but detaches from DRE under Ca 2+ stimulation, allowing gene expression. The Ca 2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca 2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca 2+ . Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca 2+ -binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca 2+ signal to CREB-mediated gene transcription.

Identification of cis-elements and evaluation of upstream regulatory region of a rice anther-specific gene, OSIPP3, conferring pollen-specific expression in Oryza sativa (L.) ssp. indica.

PubMed

Manimaran, P; Raghurami Reddy, M; Bhaskar Rao, T; Mangrauthia, Satendra K; Sundaram, R M; Balachandran, S M

2015-12-01

Pollen-specific expression. Promoters comprise of various cis-regulatory elements which control development and physiology of plants by regulating gene expression. To understand the promoter specificity and also identification of functional cis-acting elements, progressive 5' deletion analysis of the promoter fragments is widely used. We have evaluated the activity of regulatory elements of 5' promoter deletion sequences of anther-specific gene OSIPP3, viz. OSIPP3-∆1 (1504 bp), OSIPP3-∆2 (968 bp), OSIPP3-∆3 (388 bp) and OSIPP3-∆4 (286 bp) through the expression of transgene GUS in rice. In silico analysis of 1504-bp sequence harboring different copy number of cis-acting regulatory elements such as POLLENLELAT52, GTGANTG10, enhancer element of LAT52 and LAT56 indicated that they were essential for high level of expression in pollen. Histochemical GUS analysis of the transgenic plants revealed that 1504- and 968-bp fragments directed GUS expression in roots and anthers, while the 388- and 286-bp fragments restricted the GUS expression to only pollen, of which 388 bp conferred strong GUS expression. Further, GUS staining analysis of different panicle development stages (P1-P6) confirmed that the GUS gene was preferentially expressed only at P6 stage (late pollen stage). The qRT-PCR analysis of GUS transcript revealed 23-fold higher expression of GUS transcript in OSIPP3-Δ1 followed by OSIPP3-Δ2 (eightfold) and OSIPP3-Δ3 (threefold) when compared to OSIPP3-Δ4. Based on our results, we proposed that among the two smaller fragments, the 388-bp upstream regulatory region could be considered as a promising candidate for pollen-specific expression of agronomically important transgenes in rice.

Elements in the transcriptional regulatory region flanking herpes simplex virus type 1 oriS stimulate origin function.

PubMed

Wong, S W; Schaffer, P A

1991-05-01

Like other DNA-containing viruses, the three origins of herpes simplex virus type 1 (HSV-1) DNA replication are flanked by sequences containing transcriptional regulatory elements. In a transient plasmid replication assay, deletion of sequences comprising the transcriptional regulatory elements of ICP4 and ICP22/47, which flank oriS, resulted in a greater than 80-fold decrease in origin function compared with a plasmid, pOS-822, which retains these sequences. In an effort to identify specific cis-acting elements responsible for this effect, we conducted systematic deletion analysis of the flanking region with plasmid pOS-822 and tested the resulting mutant plasmids for origin function. Stimulation by cis-acting elements was shown to be both distance and orientation dependent, as changes in either parameter resulted in a decrease in oriS function. Additional evidence for the stimulatory effect of flanking sequences on origin function was demonstrated by replacement of these sequences with the cytomegalovirus immediate-early promoter, resulting in nearly wild-type levels of oriS function. In competition experiments, cotransfection of cells with the test plasmid, pOS-822, and increasing molar concentrations of a competitor plasmid which contained the ICP4 and ICP22/47 transcriptional regulatory regions but lacked core origin sequences resulted in a significant reduction in the replication efficiency of pOS-822, demonstrating that factors which bind specifically to the oriS-flanking sequences are likely involved as auxiliary proteins in oriS function. Together, these studies demonstrate that trans-acting factors and the sites to which they bind play a critical role in the efficiency of HSV-1 DNA replication from oriS in transient-replication assays.

Enhancer elements upstream of the SHOX gene are active in the developing limb.

PubMed

Durand, Claudia; Bangs, Fiona; Signolet, Jason; Decker, Eva; Tickle, Cheryll; Rappold, Gudrun

2010-05-01

Léri-Weill Dyschondrosteosis (LWD) is a dominant skeletal disorder characterized by short stature and distinct bone anomalies. SHOX gene mutations and deletions of regulatory elements downstream of SHOX resulting in haploinsufficiency have been found in patients with LWD. SHOX encodes a homeodomain transcription factor and is known to be expressed in the developing limb. We have now analyzed the regulatory significance of the region upstream of the SHOX gene. By comparative genomic analyses, we identified several conserved non-coding elements, which subsequently were tested in an in ovo enhancer assay in both chicken limb bud and cornea, where SHOX is also expressed. In this assay, we found three enhancers to be active in the developing chicken limb, but none were functional in the developing cornea. A screening of 60 LWD patients with an intact SHOX coding and downstream region did not yield any deletion of the upstream enhancer region. Thus, we speculate that SHOX upstream deletions occur at a lower frequency because of the structural organization of this genomic region and/or that SHOX upstream deletions may cause a phenotype that differs from the one observed in LWD.

Enhancer elements upstream of the SHOX gene are active in the developing limb

PubMed Central

Durand, Claudia; Bangs, Fiona; Signolet, Jason; Decker, Eva; Tickle, Cheryll; Rappold, Gudrun

2010-01-01

Léri-Weill Dyschondrosteosis (LWD) is a dominant skeletal disorder characterized by short stature and distinct bone anomalies. SHOX gene mutations and deletions of regulatory elements downstream of SHOX resulting in haploinsufficiency have been found in patients with LWD. SHOX encodes a homeodomain transcription factor and is known to be expressed in the developing limb. We have now analyzed the regulatory significance of the region upstream of the SHOX gene. By comparative genomic analyses, we identified several conserved non-coding elements, which subsequently were tested in an in ovo enhancer assay in both chicken limb bud and cornea, where SHOX is also expressed. In this assay, we found three enhancers to be active in the developing chicken limb, but none were functional in the developing cornea. A screening of 60 LWD patients with an intact SHOX coding and downstream region did not yield any deletion of the upstream enhancer region. Thus, we speculate that SHOX upstream deletions occur at a lower frequency because of the structural organization of this genomic region and/or that SHOX upstream deletions may cause a phenotype that differs from the one observed in LWD. PMID:19997128

Two negative cis-regulatory regions involved in fruit-specific promoter activity from watermelon (Citrullus vulgaris S.).

PubMed

Yin, Tao; Wu, Hanying; Zhang, Shanglong; Lu, Hongyu; Zhang, Lingxiao; Xu, Yong; Chen, Daming; Liu, Jingmei

2009-01-01

A 1.8 kb 5'-flanking region of the large subunit of ADP-glucose pyrophosphorylase, isolated from watermelon (Citrullus vulgaris S.), has fruit-specific promoter activity in transgenic tomato plants. Two negative regulatory regions, from -986 to -959 and from -472 to -424, were identified in this promoter region by fine deletion analyses. Removal of both regions led to constitutive expression in epidermal cells. Gain-of-function experiments showed that these two regions were sufficient to inhibit RFP (red fluorescent protein) expression in transformed epidermal cells when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter. Gel mobility shift experiments demonstrated the presence of leaf nuclear factors that interact with these two elements. A TCCAAAA motif was identified in these two regions, as well as one in the reverse orientation, which was confirmed to be a novel specific cis-element. A quantitative beta-glucuronidase (GUS) activity assay of stable transgenic tomato plants showed that the activities of chimeric promoters harbouring only one of the two cis-elements, or both, were approximately 10-fold higher in fruits than in leaves. These data confirm that the TCCAAAA motif functions as a fruit-specific element by inhibiting gene expression in leaves.

Two negative cis-regulatory regions involved in fruit-specific promoter activity from watermelon (Citrullus vulgaris S.)

PubMed Central

Yin, Tao; Wu, Hanying; Zhang, Shanglong; Liu, Jingmei; Lu, Hongyu; Zhang, Lingxiao; Xu, Yong; Chen, Daming

2009-01-01

A 1.8 kb 5′-flanking region of the large subunit of ADP-glucose pyrophosphorylase, isolated from watermelon (Citrullus vulgaris S.), has fruit-specific promoter activity in transgenic tomato plants. Two negative regulatory regions, from –986 to –959 and from –472 to –424, were identified in this promoter region by fine deletion analyses. Removal of both regions led to constitutive expression in epidermal cells. Gain-of-function experiments showed that these two regions were sufficient to inhibit RFP (red fluorescent protein) expression in transformed epidermal cells when fused to the cauliflower mosaic virus (CaMV) 35S minimal promoter. Gel mobility shift experiments demonstrated the presence of leaf nuclear factors that interact with these two elements. A TCCAAAA motif was identified in these two regions, as well as one in the reverse orientation, which was confirmed to be a novel specific cis-element. A quantitative β-glucuronidase (GUS) activity assay of stable transgenic tomato plants showed that the activities of chimeric promoters harbouring only one of the two cis-elements, or both, were ∼10-fold higher in fruits than in leaves. These data confirm that the TCCAAAA motif functions as a fruit-specific element by inhibiting gene expression in leaves. PMID:19073962

Cell-type-specific enrichment of risk-associated regulatory elements at ovarian cancer susceptibility loci.

PubMed

Coetzee, Simon G; Shen, Howard C; Hazelett, Dennis J; Lawrenson, Kate; Kuchenbaecker, Karoline; Tyrer, Jonathan; Rhie, Suhn K; Levanon, Keren; Karst, Alison; Drapkin, Ronny; Ramus, Susan J; Couch, Fergus J; Offit, Kenneth; Chenevix-Trench, Georgia; Monteiro, Alvaro N A; Antoniou, Antonis; Freedman, Matthew; Coetzee, Gerhard A; Pharoah, Paul D P; Noushmehr, Houtan; Gayther, Simon A

2015-07-01

Understanding the regulatory landscape of the human genome is a central question in complex trait genetics. Most single-nucleotide polymorphisms (SNPs) associated with cancer risk lie in non-protein-coding regions, implicating regulatory DNA elements as functional targets of susceptibility variants. Here, we describe genome-wide annotation of regions of open chromatin and histone modification in fallopian tube and ovarian surface epithelial cells (FTSECs, OSECs), the debated cellular origins of high-grade serous ovarian cancers (HGSOCs) and in endometriosis epithelial cells (EECs), the likely precursor of clear cell ovarian carcinomas (CCOCs). The regulatory architecture of these cell types was compared with normal human mammary epithelial cells and LNCaP prostate cancer cells. We observed similar positional patterns of global enhancer signatures across the three different ovarian cancer precursor cell types, and evidence of tissue-specific regulatory signatures compared to non-gynecological cell types. We found significant enrichment for risk-associated SNPs intersecting regulatory biofeatures at 17 known HGSOC susceptibility loci in FTSECs (P = 3.8 × 10(-30)), OSECs (P = 2.4 × 10(-23)) and HMECs (P = 6.7 × 10(-15)) but not for EECs (P = 0.45) or LNCaP cells (P = 0.88). Hierarchical clustering of risk SNPs conditioned on the six different cell types indicates FTSECs and OSECs are highly related (96% of samples using multi-scale bootstrapping) suggesting both cell types may be precursors of HGSOC. These data represent the first description of regulatory catalogues of normal precursor cells for different ovarian cancer subtypes, and provide unique insights into the tissue specific regulatory variation with respect to the likely functional targets of germline genetic susceptibility variants for ovarian cancer. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Casein kinase 1 regulates sterol regulatory element-binding protein (SREBP) to control sterol homeostasis.

PubMed

Brookheart, Rita T; Lee, Chih-Yung S; Espenshade, Peter J

2014-01-31

Sterol homeostasis is tightly controlled by the sterol regulatory element-binding protein (SREBP) transcription factor that is highly conserved from fungi to mammals. In fission yeast, SREBP functions in an oxygen-sensing pathway to promote adaptation to decreased oxygen supply that limits oxygen-dependent sterol synthesis. Low oxygen stimulates proteolytic cleavage of the SREBP homolog Sre1, generating the active transcription factor Sre1N that drives expression of sterol biosynthetic enzymes. In addition, low oxygen increases the stability and DNA binding activity of Sre1N. To identify additional signals controlling Sre1 activity, we conducted a genetic overexpression screen. Here, we describe our isolation and characterization of the casein kinase 1 family member Hhp2 as a novel regulator of Sre1N. Deletion of Hhp2 increases Sre1N protein stability and ergosterol levels in the presence of oxygen. Hhp2-dependent Sre1N degradation by the proteasome requires Hhp2 kinase activity, and Hhp2 binds and phosphorylates Sre1N at specific residues. Our results describe a role for casein kinase 1 as a direct regulator of sterol homeostasis. Given the role of mammalian Hhp2 homologs, casein kinase 1δ and 1ε, in regulation of the circadian clock, these findings may provide a mechanism for coordinating circadian rhythm and lipid metabolism.

The impact of transposable elements on mammalian development

PubMed Central

Garcia-Perez, Jose L.; Widmann, Thomas J.; Adams, Ian R.

2018-01-01

Summary Despite often being classified as selfish or junk DNA, transposable elements (TEs) are a group of abundant genetic sequences that significantly impact on mammalian development and genome regulation. In recent years, our understanding of how pre-existing TEs affect genome architecture, gene regulatory networks and protein function during mammalian embryogenesis has dramatically expanded. In addition, the mobilization of active TEs in selected cell types has been shown to generate genetic variation during development and in fully differentiated tissues. Importantly, the ongoing domestication and evolution of TEs appears to provide a rich source of regulatory elements, functional modules and genetic variation that fuels the evolution of mammalian developmental processes. Here, we review the functional impact that TEs exert on mammalian developmental processes and how the somatic activity of TEs can influence gene regulatory networks. PMID:27875251

Ginsenoside F2 reduces hair loss by controlling apoptosis through the sterol regulatory element-binding protein cleavage activating protein and transforming growth factor-β pathways in a dihydrotestosterone-induced mouse model.

PubMed

Shin, Heon-Sub; Park, Sang-Yong; Hwang, Eun-Son; Lee, Don-Gil; Mavlonov, Gafurjon Turdalievich; Yi, Tae-Hoo

2014-01-01

This study was conducted to test whether ginsenoside F2 can reduce hair loss by influencing sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and the transforming growth factor beta (TGF-β) pathway of apoptosis in dihydrotestosterone (DHT)-treated hair cells and in a DHT-induced hair loss model in mice. Results for ginsenoside F2 were compared with finasteride. DHT inhibits proliferation of hair cells and induces androgenetic alopecia and was shown to activate an apoptosis signal pathway both in vitro and in vivo. The cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation rates of DHT-treated human hair dermal papilla cells (HHDPCs) and HaCaTs increased by 48% in the ginsenoside F2-treated group and by 12% in the finasteride-treated group. Western blot analysis showed that ginsenoside F2 decreased expression of TGF-β2 related factors involved in hair loss. The present study suggested a hair loss related pathway by changing SCAP related apoptosis pathway, which has been known to control cholesterol metabolism. SCAP, sterol regulatory element-binding protein (SREBP) and caspase-12 expression in the ginsenoside F2-treated group were decreased compared to the DHT and finasteride-treated group. C57BL/6 mice were also prepared by injection with DHT and then treated with ginsenoside F2 or finasteride. Hair growth rate, density, thickness measurements and tissue histotological analysis in these groups suggested that ginsenoside F2 suppressed hair cell apoptosis and premature entry to catagen more effectively than finasteride. Our results indicated that ginsenoside F2 decreased the expression of TGF-β2 and SCAP proteins, which have been suggested to be involved in apoptosis and entry into catagen. This study provides evidence those factors in the SCAP pathway could be targets for hair loss prevention drugs.

The transcriptional regulatory network mediated by banana (Musa acuminata) dehydration-responsive element binding (MaDREB) transcription factors in fruit ripening.

PubMed

Kuang, Jian-Fei; Chen, Jian-Ye; Liu, Xun-Cheng; Han, Yan-Chao; Xiao, Yun-Yi; Shan, Wei; Tang, Yang; Wu, Ke-Qiang; He, Jun-Xian; Lu, Wang-Jin

2017-04-01

Fruit ripening is a complex, genetically programmed process involving the action of critical transcription factors (TFs). Despite the established significance of dehydration-responsive element binding (DREB) TFs in plant abiotic stress responses, the involvement of DREBs in fruit ripening is yet to be determined. Here, we identified four genes encoding ripening-regulated DREB TFs in banana (Musa acuminata), MaDREB1, MaDREB2, MaDREB3, and MaDREB4, and demonstrated that they play regulatory roles in fruit ripening. We showed that MaDREB1-MaDREB4 are nucleus-localized, induced by ethylene and encompass transcriptional activation activities. We performed a genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) experiment for MaDREB2 and identified 697 genomic regions as potential targets of MaDREB2. MaDREB2 binds to hundreds of loci with diverse functions and its binding sites are distributed in the promoter regions proximal to the transcriptional start site (TSS). Most of the MaDREB2-binding targets contain the conserved (A/G)CC(G/C)AC motif and MaDREB2 appears to directly regulate the expression of a number of genes involved in fruit ripening. In combination with transcriptome profiling (RNA sequencing) data, our results indicate that MaDREB2 may serve as both transcriptional activator and repressor during banana fruit ripening. In conclusion, our study suggests a hierarchical regulatory model of fruit ripening in banana and that the MaDREB TFs may act as transcriptional regulators in the regulatory network. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Retinal Expression of the Drosophila eyes absent Gene Is Controlled by Several Cooperatively Acting Cis-regulatory Elements

PubMed Central

Neuman, Sarah D.; Bashirullah, Arash; Kumar, Justin P.

2016-01-01

The eyes absent (eya) gene of the fruit fly, Drosophila melanogaster, is a member of an evolutionarily conserved gene regulatory network that controls eye formation in all seeing animals. The loss of eya leads to the complete elimination of the compound eye while forced expression of eya in non-retinal tissues is sufficient to induce ectopic eye formation. Within the developing retina eya is expressed in a dynamic pattern and is involved in tissue specification/determination, cell proliferation, apoptosis, and cell fate choice. In this report we explore the mechanisms by which eya expression is spatially and temporally governed in the developing eye. We demonstrate that multiple cis-regulatory elements function cooperatively to control eya transcription and that spacing between a pair of enhancer elements is important for maintaining correct gene expression. Lastly, we show that the loss of eya expression in sine oculis (so) mutants is the result of massive cell death and a progressive homeotic transformation of retinal progenitor cells into head epidermis. PMID:27930646

Modeling the evolution of regulatory elements by simultaneous detection and alignment with phylogenetic pair HMMs.

PubMed

Majoros, William H; Ohler, Uwe

2010-12-16

The computational detection of regulatory elements in DNA is a difficult but important problem impacting our progress in understanding the complex nature of eukaryotic gene regulation. Attempts to utilize cross-species conservation for this task have been hampered both by evolutionary changes of functional sites and poor performance of general-purpose alignment programs when applied to non-coding sequence. We describe a new and flexible framework for modeling binding site evolution in multiple related genomes, based on phylogenetic pair hidden Markov models which explicitly model the gain and loss of binding sites along a phylogeny. We demonstrate the value of this framework for both the alignment of regulatory regions and the inference of precise binding-site locations within those regions. As the underlying formalism is a stochastic, generative model, it can also be used to simulate the evolution of regulatory elements. Our implementation is scalable in terms of numbers of species and sequence lengths and can produce alignments and binding-site predictions with accuracy rivaling or exceeding current systems that specialize in only alignment or only binding-site prediction. We demonstrate the validity and power of various model components on extensive simulations of realistic sequence data and apply a specific model to study Drosophila enhancers in as many as ten related genomes and in the presence of gain and loss of binding sites. Different models and modeling assumptions can be easily specified, thus providing an invaluable tool for the exploration of biological hypotheses that can drive improvements in our understanding of the mechanisms and evolution of gene regulation.

The impact of transposable elements on mammalian development.

PubMed

Garcia-Perez, Jose L; Widmann, Thomas J; Adams, Ian R

2016-11-15

Despite often being classified as selfish or junk DNA, transposable elements (TEs) are a group of abundant genetic sequences that have a significant impact on mammalian development and genome regulation. In recent years, our understanding of how pre-existing TEs affect genome architecture, gene regulatory networks and protein function during mammalian embryogenesis has dramatically expanded. In addition, the mobilization of active TEs in selected cell types has been shown to generate genetic variation during development and in fully differentiated tissues. Importantly, the ongoing domestication and evolution of TEs appears to provide a rich source of regulatory elements, functional modules and genetic variation that fuels the evolution of mammalian developmental processes. Here, we review the functional impact that TEs exert on mammalian developmental processes and discuss how the somatic activity of TEs can influence gene regulatory networks. © 2016. Published by The Company of Biologists Ltd.

Characterization of noncoding regulatory DNA in the human genome.

PubMed

Elkon, Ran; Agami, Reuven

2017-08-08

Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

Deciphering the transcriptional cis-regulatory code.

PubMed

Yáñez-Cuna, J Omar; Kvon, Evgeny Z; Stark, Alexander

2013-01-01

Information about developmental gene expression resides in defined regulatory elements, called enhancers, in the non-coding part of the genome. Although cells reliably utilize enhancers to orchestrate gene expression, a cis-regulatory code that would allow their interpretation has remained one of the greatest challenges of modern biology. In this review, we summarize studies from the past three decades that describe progress towards revealing the properties of enhancers and discuss how recent approaches are providing unprecedented insights into regulatory elements in animal genomes. Over the next years, we believe that the functional characterization of regulatory sequences in entire genomes, combined with recent computational methods, will provide a comprehensive view of genomic regulatory elements and their building blocks and will enable researchers to begin to understand the sequence basis of the cis-regulatory code. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of cis-acting regulatory elements in the human oxytocin gene promoter.

PubMed

Richard, S; Zingg, H H

1991-12-01

The expression of hormone-inducible genes is determined by the interaction of trans-acting factors with hormone-inducible elements and elements mediating basal and cell-specific expression. We have shown earlier that the gene encoding the hypothalamic nonapeptide oxytocin (OT) is under the control of an estrogen response element (ERE). The present study was aimed at identifying cis-acting elements mediating basal expression of the OT gene. A construct containing sequences -381 to +36 of the human OT gene was linked to a reporter gene and transiently transfected into a series of neuronal and nonneuronal cell lines. Expression of this construct was cell specific: it was highest in the neuroblastoma-derived cell line, Neuro-2a, and lowest in NIH 3T3 and JEG-3 cells. By 5' deletion analysis, we determined that a segment from -49 to +36 was capable of mediating cells-pecific promoter activity. Within this segment, we identified three proximal promoter elements (PPE-1, PPE-2, and PPE-3) that are each required for promoter activity. Most notably, mutation of a conserved purine-rich element (GAGAGA) contained within PPE-2 leads to a 10-fold decrease in promoter strength. Gel mobility shift analysis with three different double-stranded oligonucleotides demonstrated that each proximal promoter element binds distinct nuclear factors. In each case, only the homologous oligonucleotide, but neither of the oligonucleotides corresponding to adjacent elements, was able to act as a competitor. Thus, a different set of factors appears to bind independently to each element. By reinserting the homologous ERE or a heterologous glucocorticoid response element upstream of intact or altered proximal promoter segments we determined that removal or mutation of proximal promoter elements decreases basal expression, but does not abrogate the hormone responsiveness of the promoter. In conclusion, these results indicate that an important component of the transcriptional activity of the OT

Sterol regulatory element-binding protein 1 inhibitors decrease pancreatic cancer cell viability and proliferation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Siqingaowa,; Sekar, Sathiya; Gopalakrishnan, Venkat

Sterol regulatory element-binding protein1 (SREBP1) is a key regulatory factor that controls lipid homeostasis. Overactivation of SREBP1 and elevated lipid biogenesis are considered the major characteristics in malignancies of prostate cancer, endometrial cancer, and glioblastoma. However, the impact of SREBP1 activation in the progression of pancreatic cancer has not been explored. The present study examines the effect of suppression of SREBP1 activation by its inhibitors like fatostatin and PF429242 besides analyzing the impact of inhibitory effects on SREBP1 downstream signaling cascade such as fatty acid synthase (FAS), hydroxymethylglutaryl-CoA reductase (HMGCoAR), stearoyl-CoA desaturase-1 (SCD-1), and tumor suppressor protein p53 in MIAmore » PaCa-2 pancreatic cancer cells. Both fatostatin and PF429242 inhibited the growth of MIA PaCa-2 cells in a time and concentration-dependent manner with maximal inhibition attained at 72 h time period with IC{sub 50} values of 14.5 μM and 24.5 μM respectively. Detailed Western blot analysis performed using fatostatin and PF429242 at 72 h time point led to significant decrease in the levels of the active form of SREBP1 and its downstream signaling proteins such as FAS, SCD-1 and HMGCoAR and the mutant form of tumor suppressor protein, p53, levels in comparison to the levels observed in vehicle treated control group of MIA PaCa-2 pancreatic cells over the same time period. Our in vitro data suggest that SREBP1 may contribute to pancreatic tumor growth and its inhibitors could be considered as a potential target in the management of pancreatic cancer cell proliferation. - Highlights: • A significant increase in SREBP1 levels was observed in MIA PaCa-2 cells. • Fatostatin and PF429242 suppress SREBP1 activation and its downstream signaling proteins expression. • The inhibition of SREBP1reduces tumor suppressor protein p53 in MIA PaCa-2 cells. • SREBP1 inhibition may be beneficial in treatment of

Soy protein supports cardiovascular health by downregulating hydroxymethylglutaryl-coenzyme A reductase and sterol regulatory element-binding protein-2 and increasing antioxidant enzyme activity in rats with dextran sodium sulfate-induced mild systemic inflammation.

PubMed

Marsh, Tanya G; Straub, Rachel K; Villalobos, Fatima; Hong, Mee Young

2011-12-01

Animal and human studies have indicated that the presence of soy in the diet improves cardiovascular health. Inflammation plays a pivotal role in the progression of cardiovascular disease (CVD). However, little is known about how dextran sodium sulfate (DSS)-induced systemic inflammation impacts overall heart health and, correspondingly, how soy protein modulates risk of CVD development in DSS-induced systemic inflammation. We hypothesized that soy protein-fed rats would have a lower risk of CVD by beneficial alteration of gene expression involving lipid metabolism and antioxidant capacity in DSS-induced systemic inflammation. Forty Sprague-Dawley rats were divided into 4 groups: casein, casein + DSS, soy protein, and soy protein + DSS. After 26 days, inflammation was induced in one group from each diet by incorporating 3% DSS in drinking water for 48 hours. Soy protein-fed rats had lower final body weights (P = .010), epididymal fat weights (P = .049), total cholesterol (P < .001), and low-density lipoprotein cholesterol (P < .001). In regard to gene expression, soy protein-fed rats had lower sterol regulatory element-binding protein-2 (P = .032) and hydroxymethylglutaryl-coenzyme A reductase (P = .028) levels and higher low-density lipoprotein receptor levels (P = .036). Antioxidant enzyme activity of superoxide dismutase and catalase was higher among the soy protein groups (P = .037 and P = .002, respectively). These results suggest that soy protein positively influences cardiovascular health by regulating serum lipids through modified expression of sterol regulatory element-binding protein-2 and its downstream genes (ie, hydroxymethylglutaryl-coenzyme A reductase and low-density lipoprotein receptor) and by promoting the antioxidant enzyme activity of superoxide dismutase and catalase. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification of a distant cis-regulatory element controlling pharyngeal arch-specific expression of zebrafish gdf6a/radar

PubMed Central

Reed, Nykolaus P.; Mortlock, Douglas P.

2011-01-01

Skeletal formation is an essential and intricately regulated part of vertebrate development. Humans and mice deficient in Growth and Differentiation Factor 6 (Gdf6) have numerous skeletal abnormalities including joint fusions and cartilage reductions. The expression of Gdf6 is dynamic and in part regulated by distant evolutionarily conserved cis-regulatory elements. radar/gdf6a is a zebrafish ortholog of Gdf6 and has an essential role in embryonic patterning. Here we show that radar is transcribed in the cells surrounding and between the developing cartilages of the ventral pharyngeal arches, similar to mouse Gdf6. A 312 bp evolutionarily conserved region (ECR5), 122 kilobases downstream, drives expression in a pharyngeal arch-specific manner similar to endogenous radar/gdf6a. Deletion analysis identified a 78 bp region within ECR5 that is essential for transgene activity. This work illustrates that radar is regulated in the pharyngeal arches by a distant conserved element and suggests radar has similar functions in skeletal development in fish and mammals. PMID:20201106

Variant-aware saturating mutagenesis using multiple Cas9 nucleases identifies regulatory elements at trait-associated loci.

PubMed

Canver, Matthew C; Lessard, Samuel; Pinello, Luca; Wu, Yuxuan; Ilboudo, Yann; Stern, Emily N; Needleman, Austen J; Galactéros, Frédéric; Brugnara, Carlo; Kutlar, Abdullah; McKenzie, Colin; Reid, Marvin; Chen, Diane D; Das, Partha Pratim; A Cole, Mitchel; Zeng, Jing; Kurita, Ryo; Nakamura, Yukio; Yuan, Guo-Cheng; Lettre, Guillaume; Bauer, Daniel E; Orkin, Stuart H

2017-04-01

Cas9-mediated, high-throughput, saturating in situ mutagenesis permits fine-mapping of function across genomic segments. Disease- and trait-associated variants identified in genome-wide association studies largely cluster at regulatory loci. Here we demonstrate the use of multiple designer nucleases and variant-aware library design to interrogate trait-associated regulatory DNA at high resolution. We developed a computational tool for the creation of saturating-mutagenesis libraries with single or multiple nucleases with incorporation of variants. We applied this methodology to the HBS1L-MYB intergenic region, which is associated with red-blood-cell traits, including fetal hemoglobin levels. This approach identified putative regulatory elements that control MYB expression. Analysis of genomic copy number highlighted potential false-positive regions, thus emphasizing the importance of off-target analysis in the design of saturating-mutagenesis experiments. Together, these data establish a widely applicable high-throughput and high-resolution methodology to identify minimal functional sequences within large disease- and trait-associated regions.

Conserved Non-Coding Regulatory Signatures in Arabidopsis Co-Expressed Gene Modules

PubMed Central

Spangler, Jacob B.; Ficklin, Stephen P.; Luo, Feng; Freeling, Michael; Feltus, F. Alex

2012-01-01

Complex traits and other polygenic processes require coordinated gene expression. Co-expression networks model mRNA co-expression: the product of gene regulatory networks. To identify regulatory mechanisms underlying coordinated gene expression in a tissue-enriched context, ten Arabidopsis thaliana co-expression networks were constructed after manually sorting 4,566 RNA profiling datasets into aerial, flower, leaf, root, rosette, seedling, seed, shoot, whole plant, and global (all samples combined) groups. Collectively, the ten networks contained 30% of the measurable genes of Arabidopsis and were circumscribed into 5,491 modules. Modules were scrutinized for cis regulatory mechanisms putatively encoded in conserved non-coding sequences (CNSs) previously identified as remnants of a whole genome duplication event. We determined the non-random association of 1,361 unique CNSs to 1,904 co-expression network gene modules. Furthermore, the CNS elements were placed in the context of known gene regulatory networks (GRNs) by connecting 250 CNS motifs with known GRN cis elements. Our results provide support for a regulatory role of some CNS elements and suggest the functional consequences of CNS activation of co-expression in specific gene sets dispersed throughout the genome. PMID:23024789

Conserved non-coding regulatory signatures in Arabidopsis co-expressed gene modules.

PubMed

Spangler, Jacob B; Ficklin, Stephen P; Luo, Feng; Freeling, Michael; Feltus, F Alex

2012-01-01

Complex traits and other polygenic processes require coordinated gene expression. Co-expression networks model mRNA co-expression: the product of gene regulatory networks. To identify regulatory mechanisms underlying coordinated gene expression in a tissue-enriched context, ten Arabidopsis thaliana co-expression networks were constructed after manually sorting 4,566 RNA profiling datasets into aerial, flower, leaf, root, rosette, seedling, seed, shoot, whole plant, and global (all samples combined) groups. Collectively, the ten networks contained 30% of the measurable genes of Arabidopsis and were circumscribed into 5,491 modules. Modules were scrutinized for cis regulatory mechanisms putatively encoded in conserved non-coding sequences (CNSs) previously identified as remnants of a whole genome duplication event. We determined the non-random association of 1,361 unique CNSs to 1,904 co-expression network gene modules. Furthermore, the CNS elements were placed in the context of known gene regulatory networks (GRNs) by connecting 250 CNS motifs with known GRN cis elements. Our results provide support for a regulatory role of some CNS elements and suggest the functional consequences of CNS activation of co-expression in specific gene sets dispersed throughout the genome.

Favorable genomic environments for cis-regulatory evolution: A novel theoretical framework.

PubMed

Maeso, Ignacio; Tena, Juan J

2016-09-01

Cis-regulatory changes are arguably the primary evolutionary source of animal morphological diversity. With the recent explosion of genome-wide comparisons of the cis-regulatory content in different animal species is now possible to infer general principles underlying enhancer evolution. However, these studies have also revealed numerous discrepancies and paradoxes, suggesting that the mechanistic causes and modes of cis-regulatory evolution are still not well understood and are probably much more complex than generally appreciated. Here, we argue that the mutational mechanisms and genomic regions generating new regulatory activities must comply with the constraints imposed by the molecular properties of cis-regulatory elements (CREs) and the organizational features of long-range chromatin interactions. Accordingly, we propose a new integrative evolutionary framework for cis-regulatory evolution based on two major premises for the origin of novel enhancer activity: (i) an accessible chromatin environment and (ii) compatibility with the 3D structure and interactions of pre-existing CREs. Mechanisms and DNA sequences not fulfilling these premises, will be less likely to have a measurable impact on gene expression and as such, will have a minor contribution to the evolution of gene regulation. Finally, we discuss current comparative cis-regulatory data under the light of this new evolutionary model, and propose that the two most prominent mechanisms for the evolution of cis-regulatory changes are the overprinting of ancestral CREs and the exaptation of transposable elements. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transcriptional regulatory elements in the noncoding region of human papillomavirus type 6

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Tzyy-Choou.

1989-01-01

The structure and function of the transcriptional regulatory region of human papillomavirus type 6 (HPV-6) has been investigated. To investigate tissue specific gene expression, a sensitive method to detect and localize HPV-6 viral DNA, mRNA and protein in plastic-embedded tissue sections of genital and respiratory tract papillomata by using in situ hybridization and immunoperoxidase assays has been developed. This method, using ultrathin sections and strand-specific {sup 3}H labeled riboprobes, offers the advantages of superior morphological preservation and detection of viral genomes at low copy number with good resolution, and the modified immunocytochemistry provides better sensitivity. The results suggest that genitalmore » tract epithelium is more permissive for HPV-6 replication than respiratory tract epithelium. To study the tissue tropism of HPV-6 at the level of regulation of viral gene expression, the polymerase chain reaction was used to isolate the noncoding region (NCR) of HPV-6 in independent isolates. Nucleotide sequence analysis of molecularly cloned DNA identified base substitutions, deletions/insertions and tandem duplications. Transcriptional regulatory elements in the NCR were assayed in recombinant plasmids containing the bacterial gene for chloramphenicol acetyl transferase.« less

Transposable elements as genetic regulatory substrates in early development.

PubMed

Gifford, Wesley D; Pfaff, Samuel L; Macfarlan, Todd S

2013-05-01

The abundance and ancient origins of transposable elements (TEs) in eukaryotic genomes has spawned research into the potential symbiotic relationship between these elements and their hosts. In this review, we introduce the diversity of TEs, discuss how distinct classes are uniquely regulated in development, and describe how they appear to have been coopted for the purposes of gene regulation and the orchestration of a number of processes during early embryonic development. Although young, active TEs play an important role in somatic tissues and evolution, we focus mostly on the contributions of the older, fixed elements in mammalian genomes. We also discuss major challenges inherent in the study of TEs and contemplate future experimental approaches to further investigate how they coordinate developmental processes. Published by Elsevier Ltd.

Transposable elements as genetic regulatory substrates in early development

PubMed Central

Gifford, Wesley D.; Pfaff, Samuel L.; Macfarlan, Todd S.

2014-01-01

The abundance and ancient origins of transposable elements (TEs) in eukaryotic genomes has spawned research into the potential symbiotic relationship between these elements and their hosts. In this review, we introduce the diversity of TEs, discuss how distinct classes are uniquely regulated in development, and describe how they appear to have been coopted for the purposes of gene regulation and the orchestration of a number of processes during early embryonic development. Although young, active TEs play an important role in somatic tissues and evolution, we focus mostly on the contributions of the older, fixed elements in mammalian genomes. We also discuss major challenges inherent in the study of TEs and contemplate future experimental approaches to further investigate how they coordinate developmental processes. PMID:23411159

Parallel evolution of chordate cis-regulatory code for development.

PubMed

Doglio, Laura; Goode, Debbie K; Pelleri, Maria C; Pauls, Stefan; Frabetti, Flavia; Shimeld, Sebastian M; Vavouri, Tanya; Elgar, Greg

2013-11-01

Urochordates are the closest relatives of vertebrates and at the larval stage, possess a characteristic bilateral chordate body plan. In vertebrates, the genes that orchestrate embryonic patterning are in part regulated by highly conserved non-coding elements (CNEs), yet these elements have not been identified in urochordate genomes. Consequently the evolution of the cis-regulatory code for urochordate development remains largely uncharacterised. Here, we use genome-wide comparisons between C. intestinalis and C. savignyi to identify putative urochordate cis-regulatory sequences. Ciona conserved non-coding elements (ciCNEs) are associated with largely the same key regulatory genes as vertebrate CNEs. Furthermore, some of the tested ciCNEs are able to activate reporter gene expression in both zebrafish and Ciona embryos, in a pattern that at least partially overlaps that of the gene they associate with, despite the absence of sequence identity. We also show that the ability of a ciCNE to up-regulate gene expression in vertebrate embryos can in some cases be localised to short sub-sequences, suggesting that functional cross-talk may be defined by small regions of ancestral regulatory logic, although functional sub-sequences may also be dispersed across the whole element. We conclude that the structure and organisation of cis-regulatory modules is very different between vertebrates and urochordates, reflecting their separate evolutionary histories. However, functional cross-talk still exists because the same repertoire of transcription factors has likely guided their parallel evolution, exploiting similar sets of binding sites but in different combinations.

Regulatory elements involved in constitutive and phorbol ester-inducible expression of the plasminogen activator inhibitor type 2 gene promoter.

PubMed Central

Cousin, E; Medcalf, R L; Bergonzelli, G E; Kruithof, E K

1991-01-01

Gene transcription rates and mRNA levels of plasminogen activator inhibitor type 2 (PAI-2) are markedly induced by the tumor promoting agent phorbol 12-myristate 13-acetate (PMA) in human HT1080 fibrosarcoma cells. To identify promoter elements required for basal-, and phorbol ester-inducible expression, deletion mutants of the PAI-1 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene, were transiently expressed in HT1080 cells. Constitutive CAT activity was expressed from constructs containing more than 215 bp of promoter sequence, whereas deletion to position -91 bp abolished CAT gene expression. Treatment of transfected cells with PMA resulted in a three- to ten-fold increase in CAT expression from all constructs except from the construct shortened to position -91. DNAse1 protection analysis of the promoter region between -215 and the transcription initiation site revealed numerous protected regions, including two AP1-like binding sites (AP1a and AP1b) and one CRE-like element. Site-directed mutagenesis of the AP1a site or of the CRE-like site resulted in the loss of basal CAT activity and abolished the PMA effect, whereas mutagenesis of AP1b only partially inhibited basal and PMA-mediated expression. Our results suggest that the PAI-2 promoter contains at least two elements required for basal gene transcription and PMA-mediated induction. Images PMID:1650454

Cis-regulatory element based targeted gene finding: genome-wide identification of abscisic acid- and abiotic stress-responsive genes in Arabidopsis thaliana.

PubMed

Zhang, Weixiong; Ruan, Jianhua; Ho, Tuan-Hua David; You, Youngsook; Yu, Taotao; Quatrano, Ralph S

2005-07-15

A fundamental problem of computational genomics is identifying the genes that respond to certain endogenous cues and environmental stimuli. This problem can be referred to as targeted gene finding. Since gene regulation is mainly determined by the binding of transcription factors and cis-regulatory DNA sequences, most existing gene annotation methods, which exploit the conservation of open reading frames, are not effective in finding target genes. A viable approach to targeted gene finding is to exploit the cis-regulatory elements that are known to be responsible for the transcription of target genes. Given such cis-elements, putative target genes whose promoters contain the elements can be identified. As a case study, we apply the above approach to predict the genes in model plant Arabidopsis thaliana which are inducible by a phytohormone, abscisic acid (ABA), and abiotic stress, such as drought, cold and salinity. We first construct and analyze two ABA specific cis-elements, ABA-responsive element (ABRE) and its coupling element (CE), in A.thaliana, based on their conservation in rice and other cereal plants. We then use the ABRE-CE module to identify putative ABA-responsive genes in A.thaliana. Based on RT-PCR verification and the results from literature, this method has an accuracy rate of 67.5% for the top 40 predictions. The cis-element based targeted gene finding approach is expected to be widely applicable since a large number of cis-elements in many species are available.

A Field Experiment Testing the Utility of Regulatory Fit Messages for Promoting Physical Activity.

PubMed

Latimer, Amy E; Rivers, Susan E; Rench, Tara A; Katulak, Nicole A; Hicks, Althea; Hodorowski, Julie Keany; Higgins, E Tory; Salovey, Peter

2008-05-01

Guided by regulatory focus theory, we examined whether messages tailored to individuals' promotion- or prevention-goal orientation (regulatory focus) elicit positive thoughts and feelings about physical activity and increase participation in physical activity. Inactive participants (N = 206) were assigned randomly to receive either promotion-focused or prevention-focused messages encouraging physical activity. Two weeks after message exposure, we assessed participants' thoughts and feelings about physical activity and physical activity behavior. Tailored messages that fit individuals' regulatory focus led to greater physical activity participation and more positive feelings than non-fit messages, particularly in the promotion-focused condition. Furthermore, positive retrospective feelings about physical activity mediated the effects of the tailored messages on behavior. These findings provide support for regulatory focus theory and direction for enhancing the effectiveness of messages encouraging physical activity and other health behaviors.

Identification of active miRNA and transcription factor regulatory pathways in human obesity-related inflammation.

PubMed

Zhang, Xi-Mei; Guo, Lin; Chi, Mei-Hua; Sun, Hong-Mei; Chen, Xiao-Wen

2015-03-07

Obesity-induced chronic inflammation plays a fundamental role in the pathogenesis of metabolic syndrome (MS). Recently, a growing body of evidence supports that miRNAs are largely dysregulated in obesity and that specific miRNAs regulate obesity-associated inflammation. We applied an approach aiming to identify active miRNA-TF-gene regulatory pathways in obesity. Firstly, we detected differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRs) from mRNA and miRNA expression profiles, respectively. Secondly, by mapping the DEGs and DEmiRs to the curated miRNA-TF-gene regulatory network as active seed nodes and connect them with their immediate neighbors, we obtained the potential active miRNA-TF-gene regulatory subnetwork in obesity. Thirdly, using a Breadth-First-Search (BFS) algorithm, we identified potential active miRNA-TF-gene regulatory pathways in obesity. Finally, through the hypergeometric test, we identified the active miRNA-TF-gene regulatory pathways that were significantly related to obesity. The potential active pathways with FDR < 0.0005 were considered to be the active miRNA-TF regulatory pathways in obesity. The union of the active pathways is visualized and identical nodes of the active pathways were merged. We identified 23 active miRNA-TF-gene regulatory pathways that were significantly related to obesity-related inflammation.

Coordinate Regulation of Yeast Sterol Regulatory Element-binding Protein (SREBP) and Mga2 Transcription Factors.

PubMed

Burr, Risa; Stewart, Emerson V; Espenshade, Peter J

2017-03-31

The Mga2 and Sre1 transcription factors regulate oxygen-responsive lipid homeostasis in the fission yeast Schizosaccharomyces pombe in a manner analogous to the mammalian sterol regulatory element-binding protein (SREBP)-1 and SREBP-2 transcription factors. Mga2 and SREBP-1 regulate triacylglycerol and glycerophospholipid synthesis, whereas Sre1 and SREBP-2 regulate sterol synthesis. In mammals, a shared activation mechanism allows for coordinate regulation of SREBP-1 and SREBP-2. In contrast, distinct pathways activate fission yeast Mga2 and Sre1. Therefore, it is unclear whether and how these two related pathways are coordinated to maintain lipid balance in fission yeast. Previously, we showed that Sre1 cleavage is defective in the absence of mga2 Here, we report that this defect is due to deficient unsaturated fatty acid synthesis, resulting in aberrant membrane transport. This defect is recapitulated by treatment with the fatty acid synthase inhibitor cerulenin and is rescued by addition of exogenous unsaturated fatty acids. Furthermore, sterol synthesis inhibition blocks Mga2 pathway activation. Together, these data demonstrate that Sre1 and Mga2 are each regulated by the lipid product of the other transcription factor pathway, providing a source of coordination for these two branches of lipid synthesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

[Analysis of cis-regulatory element distribution in gene promoters of Gossypium raimondii and Arabidopsis thaliana].

PubMed

Sun, Gao-Fei; He, Shou-Pu; Du, Xiong-Ming

2013-10-01

Cotton genomic studies have boomed since the release of Gossypium raimondii draft genome. In this study, cis-regulatory element (CRE) in 1 kb length sequence upstream 5' UTR of annotated genes were selected and scanned in the Arabidopsis thaliana (At) and Gossypium raimondii (Gr) genomes, based on the database of PLACE (Plant cis-acting Regulatory DNA Elements). According to the definition of this study, 44 (12.3%) and 57 (15.5%) CREs presented "peak-like" distribution in the 1 kb selected sequences of both genomes, respectively. Thirty-four of them were peak-like distributed in both genomes, which could be further categorized into 4 types based on their core sequences. The coincidence of TATABOX peak position and their actual position ((-) -30 bp) indicated that the position of a common CRE was conservative in different genes, which suggested that the peak position of these CREs was their possible actual position of transcription factors. The position of a common CRE was also different between the two genomes due to stronger length variation of 5' UTR in Gr than At. Furthermore, most of the peak-like CREs were located in the region of -110 bp-0 bp, which suggested that concentrated distribution might be conductive to the interaction of transcription factors, and then regulate the gene expression in downstream.

Identification of polycomb and trithorax group responsive elements in the regulatory region of the Drosophila homeotic gene Sex combs reduced

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gindhart, J.G. Jr.; Kaufman, T.C.

1995-02-01

The Drosophilia homeotic gene Sex combs reduced (Scr) is necessary for the establishment and maintenance of the morphological identity of the labial and prothoracic segments. In the early embryo, its expression pattern is established through the activity of several gap and segmentation gene products, as well as other transcription factors. Once established, the Polycomb group (Pc-G) and trithorax group (trx-G) gene products maintain the spatial pattern of Scr expression for the remainder of development. We report the identification of DNA fragments in the Scr regulatory region that may be important for its regulation by Polycomb and trithorax group gene products.more » When DNA fragments containing these regulatory sequences are subcloned into P-element vectors containing a white minigene, transformants containing these constructs exhibit mosaic patterns of pigmentation in the adult eye, indicating that white minigene expression is repressed in a clonally heritable manner. The size of pigmented and nonpigmented clones in the adult eye suggests that the event determining whether a cell in the eye anlagen will express white occurs at least as early as the first larval instar. The amount of white minigene repression is reduced in some Polycomb group mutants, whereas repression is enhanced in flies mutant for a subset of trithorax group loci. The repressor activity of one fragment, normally located in Scr Intron 2, is increased when it is able to homologously pair, a property consistent with genetic data suggesting that Scr exhibits transvection. Another Scr regulatory fragment, normally located 40 kb upstream of the Scr promoter, silences ectopic expression of an Scr-lacZ fusion gene in the embryo and does so in a Polycomb-dependent manner. We propose that the regulatory sequences located within these DNA fragments may normally mediate the regulation of Scr by proteins encoded by members of Polycomb and trithorax group loci. 98 refs., 6 figs., 4 tabs.« less

Identification and characterization of a silencer regulatory element in the 3'-flanking region of the murine CD46 gene.

PubMed Central

Nomura, M; Tsujimura, A; Begum, N A; Matsumoto, M; Wabiko, H; Toyoshima, K; Seya, T

2000-01-01

The murine membrane cofactor protein (CD46) gene is expressed exclusively in testis, in contrast to human CD46, which is expressed ubiquitously. To elucidate the mechanism of differential CD46 gene expression among species, we cloned entire murine CD46 genomic DNA and possible regulatory regions were placed in the flanking region of the luciferase reporter gene. The reporter gene assay revealed a silencing activity not in the promoter, but in the 3'-flanking region of the gene and the silencer-like element was identified within a 0.2-kb region between 0.6 and 0.8 kb downstream of the stop codon. This silencer-like element was highly similar to that of the pig MHC class-I gene. The introduction of a mutation into this putative silencer element of murine CD46 resulted in an abrogation of the silencing effect. Electrophoretic mobility-shift assay indicated the presence of the binding molecule(s) for this silencer sequence in murine cell lines and tissues. A size difference of the protein-silencer-element complex was observed depending upon the solubilizers used for preparation of the nuclear extracts. A mutated silencer sequence failed to interact with the binding molecules. The level of the binding factor was lower in the testicular germ cells compared with other organs. Thus the silencer element and its binding factor may play a role in transcriptional regulation of murine CD46 gene expression. These results imply that the effects of the CD46 silencer element encompass the innate immune and reproductive systems, and in mice may determine the testicular germ-cell-dominant expression of CD46. PMID:11023821

A novel processing system of sterol regulatory element-binding protein-1c regulated by polyunsaturated fatty acid.

PubMed

Nakakuki, Masanori; Kawano, Hiroyuki; Notsu, Tatsuto; Imada, Kazunori; Mizuguchi, Kiyoshi; Shimano, Hitoshi

2014-05-01

The proteolytic cascade is the key step in transactivation of sterol regulatory element-binding proteins (SREBPs), a transcriptional factor of lipid synthesis. Proteolysis of SREBP-2 is strictly regulated by sterols, but that of SREBP-1c was not strongly sterol-regulated, but inhibited by polyunsaturated fatty acids (PUFAs). In this study, the proteolytic processing of SREBP-1 and -2 was examined by transfection studies of cDNA-encoding mutants in which all the known cleavage sites were disrupted. In cultured cells, sterol-regulated SREBP-2 processing was completely eliminated by mutation of cleavage sites. In contrast, the corresponding SREBP-1c mutants as well as wild type exhibited large amounts of cleaved products in the nuclear extracts from culture cells and murine liver in vivo. The nuclear form of the mutant SREBP-1c was induced by delipidated condition and suppressed by eicosapentaenoic acid, an n-3 PUFA, but not by sterols. This novel processing mechanism was affected by neither SREBP cleavage-activating protein (SCAP) nor insulin-induced gene (Insig)-1, unlike SREBP-2, but abolished by a serine protease inhibitor. Through analysis of deletion mutant, a site-2 protease recognition sequence (DRSR) was identified to be involved in this novel processing. These findings suggest that SREBP-1c cleavage could be subjected to a novel PUFA-regulated cleavage system in addition to the sterol-regulatory SCAP/Insig system.

SORPTION OF ELEMENTAL MERCURY BY ACTIVATED CARBONS

EPA Science Inventory

The mechanisms and rate of elemental mercury (HgO) capture by activated carbons have been studied using a bench-scale apparatus. Three types of activated carbons, two of which are thermally activated (PC-100 and FGD) and one with elemental sulfur (S) impregnated in it (HGR), were...

Activity-Dependent Human Brain Coding/Noncoding Gene Regulatory Networks

PubMed Central

Lipovich, Leonard; Dachet, Fabien; Cai, Juan; Bagla, Shruti; Balan, Karina; Jia, Hui; Loeb, Jeffrey A.

2012-01-01

While most gene transcription yields RNA transcripts that code for proteins, a sizable proportion of the genome generates RNA transcripts that do not code for proteins, but may have important regulatory functions. The brain-derived neurotrophic factor (BDNF) gene, a key regulator of neuronal activity, is overlapped by a primate-specific, antisense long noncoding RNA (lncRNA) called BDNFOS. We demonstrate reciprocal patterns of BDNF and BDNFOS transcription in highly active regions of human neocortex removed as a treatment for intractable seizures. A genome-wide analysis of activity-dependent coding and noncoding human transcription using a custom lncRNA microarray identified 1288 differentially expressed lncRNAs, of which 26 had expression profiles that matched activity-dependent coding genes and an additional 8 were adjacent to or overlapping with differentially expressed protein-coding genes. The functions of most of these protein-coding partner genes, such as ARC, include long-term potentiation, synaptic activity, and memory. The nuclear lncRNAs NEAT1, MALAT1, and RPPH1, composing an RNAse P-dependent lncRNA-maturation pathway, were also upregulated. As a means to replicate human neuronal activity, repeated depolarization of SY5Y cells resulted in sustained CREB activation and produced an inverse pattern of BDNF-BDNFOS co-expression that was not achieved with a single depolarization. RNAi-mediated knockdown of BDNFOS in human SY5Y cells increased BDNF expression, suggesting that BDNFOS directly downregulates BDNF. Temporal expression patterns of other lncRNA-messenger RNA pairs validated the effect of chronic neuronal activity on the transcriptome and implied various lncRNA regulatory mechanisms. lncRNAs, some of which are unique to primates, thus appear to have potentially important regulatory roles in activity-dependent human brain plasticity. PMID:22960213

Evaluation of Downstream Regulatory Element Antagonistic Modulator Gene in Human Multinodular Goiter

PubMed Central

Shinzato, Amanda; Lerario, Antonio M.; Lin, Chin J.; Danilovic, Debora S.; Marui, Suemi; Trarbach, Ericka B.

2015-01-01

Background DREAM (Downstream Regulatory Element Antagonistic Modulator) is a neuronal calcium sensor that was suggested to modulate TSH receptor activity and whose overexpression provokes an enlargement of the thyroid gland in transgenic mice. The aim of this study was to investigate somatic mutations and DREAM gene expression in human multinodular goiter (MNG). Material/Methods DNA and RNA samples were obtained from hyperplastic thyroid glands of 60 patients (54 females) with benign MNG. DREAM mutations were evaluated by PCR and direct automatic sequencing, whereas relative quantification of mRNA was performed by real-time PCR. Over- and under-expression were defined as a 2-fold increase and decrease in comparison to normal thyroid tissue, respectively. RQ M (relative quantification mean); SD (standard deviation). Results DREAM expression was detected in all nodules evaluated. DREAM mRNA was overexpressed in 31.7% of MNG (RQ M=6.26; SD=5.08), whereas 53.3% and 15% had either normal (RQ M=1.16; SD=0.46) or underexpression (RQ M=0.30; SD=0.10), respectively. Regarding DREAM mutations analysis, only previously described intronic polymorphisms were observed. Conclusions We report DREAM gene expression in the hyperplastic thyroid gland of MNG patients. However, DREAM expression did not vary significantly, and was somewhat underexpressed in most patients, suggesting that DREAM upregulation does not significantly affect nodular development in human goiter. PMID:26319784

Transposable Elements and DNA Methylation Create in Embryonic Stem Cells Human-Specific Regulatory Sequences Associated with Distal Enhancers and Noncoding RNAs

PubMed Central

Glinsky, Gennadi V.

2015-01-01

Despite significant progress in the structural and functional characterization of the human genome, understanding of the mechanisms underlying the genetic basis of human phenotypic uniqueness remains limited. Here, I report that transposable element-derived sequences, most notably LTR7/HERV-H, LTR5_Hs, and L1HS, harbor 99.8% of the candidate human-specific regulatory loci (HSRL) with putative transcription factor-binding sites in the genome of human embryonic stem cells (hESC). A total of 4,094 candidate HSRL display selective and site-specific binding of critical regulators (NANOG [Nanog homeobox], POU5F1 [POU class 5 homeobox 1], CCCTC-binding factor [CTCF], Lamin B1), and are preferentially located within the matrix of transcriptionally active DNA segments that are hypermethylated in hESC. hESC-specific NANOG-binding sites are enriched near the protein-coding genes regulating brain size, pluripotency long noncoding RNAs, hESC enhancers, and 5-hydroxymethylcytosine-harboring regions immediately adjacent to binding sites. Sequences of only 4.3% of hESC-specific NANOG-binding sites are present in Neanderthals’ genome, suggesting that a majority of these regulatory elements emerged in Modern Humans. Comparisons of estimated creation rates of novel TF-binding sites revealed that there was 49.7-fold acceleration of creation rates of NANOG-binding sites in genomes of Chimpanzees compared with the mouse genomes and further 5.7-fold acceleration in genomes of Modern Humans compared with the Chimpanzees genomes. Preliminary estimates suggest that emergence of one novel NANOG-binding site detectable in hESC required 466 years of evolution. Pathway analysis of coding genes that have hESC-specific NANOG-binding sites within gene bodies or near gene boundaries revealed their association with physiological development and functions of nervous and cardiovascular systems, embryonic development, behavior, as well as development of a diverse spectrum of pathological conditions

Emerging principles of regulatory evolution.

PubMed

Prud'homme, Benjamin; Gompel, Nicolas; Carroll, Sean B

2007-05-15

Understanding the genetic and molecular mechanisms governing the evolution of morphology is a major challenge in biology. Because most animals share a conserved repertoire of body-building and -patterning genes, morphological diversity appears to evolve primarily through changes in the deployment of these genes during development. The complex expression patterns of developmentally regulated genes are typically controlled by numerous independent cis-regulatory elements (CREs). It has been proposed that morphological evolution relies predominantly on changes in the architecture of gene regulatory networks and in particular on functional changes within CREs. Here, we discuss recent experimental studies that support this hypothesis and reveal some unanticipated features of how regulatory evolution occurs. From this growing body of evidence, we identify three key operating principles underlying regulatory evolution, that is, how regulatory evolution: (i) uses available genetic components in the form of preexisting and active transcription factors and CREs to generate novelty; (ii) minimizes the penalty to overall fitness by introducing discrete changes in gene expression; and (iii) allows interactions to arise among any transcription factor and downstream CRE. These principles endow regulatory evolution with a vast creative potential that accounts for both relatively modest morphological differences among closely related species and more profound anatomical divergences among groups at higher taxonomical levels.

MIRA: An R package for DNA methylation-based inference of regulatory activity.

PubMed

Lawson, John T; Tomazou, Eleni M; Bock, Christoph; Sheffield, Nathan C

2018-03-01

DNA methylation contains information about the regulatory state of the cell. MIRA aggregates genome-scale DNA methylation data into a DNA methylation profile for independent region sets with shared biological annotation. Using this profile, MIRA infers and scores the collective regulatory activity for each region set. MIRA facilitates regulatory analysis in situations where classical regulatory assays would be difficult and allows public sources of open chromatin and protein binding regions to be leveraged for novel insight into the regulatory state of DNA methylation datasets. R package available on Bioconductor: http://bioconductor.org/packages/release/bioc/html/MIRA.html. nsheffield@virginia.edu.

Fanconi Anemia Core Complex Gene Promoters Harbor Conserved Transcription Regulatory Elements

PubMed Central

Meier, Daniel; Schindler, Detlev

2011-01-01

The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5′ region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3′ regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters. PMID:21826217

Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

PubMed

Meier, Daniel; Schindler, Detlev

2011-01-01

The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

Regulatory elements of Caenorhabditis elegans ribosomal protein genes

PubMed Central

2012-01-01

Background Ribosomal protein genes (RPGs) are essential, tightly regulated, and highly expressed during embryonic development and cell growth. Even though their protein sequences are strongly conserved, their mechanism of regulation is not conserved across yeast, Drosophila, and vertebrates. A recent investigation of genomic sequences conserved across both nematode species and associated with different gene groups indicated the existence of several elements in the upstream regions of C. elegans RPGs, providing a new insight regarding the regulation of these genes in C. elegans. Results In this study, we performed an in-depth examination of C. elegans RPG regulation and found nine highly conserved motifs in the upstream regions of C. elegans RPGs using the motif discovery algorithm DME. Four motifs were partially similar to transcription factor binding sites from C. elegans, Drosophila, yeast, and human. One pair of these motifs was found to co-occur in the upstream regions of 250 transcripts including 22 RPGs. The distance between the two motifs displayed a complex frequency pattern that was related to their relative orientation. We tested the impact of three of these motifs on the expression of rpl-2 using a series of reporter gene constructs and showed that all three motifs are necessary to maintain the high natural expression level of this gene. One of the motifs was similar to the binding site of an orthologue of POP-1, and we showed that RNAi knockdown of pop-1 impacts the expression of rpl-2. We further determined the transcription start site of rpl-2 by 5’ RACE and found that the motifs lie 40–90 bases upstream of the start site. We also found evidence that a noncoding RNA, contained within the outron of rpl-2, is co-transcribed with rpl-2 and cleaved during trans-splicing. Conclusions Our results indicate that C. elegans RPGs are regulated by a complex novel series of regulatory elements that is evolutionarily distinct from those of all other species

Changes in cis-regulatory elements of a key floral regulator are associated with divergence of inflorescence architectures.

PubMed

Kusters, Elske; Della Pina, Serena; Castel, Rob; Souer, Erik; Koes, Ronald

2015-08-15

Higher plant species diverged extensively with regard to the moment (flowering time) and position (inflorescence architecture) at which flowers are formed. This seems largely caused by variation in the expression patterns of conserved genes that specify floral meristem identity (FMI), rather than changes in the encoded proteins. Here, we report a functional comparison of the promoters of homologous FMI genes from Arabidopsis, petunia, tomato and Antirrhinum. Analysis of promoter-reporter constructs in petunia and Arabidopsis, as well as complementation experiments, showed that the divergent expression of leafy (LFY) and the petunia homolog aberrant leaf and flower (ALF) results from alterations in the upstream regulatory network rather than cis-regulatory changes. The divergent expression of unusual floral organs (UFO) from Arabidopsis, and the petunia homolog double top (DOT), however, is caused by the loss or gain of cis-regulatory promoter elements, which respond to trans-acting factors that are expressed in similar patterns in both species. Introduction of pUFO:UFO causes no obvious defects in Arabidopsis, but in petunia it causes the precocious and ectopic formation of flowers. This provides an example of how a change in a cis-regulatory region can account for a change in the plant body plan. © 2015. Published by The Company of Biologists Ltd.

Moving through the Stressed Genome: Emerging Regulatory Roles for Transposons in Plant Stress Response.

PubMed

Negi, Pooja; Rai, Archana N; Suprasanna, Penna

2016-01-01

The recognition of a positive correlation between organism genome size with its transposable element (TE) content, represents a key discovery of the field of genome biology. Considerable evidence accumulated since then suggests the involvement of TEs in genome structure, evolution and function. The global genome reorganization brought about by transposon activity might play an adaptive/regulatory role in the host response to environmental challenges, reminiscent of McClintock's original 'Controlling Element' hypothesis. This regulatory aspect of TEs is also garnering support in light of the recent evidences, which project TEs as "distributed genomic control modules." According to this view, TEs are capable of actively reprogramming host genes circuits and ultimately fine-tuning the host response to specific environmental stimuli. Moreover, the stress-induced changes in epigenetic status of TE activity may allow TEs to propagate their stress responsive elements to host genes; the resulting genome fluidity can permit phenotypic plasticity and adaptation to stress. Given their predominating presence in the plant genomes, nested organization in the genic regions and potential regulatory role in stress response, TEs hold unexplored potential for crop improvement programs. This review intends to present the current information about the roles played by TEs in plant genome organization, evolution, and function and highlight the regulatory mechanisms in plant stress responses. We will also briefly discuss the connection between TE activity, host epigenetic response and phenotypic plasticity as a critical link for traversing the translational bridge from a purely basic study of TEs, to the applied field of stress adaptation and crop improvement.

Liver X receptor regulates hepatic nuclear O-GlcNAc signaling and carbohydrate responsive element-binding protein activity[S

PubMed Central

Bindesbøll, Christian; Fan, Qiong; Nørgaard, Rikke C.; MacPherson, Laura; Ruan, Hai-Bin; Wu, Jing; Pedersen, Thomas Å.; Steffensen, Knut R.; Yang, Xiaoyong; Matthews, Jason; Mandrup, Susanne; Nebb, Hilde I.; Grønning-Wang, Line M.

2015-01-01

Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/β+/+ and LXRα/β−/− mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPβ. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity. PMID:25724563

Cis-regulatory landscapes of four cell types of the retina

PubMed Central

Hartl, Dominik; Jüttner, Josephine

2017-01-01

Abstract The retina is composed of ∼50 cell-types with specific functions for the process of vision. Identification of the cis-regulatory elements active in retinal cell-types is key to elucidate the networks controlling this diversity. Here, we combined transcriptome and epigenome profiling to map the regulatory landscape of four cell-types isolated from mouse retinas including rod and cone photoreceptors as well as rare inter-neuron populations such as horizontal and starburst amacrine cells. Integration of this information reveals sequence determinants and candidate transcription factors for controlling cellular specialization. Additionally, we refined parallel reporter assays to enable studying the transcriptional activity of large collection of sequences in individual cell-types isolated from a tissue. We provide proof of concept for this approach and its scalability by characterizing the transcriptional capacity of several hundred putative regulatory sequences within individual retinal cell-types. This generates a catalogue of cis-regulatory regions active in retinal cell types and we further demonstrate their utility as potential resource for cellular tagging and manipulation. PMID:29059322

Activation of IFN-beta element by IRF-1 requires a posttranslational event in addition to IRF-1 synthesis.

PubMed Central

Watanabe, N; Sakakibara, J; Hovanessian, A G; Taniguchi, T; Fujita, T

1991-01-01

Expression of the Type I IFN (i.e., IFN-alpha s and IFN-beta) genes is efficiently induced by viruses at the transcriptional level. This induction is mediated by at least two types of positive regulatory elements located in the human IFN-beta gene promoter: (1) the repeated elements which bind both the transcriptional activator IRF-1 and the repressor IRF-2 (IRF-elements; IRF-Es), and (2) the kappa B element (kappa B-E), which binds NF kappa B and is located between the IRF-Es and the TATA box. In this study we demonstrate that a promoter containing synthetic IRF-E, which displays high affinity for the IRFs can be efficiently activated by Newcastle disease virus (NDV). In contrast, such activation was either very weak or nil when cells were treated by IFN-beta or tumor necrosis factor-alpha (TNF-alpha), despite the fact they both efficiently induce de novo synthesis of the short-lived IRF-1 in L929 cells. In fact, efficient activation of the IRF-E apparently requires an event in addition to de novo IRF-1 induction, which can be elicited by NDV even in the presence of protein synthesis inhibitor, cycloheximide. Moreover, efficient activation of the IRF-E by NDV is specifically inhibited by the protein kinase inhibitor, Staurosporin. Hence our results suggest the importance of IRF-1 synthesis and post-translational modification event(s), possibly phosphorylation for the efficient activation of IRF-Es, which are otherwise under negative regulation by IRF-2. Images PMID:1886766

Proteome-wide Structural Analysis of PTM Hotspots Reveals Regulatory Elements Predicted to Impact Biological Function and Disease.

PubMed

Torres, Matthew P; Dewhurst, Henry; Sundararaman, Niveda

2016-11-01

Post-translational modifications (PTMs) regulate protein behavior through modulation of protein-protein interactions, enzymatic activity, and protein stability essential in the translation of genotype to phenotype in eukaryotes. Currently, less than 4% of all eukaryotic PTMs are reported to have biological function - a statistic that continues to decrease with an increasing rate of PTM detection. Previously, we developed SAPH-ire (Structural Analysis of PTM Hotspots) - a method for the prioritization of PTM function potential that has been used effectively to reveal novel PTM regulatory elements in discrete protein families (Dewhurst et al., 2015). Here, we apply SAPH-ire to the set of eukaryotic protein families containing experimental PTM and 3D structure data - capturing 1,325 protein families with 50,839 unique PTM sites organized into 31,747 modified alignment positions (MAPs), of which 2010 (∼6%) possess known biological function. Here, we show that using an artificial neural network model (SAPH-ire NN) trained to identify MAP hotspots with biological function results in prediction outcomes that far surpass the use of single hotspot features, including nearest neighbor PTM clustering methods. We find the greatest enhancement in prediction for positions with PTM counts of five or less, which represent 98% of all MAPs in the eukaryotic proteome and 90% of all MAPs found to have biological function. Analysis of the top 1092 MAP hotspots revealed 267 of truly unknown function (containing 5443 distinct PTMs). Of these, 165 hotspots could be mapped to human KEGG pathways for normal and/or disease physiology. Many high-ranking hotspots were also found to be disease-associated pathogenic sites of amino acid substitution despite the lack of observable PTM in the human protein family member. Taken together, these experiments demonstrate that the functional relevance of a PTM can be predicted very effectively by neural network models, revealing a large but testable

Proteome-wide Structural Analysis of PTM Hotspots Reveals Regulatory Elements Predicted to Impact Biological Function and Disease*

PubMed Central

Dewhurst, Henry; Sundararaman, Niveda

2016-01-01

Post-translational modifications (PTMs) regulate protein behavior through modulation of protein-protein interactions, enzymatic activity, and protein stability essential in the translation of genotype to phenotype in eukaryotes. Currently, less than 4% of all eukaryotic PTMs are reported to have biological function - a statistic that continues to decrease with an increasing rate of PTM detection. Previously, we developed SAPH-ire (Structural Analysis of PTM Hotspots) - a method for the prioritization of PTM function potential that has been used effectively to reveal novel PTM regulatory elements in discrete protein families (Dewhurst et al., 2015). Here, we apply SAPH-ire to the set of eukaryotic protein families containing experimental PTM and 3D structure data - capturing 1,325 protein families with 50,839 unique PTM sites organized into 31,747 modified alignment positions (MAPs), of which 2010 (∼6%) possess known biological function. Here, we show that using an artificial neural network model (SAPH-ire NN) trained to identify MAP hotspots with biological function results in prediction outcomes that far surpass the use of single hotspot features, including nearest neighbor PTM clustering methods. We find the greatest enhancement in prediction for positions with PTM counts of five or less, which represent 98% of all MAPs in the eukaryotic proteome and 90% of all MAPs found to have biological function. Analysis of the top 1092 MAP hotspots revealed 267 of truly unknown function (containing 5443 distinct PTMs). Of these, 165 hotspots could be mapped to human KEGG pathways for normal and/or disease physiology. Many high-ranking hotspots were also found to be disease-associated pathogenic sites of amino acid substitution despite the lack of observable PTM in the human protein family member. Taken together, these experiments demonstrate that the functional relevance of a PTM can be predicted very effectively by neural network models, revealing a large but testable

17 CFR 1.59 - Activities of self-regulatory organization employees, governing board members, committee members...

Code of Federal Regulations, 2012 CFR

2012-04-01

... COMMODITY EXCHANGE ACT Miscellaneous § 1.59 Activities of self-regulatory organization employees, governing...) Self-regulatory organization means “self-regulatory organization,” as defined in Commission regulation... governors of a self-regulatory organization. (3) Committee member means a member, or functional equivalent...

17 CFR 1.59 - Activities of self-regulatory organization employees, governing board members, committee members...

Code of Federal Regulations, 2011 CFR

2011-04-01

... COMMODITY EXCHANGE ACT Miscellaneous § 1.59 Activities of self-regulatory organization employees, governing...) Self-regulatory organization means “self-regulatory organization,” as defined in Commission regulation... governors of a self-regulatory organization. (3) Committee member means a member, or functional equivalent...

17 CFR 1.59 - Activities of self-regulatory organization employees, governing board members, committee members...

Code of Federal Regulations, 2010 CFR

2010-04-01

... COMMODITY EXCHANGE ACT Miscellaneous § 1.59 Activities of self-regulatory organization employees, governing...) Self-regulatory organization means “self-regulatory organization,” as defined in Commission regulation... governors of a self-regulatory organization. (3) Committee member means a member, or functional equivalent...

The Evolution of Lineage-Specific Regulatory Activities in the Human Embryonic Limb

PubMed Central

Cotney, Justin; Leng, Jing; Yin, Jun; Reilly, Steven K.; DeMare, Laura E.; Emera, Deena; Ayoub, Albert E.; Rakic, Pasko; Noonan, James P.

2013-01-01

SUMMARY The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find that 13% of promoters and 11% of enhancers have gained activity on the human lineage since the human-rhesus divergence. These gains largely arose by modification of ancestral regulatory activities in the limb or potential co-option from other tissues and are likely to have heterogeneous genetic causes. Most enhancers that exhibit gain of activity in humans originated in mammals. Gains at promoters and enhancers in the human limb are associated with increased gene expression, suggesting they include molecular drivers of human morphological evolution. PMID:23827682

17 CFR 1.59 - Activities of self-regulatory organization employees, governing board members, committee members...

Code of Federal Regulations, 2014 CFR

2014-04-01

... COMMODITY EXCHANGE ACT Miscellaneous § 1.59 Activities of self-regulatory organization employees, governing...) Self-regulatory organization means “self-regulatory organization,” as defined in § 1.3(ee), and... member, or functional equivalent thereof, of the board of governors of a self-regulatory organization. (3...

17 CFR 1.59 - Activities of self-regulatory organization employees, governing board members, committee members...

Code of Federal Regulations, 2013 CFR

2013-04-01

... COMMODITY EXCHANGE ACT Miscellaneous § 1.59 Activities of self-regulatory organization employees, governing...) Self-regulatory organization means “self-regulatory organization,” as defined in § 1.3(ee), and... member, or functional equivalent thereof, of the board of governors of a self-regulatory organization. (3...

Two distinct auto-regulatory loops operate at the PU.1 locus in B cells and myeloid cells

PubMed Central

Leddin, Mathias; Perrod, Chiara; Hoogenkamp, Maarten; Ghani, Saeed; Assi, Salam; Heinz, Sven; Wilson, Nicola K.; Follows, George; Schönheit, Jörg; Vockentanz, Lena; Mosammam, Ali M.; Chen, Wei; Tenen, Daniel G.; Westhead, David R.; Göttgens, Berthold

2011-01-01

The transcription factor PU.1 occupies a central role in controlling myeloid and early B-cell development, and its correct lineage-specific expression is critical for the differentiation choice of hematopoietic progenitors. However, little is known of how this tissue-specific pattern is established. We previously identified an upstream regulatory cis element whose targeted deletion in mice decreases PU.1 expression and causes leukemia. We show here that the upstream regulatory cis element alone is insufficient to confer physiologic PU.1 expression in mice but requires the cooperation with other, previously unidentified elements. Using a combination of transgenic studies, global chromatin assays, and detailed molecular analyses we present evidence that PU.1 is regulated by a novel mechanism involving cross talk between different cis elements together with lineage-restricted autoregulation. In this model, PU.1 regulates its expression in B cells and macrophages by differentially associating with cell type–specific transcription factors at one of its cis-regulatory elements to establish differential activity patterns at other elements. PMID:21239694

Transcriptional activity of transposable elements in coelacanth.

PubMed

Forconi, Mariko; Chalopin, Domitille; Barucca, Marco; Biscotti, Maria Assunta; De Moro, Gianluca; Galiana, Delphine; Gerdol, Marco; Pallavicini, Alberto; Canapa, Adriana; Olmo, Ettore; Volff, Jean-Nicolas

2014-09-01

The morphological stasis of coelacanths has long suggested a slow evolutionary rate. General genomic stasis might also imply a decrease of transposable elements activity. To evaluate the potential activity of transposable elements (TEs) in "living fossil" species, transcriptomic data of Latimeria chalumnae and its Indonesian congener Latimeria menadoensis were compared through the RNA-sequencing mapping procedures in three different organs (liver, testis, and muscle). The analysis of coelacanth transcriptomes highlights a significant percentage of transcribed TEs in both species. Major contributors are LINE retrotransposons, especially from the CR1 family. Furthermore, some particular elements such as a LF-SINE and a LINE2 sequences seem to be more expressed than other elements. The amount of TEs expressed in testis suggests possible transposition burst in incoming generations. Moreover, significant amount of TEs in liver and muscle transcriptomes were also observed. Analyses of elements displaying marked organ-specific expression gave us the opportunity to highlight exaptation cases, that is, the recruitment of TEs as new cellular genes, but also to identify a new Latimeria-specific family of Short Interspersed Nuclear Elements called CoeG-SINEs. Overall, transcriptome results do not seem to be in line with a slow-evolving genome with poor TE activity. © 2013 Wiley Periodicals, Inc.

Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element.

PubMed

Lim, Chun Shen; Brown, Chris M

2016-09-01

Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel.

Hepatitis B virus nuclear export elements: RNA stem-loop α and β, key parts of the HBV post-transcriptional regulatory element

PubMed Central

Lim, Chun Shen; Brown, Chris M.

2016-01-01

ABSTRACT Many viruses contain RNA elements that modulate splicing and/or promote nuclear export of their RNAs. The RNAs of the major human pathogen, hepatitis B virus (HBV) contain a large (~600 bases) composite cis-acting 'post-transcriptional regulatory element' (PRE). This element promotes expression from these naturally intronless transcripts. Indeed, the related woodchuck hepadnavirus PRE (WPRE) is used to enhance expression in gene therapy and other expression vectors. These PRE are likely to act through a combination of mechanisms, including promotion of RNA nuclear export. Functional components of both the HBV PRE and WPRE are 2 conserved RNA cis-acting stem-loop (SL) structures, SLα and SLβ. They are within the coding regions of polymerase (P) gene, and both P and X genes, respectively. Based on previous studies using mutagenesis and/or nuclear magnetic resonance (NMR), here we propose 2 covariance models for SLα and SLβ. The model for the 30-nucleotide SLα contains a G-bulge and a CNGG(U) apical loop of which the first and the fourth loop residues form a CG pair and the fifth loop residue is bulged out, as observed in the NMR structure. The model for the 23-nucleotide SLβ contains a 7-base-pair stem and a 9-nucleotide loop. Comparison of the models with other RNA structural elements, as well as similarity searches of human transcriptome and viral genomes demonstrate that SLα and SLβ are specific to HBV transcripts. However, they are well conserved among the hepadnaviruses of non-human primates, the woodchuck and ground squirrel. PMID:27031749

The commercial development of space: is an international regulatory framework needed?

PubMed

Contant, Corinne M; Logsdon, John M

2004-04-01

The commercial space sector to date has failed to develop comprehensive regulations--"rules of the road"--for its international activities. Within the next 5 years, conflicts with respect to international trade in satellite sales and launch services could emerge, highlighting the need for such a regulatory framework. If the commercial space sector is to continue to develop, it is important to begin discussions now, before these conflicts become significant, on the elements of an appropriate international regulatory framework. The existing framework for space activities was developed when government, not commercial, space activities were dominant, or was adapted from regulations in other sectors such as terrestrial telecommunications. c2003 Elsevier Ltd. All rights reserved.

FARE-CAFE: a database of functional and regulatory elements of cancer-associated fusion events.

PubMed

Korla, Praveen Kumar; Cheng, Jack; Huang, Chien-Hung; Tsai, Jeffrey J P; Liu, Yu-Hsuan; Kurubanjerdjit, Nilubon; Hsieh, Wen-Tsong; Chen, Huey-Yi; Ng, Ka-Lok

2015-01-01

Chromosomal translocation (CT) is of enormous clinical interest because this disorder is associated with various major solid tumors and leukemia. A tumor-specific fusion gene event may occur when a translocation joins two separate genes. Currently, various CT databases provide information about fusion genes and their genomic elements. However, no database of the roles of fusion genes, in terms of essential functional and regulatory elements in oncogenesis, is available. FARE-CAFE is a unique combination of CTs, fusion proteins, protein domains, domain-domain interactions, protein-protein interactions, transcription factors and microRNAs, with subsequent experimental information, which cannot be found in any other CT database. Genomic DNA information including, for example, manually collected exact locations of the first and second break points, sequences and karyotypes of fusion genes are included. FARE-CAFE will substantially facilitate the cancer biologist's mission of elucidating the pathogenesis of various types of cancer. This database will ultimately help to develop 'novel' therapeutic approaches. Database URL: http://ppi.bioinfo.asia.edu.tw/FARE-CAFE. © The Author(s) 2015. Published by Oxford University Press.

FARE-CAFE: a database of functional and regulatory elements of cancer-associated fusion events

PubMed Central

Korla, Praveen Kumar; Cheng, Jack; Huang, Chien-Hung; Tsai, Jeffrey J. P.; Liu, Yu-Hsuan; Kurubanjerdjit, Nilubon; Hsieh, Wen-Tsong; Chen, Huey-Yi; Ng, Ka-Lok

2015-01-01

Chromosomal translocation (CT) is of enormous clinical interest because this disorder is associated with various major solid tumors and leukemia. A tumor-specific fusion gene event may occur when a translocation joins two separate genes. Currently, various CT databases provide information about fusion genes and their genomic elements. However, no database of the roles of fusion genes, in terms of essential functional and regulatory elements in oncogenesis, is available. FARE-CAFE is a unique combination of CTs, fusion proteins, protein domains, domain–domain interactions, protein–protein interactions, transcription factors and microRNAs, with subsequent experimental information, which cannot be found in any other CT database. Genomic DNA information including, for example, manually collected exact locations of the first and second break points, sequences and karyotypes of fusion genes are included. FARE-CAFE will substantially facilitate the cancer biologist’s mission of elucidating the pathogenesis of various types of cancer. This database will ultimately help to develop ‘novel’ therapeutic approaches. Database URL: http://ppi.bioinfo.asia.edu.tw/FARE-CAFE PMID:26384373

A cis-regulatory logic simulator.

PubMed

Zeigler, Robert D; Gertz, Jason; Cohen, Barak A

2007-07-27

A major goal of computational studies of gene regulation is to accurately predict the expression of genes based on the cis-regulatory content of their promoters. The development of computational methods to decode the interactions among cis-regulatory elements has been slow, in part, because it is difficult to know, without extensive experimental validation, whether a particular method identifies the correct cis-regulatory interactions that underlie a given set of expression data. There is an urgent need for test expression data in which the interactions among cis-regulatory sites that produce the data are known. The ability to rapidly generate such data sets would facilitate the development and comparison of computational methods that predict gene expression patterns from promoter sequence. We developed a gene expression simulator which generates expression data using user-defined interactions between cis-regulatory sites. The simulator can incorporate additive, cooperative, competitive, and synergistic interactions between regulatory elements. Constraints on the spacing, distance, and orientation of regulatory elements and their interactions may also be defined and Gaussian noise can be added to the expression values. The simulator allows for a data transformation that simulates the sigmoid shape of expression levels from real promoters. We found good agreement between sets of simulated promoters and predicted regulatory modules from real expression data. We present several data sets that may be useful for testing new methodologies for predicting gene expression from promoter sequence. We developed a flexible gene expression simulator that rapidly generates large numbers of simulated promoters and their corresponding transcriptional output based on specified interactions between cis-regulatory sites. When appropriate rule sets are used, the data generated by our simulator faithfully reproduces experimentally derived data sets. We anticipate that using simulated

Deficient Activity in the Neural Systems That Mediate Self-regulatory Control in Bulimia Nervosa

PubMed Central

Marsh, Rachel; Steinglass, Joanna E.; Gerber, Andrew J.; O’Leary, Kara Graziano; Wang, Zhishun; Murphy, David; Walsh, B. Timothy; Peterson, Bradley S.

2009-01-01

Context Disturbances in neural systems that mediate voluntary self-regulatory processes may contribute to bulimia nervosa (BN) by releasing feeding behaviors from regulatory control. Objective To study the functional activity in neural circuits that subserve self-regulatory control in women with BN. Design We compared functional magnetic resonance imaging blood oxygenation level–dependent responses in patients with BN with healthy controls during performance of the Simon Spatial Incompatibility task. Setting University research institute. Participants Forty women: 20 patients with BN and 20 healthy control participants. Main Outcome Measure We used general linear modeling of Simon Spatial Incompatibility task–related activations to compare groups on their patterns of brain activation associated with the successful or unsuccessful engagement of self-regulatory control. Results Patients with BN responded more impulsively and made more errors on the task than did healthy controls; patients with the most severe symptoms made the most errors. During correct responding on incongruent trials, patients failed to activate frontostriatal circuits to the same degree as healthy controls in the left inferolateral prefrontal cortex (Brodmann area [BA] 45), bilateral inferior frontal gyrus (BA 44), lenticular and caudate nuclei, and anterior cingulate cortex (BA 24/32). Patients activated the dorsal anterior cingulate cortex (BA 32) more when making errors than when responding correctly. In contrast, healthy participants activated the anterior cingulate cortex more during correct than incorrect responses, and they activated the striatum more when responding incorrectly, likely reflecting an automatic response tendency that, in the absence of concomitant anterior cingulate cortex activity, produced incorrect responses. Conclusions Self-regulatory processes are impaired in women with BN, likely because of their failure to engage frontostriatal circuits appropriately. These

Cis-regulatory landscapes of four cell types of the retina.

PubMed

Hartl, Dominik; Krebs, Arnaud R; Jüttner, Josephine; Roska, Botond; Schübeler, Dirk

2017-11-16

The retina is composed of ∼50 cell-types with specific functions for the process of vision. Identification of the cis-regulatory elements active in retinal cell-types is key to elucidate the networks controlling this diversity. Here, we combined transcriptome and epigenome profiling to map the regulatory landscape of four cell-types isolated from mouse retinas including rod and cone photoreceptors as well as rare inter-neuron populations such as horizontal and starburst amacrine cells. Integration of this information reveals sequence determinants and candidate transcription factors for controlling cellular specialization. Additionally, we refined parallel reporter assays to enable studying the transcriptional activity of large collection of sequences in individual cell-types isolated from a tissue. We provide proof of concept for this approach and its scalability by characterizing the transcriptional capacity of several hundred putative regulatory sequences within individual retinal cell-types. This generates a catalogue of cis-regulatory regions active in retinal cell types and we further demonstrate their utility as potential resource for cellular tagging and manipulation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28.

PubMed

Pla, M; Vilardell, J; Guiltinan, M J; Marcotte, W R; Niogret, M F; Quatrano, R S; Pagès, M

1993-01-01

The maize gene rab28 has been identified as ABA-inducible in embryos and vegetative tissues. It is also induced by water stress in young leaves. The proximal promoter region contains the conserved cis-acting element CCACGTGG (ABRE) reported for ABA induction in other plant genes. Transient expression assays in rice protoplasts indicate that a 134 bp fragment (-194 to -60 containing the ABRE) fused to a truncated cauliflower mosaic virus promoter (35S) is sufficient to confer ABA-responsiveness upon the GUS reporter gene. Gel retardation experiments indicate that nuclear proteins from tissues in which the rab28 gene is expressed can interact specifically with this 134 bp DNA fragment. Nuclear protein extracts from embryo and water-stressed leaves generate specific complexes of different electrophoretic mobility which are stable in the presence of detergent and high salt. However, by DMS footprinting the same guanine-specific contacts with the ABRE in both the embryo and leaf binding activities were detected. These results indicate that the rab28 promoter sequence CCACGTGG is a functional ABA-responsive element, and suggest that distinct regulatory factors with apparent similar affinity for the ABRE sequence may be involved in the hormone action during embryo development and in vegetative tissues subjected to osmotic stress.

Bladder inflammatory transcriptome in response to tachykinins: Neurokinin 1 receptor-dependent genes and transcription regulatory elements

PubMed Central

Saban, Ricardo; Simpson, Cindy; Vadigepalli, Rajanikanth; Memet, Sylvie; Dozmorov, Igor; Saban, Marcia R

2007-01-01

Background Tachykinins (TK), such as substance P, and their neurokinin receptors which are ubiquitously expressed in the human urinary tract, represent an endogenous system regulating bladder inflammatory, immune responses, and visceral hypersensitivity. Increasing evidence correlates alterations in the TK system with urinary tract diseases such as neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis. However, despite promising effects in animal models, there seems to be no published clinical study showing that NK-receptor antagonists are an effective treatment of pain in general or urinary tract disorders, such as detrusor overactivity. In order to search for therapeutic targets that could block the tachykinin system, we set forth to determine the regulatory network downstream of NK1 receptor activation. First, NK1R-dependent transcripts were determined and used to query known databases for their respective transcription regulatory elements (TREs). Methods An expression analysis was performed using urinary bladders isolated from sensitized wild type (WT) and NK1R-/- mice that were stimulated with saline, LPS, or antigen to provoke inflammation. Based on cDNA array results, NK1R-dependent genes were selected. PAINT software was used to query TRANSFAC database and to retrieve upstream TREs that were confirmed by electrophoretic mobility shift assays. Results The regulatory network of TREs driving NK1R-dependent genes presented cRel in a central position driving 22% of all genes, followed by AP-1, NF-kappaB, v-Myb, CRE-BP1/c-Jun, USF, Pax-6, Efr-1, Egr-3, and AREB6. A comparison between NK1R-dependent and NK1R-independent genes revealed Nkx-2.5 as a unique discriminator. In the presence of NK1R, Nkx2-5 _01 was significantly correlated with 36 transcripts which included several candidates for mediating bladder development (FGF) and inflammation (PAR-3, IL-1R, IL-6, α-NGF, TSP2). In the absence of NK1R, the matrix Nkx2

A novel E2 box-GATA element modulates Cdc6 transcription during human cells polyploidization

PubMed Central

Vilaboa, Nuria; Bermejo, Rodrigo; Martinez, Pilar; Bornstein, Rafael; Calés, Carmela

2004-01-01

Cdc6 is a key regulator of the strict alternation of S and M phases during the mitotic cell cycle. In mammalian and plant cells that physiologically become polyploid, cdc6 is transcriptionally and post-translationally regulated. We have recently reported that Cdc6 levels are maintained in megakaryoblastic HEL cells, but severely downregulated by ectopic expression of transcriptional repressor Drosophila melanogaster escargot. Here, we show that cdc6 promoter activity is upregulated during megakaryocytic differentiation of HEL endoreplicating cells, and that Escargot interferes with such activation. Transactivation experiments showed that a 1.7 kb region located at 2800 upstream cdc6 transcription initiation site behaved as a potent enhancer in endoreplicating cells only. This activity was mainly dependent on a novel cis-regulatory element composed by an E2 box overlapping a GATA motif. Ectopic Escargot could bind this regulatory element in vitro and endogenous GATA-1 and E2A formed specific complexes in megakaryoblastic cells as well as in primary megakaryocytes. Chromatin Immunoprecipitation analysis revealed that both transcription factors were occupying the E2 box/GATA site in vivo. Altogether, these data suggest that cdc6 expression could be actively maintained during megakaryocytic differentiation through transcriptional mechanisms involving specific cis- and trans-regulatory elements. PMID:15590906

Fibroblast growth factor and cyclic AMP (cAMP) synergistically activate gene expression at a cAMP response element.

PubMed Central

Tan, Y; Low, K G; Boccia, C; Grossman, J; Comb, M J

1994-01-01

Growth factors and cyclic AMP (cAMP) are known to activate distinct intracellular signaling pathways. Fibroblast growth factor (FGF) activates ras-dependent kinase cascades, resulting in the activation of MAP kinases, whereas cAMP activates protein kinase A. In this study, we report that growth factors and cAMP act synergistically to stimulate proenkephalin gene expression. Positive synergy between growth factor- and cAMP-activated signaling pathways on gene expression has not been previously reported, and we suggest that these synergistic interactions represent a useful model for analyzing interactions between these pathways. Transfection and mutational studies indicate that both FGF-dependent gene activation and cAMP-dependent gene activation require cAMP response element 2 (CRE-2), a previously characterized cAMP-dependent regulatory element. Furthermore, multiple copies of this element are sufficient to confer FGF regulation upon a minimal promoter, indicating that FGF and cAMP signaling converge upon transcription factors acting at CRE-2. Among many different ATF/AP-1 factors tested, two factors, ATF-3 and c-Jun, stimulate proenkephalin transcription in an FGF- or Ras-dependent fashion. Finally, we show that ATF-3 and c-Jun form heterodimeric complexes in SK-N-MC cells and that the levels of both proteins are increased in response to FGF but not cAMP. Together, these results indicate that growth factor- and cAMP-dependent signaling pathways converge at CRE-2 to synergistically stimulate gene expression and that ATF-3 and c-Jun regulate proenkephalin transcription in response to both growth factor- and cAMP-dependent intracellular signaling pathways. Images PMID:7935470

n-Alkane and clofibrate, a peroxisome proliferator, activate transcription of ALK2 gene encoding cytochrome P450alk2 through distinct cis-acting promoter elements in Candida maltosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kogure, Takahisa; Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Higashijima 265-1, Niitsu, Niigata 956-8603; Takagi, Masamichi

2005-04-01

The ALK2 gene, encoding one of the n-alkane-hydroxylating cytochromes P450 in Candida maltosa, is induced by n-alkanes and a peroxisome proliferator, clofibrate. Deletion analysis of this gene's promoter revealed two cis-acting elements-an n-alkane-responsive element (ARE2) and a clofibrate-responsive element (CRE2)-that partly overlap in sequence but have distinct functions. ARE2-mediated activation responded to n-alkanes but not to clofibrate and was repressed by glucose. CRE2-mediated activation responded to polyunsaturated fatty acids and steroid hormones as well as to peroxisome proliferators but not to n-alkanes, and it was not repressed by glucose. Both elements mediated activation by oleic acid. Mutational analysis demonstrated thatmore » three CCG sequences in CRE2 were critical to the activation by clofibrate as well as to the in vitro binding of a specific protein to this element. These findings suggest that ALK2 is induced by peroxisome proliferators and steroid hormones through a specific CRE2-mediated regulatory mechanism.« less

The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers

PubMed Central

2016-01-01

Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains. PMID:26252467

Regulation of P-element transposase activity in Drosophila melanogaster by hobo transgenes that contain KP elements.

PubMed Central

Simmons, Michael J; Haley, Kevin J; Grimes, Craig D; Raymond, John D; Fong, Joseph C L

2002-01-01

Fusions between the Drosophila hsp70 promoter and three different incomplete P elements, KP, SP, and BP1, were inserted into the Drosophila genome by means of hobo transformation vectors and the resulting transgenic stocks were tested for repression of P-element transposase activity. Only the H(hsp/KP) transgenes repressed transposase activity, and the degree of repression was comparable to that of a naturally occurring KP element. The KP transgenes repressed transposase activity both with and without heat-shock treatments. Both the KP element and H(hsp/KP) transgenes repressed the transposase activity encoded by the modified P element in the P(ry(+), Delta2-3)99B transgene more effectively than that encoded by the complete P element in the H(hsp/CP)2 transgene even though the P(ry(+), Delta2-3)99B transgene was the stronger transposase source. Repression of both transposase sources appeared to be due to a zygotic effect of the KP element or transgene. There was no evidence for repression by a strictly maternal effect; nor was there any evidence for enhancement of KP repression by the joint maternal transmission of H(hsp/KP) and H(hsp/CP) transgenes. These results are consistent with the idea that KP-mediated repression of P-element activity involves a KP-repressor polypeptide that is not maternally transmitted and that KP-mediated repression is not strengthened by the 66-kD repressor produced by complete P elements through alternate splicing of their RNA. PMID:12019235

Regulatory analysis of the mouse Hoxb3 gene: multiple elements work in concert to direct temporal and spatial patterns of expression.

PubMed

Kwan, C T; Tsang, S L; Krumlauf, R; Sham, M H

2001-04-01

The expression pattern of the mouse Hoxb3 gene is exceptionally complex and dynamic compared with that of other members of the Hoxb cluster. There are multiple types of transcripts for Hoxb3 gene, and the anterior boundaries of its expression vary at different stages of development. Two enhancers flanking Hoxb3 on the 3' and 5' sides regulate Hoxb2 and Hoxb4, respectively, and these control regions define the two ends of a 28-kb interval in and around the Hoxb3 locus. To assay the regulatory potential of DNA fragments in this interval we have used transgenic analysis with a lacZ reporter gene to locate cis-elements for directing the dynamic patterns of Hoxb3 expression. Our detailed analysis has identified four new and widely spaced cis-acting regulatory regions that can together account for major aspects of the Hoxb3 expression pattern. Elements Ib, IIIa, and IVb control gene expression in neural and mesodermal tissues; element Va controls mesoderm-specific gene expression. The most anterior neural expression domain of Hoxb3 is controlled by an r5 enhancer (element IVa); element IIIa directs reporter expression in the anterior spinal cord and hindbrain up to r6, and the region A enhancer (in element I) mediates posterior neural expression. Hence, the regulation of segmental expression of Hoxb3 in the hindbrain is different from that of Hoxa3, as two separate enhancer elements contribute to expression in r5 and r6. The mesoderm-specific element (Va) directs reporter expression to prevertebra C1 at 12.5 dpc, which is the anterior limit of paraxial mesoderm expression for Hoxb3. When tested in combinations, these cis-elements appear to work as modules in an additive manner to recapitulate the major endogenous expression patterns of Hoxb3 during embryogenesis. Together our study shows that multiple control elements direct reporter gene expression in diverse tissue-, temporal-, and spatially restricted subset of the endogenous Hoxb3 expression domains and work in

Antiviral Activity of Porcine Interferon Regulatory Factor 1 against Swine Viruses in Cell Culture.

PubMed

Li, Yongtao; Chang, Hongtao; Yang, Xia; Zhao, Yongxiang; Chen, Lu; Wang, Xinwei; Liu, Hongying; Wang, Chuanqing; Zhao, Jun

2015-11-17

Interferon regulatory factor 1 (IRF1), as an important transcription factor, is abundantly induced upon virus infections and participates in host antiviral immune responses. However, the roles of porcine IRF1 (poIRF1) in host antiviral defense remain poorly understood. In this study, we determined that poIRF1 was upregulated upon infection with viruses and distributed in nucleus in porcine PK-15 cells. Subsequently, we tested the antiviral activities of poIRF1 against several swine viruses in cells. Overexpression of poIRF1 can efficiently suppress the replication of viruses, and knockdown of poIRF1 promotes moderately viral replication. Interestingly, overexpression of poIRF1 enhances dsRNA-induced IFN-β and IFN-stimulated response element (ISRE) promoter activation, whereas knockdown of poIRF1 cannot significantly affect the activation of IFN-β promoter induced by RNA viruses. This study suggests that poIRF1 plays a significant role in cellular antiviral response against swine viruses, but might be dispensable for IFN-β induction triggered by RNA viruses in PK-15 cells. Given these results, poIRF1 plays potential roles in cellular antiviral responses against swine viruses.

Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus

PubMed Central

Sundaram, Vasavi; Choudhary, Mayank N. K.; Pehrsson, Erica; Xing, Xiaoyun; Fiore, Christopher; Pandey, Manishi; Maricque, Brett; Udawatta, Methma; Ngo, Duc; Chen, Yujie; Paguntalan, Asia; Ray, Tammy; Hughes, Ava; Cohen, Barak A.; Wang, Ting

2017-01-01

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome. PMID:28348391

76 FR 18165 - Request for Public Comments Concerning Regulatory Cooperation Activities That Would Help...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-04-01

... DEPARTMENT OF COMMERCE International Trade Administration Request for Public Comments Concerning Regulatory Cooperation Activities That Would Help Eliminate or Reduce Unnecessary Regulatory Divergences in North America That Disrupt U.S. Exports AGENCY: International Trade Administration, Commerce. ACTION...

Identification and characterization of regulatory elements in the promoter of ACVR1, the gene mutated in Fibrodysplasia Ossificans Progressiva

PubMed Central

2013-01-01

Background The ACVR1 gene encodes a type I receptor for bone morphogenetic proteins (BMPs). Mutations in the ACVR1 gene are associated with Fibrodysplasia Ossificans Progressiva (FOP), a rare and extremely disabling disorder characterized by congenital malformation of the great toes and progressive heterotopic endochondral ossification in muscles and other non-skeletal tissues. Several aspects of FOP pathophysiology are still poorly understood, including mechanisms regulating ACVR1 expression. This work aimed to identify regulatory elements that control ACVR1 gene transcription. Methods and results We first characterized the structure and composition of human ACVR1 gene transcripts by identifying the transcription start site, and then characterized a 2.9 kb upstream region. This region showed strong activating activity when tested by reporter gene assays in transfected cells. We identified specific elements within the 2.9 kb region that are important for transcription factor binding using deletion constructs, co-transfection experiments with plasmids expressing selected transcription factors, site-directed mutagenesis of consensus binding-site sequences, and by protein/DNA binding assays. We also characterized a GC-rich minimal promoter region containing binding sites for the Sp1 transcription factor. Conclusions Our results showed that several transcription factors such as Egr-1, Egr-2, ZBTB7A/LRF, and Hey1, regulate the ACVR1 promoter by binding to the -762/-308 region, which is essential to confer maximal transcriptional activity. The Sp1 transcription factor acts at the most proximal promoter segment upstream of the transcription start site. We observed significant differences in different cell types suggesting tissue specificity of transcriptional regulation. These findings provide novel insights into the molecular mechanisms that regulate expression of the ACVR1 gene and that could be targets of new strategies for future therapeutic treatments. PMID:24047559

Mouse regulatory DNA landscapes reveal global principles of cis-regulatory evolution.

PubMed

Vierstra, Jeff; Rynes, Eric; Sandstrom, Richard; Zhang, Miaohua; Canfield, Theresa; Hansen, R Scott; Stehling-Sun, Sandra; Sabo, Peter J; Byron, Rachel; Humbert, Richard; Thurman, Robert E; Johnson, Audra K; Vong, Shinny; Lee, Kristen; Bates, Daniel; Neri, Fidencio; Diegel, Morgan; Giste, Erika; Haugen, Eric; Dunn, Douglas; Wilken, Matthew S; Josefowicz, Steven; Samstein, Robert; Chang, Kai-Hsin; Eichler, Evan E; De Bruijn, Marella; Reh, Thomas A; Skoultchi, Arthur; Rudensky, Alexander; Orkin, Stuart H; Papayannopoulou, Thalia; Treuting, Piper M; Selleri, Licia; Kaul, Rajinder; Groudine, Mark; Bender, M A; Stamatoyannopoulos, John A

2014-11-21

To study the evolutionary dynamics of regulatory DNA, we mapped >1.3 million deoxyribonuclease I-hypersensitive sites (DHSs) in 45 mouse cell and tissue types, and systematically compared these with human DHS maps from orthologous compartments. We found that the mouse and human genomes have undergone extensive cis-regulatory rewiring that combines branch-specific evolutionary innovation and loss with widespread repurposing of conserved DHSs to alternative cell fates, and that this process is mediated by turnover of transcription factor (TF) recognition elements. Despite pervasive evolutionary remodeling of the location and content of individual cis-regulatory regions, within orthologous mouse and human cell types the global fraction of regulatory DNA bases encoding recognition sites for each TF has been strictly conserved. Our findings provide new insights into the evolutionary forces shaping mammalian regulatory DNA landscapes. Copyright © 2014, American Association for the Advancement of Science.

Elements of active vibration control for rotating machinery

NASA Technical Reports Server (NTRS)

Ulbrich, Heinz

1990-01-01

The success or failure of active vibration control is determined by the availability of suitable actuators, modeling of the entire system including all active elements, positioning of the actuators and sensors, and implementation of problem-adapted control concepts. All of these topics are outlined and their special problems are discussed in detail. Special attention is given to efficient modeling of systems, especially for considering the active elements. Finally, design methods for and the application of active vibration control on rotating machinery are demonstrated by several real applications.

A 3.0-kb deletion including an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene in an individual with the Bm phenotype.

PubMed

Sano, R; Kuboya, E; Nakajima, T; Takahashi, Y; Takahashi, K; Kubo, R; Kominato, Y; Takeshita, H; Yamao, H; Kishida, T; Isa, K; Ogasawara, K; Uchikawa, M

2015-04-01

We developed a sequence-specific primer PCR (SSP-PCR) for detection of a 5.8-kb deletion (B(m) 5.8) involving an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene. Using this SSP-PCR, we performed genetic analysis of 382 individuals with Bm or ABm. The 5.8-kb deletion was found in 380 individuals, and disruption of the GATA motif in the regulatory element was found in one individual. Furthermore, a novel 3.0-kb deletion involving the element (B(m) 3.0) was demonstrated in the remaining individual. Comparisons of single-nucleotide polymorphisms and microsatellites in intron 1 between B(m) 5.8 and B(m) 3.0 suggested that these deletions occurred independently. © 2014 International Society of Blood Transfusion.

The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization.

PubMed

Romero-López, Cristina; Barroso-delJesus, Alicia; Berzal-Herranz, Alfredo

2017-02-24

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3'UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2'-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome.

The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization

PubMed Central

Romero-López, Cristina; Barroso-delJesus, Alicia; Berzal-Herranz, Alfredo

2017-01-01

The RNA genome of the hepatitis C virus (HCV) establishes a network of long-distance RNA-RNA interactions that direct the progression of the infective cycle. This work shows that the dimerization of the viral genome, which is initiated at the dimer linkage sequence (DLS) within the 3′UTR, is promoted by the CRE region, while the IRES is a negative regulatory partner. Using differential 2′-acylation probing (SHAPE-dif) and molecular interference (HMX) technologies, the CRE activity was found to mainly lie in the critical 5BSL3.2 domain, while the IRES-mediated effect is dependent upon conserved residues within the essential structural elements JIIIabc, JIIIef and PK2. These findings support the idea that, along with the DLS motif, the IRES and CRE are needed to control HCV genome dimerization. They also provide evidences of a novel function for these elements as chaperone-like partners that fine-tune the architecture of distant RNA domains within the HCV genome. PMID:28233845

Identification of a cis-Regulatory Element Involved in Phytochrome Down-Regulated Expression of the Pea Small GTPase Gene pra21

PubMed Central

Inaba, Takehito; Nagano, Yukio; Sakakibara, Toshihiro; Sasaki, Yukiko

1999-01-01

The pra2 gene encodes a pea (Pisum sativum) small GTPase belonging to the YPT/rab family, and its expression is down-regulated by light, mediated by phytochrome. We have isolated and characterized a genomic clone of this gene and constructed a fusion DNA of its 5′-upstream region in front of the gene for firefly luciferase. Using this construct in a transient assay, we determined a pra2 cis-regulatory region sufficient to direct the light down-regulation of the luciferase reporter gene. Both 5′- and internal deletion analyses revealed that the 93-bp sequence between −734 and −642 from the transcriptional start site was important for phytochrome down-regulation. Gain-of-function analysis showed that this 93-bp region could confer light down-regulation when fused to the cauliflower mosaic virus 35S promoter. Furthermore, linker-scanning analysis showed that a 12-bp sequence within the 93-bp region mediated phytochrome down-regulation. Gel-retardation analysis showed the presence of a nuclear factor that was specifically bound to the 12-bp sequence in vitro. These results indicate that this element is a cis-regulatory element involved in phytochrome down-regulated expression. PMID:10364400

Mammalian genomic regulatory regions predicted by utilizing human genomics, transcriptomics, and epigenetics data

PubMed Central

Nguyen, Quan H; Tellam, Ross L; Naval-Sanchez, Marina; Porto-Neto, Laercio R; Barendse, William; Reverter, Antonio; Hayes, Benjamin; Kijas, James; Dalrymple, Brian P

2018-01-01

Abstract Genome sequences for hundreds of mammalian species are available, but an understanding of their genomic regulatory regions, which control gene expression, is only beginning. A comprehensive prediction of potential active regulatory regions is necessary to functionally study the roles of the majority of genomic variants in evolution, domestication, and animal production. We developed a computational method to predict regulatory DNA sequences (promoters, enhancers, and transcription factor binding sites) in production animals (cows and pigs) and extended its broad applicability to other mammals. The method utilizes human regulatory features identified from thousands of tissues, cell lines, and experimental assays to find homologous regions that are conserved in sequences and genome organization and are enriched for regulatory elements in the genome sequences of other mammalian species. Importantly, we developed a filtering strategy, including a machine learning classification method, to utilize a very small number of species-specific experimental datasets available to select for the likely active regulatory regions. The method finds the optimal combination of sensitivity and accuracy to unbiasedly predict regulatory regions in mammalian species. Furthermore, we demonstrated the utility of the predicted regulatory datasets in cattle for prioritizing variants associated with multiple production and climate change adaptation traits and identifying potential genome editing targets. PMID:29618048

Mammalian genomic regulatory regions predicted by utilizing human genomics, transcriptomics, and epigenetics data.

PubMed

Nguyen, Quan H; Tellam, Ross L; Naval-Sanchez, Marina; Porto-Neto, Laercio R; Barendse, William; Reverter, Antonio; Hayes, Benjamin; Kijas, James; Dalrymple, Brian P

2018-03-01

Genome sequences for hundreds of mammalian species are available, but an understanding of their genomic regulatory regions, which control gene expression, is only beginning. A comprehensive prediction of potential active regulatory regions is necessary to functionally study the roles of the majority of genomic variants in evolution, domestication, and animal production. We developed a computational method to predict regulatory DNA sequences (promoters, enhancers, and transcription factor binding sites) in production animals (cows and pigs) and extended its broad applicability to other mammals. The method utilizes human regulatory features identified from thousands of tissues, cell lines, and experimental assays to find homologous regions that are conserved in sequences and genome organization and are enriched for regulatory elements in the genome sequences of other mammalian species. Importantly, we developed a filtering strategy, including a machine learning classification method, to utilize a very small number of species-specific experimental datasets available to select for the likely active regulatory regions. The method finds the optimal combination of sensitivity and accuracy to unbiasedly predict regulatory regions in mammalian species. Furthermore, we demonstrated the utility of the predicted regulatory datasets in cattle for prioritizing variants associated with multiple production and climate change adaptation traits and identifying potential genome editing targets.

Regulatory principles governing Salmonella and Yersinia virulence

PubMed Central

Erhardt, Marc; Dersch, Petra

2015-01-01

Enteric pathogens such as Salmonella and Yersinia evolved numerous strategies to survive and proliferate in different environmental reservoirs and mammalian hosts. Deciphering common and pathogen-specific principles for how these bacteria adjust and coordinate spatiotemporal expression of virulence determinants, stress adaptation, and metabolic functions is fundamental to understand microbial pathogenesis. In order to manage sudden environmental changes, attacks by the host immune systems and microbial competition, the pathogens employ a plethora of transcriptional and post-transcriptional control elements, including transcription factors, sensory and regulatory RNAs, RNAses, and proteases, to fine-tune and control complex gene regulatory networks. Many of the contributing global regulators and the molecular mechanisms of regulation are frequently conserved between Yersinia and Salmonella. However, the interplay, arrangement, and composition of the control elements vary between these closely related enteric pathogens, which generate phenotypic differences leading to distinct pathogenic properties. In this overview we present common and different regulatory networks used by Salmonella and Yersinia to coordinate the expression of crucial motility, cell adhesion and invasion determinants, immune defense strategies, and metabolic adaptation processes. We highlight evolutionary changes of the gene regulatory circuits that result in different properties of the regulatory elements and how this influences the overall outcome of the infection process. PMID:26441883

Hypothalamic AMP-activated protein kinase mediates counter-regulatory responses to hypoglycaemia in rats.

PubMed

Han, S-M; Namkoong, C; Jang, P G; Park, I S; Hong, S W; Katakami, H; Chun, S; Kim, S W; Park, J-Y; Lee, K-U; Kim, M-S

2005-10-01

Appropriate counter-regulatory hormonal responses are essential for recovery from hypoglycaemia. Although the hypothalamus is known to be involved in these responses, the molecular mechanisms have not been fully elucidated. AMP-activated protein kinase (AMPK) functions as a cellular energy sensor, being activated during energy depletion. As AMPK is expressed in the hypothalamus, an important site of neuroendocrine regulation, the present study was undertaken to determine whether hypothalamic AMPK mediates counter-regulatory responses to hypoglycaemia. Hypoglycaemia was induced by i.p. injection of regular insulin (6 U/kg) in Sprague-Dawley rats. Hypothalamic AMPK phosphorylation and activities were determined 1 h after i.p. insulin injection. To investigate the role of hypothalamic AMPK activation in mediating counter-regulatory responses, an AMPK inhibitor, compound C, was pre-administered intracerebroventricularly (i.c.v.) or dominant-negative (DN)-AMPK was overexpressed in the hypothalamus before induction of hypoglycaemia. Insulin-induced hypoglycaemia increased hypothalamic AMPK phosphorylation and alpha2-AMPK activities in rats. The change was significant in the arcuate nucleus/ventromedial hypothalamus (ARC/VMH) and paraventricular nuclei (PVN). Prior i.c.v. administration of compound C attenuated hypoglycaemia-induced increases in plasma concentrations of corticosterone, glucagon and catecholamines, resulting in severe and prolonged hypoglycaemia. ARC/VMH DN-AMPK overexpression impaired early counter-regulation, as evidenced by reduced glucagon and catecholamine responses. In contrast, PVN DN-AMPK overexpression attenuated late counter-regulation and corticosterone responses. Systemic hypoglycaemia causes hypothalamic AMPK activation, which is important for counter-regulatory hormonal responses. Our data indicate that hypothalamic AMPK acts as a fuel gauge, sensing the whole-body energy state and regulating not only energy homeostasis but also

Moving through the Stressed Genome: Emerging Regulatory Roles for Transposons in Plant Stress Response

PubMed Central

Negi, Pooja; Rai, Archana N.; Suprasanna, Penna

2016-01-01

The recognition of a positive correlation between organism genome size with its transposable element (TE) content, represents a key discovery of the field of genome biology. Considerable evidence accumulated since then suggests the involvement of TEs in genome structure, evolution and function. The global genome reorganization brought about by transposon activity might play an adaptive/regulatory role in the host response to environmental challenges, reminiscent of McClintock's original ‘Controlling Element’ hypothesis. This regulatory aspect of TEs is also garnering support in light of the recent evidences, which project TEs as “distributed genomic control modules.” According to this view, TEs are capable of actively reprogramming host genes circuits and ultimately fine-tuning the host response to specific environmental stimuli. Moreover, the stress-induced changes in epigenetic status of TE activity may allow TEs to propagate their stress responsive elements to host genes; the resulting genome fluidity can permit phenotypic plasticity and adaptation to stress. Given their predominating presence in the plant genomes, nested organization in the genic regions and potential regulatory role in stress response, TEs hold unexplored potential for crop improvement programs. This review intends to present the current information about the roles played by TEs in plant genome organization, evolution, and function and highlight the regulatory mechanisms in plant stress responses. We will also briefly discuss the connection between TE activity, host epigenetic response and phenotypic plasticity as a critical link for traversing the translational bridge from a purely basic study of TEs, to the applied field of stress adaptation and crop improvement. PMID:27777577

A statistical method for measuring activation of gene regulatory networks.

PubMed

Esteves, Gustavo H; Reis, Luiz F L

2018-06-13

Gene expression data analysis is of great importance for modern molecular biology, given our ability to measure the expression profiles of thousands of genes and enabling studies rooted in systems biology. In this work, we propose a simple statistical model for the activation measuring of gene regulatory networks, instead of the traditional gene co-expression networks. We present the mathematical construction of a statistical procedure for testing hypothesis regarding gene regulatory network activation. The real probability distribution for the test statistic is evaluated by a permutation based study. To illustrate the functionality of the proposed methodology, we also present a simple example based on a small hypothetical network and the activation measuring of two KEGG networks, both based on gene expression data collected from gastric and esophageal samples. The two KEGG networks were also analyzed for a public database, available through NCBI-GEO, presented as Supplementary Material. This method was implemented in an R package that is available at the BioConductor project website under the name maigesPack.

Modulation of tissue repair by regeneration enhancer elements.

PubMed

Kang, Junsu; Hu, Jianxin; Karra, Ravi; Dickson, Amy L; Tornini, Valerie A; Nachtrab, Gregory; Gemberling, Matthew; Goldman, Joseph A; Black, Brian L; Poss, Kenneth D

2016-04-14

How tissue regeneration programs are triggered by injury has received limited research attention. Here we investigate the existence of enhancer regulatory elements that are activated in regenerating tissue. Transcriptomic analyses reveal that leptin b (lepb) is highly induced in regenerating hearts and fins of zebrafish. Epigenetic profiling identified a short DNA sequence element upstream and distal to lepb that acquires open chromatin marks during regeneration and enables injury-dependent expression from minimal promoters. This element could activate expression in injured neonatal mouse tissues and was divisible into tissue-specific modules sufficient for expression in regenerating zebrafish fins or hearts. Simple enhancer-effector transgenes employing lepb-linked sequences upstream of pro- or anti-regenerative factors controlled the efficacy of regeneration in zebrafish. Our findings provide evidence for 'tissue regeneration enhancer elements' (TREEs) that trigger gene expression in injury sites and can be engineered to modulate the regenerative potential of vertebrate organs.

Genome-Wide Identification of Regulatory Sequences Undergoing Accelerated Evolution in the Human Genome

PubMed Central

Dong, Xinran; Wang, Xiao; Zhang, Feng; Tian, Weidong

2016-01-01

Accelerated evolution of regulatory sequence can alter the expression pattern of target genes, and cause phenotypic changes. In this study, we used DNase I hypersensitive sites (DHSs) to annotate putative regulatory sequences in the human genome, and conducted a genome-wide analysis of the effects of accelerated evolution on regulatory sequences. Working under the assumption that local ancient repeat elements of DHSs are under neutral evolution, we discovered that ∼0.44% of DHSs are under accelerated evolution (ace-DHSs). We found that ace-DHSs tend to be more active than background DHSs, and are strongly associated with epigenetic marks of active transcription. The target genes of ace-DHSs are significantly enriched in neuron-related functions, and their expression levels are positively selected in the human brain. Thus, these lines of evidences strongly suggest that accelerated evolution on regulatory sequences plays important role in the evolution of human-specific phenotypes. PMID:27401230

Transposable elements in Drosophila.

PubMed

McCullers, Tabitha J; Steiniger, Mindy

2017-01-01

Transposable elements (TEs) are mobile genetic elements that can mobilize within host genomes. As TEs comprise more than 40% of the human genome and are linked to numerous diseases, understanding their mechanisms of mobilization and regulation is important. Drosophila melanogaster is an ideal model organism for the study of eukaryotic TEs as its genome contains a diverse array of active TEs. TEs universally impact host genome size via transposition and deletion events, but may also adopt unique functional roles in host organisms. There are 2 main classes of TEs: DNA transposons and retrotransposons. These classes are further divided into subgroups of TEs with unique structural and functional characteristics, demonstrating the significant variability among these elements. Despite this variability, D. melanogaster and other eukaryotic organisms utilize conserved mechanisms to regulate TEs. This review focuses on the transposition mechanisms and regulatory pathways of TEs, and their functional roles in D. melanogaster .

Innate immune activity conditions the effect of regulatory variants upon monocyte gene expression.

PubMed

Fairfax, Benjamin P; Humburg, Peter; Makino, Seiko; Naranbhai, Vivek; Wong, Daniel; Lau, Evelyn; Jostins, Luke; Plant, Katharine; Andrews, Robert; McGee, Chris; Knight, Julian C

2014-03-07

To systematically investigate the impact of immune stimulation upon regulatory variant activity, we exposed primary monocytes from 432 healthy Europeans to interferon-γ (IFN-γ) or differing durations of lipopolysaccharide and mapped expression quantitative trait loci (eQTLs). More than half of cis-eQTLs identified, involving hundreds of genes and associated pathways, are detected specifically in stimulated monocytes. Induced innate immune activity reveals multiple master regulatory trans-eQTLs including the major histocompatibility complex (MHC), coding variants altering enzyme and receptor function, an IFN-β cytokine network showing temporal specificity, and an interferon regulatory factor 2 (IRF2) transcription factor-modulated network. Induced eQTL are significantly enriched for genome-wide association study loci, identifying context-specific associations to putative causal genes including CARD9, ATM, and IRF8. Thus, applying pathophysiologically relevant immune stimuli assists resolution of functional genetic variants.

Computational Identification of Tissue-Specific Splicing Regulatory Elements in Human Genes from RNA-Seq Data.

PubMed

Badr, Eman; ElHefnawi, Mahmoud; Heath, Lenwood S

2016-01-01

Alternative splicing is a vital process for regulating gene expression and promoting proteomic diversity. It plays a key role in tissue-specific expressed genes. This specificity is mainly regulated by splicing factors that bind to specific sequences called splicing regulatory elements (SREs). Here, we report a genome-wide analysis to study alternative splicing on multiple tissues, including brain, heart, liver, and muscle. We propose a pipeline to identify differential exons across tissues and hence tissue-specific SREs. In our pipeline, we utilize the DEXSeq package along with our previously reported algorithms. Utilizing the publicly available RNA-Seq data set from the Human BodyMap project, we identified 28,100 differentially used exons across the four tissues. We identified tissue-specific exonic splicing enhancers that overlap with various previously published experimental and computational databases. A complicated exonic enhancer regulatory network was revealed, where multiple exonic enhancers were found across multiple tissues while some were found only in specific tissues. Putative combinatorial exonic enhancers and silencers were discovered as well, which may be responsible for exon inclusion or exclusion across tissues. Some of the exonic enhancers are found to be co-occurring with multiple exonic silencers and vice versa, which demonstrates a complicated relationship between tissue-specific exonic enhancers and silencers.

DNaseI Hypersensitivity and Ultraconservation Reveal Novel, Interdependent Long-Range Enhancers at the Complex Pax6 Cis-Regulatory Region

PubMed Central

McBride, David J.; Buckle, Adam; van Heyningen, Veronica; Kleinjan, Dirk A.

2011-01-01

The PAX6 gene plays a crucial role in development of the eye, brain, olfactory system and endocrine pancreas. Consistent with its pleiotropic role the gene exhibits a complex developmental expression pattern which is subject to strict spatial, temporal and quantitative regulation. Control of expression depends on a large array of cis-elements residing in an extended genomic domain around the coding region of the gene. The minimal essential region required for proper regulation of this complex locus has been defined through analysis of human aniridia-associated breakpoints and YAC transgenic rescue studies of the mouse smalleye mutant. We have carried out a systematic DNase I hypersensitive site (HS) analysis across 200 kb of this critical region of mouse chromosome 2E3 to identify putative regulatory elements. Mapping the identified HSs onto a percent identity plot (PIP) shows many HSs correspond to recognisable genomic features such as evolutionarily conserved sequences, CpG islands and retrotransposon derived repeats. We then focussed on a region previously shown to contain essential long range cis-regulatory information, the Pax6 downstream regulatory region (DRR), allowing comparison of mouse HS data with previous human HS data for this region. Reporter transgenic mice for two of the HS sites, HS5 and HS6, show that they function as tissue specific regulatory elements. In addition we have characterised enhancer activity of an ultra-conserved cis-regulatory region located near Pax6, termed E60. All three cis-elements exhibit multiple spatio-temporal activities in the embryo that overlap between themselves and other elements in the locus. Using a deletion set of YAC reporter transgenic mice we demonstrate functional interdependence of the elements. Finally, we use the HS6 enhancer as a marker for the migration of precerebellar neuro-epithelium cells to the hindbrain precerebellar nuclei along the posterior and anterior extramural streams allowing visualisation of

Meeting physical activity recommendations: self-regulatory efficacy characterizes differential adherence during arthritis flares.

PubMed

Gyurcsik, Nancy C; Brawley, Lawrence R; Spink, Kevin S; Sessford, James D

2013-02-01

Using social-cognitive theory, we examined whether adults who experienced an arthritis flare and met/did not meet the disease-specific public health recommended dose for physical activity differed in their self-regulatory efficacy beliefs, overall pain, and flare-related factors. Adults with arthritis (N = 56; M(age) = 49.41 ± 11.56 years) participated in this prospective study. Multivariate analysis of variance comparing groups who met or did not meet the recommended dose (n(met) = 24, ≥ 150 minutes/week vs. n(not met) = 32, < 150 min/week) on efficacy, overall pain, and flare-related factors was significant (p < .01; η(partial)² = .28). People meeting the dose had significantly greater self-regulatory efficacy to overcome arthritis barriers (M(met dose) = 7.33 ± 1.95 vs. M(did not meet dose) = 5.74 ± 2.08, η(partial)² = .14) and to schedule/plan (M(met dose) = 7.27 ± 1.80 vs. M(did not meet dose) = 5.72 ± 1.90, η(partial)² = .15). Overall pain and flare-related factors did not differ (ps > .05). During flares, individuals with greater self-regulatory efficacy to manage disease barriers and plan their physical activity were more adherent to disease-specific public health activity recommendations. This study was the first to demonstrate differences in social cognitions that characterize adherence to recommended activity among people challenged by arthritis flares. Findings support the theoretical position that self-regulatory efficacy is related to better adherence in the face of challenging disease-related circumstances. The importance of studying individual characteristics of people who succeed in being active despite such obstacles is stressed.

Proviruses with Long-Term Stable Expression Accumulate in Transcriptionally Active Chromatin Close to the Gene Regulatory Elements: Comparison of ASLV-, HIV- and MLV-Derived Vectors

PubMed Central

Miklík, Dalibor; Šenigl, Filip; Hejnar, Jiří

2018-01-01

Individual groups of retroviruses and retroviral vectors differ in their integration site preference and interaction with the host genome. Hence, immediately after infection genome-wide distribution of integrated proviruses is non-random. During long-term in vitro or persistent in vivo infection, the genomic position and chromatin environment of the provirus affects its transcriptional activity. Thus, a selection of long-term stably expressed proviruses and elimination of proviruses, which have been gradually silenced by epigenetic mechanisms, helps in the identification of genomic compartments permissive for proviral transcription. We compare here the extent and time course of provirus silencing in single cell clones of the K562 human myeloid lymphoblastoma cell line that have been infected with retroviral reporter vectors derived from avian sarcoma/leukosis virus (ASLV), human immunodeficiency virus type 1 (HIV) and murine leukaemia virus (MLV). While MLV proviruses remain transcriptionally active, ASLV proviruses are prone to rapid silencing. The HIV provirus displays gradual silencing only after an extended time period in culture. The analysis of integration sites of long-term stably expressed proviruses shows a strong bias for some genomic features—especially integration close to the transcription start sites of active transcription units. Furthermore, complex analysis of histone modifications enriched at the site of integration points to the accumulation of proviruses of all three groups in gene regulatory segments, particularly close to the enhancer loci. We conclude that the proximity to active regulatory chromatin segments correlates with stable provirus expression in various retroviral species. PMID:29517993

Evidence of reduced recombination rate in human regulatory domains.

PubMed

Liu, Yaping; Sarkar, Abhishek; Kheradpour, Pouya; Ernst, Jason; Kellis, Manolis

2017-10-20

Recombination rate is non-uniformly distributed across the human genome. The variation of recombination rate at both fine and large scales cannot be fully explained by DNA sequences alone. Epigenetic factors, particularly DNA methylation, have recently been proposed to influence the variation in recombination rate. We study the relationship between recombination rate and gene regulatory domains, defined by a gene and its linked control elements. We define these links using expression quantitative trait loci (eQTLs), methylation quantitative trait loci (meQTLs), chromatin conformation from publicly available datasets (Hi-C and ChIA-PET), and correlated activity links that we infer across cell types. Each link type shows a "recombination rate valley" of significantly reduced recombination rate compared to matched control regions. This recombination rate valley is most pronounced for gene regulatory domains of early embryonic development genes, housekeeping genes, and constitutive regulatory elements, which are known to show increased evolutionary constraint across species. Recombination rate valleys show increased DNA methylation, reduced doublestranded break initiation, and increased repair efficiency, specifically in the lineage leading to the germ line. Moreover, by using only the overlap of functional links and DNA methylation in germ cells, we are able to predict the recombination rate with high accuracy. Our results suggest the existence of a recombination rate valley at regulatory domains and provide a potential molecular mechanism to interpret the interplay between genetic and epigenetic variations.

The medical dictionary for regulatory activities (MedDRA).

PubMed

Brown, E G; Wood, L; Wood, S

1999-02-01

The International Conference on Harmonisation has agreed upon the structure and content of the Medical Dictionary for Regulatory Activities (MedDRA) version 2.0 which should become available in the early part of 1999. This medical terminology is intended for use in the pre- and postmarketing phases of the medicines regulatory process, covering diagnoses, symptoms and signs, adverse drug reactions and therapeutic indications, the names and qualitative results of investigations, surgical and medical procedures, and medical/social history. It can be used for recording adverse events and medical history in clinical trials, in the analysis and tabulations of data from these trials and in the expedited submission of safety data to government regulatory authorities, as well as in constructing standard product information and documentation for applications for marketing authorisation. After licensing of a medicine, it may be used in pharmacovigilance and is expected to be the preferred terminology for international electronic regulatory communication. MedDRA is a hierarchical terminology with 5 levels and is multiaxial: terms may exist in more than 1 vertical axis, providing specificity of terms for data entry and flexibility in data retrieval. Terms in MedDRA were derived from several sources including the WHO's adverse reaction terminology (WHO-ART), Coding Symbols for a Thesaurus of Adverse Reaction Terms (COSTART), International Classification of Diseases (ICD) 9 and ICD9-CM. It will be maintained, further developed and distributed by a Maintenance Support Services Organisation (MSSO). It is anticipated that using MedDRA will improve the quality of data captured on databases, support effective analysis by providing clinically relevant groupings of terms and facilitate electronic communication of data, although as a new tool, users will need to invest time in gaining expertise in its use.

A regulatory network to segregate the identity of neuronal subtypes.

PubMed

Lee, Seunghee; Lee, Bora; Joshi, Kaumudi; Pfaff, Samuel L; Lee, Jae W; Lee, Soo-Kyung

2008-06-01

Spinal motor neurons (MNs) and V2 interneurons (V2-INs) are specified by two related LIM-complexes, MN-hexamer and V2-tetramer, respectively. Here we show how multiple parallel and complementary feedback loops are integrated to assign these two cell fates accurately. While MN-hexamer response elements (REs) are specific to MN-hexamer, V2-tetramer-REs can bind both LIM-complexes. In embryonic MNs, however, two factors cooperatively suppress the aberrant activation of V2-tetramer-REs. First, LMO4 blocks V2-tetramer assembly. Second, MN-hexamer induces a repressor, Hb9, which binds V2-tetramer-REs and suppresses their activation. V2-INs use a similar approach; V2-tetramer induces a repressor, Chx10, which binds MN-hexamer-REs and blocks their activation. Thus, our study uncovers a regulatory network to segregate related cell fates, which involves reciprocal feedforward gene regulatory loops.

Structural and functional analysis of mouse Msx1 gene promoter: sequence conservation with human MSX1 promoter points at potential regulatory elements.

PubMed

Gonzalez, S M; Ferland, L H; Robert, B; Abdelhay, E

1998-06-01

Vertebrate Msx genes are related to one of the most divergent homeobox genes of Drosophila, the muscle segment homeobox (msh) gene, and are expressed in a well-defined pattern at sites of tissue interactions. This pattern of expression is conserved in vertebrates as diverse as quail, zebrafish, and mouse in a range of sites including neural crest, appendages, and craniofacial structures. In the present work, we performed structural and functional analyses in order to identify potential cis-acting elements that may be regulating Msx1 gene expression. To this end, a 4.9-kb segment of the 5'-flanking region was sequenced and analyzed for transcription-factor binding sites. Four regions showing a high concentration of these sites were identified. Transfection assays with fragments of regulatory sequences driving the expression of the bacterial lacZ reporter gene showed that a region of 4 kb upstream of the transcription start site contains positive and negative elements responsible for controlling gene expression. Interestingly, a fragment of 130 bp seems to contain the minimal elements necessary for gene expression, as its removal completely abolishes gene expression in cultured cells. These results are reinforced by comparison of this region with the human Msx1 gene promoter, which shows extensive conservation, including many consensus binding sites, suggesting a regulatory role for them.

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

PubMed

Keogh, M C; Chen, D; Schmitt, J F; Dennehy, U; Kakkar, V V; Lemoine, N R

1999-04-01

The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

Transforming Growth Factor β Suppresses Peroxisome Proliferator–Activated Receptor γ Expression via Both SMAD Binding and Novel TGF-β Inhibitory Elements

PubMed Central

Lakshmi, Sowmya P.; Reddy, Aravind T.; Reddy, Raju C.

2017-01-01

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other’s expression, and such PPARγ downregulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here we show that TGF-β induces association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive corepressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. PMID:28100650

Transposable elements in Drosophila

PubMed Central

McCullers, Tabitha J.; Steiniger, Mindy

2017-01-01

ABSTRACT Transposable elements (TEs) are mobile genetic elements that can mobilize within host genomes. As TEs comprise more than 40% of the human genome and are linked to numerous diseases, understanding their mechanisms of mobilization and regulation is important. Drosophila melanogaster is an ideal model organism for the study of eukaryotic TEs as its genome contains a diverse array of active TEs. TEs universally impact host genome size via transposition and deletion events, but may also adopt unique functional roles in host organisms. There are 2 main classes of TEs: DNA transposons and retrotransposons. These classes are further divided into subgroups of TEs with unique structural and functional characteristics, demonstrating the significant variability among these elements. Despite this variability, D. melanogaster and other eukaryotic organisms utilize conserved mechanisms to regulate TEs. This review focuses on the transposition mechanisms and regulatory pathways of TEs, and their functional roles in D. melanogaster. PMID:28580197

Using reporter gene assays to identify cis regulatory differences between humans and chimpanzees.

PubMed

Chabot, Adrien; Shrit, Ralla A; Blekhman, Ran; Gilad, Yoav

2007-08-01

Most phenotypic differences between human and chimpanzee are likely to result from differences in gene regulation, rather than changes to protein-coding regions. To date, however, only a handful of human-chimpanzee nucleotide differences leading to changes in gene regulation have been identified. To hone in on differences in regulatory elements between human and chimpanzee, we focused on 10 genes that were previously found to be differentially expressed between the two species. We then designed reporter gene assays for the putative human and chimpanzee promoters of the 10 genes. Of seven promoters that we found to be active in human liver cell lines, human and chimpanzee promoters had significantly different activity in four cases, three of which recapitulated the gene expression difference seen in the microarray experiment. For these three genes, we were therefore able to demonstrate that a change in cis influences expression differences between humans and chimpanzees. Moreover, using site-directed mutagenesis on one construct, the promoter for the DDA3 gene, we were able to identify three nucleotides that together lead to a cis regulatory difference between the species. High-throughput application of this approach can provide a map of regulatory element differences between humans and our close evolutionary relatives.

Initial deployment of the cardiogenic gene regulatory network in the basal chordate, Ciona intestinalis.

PubMed

Woznica, Arielle; Haeussler, Maximilian; Starobinska, Ella; Jemmett, Jessica; Li, Younan; Mount, David; Davidson, Brad

2012-08-01

The complex, partially redundant gene regulatory architecture underlying vertebrate heart formation has been difficult to characterize. Here, we dissect the primary cardiac gene regulatory network in the invertebrate chordate, Ciona intestinalis. The Ciona heart progenitor lineage is first specified by Fibroblast Growth Factor/Map Kinase (FGF/MapK) activation of the transcription factor Ets1/2 (Ets). Through microarray analysis of sorted heart progenitor cells, we identified the complete set of primary genes upregulated by FGF/Ets shortly after heart progenitor emergence. Combinatorial sequence analysis of these co-regulated genes generated a hypothetical regulatory code consisting of Ets binding sites associated with a specific co-motif, ATTA. Through extensive reporter analysis, we confirmed the functional importance of the ATTA co-motif in primary heart progenitor gene regulation. We then used the Ets/ATTA combination motif to successfully predict a number of additional heart progenitor gene regulatory elements, including an intronic element driving expression of the core conserved cardiac transcription factor, GATAa. This work significantly advances our understanding of the Ciona heart gene network. Furthermore, this work has begun to elucidate the precise regulatory architecture underlying the conserved, primary role of FGF/Ets in chordate heart lineage specification. Copyright © 2012 Elsevier Inc. All rights reserved.

Improvement of levan production in Bacillus amyloliquefaciens through metabolic optimization of regulatory elements.

PubMed

Gu, Yanyan; Zheng, Jiayi; Feng, Jun; Cao, Mingfeng; Gao, Weixia; Quan, Yufen; Dang, Yulei; Wang, Yi; Wang, Shufang; Song, Cunjiang

2017-05-01

Levan is a functional homopolymer of fructose with considerable applications in food, pharmaceutical and cosmetic industries. To improve the levan production in Bacillus amyloliquefaciens, the regulatory elements of sacB (encoding levansucrase) expression and levansucrase secretion were optimized. Four heterologous promoters were evaluated for sacB expression, and the Pgrac promoter led to the highest level for both sacB transcription and levansucrase enzyme activity. The levan production in the corresponding recombinant strain ΔLP-pHTPgrac reached 30.5 g/L, which was 114% higher than that of the control strain NK-ΔLP. In a further step, eight signal peptides were investigated (with Pgrac as the promoter for sacB expression) for their effects on the levansucrase secretion and levan production. The signal peptide yncM was identified as the optimal one, with a secretion efficiency of approximately 90%, and the levan production in the corresponding recombinant strain ΔLP-Y reached 37.4 g/L, which was 161% higher when compared with the control strains NK-ΔLP. Finally, fed-batch fermentation was carried out in 5-L bioreactors for levan production using the recombinant strain ΔLP-Y. A final levan concentration of 102 g/L was achieved, which is very close to the ever reported highest levan production level from the literature. To our best knowledge, this is the first report of the improvement of levan production through metabolic optimization for sacB expression and levansucrase secretion. The results from this study provided essential insights for systematically metabolic engineering of microbial cell factories for enhanced biochemical production.

Estrogens and Progesterone Promote Persistent CCND1 Gene Activation during G1 by Inducing Transcriptional Derepression via c-Jun/c-Fos/Estrogen Receptor (Progesterone Receptor) Complex Assembly to a Distal Regulatory Element and Recruitment of Cyclin D1 to Its Own Gene Promoter

PubMed Central

Cicatiello, Luigi; Addeo, Raffaele; Sasso, Annarita; Altucci, Lucia; Petrizzi, Valeria Belsito; Borgo, Raphaelle; Cancemi, Massimo; Caporali, Simona; Caristi, Silvana; Scafoglio, Claudio; Teti, Diana; Bresciani, Francesco; Perillo, Bruno; Weisz, Alessandro

2004-01-01

Transcriptional activation of the cyclin D1 gene (CCND1) plays a pivotal role in G1-phase progression, which is thereby controlled by multiple regulatory factors, including nuclear receptors (NRs). Appropriate CCND1 gene activity is essential for normal development and physiology of the mammary gland, where it is regulated by ovarian steroids through a mechanism(s) that is not fully elucidated. We report here that CCND1 promoter activation by estrogens in human breast cancer cells is mediated by recruitment of a c-Jun/c-Fos/estrogen receptor α complex to the tetradecanoyl phorbol acetate-responsive element of the gene, together with Oct-1 to a site immediately adjacent. This process coincides with the release from the same DNA region of a transcriptional repressor complex including Yin-Yang 1 (YY1) and histone deacetylase 1 and is sufficient to induce the assembly of the basal transcription machinery on the promoter and to lead to initial cyclin D1 accumulation in the cell. Later on in estrogen stimulation, the cyclin D1/Cdk4 holoenzyme associates with the CCND1 promoter, where E2F and pRb can also be found, contributing to the long-lasting gene enhancement required to drive G1-phase completion. Interestingly, progesterone triggers similar regulatory events through its own NRs, suggesting that the gene regulation cascade described here represents a crossroad for the transcriptional control of G1-phase progression by different classes of NRs. PMID:15282324

Genome-Wide Identification of Regulatory Sequences Undergoing Accelerated Evolution in the Human Genome.

PubMed

Dong, Xinran; Wang, Xiao; Zhang, Feng; Tian, Weidong

2016-10-01

Accelerated evolution of regulatory sequence can alter the expression pattern of target genes, and cause phenotypic changes. In this study, we used DNase I hypersensitive sites (DHSs) to annotate putative regulatory sequences in the human genome, and conducted a genome-wide analysis of the effects of accelerated evolution on regulatory sequences. Working under the assumption that local ancient repeat elements of DHSs are under neutral evolution, we discovered that ∼0.44% of DHSs are under accelerated evolution (ace-DHSs). We found that ace-DHSs tend to be more active than background DHSs, and are strongly associated with epigenetic marks of active transcription. The target genes of ace-DHSs are significantly enriched in neuron-related functions, and their expression levels are positively selected in the human brain. Thus, these lines of evidences strongly suggest that accelerated evolution on regulatory sequences plays important role in the evolution of human-specific phenotypes. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Four regulatory elements in the human c-fos promoter mediate transactivation by HTLV-1 Tax protein.

PubMed

Alexandre, C; Verrier, B

1991-04-01

Expression of the human c-fos proto-oncogene is activated in trans by the Tax protein encoded by human T-cell leukemia virus type-1 (HTLV-1). Indeed, we show here that a HeLa clone stably transfected by Tax expresses Fos at a high level. We also show that multiple elements of the human c-fos promoter, i.e. the v-sis conditioned medium inducible element (SIE), the dyad symmetry element (DSE) necessary for growth factor induction, the octanucleotide direct repeat element (DR), and the cyclic AMP response element (CRE) centred at -60, can all mediate Tax transactivation. In the DSE, the 10bp central core that binds the serum response factor (SRF) is, by itself, sufficient to mediate Tax transactivation. Moreover, a CRE-binding protein is involved in Tax activation through the CRE-60 element. Since Fos is a transregulator of cellular genes, our results suggest that the oncoprotein plays a crucial role in T-cell transformation by HTLV-1 in conjunction with other Tax-inducible genes.

Emergent self-regulatory activity among young children during scientific inquiry: An analysis of six kindergarten children

NASA Astrophysics Data System (ADS)

Lomangino, Adrienne Gelpi

2000-10-01

This qualitative investigation extends the study of self-regulation to examine young children's developing self-regulated learning competencies. The framework for this research draws upon social cognitive, developmental, and sociocultural perspectives on self-regulation and research on children's scientific thinking. Taking a multiple case study approach, this study examines six kindergarten children's emerging self-regulatory competencies during inquiry-based science instruction. Data were collected during two inquiry-based science programs of study, one pertaining to light and shadow and a second pertaining to motion on inclined planes. Data sources included: videotaped records of the instruction, transcriptions of the videotapes, interviews with the children and teacher, student work, and field notes. Taking an inductive approach to analysis, patterns in the children's activity were identified through a recursive process of defining and refining categories that characterized the children's verbal and behavioral activity. Each case study examines a child's behavior within each phase of the inquiry for evidence of emerging self-regulatory competence. Analysis revealed nascent forms of goal-setting and planning, monitoring, resource management, seeking social assistance, and evaluating. Monitoring activity occurred more frequently than planning or evaluating. For several children, animating materials served to promote motivation. Children's efforts to support peers' activity and monitor the meaning of ongoing discourse contrast with common assumptions about children's attention to others' thinking. Variations in self-regulatory activity were found across phases of instruction. The children exhibited interpersonal self-regulatory efforts, in which monitoring and control of the self was entwined with the activity of others. Joint participation also played a critical role in supporting the metacognitive demands of self-regulation and prompting metacognitive awareness

Transcription factor MBF-I interacts with metal regulatory elements of higher eucaryotic metallothionein genes.

PubMed Central

Imbert, J; Zafarullah, M; Culotta, V C; Gedamu, L; Hamer, D

1989-01-01

Metallothionein (MT) gene promoters in higher eucaryotes contain multiple metal regulatory elements (MREs) that are responsible for the metal induction of MT gene transcription. We identified and purified to near homogeneity a 74-kilodalton mouse nuclear protein that specifically binds to certain MRE sequences. This protein, MBF-I, was purified employing as an affinity reagent a trout MRE that is shown to be functional in mouse cells but which lacks the G+C-rich and SP1-like sequences found in many mammalian MT gene promoters. Using point-mutated MREs, we showed that there is a strong correlation between DNA binding in vitro and MT gene regulation in vivo, suggesting a direct role of MBF-I in MT gene transcription. We also showed that MBF-I can induce MT gene transcription in vitro in a mouse extract and that this stimulation requires zinc. Images PMID:2586522

Oxymatrine attenuates hepatic steatosis in non-alcoholic fatty liver disease rats fed with high fructose diet through inhibition of sterol regulatory element binding transcription factor 1 (Srebf1) and activation of peroxisome proliferator activated receptor alpha (Pparα).

PubMed

Shi, Li-juan; Shi, Lei; Song, Guang-yao; Zhang, He-fang; Hu, Zhi-juan; Wang, Chao; Zhang, Dong-hui

2013-08-15

The aim of this study was to examine the therapeutic effect of oxymatrine, a monomer isolated from the medicinal plant Sophora flavescens Ait, on the hepatic lipid metabolism in non-alcoholic fatty liver (NAFLD) rats and to explore the potential mechanism. Rats were fed with high fructose diet for 8 weeks to establish the NAFLD model, then were given oxymatrine treatment (40, 80, and 160 mg/kg, respectively) for another 8 weeks. Body weight gain, liver index, serum and liver lipids, and histopathological evaluation were measured. Enzymatic activity and gene expression of the key enzymes involved in the lipogenesis and fatty acid oxidation were assayed. The results showed that oxymatrine treatment reduced body weight gain, liver weight, liver index, dyslipidemia, and liver triglyceride level in a dose dependant manner. Importantly, the histopathological examination of liver confirmed that oxymatrine could decrease the liver lipid accumulation. The treatment also decreased the fatty acid synthase (FAS) enzymatic activity and increased the carnitine palmitoyltransferase 1A (CPT1A) enzymatic activity. Besides, oxymatrine treatment decreased the mRNA expression of sterol regulatory element binding transcription factor 1(Srebf1), fatty acid synthase (Fasn), and acetyl CoA carboxylase (Acc), and increased the mRNA expression of peroxisome proliferator activated receptor alpha (Pparα), carnitine palmitoyltransferase 1A (Cpt1a), and acyl CoA oxidase (Acox1) in high fructose diet induced NAFLD rats. These results suggested that the therapeutic effect of oxymatrine on the hepatic steatosis in high fructose diet induced fatty liver rats is partly due to down-regulating Srebf1 and up-regulating Pparα mediated metabolic pathways simultaneously. © 2013 Elsevier B.V. All rights reserved.

Regulatory Snapshots: integrative mining of regulatory modules from expression time series and regulatory networks.

PubMed

Gonçalves, Joana P; Aires, Ricardo S; Francisco, Alexandre P; Madeira, Sara C

2012-01-01

Explaining regulatory mechanisms is crucial to understand complex cellular responses leading to system perturbations. Some strategies reverse engineer regulatory interactions from experimental data, while others identify functional regulatory units (modules) under the assumption that biological systems yield a modular organization. Most modular studies focus on network structure and static properties, ignoring that gene regulation is largely driven by stimulus-response behavior. Expression time series are key to gain insight into dynamics, but have been insufficiently explored by current methods, which often (1) apply generic algorithms unsuited for expression analysis over time, due to inability to maintain the chronology of events or incorporate time dependency; (2) ignore local patterns, abundant in most interesting cases of transcriptional activity; (3) neglect physical binding or lack automatic association of regulators, focusing mainly on expression patterns; or (4) limit the discovery to a predefined number of modules. We propose Regulatory Snapshots, an integrative mining approach to identify regulatory modules over time by combining transcriptional control with response, while overcoming the above challenges. Temporal biclustering is first used to reveal transcriptional modules composed of genes showing coherent expression profiles over time. Personalized ranking is then applied to prioritize prominent regulators targeting the modules at each time point using a network of documented regulatory associations and the expression data. Custom graphics are finally depicted to expose the regulatory activity in a module at consecutive time points (snapshots). Regulatory Snapshots successfully unraveled modules underlying yeast response to heat shock and human epithelial-to-mesenchymal transition, based on regulations documented in the YEASTRACT and JASPAR databases, respectively, and available expression data. Regulatory players involved in functionally enriched

Regulatory Snapshots: Integrative Mining of Regulatory Modules from Expression Time Series and Regulatory Networks

PubMed Central

Gonçalves, Joana P.; Aires, Ricardo S.; Francisco, Alexandre P.; Madeira, Sara C.

2012-01-01

Explaining regulatory mechanisms is crucial to understand complex cellular responses leading to system perturbations. Some strategies reverse engineer regulatory interactions from experimental data, while others identify functional regulatory units (modules) under the assumption that biological systems yield a modular organization. Most modular studies focus on network structure and static properties, ignoring that gene regulation is largely driven by stimulus-response behavior. Expression time series are key to gain insight into dynamics, but have been insufficiently explored by current methods, which often (1) apply generic algorithms unsuited for expression analysis over time, due to inability to maintain the chronology of events or incorporate time dependency; (2) ignore local patterns, abundant in most interesting cases of transcriptional activity; (3) neglect physical binding or lack automatic association of regulators, focusing mainly on expression patterns; or (4) limit the discovery to a predefined number of modules. We propose Regulatory Snapshots, an integrative mining approach to identify regulatory modules over time by combining transcriptional control with response, while overcoming the above challenges. Temporal biclustering is first used to reveal transcriptional modules composed of genes showing coherent expression profiles over time. Personalized ranking is then applied to prioritize prominent regulators targeting the modules at each time point using a network of documented regulatory associations and the expression data. Custom graphics are finally depicted to expose the regulatory activity in a module at consecutive time points (snapshots). Regulatory Snapshots successfully unraveled modules underlying yeast response to heat shock and human epithelial-to-mesenchymal transition, based on regulations documented in the YEASTRACT and JASPAR databases, respectively, and available expression data. Regulatory players involved in functionally enriched

Perceived success/failure and attributions associated with self-regulatory efficacy to meet physical activity recommendations for women with arthritis.

PubMed

Spink, Kevin S; Brawley, Lawrence R; Gyurcsik, Nancy C

2016-10-01

The relationship between attributional dimensions women assign to the cause of their perceived success or failure at meeting the recommended physical activity dose and self-regulatory efficacy for future physical activity was examined among women with arthritis. Women (N = 117) aged 18-84 years, with self-reported medically-diagnosed arthritis, completed on-line questions in the fall of 2013 assessing endurance physical activity, perceived outcome for meeting the recommended levels of endurance activity, attributions for one's success or failure in meeting the recommendations, and self-regulatory efficacy to schedule/plan endurance activity over the next month. The main theoretically-driven finding revealed that the interaction of the stability dimension with perceived success/failure was significantly related to self-regulatory efficacy for scheduling and planning future physical activity (β = 0.35, p = .002). Outcomes attributed to more versus less stable factors accentuated differences in self-regulatory efficacy beliefs following perceived success and failure at being active. It appears that attributional dimensions were associated with self-regulatory efficacy in women with arthritis. This suggests that rather than objectively observed past mastery experience, women's subjective perceptions and explanations of their past experiences were related to efficacy beliefs, especially following a failure experience.

The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaynes, J.B.; Johnson, J.E.; Buskin, J.N.

1988-01-01

Muscle creatine kinase (MCK) is induced to high levels during skeletal muscle differentiation. The authors examined the upstream regulatory elements of the mouse MCK gene which specify its activation during myogenesis in culture. Fusion genes containing up to 3,300 nucleotides (nt) of MCK 5' flanking DNA in various positions and orientations relative to the bacterial chloramphenicol acetyltransferase (CAT) structural gene were transfected into cultured cells. Transient expression of CAT was compared between proliferating and differentiated MM14 mouse myoblasts and with nonmyogenic mouse L cells. The major effector of high-level expression was found to have the properties of a transcriptional enhancer.more » This element, located between 1,050 and 1,256 nt upstream of the transcription start site, was also found to have a major influence on the tissue and differentiation specificity of MCK expression; it activated either the MCK promoter or heterologous promoters only in differentiated muscle cells. Comparisons of viral and cellular enhancer sequences with the MCK enhancer revealed some similarities to essential regions of the simian virus 40 enhancer as well as to a region of the immunoglobulin heavy-chain enhancer, which has been implicated in tissue-specific protein binding. Even in the absence of the enhancer, low-level expression from a 776-nt MCK promoter retained differentiation specificity. In addition to positive regulatory elements, our data provide some evidence for negative regulatory elements with activity in myoblasts. These may contribute to the cell type and differentiation specificity of MCK expression.« less

Cloning and Characterization of 5′ Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

PubMed Central

Tang, Guiying; Xu, Pingli; Liu, Wei; Liu, Zhanji; Shan, Lei

2015-01-01

LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5′ flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5′ terminal and/or 3′ terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65bp proximal promoter region and the 52bp 5′ UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements. PMID:26426444

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis.

PubMed

Kague, Erika; Bessling, Seneca L; Lee, Josephine; Hu, Gui; Passos-Bueno, Maria Rita; Fisher, Shannon

2010-01-15

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. Copyright 2009 Elsevier Inc. All rights reserved.

Transposable elements contribute to activation of maize genes in response to abiotic stress.

PubMed

Makarevitch, Irina; Waters, Amanda J; West, Patrick T; Stitzer, Michelle; Hirsch, Candice N; Ross-Ibarra, Jeffrey; Springer, Nathan M

2015-01-01

Transposable elements (TEs) account for a large portion of the genome in many eukaryotic species. Despite their reputation as "junk" DNA or genomic parasites deleterious for the host, TEs have complex interactions with host genes and the potential to contribute to regulatory variation in gene expression. It has been hypothesized that TEs and genes they insert near may be transcriptionally activated in response to stress conditions. The maize genome, with many different types of TEs interspersed with genes, provides an ideal system to study the genome-wide influence of TEs on gene regulation. To analyze the magnitude of the TE effect on gene expression response to environmental changes, we profiled gene and TE transcript levels in maize seedlings exposed to a number of abiotic stresses. Many genes exhibit up- or down-regulation in response to these stress conditions. The analysis of TE families inserted within upstream regions of up-regulated genes revealed that between four and nine different TE families are associated with up-regulated gene expression in each of these stress conditions, affecting up to 20% of the genes up-regulated in response to abiotic stress, and as many as 33% of genes that are only expressed in response to stress. Expression of many of these same TE families also responds to the same stress conditions. The analysis of the stress-induced transcripts and proximity of the transposon to the gene suggests that these TEs may provide local enhancer activities that stimulate stress-responsive gene expression. Our data on allelic variation for insertions of several of these TEs show strong correlation between the presence of TE insertions and stress-responsive up-regulation of gene expression. Our findings suggest that TEs provide an important source of allelic regulatory variation in gene response to abiotic stress in maize.

Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

PubMed Central

2010-01-01

Background Regulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken. Results We have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines. Conclusions From these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species. PMID:20184756

Transforming growth factor β suppresses peroxisome proliferator-activated receptor γ expression via both SMAD binding and novel TGF-β inhibitory elements.

PubMed

Lakshmi, Sowmya P; Reddy, Aravind T; Reddy, Raju C

2017-04-24

Transforming growth factor β (TGF-β) contributes to wound healing and, when dysregulated, to pathological fibrosis. TGF-β and the anti-fibrotic nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) repress each other's expression, and such PPARγ down-regulation is prominent in fibrosis and mediated, via previously unknown SMAD-signaling mechanisms. Here, we show that TGF-β induces the association of SMAD3 with both SMAD4, needed for translocation of the complex into the nucleus, and the essential context-sensitive co-repressors E2F4 and p107. The complex mediates TGF-β-induced repression by binding to regulatory elements in the target promoter. In the PPARG promoter, we found that the SMAD3-SMAD4 complex binds both to a previously unknown consensus TGF-β inhibitory element (TIE) and also to canonical SMAD-binding elements (SBEs). Furthermore, the TIE and SBEs independently mediated the partial repression of PPARG transcription, the first demonstration of a TIE and SBEs functioning within the same promoter. Also, TGF-β-treated fibroblasts contained SMAD complexes that activated a SMAD target gene in addition to those repressing PPARG transcription, the first finding of such dual activity within the same cell. These findings describe in detail novel mechanisms by which TGF-β represses PPARG transcription, thereby facilitating its own pro-fibrotic activity. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Matching Element Symbols with State Abbreviations: A Fun Activity for Browsing the Periodic Table of Chemical Elements

ERIC Educational Resources Information Center

Woelk, Klaus

2009-01-01

A classroom activity is presented in which students are challenged to find matches between the United States two-letter postal abbreviations for states and chemical element symbols. The activity aims to lessen negative apprehensions students might have when the periodic table of the elements with its more than 100 combinations of letters is first…

Unexpected T cell regulatory activity of anti-histone H1 autoantibody: Its mode of action in regulatory T cell-dependent and -independent manners

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takaoka, Yuki; Kawamoto, Seiji, E-mail: skawa@hiroshima-u.ac.jp; Katayama, Akiko

2013-02-08

Highlights: ► Anti-histone H1 autoantibody (anti-H1) acts on T cells to inhibit their activation. ► Anti-H1 suppresses T cell activation in Treg cell-dependent and -independent manners. ► Suboptimal dose of anti-H1 enhances suppressor function of Treg cells. ► High dose of anti-H1 directly inhibits T cell receptor signaling. -- Abstract: Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Heremore » we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4{sup +}Foxp3{sup +} Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.« less

78 FR 13712 - U.S. Nuclear Regulatory Commission Planned Monitoring Activities for F-Area Tank Farm at the...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-28

... Monitoring Activities for F-Area Tank Farm at the Savannah River Site, Revision 0 AGENCY: Nuclear Regulatory... carrying out its responsibilities for monitoring DOE's waste disposal activities at the F-Area Tank Farm at... the availability of ``U.S. Nuclear Regulatory Commission Plan for Monitoring Disposal Actions Taken by...

Finite-element model of the active organ of Corti

PubMed Central

Elliott, Stephen J.; Baumgart, Johannes

2016-01-01

The cochlear amplifier that provides our hearing with its extraordinary sensitivity and selectivity is thought to be the result of an active biomechanical process within the sensory auditory organ, the organ of Corti. Although imaging techniques are developing rapidly, it is not currently possible, in a fully active cochlea, to obtain detailed measurements of the motion of individual elements within a cross section of the organ of Corti. This motion is predicted using a two-dimensional finite-element model. The various solid components are modelled using elastic elements, the outer hair cells (OHCs) as piezoelectric elements and the perilymph and endolymph as viscous and nearly incompressible fluid elements. The model is validated by comparison with existing measurements of the motions within the passive organ of Corti, calculated when it is driven either acoustically, by the fluid pressure or electrically, by excitation of the OHCs. The transverse basilar membrane (BM) motion and the shearing motion between the tectorial membrane and the reticular lamina are calculated for these two excitation modes. The fully active response of the BM to acoustic excitation is predicted using a linear superposition of the calculated responses and an assumed frequency response for the OHC feedback. PMID:26888950

Sterol regulatory element-binding protein-1 participates in the regulation of fatty acid synthase expression in colorectal neoplasia.

PubMed

Li, J N; Mahmoud, M A; Han, W F; Ripple, M; Pizer, E S

2000-11-25

Endogenous fatty acid synthesis has been observed in certain rapidly proliferating normal and neoplastic tissues. Sterol regulatory element-binding proteins (SREBPs) are transcription factors that regulate the expression of lipogenic genes including fatty acid synthase (FAS), the major biosynthetic enzyme for fatty acid synthesis. We have previously shown that SREBP-1, FAS, and Ki-67, a proliferation marker, colocalized in the crypts of the fetal gastrointestinal tract epithelium. This study sought to determine whether SREBP-1 participates in the regulation of proliferation-associated fatty acid synthesis in colorectal neoplasia. An immunohistochemical analysis of SREBP-1, FAS, and Ki-67 expression in 25 primary human colorectal carcinoma specimens showed colocalization in 22 of these. To elucidate a functional linkage between SREBP-1 activation and proliferation-associated FA synthesis, SREBP-1 and FAS content were assayed during the adaptive response of cultured HCT116 colon carcinoma cells to pharmacological inhibition of FA synthesis. Cerulenin and TOFA each inhibited the endogenous synthesis of fatty acids in a dose-dependent manner and each induced increases in both precursor and mature forms of SREBP-1. Subsequently, both the transcriptional activity of the FAS promoter in a luciferase reporter gene construct and the FAS expression increased. These results demonstrate that tumor cells recognize and respond to a deficiency in endogenous fatty acid synthesis by upregulating both SREBP-1 and FAS expression and support the model that SREBP-1 participates in the transcriptional regulation of lipogenic genes in colorectal neoplasia. Copyright 2000 Academic Press.

Hypoxia-induced endothelial NO synthase gene transcriptional activation is mediated through the tax-responsive element in endothelial cells.

PubMed

Min, Jiho; Jin, Yoon-Mi; Moon, Je-Sung; Sung, Min-Sun; Jo, Sangmee Ahn; Jo, Inho

2006-06-01

Although hypoxia is known to induce upregulation of endothelial NO synthase (eNOS) gene expression, the underlying mechanism is largely unclear. In this study, we show that hypoxia increases eNOS gene expression through the binding of phosphorylated cAMP-responsive element binding (CREB) protein (pCREB) to the eNOS gene promoter. Hypoxia (1% O2) increased both eNOS expression and NO production, peaking at 24 hours, in bovine aortic endothelial cells, and these increases were accompanied by increases in pCREB. Treatment with the protein kinase A inhibitor H-89 or transfection with dominant-negative inhibitor of CREB reversed the hypoxia-induced increases in eNOS expression and NO production, with concomitant inhibition of the phosphorylation of CREB induced by hypoxia, suggesting an involvement of protein kinase A/pCREB-mediated pathway. To map the regulatory elements of the eNOS gene responsible for pCREB binding under hypoxia, we constructed an eNOS gene promoter (-1600 to +22 nucleotides) fused with a luciferase reporter gene [pGL2-eNOS(-1600)]. Hypoxia (for 24-hour incubation) increased the promoter activity by 2.36+/-0.18-fold in the bovine aortic endothelial cells transfected with pGL2-eNOS(-1600). However, progressive 5'-deletion from -1600 to -873 completely attenuated the hypoxia-induced increase in promoter activity. Electrophoretic mobility shift, anti-pCREB antibody supershift, and site-specific mutation analyses showed that pCREB is bound to the Tax-responsive element (TRE) site, a cAMP-responsive element-like site, located at -924 to -921 of the eNOS promoter. Our data demonstrate that the interaction between pCREB and the Tax-responsive element site within the eNOS promoter may represent a novel mechanism for the mediation of hypoxia-stimulated eNOS gene expression.

Succession of splicing regulatory elements determines cryptic 5΄ss functionality

PubMed Central

Brillen, Anna-Lena; Schöneweis, Katrin; Walotka, Lara; Hartmann, Linda; Müller, Lisa; Ptok, Johannes; Kaisers, Wolfgang; Poschmann, Gereon; Stühler, Kai; Buratti, Emanuele

2017-01-01

Abstract A critical step in exon definition is the recognition of a proper splice donor (5΄ss) by the 5’ end of U1 snRNA. In the selection of appropriate 5΄ss, cis-acting splicing regulatory elements (SREs) are indispensable. As a model for 5΄ss recognition, we investigated cryptic 5΄ss selection within the human fibrinogen Bβ-chain gene (FGB) exon 7, where we identified several exonic SREs that simultaneously acted on up- and downstream cryptic 5΄ss. In the FGB exon 7 model system, 5΄ss selection iteratively proceeded along an alternating sequence of U1 snRNA binding sites and interleaved SREs which in principle supported different 3’ exon ends. Like in a relay race, SREs either suppressed a potential 5΄ss and passed the splicing baton on or splicing actually occurred. From RNA-Seq data, we systematically selected 19 genes containing exons with silent U1 snRNA binding sites competing with nearby highly used 5΄ss. Extensive SRE analysis by different algorithms found authentic 5΄ss significantly more supported by SREs than silent U1 snRNA binding sites, indicating that our concept may permit generalization to a model for 5΄ss selection and 3’ exon end definition. PMID:28039323

Mapping cis-Regulatory Domains in the Human Genome UsingMulti-Species Conservation of Synteny

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahituv, Nadav; Prabhakar, Shyam; Poulin, Francis

2005-06-13

Our inability to associate distant regulatory elements with the genes that they regulate has largely precluded their examination for sequence alterations contributing to human disease. One major obstacle is the large genomic space surrounding targeted genes in which such elements could potentially reside. In order to delineate gene regulatory boundaries we used whole-genome human-mouse-chicken (HMC) and human-mouse-frog (HMF) multiple alignments to compile conserved blocks of synteny (CBS), under the hypothesis that these blocks have been kept intact throughout evolution at least in part by the requirement of regulatory elements to stay linked to the genes that they regulate. A totalmore » of 2,116 and 1,942 CBS>200 kb were assembled for HMC and HMF respectively, encompassing 1.53 and 0.86 Gb of human sequence. To support the existence of complex long-range regulatory domains within these CBS we analyzed the prevalence and distribution of chromosomal aberrations leading to position effects (disruption of a genes regulatory environment), observing a clear bias not only for mapping onto CBS but also for longer CBS size. Our results provide a genome wide data set characterizing the regulatory domains of genes and the conserved regulatory elements within them.« less

Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins.

PubMed

Niles, Brad J; Clegg, Michael S; Hanna, Lynn A; Chou, Susan S; Momma, Tony Y; Hong, Heeok; Keen, Carl L

2008-02-22

One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.

Dietary Apigenin Exerts Immune-Regulatory Activity in Vivo by Reducing NF-κB Activity, Halting Leukocyte Infiltration and Restoring Normal Metabolic Function

PubMed Central

Cardenas, Horacio; Arango, Daniel; Nicholas, Courtney; Duarte, Silvia; Nuovo, Gerard J.; He, Wei; Voss, Oliver H.; Gonzalez-Mejia, M. Elba; Guttridge, Denis C.; Grotewold, Erich; Doseff, Andrea I.

2016-01-01

The increasing prevalence of inflammatory diseases and the adverse effects associated with the long-term use of current anti-inflammatory therapies prompt the identification of alternative approaches to reestablish immune balance. Apigenin, an abundant dietary flavonoid, is emerging as a potential regulator of inflammation. Here, we show that apigenin has immune-regulatory activity in vivo. Apigenin conferred survival to mice treated with a lethal dose of Lipopolysaccharide (LPS) restoring normal cardiac function and heart mitochondrial Complex I activity. Despite the adverse effects associated with high levels of splenocyte apoptosis in septic models, apigenin had no effect on reducing cell death. However, we found that apigenin decreased LPS-induced apoptosis in lungs, infiltration of inflammatory cells and chemotactic factors’ accumulation, re-establishing normal lung architecture. Using NF-κB luciferase transgenic mice, we found that apigenin effectively modulated NF-κB activity in the lungs, suggesting the ability of dietary compounds to exert immune-regulatory activity in an organ-specific manner. Collectively, these findings provide novel insights into the underlying immune-regulatory mechanisms of dietary nutraceuticals in vivo. PMID:26938530

Dietary Apigenin Exerts Immune-Regulatory Activity in Vivo by Reducing NF-κB Activity, Halting Leukocyte Infiltration and Restoring Normal Metabolic Function.

PubMed

Cardenas, Horacio; Arango, Daniel; Nicholas, Courtney; Duarte, Silvia; Nuovo, Gerard J; He, Wei; Voss, Oliver H; Gonzalez-Mejia, M Elba; Guttridge, Denis C; Grotewold, Erich; Doseff, Andrea I

2016-03-01

The increasing prevalence of inflammatory diseases and the adverse effects associated with the long-term use of current anti-inflammatory therapies prompt the identification of alternative approaches to reestablish immune balance. Apigenin, an abundant dietary flavonoid, is emerging as a potential regulator of inflammation. Here, we show that apigenin has immune-regulatory activity in vivo. Apigenin conferred survival to mice treated with a lethal dose of Lipopolysaccharide (LPS) restoring normal cardiac function and heart mitochondrial Complex I activity. Despite the adverse effects associated with high levels of splenocyte apoptosis in septic models, apigenin had no effect on reducing cell death. However, we found that apigenin decreased LPS-induced apoptosis in lungs, infiltration of inflammatory cells and chemotactic factors' accumulation, re-establishing normal lung architecture. Using NF-κB luciferase transgenic mice, we found that apigenin effectively modulated NF-κB activity in the lungs, suggesting the ability of dietary compounds to exert immune-regulatory activity in an organ-specific manner. Collectively, these findings provide novel insights into the underlying immune-regulatory mechanisms of dietary nutraceuticals in vivo.

Regulatory T cells ameliorate tissue plasminogen activator-induced brain haemorrhage after stroke.

PubMed

Mao, Leilei; Li, Peiying; Zhu, Wen; Cai, Wei; Liu, Zongjian; Wang, Yanling; Luo, Wenli; Stetler, Ruth A; Leak, Rehana K; Yu, Weifeng; Gao, Yanqin; Chen, Jun; Chen, Gang; Hu, Xiaoming

2017-07-01

Delayed thrombolytic treatment with recombinant tissue plasminogen activator (tPA) may exacerbate blood-brain barrier breakdown after ischaemic stroke and lead to lethal haemorrhagic transformation. The immune system is a dynamic modulator of stroke response, and excessive immune cell accumulation in the cerebral vasculature is associated with compromised integrity of the blood-brain barrier. We previously reported that regulatory T cells, which function to suppress excessive immune responses, ameliorated blood-brain barrier damage after cerebral ischaemia. This study assessed the impact of regulatory T cells in the context of tPA-induced brain haemorrhage and investigated the underlying mechanisms of action. The number of circulating regulatory T cells in stroke patients was dramatically reduced soon after stroke onset (84 acute ischaemic stroke patients with or without intravenous tPA treatment, compared to 115 age and gender-matched healthy controls). Although stroke patients without tPA treatment gradually repopulated the numbers of circulating regulatory T cells within the first 7 days after stroke, post-ischaemic tPA treatment led to sustained suppression of regulatory T cells in the blood. We then used the murine suture and embolic middle cerebral artery occlusion models of stroke to investigate the therapeutic potential of adoptive regulatory T cell transfer against tPA-induced haemorrhagic transformation. Delayed administration of tPA (10 mg/kg) resulted in haemorrhagic transformation in the ischaemic territory 1 day after ischaemia. When regulatory T cells (2 × 106/mouse) were intravenously administered immediately after delayed tPA treatment in ischaemic mice, haemorrhagic transformation was significantly decreased, and this was associated with improved sensorimotor functions. Blood-brain barrier disruption and tight junction damages were observed in the presence of delayed tPA after stroke, but were mitigated by regulatory T cell transfer. Mechanistic

Allowance trading activity and state regulatory rulings: Evidence from the US Acid Rain Program

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bailey, E.M.

1997-12-31

The US Acid Rain Program is one of the first, and by far the most extensive, applications of a market based approach to pollution control. From the beginning, there has been concern whether utilities would participate in allowance trading, and whether regulatory activity at the state level would further complicate utilities` decision to trade allowances. This paper finds that public utility commission regulation has encouraged allowance trading activity in states with regulatory rulings, but that allowance trading activity has not been limited to states issuing regulations. Until there is evidence suggesting that significant additional cost savings could have been obtainedmore » if additional allowance trading activity had occurred in states without regulations or that utilities in states with regulations are still not taking advantage of all cost saving trading opportunities, this analysis suggests that there is little reason to believe that allowance trading activity is impeded by public utility commission regulations.« less

CRX ChIP-seq reveals the cis-regulatory architecture of mouse photoreceptors

PubMed Central

Corbo, Joseph C.; Lawrence, Karen A.; Karlstetter, Marcus; Myers, Connie A.; Abdelaziz, Musa; Dirkes, William; Weigelt, Karin; Seifert, Martin; Benes, Vladimir; Fritsche, Lars G.; Weber, Bernhard H.F.; Langmann, Thomas

2010-01-01

Approximately 98% of mammalian DNA is noncoding, yet we understand relatively little about the function of this enigmatic portion of the genome. The cis-regulatory elements that control gene expression reside in noncoding regions and can be identified by mapping the binding sites of tissue-specific transcription factors. Cone-rod homeobox (CRX) is a key transcription factor in photoreceptor differentiation and survival, but its in vivo targets are largely unknown. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) on CRX to identify thousands of cis-regulatory regions around photoreceptor genes in adult mouse retina. CRX directly regulates downstream photoreceptor transcription factors and their target genes via a network of spatially distributed regulatory elements around each locus. CRX-bound regions act in a synergistic fashion to activate transcription and contain multiple CRX binding sites which interact in a spacing- and orientation-dependent manner to fine-tune transcript levels. CRX ChIP-seq was also performed on Nrl−/− retinas, which represent an enriched source of cone photoreceptors. Comparison with the wild-type ChIP-seq data set identified numerous rod- and cone-specific CRX-bound regions as well as many shared elements. Thus, CRX combinatorially orchestrates the transcriptional networks of both rods and cones by coordinating the expression of photoreceptor genes including most retinal disease genes. In addition, this study pinpoints thousands of noncoding regions of relevance to both Mendelian and complex retinal disease. PMID:20693478

Germinal and Somatic Activity of the Maize Element Activator (Ac) in Arabidopsis

PubMed Central

Keller, J.; Lim, E.; James-Jr., D. W.; Dooner, H. K.

1992-01-01

We have investigated the germinal and somatic activity of the maize Activator (Ac) element in Arabidopsis with the objective of developing an efficient transposon-based system for gene isolation in that plant. Transposition activity was assayed with a chimeric marker that consists of the cauliflower mosaic virus 35S promoter and a bacterial streptomycin phosphotransferase gene (SPT). Somatic activity was detected in seedlings germinated on plates containing streptomycin as green-resistant sectors against a background of white-sensitive cells. Germinal excisions resulted in fully green seedlings. The transposition frequency was extremely low when a single copy of the transposon was present, but appeared to increase with an increase in Ac copy number. Plants that were selected as variegated produced an increased number of green progeny. The methylation state of the Ac elements in lines with either low or high levels of excision was assessed by restriction analysis. No difference was found between these lines, indicating that the degree of methylation did not contribute to the level of Ac activity. Germinal excision events were analyzed molecularly and shown to carry reinserted transposons in about 50% of the cases. In several instances, streptomycin-resistant siblings carried the same transposed Ac element, indicating that excision had occurred prior to meiosis in the parent. We discuss parameters that need to be considered to optimize the use of Ac as a transposon tag in Arabidopsis. PMID:1322854

Feather Development Genes and Associated Regulatory Innovation Predate the Origin of Dinosauria

PubMed Central

Lowe, Craig B.; Clarke, Julia A.; Baker, Allan J.; Haussler, David; Edwards, Scott V.

2015-01-01

The evolution of avian feathers has recently been illuminated by fossils and the identification of genes involved in feather patterning and morphogenesis. However, molecular studies have focused mainly on protein-coding genes. Using comparative genomics and more than 600,000 conserved regulatory elements, we show that patterns of genome evolution in the vicinity of feather genes are consistent with a major role for regulatory innovation in the evolution of feathers. Rates of innovation at feather regulatory elements exhibit an extended period of innovation with peaks in the ancestors of amniotes and archosaurs. We estimate that 86% of such regulatory elements and 100% of the nonkeratin feather gene set were present prior to the origin of Dinosauria. On the branch leading to modern birds, we detect a strong signal of regulatory innovation near insulin-like growth factor binding protein (IGFBP) 2 and IGFBP5, which have roles in body size reduction, and may represent a genomic signature for the miniaturization of dinosaurian body size preceding the origin of flight. PMID:25415961

Wnt6 activates endoderm in the sea urchin gene regulatory network

PubMed Central

Croce, Jenifer; Range, Ryan; Wu, Shu-Yu; Miranda, Esther; Lhomond, Guy; Peng, Jeff Chieh-fu; Lepage, Thierry; McClay, David R.

2011-01-01

In the sea urchin, entry of β-catenin into the nuclei of the vegetal cells at 4th and 5th cleavages is necessary for activation of the endomesoderm gene regulatory network. Beyond that, little is known about how the embryo uses maternal information to initiate specification. Here, experiments establish that of the three maternal Wnts in the egg, Wnt6 is necessary for activation of endodermal genes in the endomesoderm GRN. A small region of the vegetal cortex is shown to be necessary for activation of the endomesoderm GRN. If that cortical region of the egg is removed, addition of Wnt6 rescues endoderm. At a molecular level, the vegetal cortex region contains a localized concentration of Dishevelled (Dsh) protein, a transducer of the canonical Wnt pathway; however, Wnt6 mRNA is not similarly localized. Ectopic activation of the Wnt pathway, through the expression of an activated form of β-catenin, of a dominant-negative variant of GSK-3β or of Dsh itself, rescues endomesoderm specification in eggs depleted of the vegetal cortex. Knockdown experiments in whole embryos show that absence of Wnt6 produces embryos that lack endoderm, but those embryos continue to express a number of mesoderm markers. Thus, maternal Wnt6 plus a localized vegetal cortical molecule, possibly Dsh, is necessary for endoderm specification; this has been verified in two species of sea urchin. The data also show that Wnt6 is only one of what are likely to be multiple components that are necessary for activation of the entire endomesoderm gene regulatory network. PMID:21750039

Functional analysis of a large set of BRCA2 exon 7 variants highlights the predictive value of hexamer scores in detecting alterations of exonic splicing regulatory elements.

PubMed

Di Giacomo, Daniela; Gaildrat, Pascaline; Abuli, Anna; Abdat, Julie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

2013-11-01

Exonic variants can alter pre-mRNA splicing either by changing splice sites or by modifying splicing regulatory elements. Often these effects are difficult to predict and are only detected by performing RNA analyses. Here, we analyzed, in a minigene assay, 26 variants identified in the exon 7 of BRCA2, a cancer predisposition gene. Our results revealed eight new exon skipping mutations in this exon: one directly altering the 5' splice site and seven affecting potential regulatory elements. This brings the number of splicing regulatory mutations detected in BRCA2 exon 7 to a total of 11, a remarkably high number considering the total number of variants reported in this exon (n = 36), all tested in our minigene assay. We then exploited this large set of splicing data to test the predictive value of splicing regulator hexamers' scores recently established by Ke et al. (). Comparisons of hexamer-based predictions with our experimental data revealed high sensitivity in detecting variants that increased exon skipping, an important feature for prescreening variants before RNA analysis. In conclusion, hexamer scores represent a promising tool for predicting the biological consequences of exonic variants and may have important applications for the interpretation of variants detected by high-throughput sequencing. © 2013 WILEY PERIODICALS, INC.

IFN-α and CD46 stimulation are associated with active lupus and skew natural T regulatory cell differentiation to type 1 regulatory T (Tr1) cells

PubMed Central

Le Buanec, Hélène; Gougeon, Marie-Lise; Mathian, Alexis; Lebon, Pierre; Dupont, Jean-Michel; Peltre, Gabriel; Hemon, Patrice; Schmid, Michel; Bizzini, Bernard; Künding, Thomas; Burny, Arsène; Bensussan, Armand; Amoura, Zahir; Gallo, Robert C.; Zagury, Daniel

2011-01-01

Immune suppressive activities exerted by regulatory T-cell subsets have several specific functions, including self-tolerance and regulation of adaptive immune reactions, and their dysfunction can lead to autoimmune diseases and contribute to AIDS and cancer. Two functionally distinct regulatory T-cell subsets are currently identified in peripheral tissues: thymus-developed natural T regulatory cells (nTregs) controlling self-tolerance and antiinflammatory IL-10–secreting type 1 regulatory T cells (Tr1) derived from Ag-stimulated T cells, which regulate inflammation-dependent adaptive immunity and minimize immunopathology. We establish herein that cell contact-mediated nTreg regulatory function is inhibited by inflammation, especially in the presence of the complement C3b receptor (CD46). Instead, as with other T-cell subsets, the latter inflammatory conditions of stimulation skew nTreg differentiation to Tr1 cells secreting IL-10, an effect potentiated by IFN-α. The clinical relevance of these findings was verified in a study of 152 lupus patients, in which we showed that lupus nTreg dysfunction is not due to intrinsic defects but is rather induced by C3b stimulation of CD46 and IFN-α and that these immune components of inflammation are directly associated with active lupus. These results provide a rationale for using anti–IFN-α Ab immunotherapy in lupus patients. PMID:22065791

ESPERR: learning strong and weak signals in genomic sequence alignments to identify functional elements.

PubMed

Taylor, James; Tyekucheva, Svitlana; King, David C; Hardison, Ross C; Miller, Webb; Chiaromonte, Francesca

2006-12-01

Genomic sequence signals - such as base composition, presence of particular motifs, or evolutionary constraint - have been used effectively to identify functional elements. However, approaches based only on specific signals known to correlate with function can be quite limiting. When training data are available, application of computational learning algorithms to multispecies alignments has the potential to capture broader and more informative sequence and evolutionary patterns that better characterize a class of elements. However, effective exploitation of patterns in multispecies alignments is impeded by the vast number of possible alignment columns and by a limited understanding of which particular strings of columns may characterize a given class. We have developed a computational method, called ESPERR (evolutionary and sequence pattern extraction through reduced representations), which uses training examples to learn encodings of multispecies alignments into reduced forms tailored for the prediction of chosen classes of functional elements. ESPERR produces a greatly improved Regulatory Potential score, which can discriminate regulatory regions from neutral sites with excellent accuracy ( approximately 94%). This score captures strong signals (GC content and conservation), as well as subtler signals (with small contributions from many different alignment patterns) that characterize the regulatory elements in our training set. ESPERR is also effective for predicting other classes of functional elements, as we show for DNaseI hypersensitive sites and highly conserved regions with developmental enhancer activity. Our software, training data, and genome-wide predictions are available from our Web site (http://www.bx.psu.edu/projects/esperr).

A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury.

PubMed

Guenther, Catherine A; Wang, Zhen; Li, Emma; Tran, Misha C; Logan, Catriona Y; Nusse, Roel; Pantalena-Filho, Luiz; Yang, George P; Kingsley, David M

2015-08-01

Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an "injury response" control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals. Copyright © 2015 Elsevier Inc. All rights reserved.

Self-Regulatory Imagery and Physical Activity in Middle-Aged and Older Adults: A Social-Cognitive Perspective.

PubMed

Kosteli, Maria-Christina; Cumming, Jennifer; Williams, Sarah E

2018-01-01

Limited research has investigated exercise imagery use in middle-aged and older adults and its relationship with affective and behavioral correlates. The study examined the association between self-regulatory imagery and physical activity (PA) through key social cognitive variables. Middle-aged and older adults (N = 299; M age = 59.73 years, SD = 7.73, range = 50 to 80) completed self-report measures assessing self-regulatory imagery use, self-efficacy, outcome expectations, perceived barriers, self-regulatory behavior, enjoyment, and PA levels. Path analysis supported a model (χ² [14] = 21.76, p = .08, CFI = .99, TLI = .97, SRMR = .03, RMSEA = .04) whereby self-regulatory imagery positively predicted self-efficacy, outcome expectations, and self-regulatory behaviors. Furthermore, self-regulatory imagery indirectly predicted barriers, outcome expectations, self-regulation, enjoyment, and PA. This research highlights self-regulatory imagery as an effective strategy in modifying exercise-related cognitions and behaviors. Incorporating social cognitive constructs into the design of imagery interventions may increase PA engagement.

Evaluation of phylogenetic footprint discovery for predicting bacterial cis-regulatory elements and revealing their evolution.

PubMed

Janky, Rekin's; van Helden, Jacques

2008-01-23

The detection of conserved motifs in promoters of orthologous genes (phylogenetic footprints) has become a common strategy to predict cis-acting regulatory elements. Several software tools are routinely used to raise hypotheses about regulation. However, these tools are generally used as black boxes, with default parameters. A systematic evaluation of optimal parameters for a footprint discovery strategy can bring a sizeable improvement to the predictions. We evaluate the performances of a footprint discovery approach based on the detection of over-represented spaced motifs. This method is particularly suitable for (but not restricted to) Bacteria, since such motifs are typically bound by factors containing a Helix-Turn-Helix domain. We evaluated footprint discovery in 368 Escherichia coli K12 genes with annotated sites, under 40 different combinations of parameters (taxonomical level, background model, organism-specific filtering, operon inference). Motifs are assessed both at the levels of correctness and significance. We further report a detailed analysis of 181 bacterial orthologs of the LexA repressor. Distinct motifs are detected at various taxonomical levels, including the 7 previously characterized taxon-specific motifs. In addition, we highlight a significantly stronger conservation of half-motifs in Actinobacteria, relative to Firmicutes, suggesting an intermediate state in specificity switching between the two Gram-positive phyla, and thereby revealing the on-going evolution of LexA auto-regulation. The footprint discovery method proposed here shows excellent results with E. coli and can readily be extended to predict cis-acting regulatory signals and propose testable hypotheses in bacterial genomes for which nothing is known about regulation.

Regulatory Mechanisms That Prevent Re-initiation of DNA Replication Can Be Locally Modulated at Origins by Nearby Sequence Elements

PubMed Central

Richardson, Christopher D.; Li, Joachim J.

2014-01-01

Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, origins re-initiate with diverse efficiencies when these mechanisms are disabled, and this diversity cannot be explained by differences in the efficiency or timing of origin initiation during normal S phase replication. This observation raises the possibility of an additional layer of replication control that can differentially regulate re-initiation at distinct origins. We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs). We have mapped the two RIPs to ∼60 bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 reassociates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Our findings identify a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are compromised. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some regions of the genome

Alu-mediated deletion of SOX10 regulatory elements in Waardenburg syndrome type 4

PubMed Central

Bondurand, Nadége; Fouquet, Virginie; Baral, Viviane; Lecerf, Laure; Loundon, Natalie; Goossens, Michel; Duriez, Benedicte; Labrune, Philippe; Pingault, Veronique

2012-01-01

Waardenburg syndrome type 4 (WS4) is a rare neural crest disorder defined by the combination of Waardenburg syndrome (sensorineural hearing loss and pigmentation defects) and Hirschsprung disease (intestinal aganglionosis). Three genes are known to be involved in this syndrome, that is, EDN3 (endothelin-3), EDNRB (endothelin receptor type B), and SOX10. However, 15–35% of WS4 remains unexplained at the molecular level, suggesting that other genes could be involved and/or that mutations within known genes may have escaped previous screenings. Here, we searched for deletions within recently identified SOX10 regulatory sequences and describe the first characterization of a WS4 patient presenting with a large deletion encompassing three of these enhancers. Analysis of the breakpoint region suggests a complex rearrangement involving three Alu sequences that could be mediated by a FosTes/MMBIR replication mechanism. Taken together with recent reports, our results demonstrate that the disruption of highly conserved non-coding elements located within or at a long distance from the coding sequences of key genes can result in several neurocristopathies. This opens up new routes to the molecular dissection of neural crest disorders. PMID:22378281

Alu-mediated deletion of SOX10 regulatory elements in Waardenburg syndrome type 4.

PubMed

Bondurand, Nadége; Fouquet, Virginie; Baral, Viviane; Lecerf, Laure; Loundon, Natalie; Goossens, Michel; Duriez, Benedicte; Labrune, Philippe; Pingault, Veronique

2012-09-01

Waardenburg syndrome type 4 (WS4) is a rare neural crest disorder defined by the combination of Waardenburg syndrome (sensorineural hearing loss and pigmentation defects) and Hirschsprung disease (intestinal aganglionosis). Three genes are known to be involved in this syndrome, that is, EDN3 (endothelin-3), EDNRB (endothelin receptor type B), and SOX10. However, 15-35% of WS4 remains unexplained at the molecular level, suggesting that other genes could be involved and/or that mutations within known genes may have escaped previous screenings. Here, we searched for deletions within recently identified SOX10 regulatory sequences and describe the first characterization of a WS4 patient presenting with a large deletion encompassing three of these enhancers. Analysis of the breakpoint region suggests a complex rearrangement involving three Alu sequences that could be mediated by a FosTes/MMBIR replication mechanism. Taken together with recent reports, our results demonstrate that the disruption of highly conserved non-coding elements located within or at a long distance from the coding sequences of key genes can result in several neurocristopathies. This opens up new routes to the molecular dissection of neural crest disorders.

[Regulatory T cells].

PubMed

Marinić, Igor; Gagro, Alenka; Rabatić, Sabina

2006-12-01

Regulatory T-cells are a subset of T cells that have beene extensively studied in modern immunology. They are important for the maintenance of peripheral tolerance, and have an important role in various clinical conditions such as allergy, autoimmune disorders, tumors, infections, and in transplant medicine. Basically, this population has a suppressive effect on the neighboring immune cells, thus contributing to the local modulation and control of immune response. There are two main populations of regulatory T cells - natural regulatory T cells, which form a distinct cellular lineage, develop in thymus and perform their modulatory action through direct intercellular contact, along with the secreted cytokines; and inducible regulatory T cells, which develop in the periphery after contact with the antigen that is presented on the antigen presenting cell, and their primary mode of action is through the interleukin 10 (IL-10) and transforming growth factor beta (TGF-alpha) cytokines. Natural regulatory T cells are activated through T cell receptor after contact with specific antigen and inhibit proliferation of other T cells in an antigen independent manner. One of the major difficulties in the research of regulatory T cells is the lack of specific molecular markers that would identify these cells. Natural regulatory T cells constitutively express surface molecule CD25, but many other surface and intracellular molecules (HLA-DR, CD122, CD45RO, CD62, CTLA-4, GITR, PD-1, Notch, FOXP3, etc.) are being investigated for further phenotypic characterization of these cells. Because regulatory T cells have an important role in establishing peripheral tolerance, their importance is manifested in a number of clinical conditions. In the IPEX syndrome (immunodysregulation, polyendocrinopathy and enteropathy, X-linked), which is caused by mutation in Foxp3 gene that influences the development and function of regulatory T cells, patients develop severe autoimmune reactions that

Changes in FDA enforcement activities following changes in federal administration: the case of regulatory letters released to pharmaceutical companies.

PubMed

Nguyen, Diane; Seoane-Vazquez, Enrique; Rodriguez-Monguio, Rosa; Montagne, Michael

2013-01-22

The United States (US) Food and Drug Administration (FDA) is responsible for the protection of the public health by assuring the safety, effectiveness and security of human drugs and biological products through the enforcement of the Federal Food, Drug and Cosmetic Act (FDCA) and related regulations. These enforcement activities include regulatory letters (i.e. warning letters and notice of violation) to pharmaceutical companies. A regulatory letter represents the FDA's first official notification to a pharmaceutical company that the FDA has discovered a product or activity in violation of the FDCA.This study analyzed trends in the pharmaceutical-related regulatory letters released by the FDA during the period 1997-2011 and assessed differences in the average number and type of regulatory letters released during the last four federal administrations. Data derived from the FDA webpage. Information about the FDA office releasing the letter, date, company, and drug-related violation was collected. Regulatory letters were classified by federal administration. Descriptive statistics were performed for the analysis. Between 1997 and 2011 the FDA released 2,467 regulatory letters related to pharmaceuticals. FDA headquarters offices released 50.6% and district offices 49.4% of the regulatory letters. The Office of Prescription Drug Promotion released the largest number of regulatory letters (850; 34.5% of the total), followed by the Office of Scientific Investigations (131; 5.3%), and the Office of Compliance (105; 4.3%). During the 2nd Clinton Administration (1997-2000) the average number of regulatory letters per year was 242.8 ± 45.6, during the Bush Administration (2001-2008) it was 120.4 ± 33.7, and during the first three years of the Obama administration (2009-2011) it was 177.7.0 ± 17.0. The average number of regulatory letters released by the Office of Prescription Drug Promotion also varied by administration: Clinton (122.3 ± 36.4), Bush (29.5�

Scanning sequences after Gibbs sampling to find multiple occurrences of functional elements

PubMed Central

Tharakaraman, Kannan; Mariño-Ramírez, Leonardo; Sheetlin, Sergey L; Landsman, David; Spouge, John L

2006-01-01

Background Many DNA regulatory elements occur as multiple instances within a target promoter. Gibbs sampling programs for finding DNA regulatory elements de novo can be prohibitively slow in locating all instances of such an element in a sequence set. Results We describe an improvement to the A-GLAM computer program, which predicts regulatory elements within DNA sequences with Gibbs sampling. The improvement adds an optional "scanning step" after Gibbs sampling. Gibbs sampling produces a position specific scoring matrix (PSSM). The new scanning step resembles an iterative PSI-BLAST search based on the PSSM. First, it assigns an "individual score" to each subsequence of appropriate length within the input sequences using the initial PSSM. Second, it computes an E-value from each individual score, to assess the agreement between the corresponding subsequence and the PSSM. Third, it permits subsequences with E-values falling below a threshold to contribute to the underlying PSSM, which is then updated using the Bayesian calculus. A-GLAM iterates its scanning step to convergence, at which point no new subsequences contribute to the PSSM. After convergence, A-GLAM reports predicted regulatory elements within each sequence in order of increasing E-values, so users have a statistical evaluation of the predicted elements in a convenient presentation. Thus, although the Gibbs sampling step in A-GLAM finds at most one regulatory element per input sequence, the scanning step can now rapidly locate further instances of the element in each sequence. Conclusion Datasets from experiments determining the binding sites of transcription factors were used to evaluate the improvement to A-GLAM. Typically, the datasets included several sequences containing multiple instances of a regulatory motif. The improvements to A-GLAM permitted it to predict the multiple instances. PMID:16961919

Prospective Activities outlined for Regulatory Approval in Ghana Overview

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abrefah, R.G.; Odoi, H.C.; Mo, S.C.

The Ghana Research Reactor-1 (GHARR-1) is one of Chinese’s Miniature Neutron Source Reactor (MNSR) which was purchased under a tripartite agreement between Ghana, China and the IAEA. The reactor was installed in 1994 and has since been in operation without any incident. It has been used chiefly for Neutron Activation Analysis (NAA) and Training of students in the field of Nuclear Engineering. The GHARR-1 has been earmarked for the Conversion of Core from HEU to LEU which is in accordance with the GTRI program and other related and/or associated programs. Over the past few years the National Nuclear Research Institutemore » (NNRI), the Operating Organization of the Research Reactor for the Ghana Atomic Energy Commission (GAEC), has undertaken various tasks in order to implement the replacement of the reactor core. After completion, of the neutronic calculations, results showed that that an LEU fuel of 12.5% enrichment was desirable. However, recent developments have shown that an LEU fuel with 13% enrichment will be fabricated by the manufacturers, which is captured in a fuel specification document sent to NNRI by the CIAE. It is therefore imperative that all neutronic and thermal hydraulic calculation be done again to help acquire regulatory approval. Furthermore, the radiation exposure to personnel involved in the conversion must be estimated to help convince our regulators. This paper outlines the processes and activities that will enable us meet regulatory requirements.« less

Unraveling transcriptional control and cis-regulatory codes using the software suite GeneACT

PubMed Central

Cheung, Tom Hiu; Kwan, Yin Lam; Hamady, Micah; Liu, Xuedong

2006-01-01

Deciphering gene regulatory networks requires the systematic identification of functional cis-acting regulatory elements. We present a suite of web-based bioinformatics tools, called GeneACT , that can rapidly detect evolutionarily conserved transcription factor binding sites or microRNA target sites that are either unique or over-represented in differentially expressed genes from DNA microarray data. GeneACT provides graphic visualization and extraction of common regulatory sequence elements in the promoters and 3'-untranslated regions that are conserved across multiple mammalian species. PMID:17064417

Feather development genes and associated regulatory innovation predate the origin of Dinosauria.

PubMed

Lowe, Craig B; Clarke, Julia A; Baker, Allan J; Haussler, David; Edwards, Scott V

2015-01-01

The evolution of avian feathers has recently been illuminated by fossils and the identification of genes involved in feather patterning and morphogenesis. However, molecular studies have focused mainly on protein-coding genes. Using comparative genomics and more than 600,000 conserved regulatory elements, we show that patterns of genome evolution in the vicinity of feather genes are consistent with a major role for regulatory innovation in the evolution of feathers. Rates of innovation at feather regulatory elements exhibit an extended period of innovation with peaks in the ancestors of amniotes and archosaurs. We estimate that 86% of such regulatory elements and 100% of the nonkeratin feather gene set were present prior to the origin of Dinosauria. On the branch leading to modern birds, we detect a strong signal of regulatory innovation near insulin-like growth factor binding protein (IGFBP) 2 and IGFBP5, which have roles in body size reduction, and may represent a genomic signature for the miniaturization of dinosaurian body size preceding the origin of flight. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

75 FR 58004 - Self-Regulatory Organizations; Financial Industry Regulatory Authority, Inc.; Notice of Filing of...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-09-23

... By-Laws to remove the exemption from the trading activity fee (``TAF'') for transactions in exchange... TAF is one of three member regulatory fees FINRA uses to fund its member regulation activities, which... activities.\\3\\ FINRA initially adopted the TAF in 2002 as a replacement for an earlier regulatory fee based...

Determination of elements in hospital waste with neutron activation analysis method

NASA Astrophysics Data System (ADS)

Dwijananti, P.; Astuti, B.; Alwiyah; Fianti

2018-03-01

The producer of the biggest B3 waste is hospital. The waste is from medical and laboratory activities. The purpose of this study is to determine the elements contained in the liquid waste from hospital and calculate the levels of these elements. This research was done by analysis of the neutron activation conducted at BATAN Yogyakarta. The neutron activation analysis is divided into two stages: activation of the samples using neutron sources of reactor Kartini, then chopping by using a set of tools, gamma spectrometer with HPGe detector. Qualitative and quantitative analysis were done by matching the gamma spectrum peak to the Neutron Activation Table. The sample was taken from four points of the liquid waste treatment plant (WWTP) Bhakti Wira Tamtama Semarang hospital. The results showed that the samples containing elements of Cr, Zn, Fe, Co, and Na, with the levels of each element is Cr (0.033 - 0.075) mg/L, Zn (0.090 - 1.048) mg/L, Fe (2.937-37.743) mg/L, Co (0.005-0.023) mg/L, and Na (61.088-116.330) mg/L. Comparing to the standard value, the liquid is safe to the environment.

Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Jinsil; Ha, Hye-Jeong; Kim, Sujin

Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that anmore » rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.« less

Functional and topological characteristics of mammalian regulatory domains

PubMed Central

Symmons, Orsolya; Uslu, Veli Vural; Tsujimura, Taro; Ruf, Sandra; Nassari, Sonya; Schwarzer, Wibke; Ettwiller, Laurence; Spitz, François

2014-01-01

Long-range regulatory interactions play an important role in shaping gene-expression programs. However, the genomic features that organize these activities are still poorly characterized. We conducted a large operational analysis to chart the distribution of gene regulatory activities along the mouse genome, using hundreds of insertions of a regulatory sensor. We found that enhancers distribute their activities along broad regions and not in a gene-centric manner, defining large regulatory domains. Remarkably, these domains correlate strongly with the recently described TADs, which partition the genome into distinct self-interacting blocks. Different features, including specific repeats and CTCF-binding sites, correlate with the transition zones separating regulatory domains, and may help to further organize promiscuously distributed regulatory influences within large domains. These findings support a model of genomic organization where TADs confine regulatory activities to specific but large regulatory domains, contributing to the establishment of specific gene expression profiles. PMID:24398455

Regulatory T Cell Responses to High-Dose Methylprednisolone in Active Systemic Lupus Erythematosus.

PubMed

Mathian, Alexis; Jouenne, Romain; Chader, Driss; Cohen-Aubart, Fleur; Haroche, Julien; Fadlallah, Jehane; Claër, Laetitia; Musset, Lucile; Gorochov, Guy; Amoura, Zahir; Miyara, Makoto

2015-01-01

A slight increase in the proportion of circulating regulatory T (Treg) cells has been reported in systemic lupus erythematosus (SLE) patients taking oral prednisone. The effects of intravenous (IV) high dose methylprednisolone (MP) on Tregs have not yet been described, especially in active SLE. We prospectively analyzed the proportion of circulating CD4+ Treg cell subsets defined as follows: (1) naïve Treg (nTreg) FoxP3lowCD45RA+ cells; (2) effector Treg (eTreg) FoxP3highCD45RA- cells; and (3) non-suppressive FoxP3lowCD45RA- cells (non-regulatory Foxp3low T cells). Peripheral blood mononuclear cells of patients with active SLE were analyzed before the first infusion of IV high dose MP (day 0) and the following days (day 1, day 2, ±day 3 and ±day 8). The activity of SLE was assessed by the SLEDAI score. Seventeen patients were included. Following MP infusions, the median (range) percentage of eTregs significantly increased from 1.62% (0.53-8.43) at day 0 to 2.80% (0.83-14.60) at day 1 (p = 0.003 versus day 0), 4.64% (0.50-12.40) at day 2 (p = 0.06 versus day 1) and 7.50% (1.02-20.70) at day 3 (p = 0.008 versus day 2), and declined to baseline values at day 8. Expanding eTreg cells were actively proliferating, as they expressed Ki-67. The frequency of non-regulatory FoxP3low T cells decreased from 6.39% (3.20-17.70) at day 0 to 4.74% (1.03-9.72) at day 2 (p = 0.005); nTreg frequency did not change. All patients clinically improved immediately after MP pulses. The absence of flare after one year of follow up was associated with a higher frequency of eTregs at day 2. IV high dose MP induces a rapid, dramatic and transient increase in circulating regulatory T cells. This increase may participate in the preventive effect of MP on subsequent flares in SLE.

Regulatory T Cell Responses to High-Dose Methylprednisolone in Active Systemic Lupus Erythematosus

PubMed Central

Chader, Driss; Cohen-Aubart, Fleur; Haroche, Julien; Fadlallah, Jehane; Claër, Laetitia; Musset, Lucile; Gorochov, Guy; Amoura, Zahir; Miyara, Makoto

2015-01-01

Background/Purpose A slight increase in the proportion of circulating regulatory T (Treg) cells has been reported in systemic lupus erythematosus (SLE) patients taking oral prednisone. The effects of intravenous (IV) high dose methylprednisolone (MP) on Tregs have not yet been described, especially in active SLE. Methods We prospectively analyzed the proportion of circulating CD4+ Treg cell subsets defined as follows: (1) naïve Treg (nTreg) FoxP3lowCD45RA+ cells; (2) effector Treg (eTreg) FoxP3highCD45RA− cells; and (3) non-suppressive FoxP3lowCD45RA− cells (non-regulatory Foxp3low T cells). Peripheral blood mononuclear cells of patients with active SLE were analyzed before the first infusion of IV high dose MP (day 0) and the following days (day 1, day 2, ±day 3 and ±day 8). The activity of SLE was assessed by the SLEDAI score. Results Seventeen patients were included. Following MP infusions, the median (range) percentage of eTregs significantly increased from 1.62% (0.53–8.43) at day 0 to 2.80% (0.83–14.60) at day 1 (p = 0.003 versus day 0), 4.64% (0.50–12.40) at day 2 (p = 0.06 versus day 1) and 7.50% (1.02–20.70) at day 3 (p = 0.008 versus day 2), and declined to baseline values at day 8. Expanding eTreg cells were actively proliferating, as they expressed Ki-67. The frequency of non-regulatory FoxP3low T cells decreased from 6.39% (3.20–17.70) at day 0 to 4.74% (1.03–9.72) at day 2 (p = 0.005); nTreg frequency did not change. All patients clinically improved immediately after MP pulses. The absence of flare after one year of follow up was associated with a higher frequency of eTregs at day 2. Conclusion IV high dose MP induces a rapid, dramatic and transient increase in circulating regulatory T cells. This increase may participate in the preventive effect of MP on subsequent flares in SLE. PMID:26629828

EPA ACTIVITIES TO PREPARE FOR REGULATORY AND RISK ASSESSMENT APPLICATIONS OF GENOMICS INFORMATION

EPA Science Inventory

Genomics will have significant implications for risk assessment and regulatory decision making. Since 2002, the U.S. EPA has undertaken a number of cross-Agency activities to further prepare itself to receive,interpret and apply genomics information for risk assessment and regul...

Antioxidant response elements: Discovery, classes, regulation and potential applications.

PubMed

Raghunath, Azhwar; Sundarraj, Kiruthika; Nagarajan, Raju; Arfuso, Frank; Bian, Jinsong; Kumar, Alan P; Sethi, Gautam; Perumal, Ekambaram

2018-07-01

Exposure to antioxidants and xenobiotics triggers the expression of a myriad of genes encoding antioxidant proteins, detoxifying enzymes, and xenobiotic transporters to offer protection against oxidative stress. This articulated universal mechanism is regulated through the cis-acting elements in an array of Nrf2 target genes called antioxidant response elements (AREs), which play a critical role in redox homeostasis. Though the Keap1/Nrf2/ARE system involves many players, AREs hold the key in transcriptional regulation of cytoprotective genes. ARE-mediated reporter constructs have been widely used, including xenobiotics profiling and Nrf2 activator screening. The complexity of AREs is brought by the presence of other regulatory elements within the AREs. The diversity in the ARE sequences not only bring regulatory selectivity of diverse transcription factors, but also confer functional complexity in the Keap1/Nrf2/ARE pathway. The different transcription factors either homodimerize or heterodimerize to bind the AREs. Depending on the nature of partners, they may activate or suppress the transcription. Attention is required for deeper mechanistic understanding of ARE-mediated gene regulation. The computational methods of identification and analysis of AREs are still in their infancy. Investigations are required to know whether epigenetics mechanism plays a role in the regulation of genes mediated through AREs. The polymorphisms in the AREs leading to oxidative stress related diseases are warranted. A thorough understanding of AREs will pave the way for the development of therapeutic agents against cancer, neurodegenerative, cardiovascular, metabolic and other diseases with oxidative stress. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Immortalization-susceptible elements and their binding factors mediate rejuvenation of regulation of the type I collagenase gene in simian virus 40 large T antigen-transformed immortal human fibroblasts.

PubMed Central

Imai, S; Fujino, T; Nishibayashi, S; Manabe, T; Takano, T

1994-01-01

Dramatic changes occur in expression of the type I collagenase gene during the process of immortalization in simian virus 40 large T antigen-transformed human fibroblasts (S. Imai and T. Takano, Biochem. Biophys. Res. Commun. 189:148-153, 1992). From transient transfection assays, it was determined that these changes involved the functions of two immortalization-susceptible cis-acting elements, ISE1 and ISE2, located in a 100-bp region about 1.7 kb upstream. The profiles of binding of an activator, Proserpine, to the enhancer ISE1 were similar in the extracts of young, senescent preimmortalized and immortalized cells. ISE2 contained both negative and positive regulatory elements located adjacent to each other. The positive regulatory element consisted of a tandem array of putative Ets family- and AP-1-binding sites. An activator, Pluto, interacted with this positive regulatory element and had an AP-1-related component as a complex. The binding activity of Pluto was predominantly detected only in the extract from senescent preimmortalized cells. In contrast, a repressor, Orpheus, which bound to the ATG-rich negative regulatory element of ISE2, was prominently detected in extracts from both young preimmortalized and immortalized cells and appeared to suppress transcription in an orientation-dependent manner. Thus, the interplay of Pluto and Orpheus was suggested to be crucial for regulation of the collagenase gene accompanying in vitro aging and immortalization. Proserpine seemed to interact with Pluto to mediate strong expression of the collagenase gene in cellular senescence. On the basis of these results, we propose a model for regulation of the collagenase gene during in vitro aging and immortalization. Images PMID:7935433

On the Concept of Cis-regulatory Information: From Sequence Motifs to Logic Functions

NASA Astrophysics Data System (ADS)

Tarpine, Ryan; Istrail, Sorin

The regulatory genome is about the “system level organization of the core genomic regulatory apparatus, and how this is the locus of causality underlying the twin phenomena of animal development and animal evolution” (E.H. Davidson. The Regulatory Genome: Gene Regulatory Networks in Development and Evolution, Academic Press, 2006). Information processing in the regulatory genome is done through regulatory states, defined as sets of transcription factors (sequence-specific DNA binding proteins which determine gene expression) that are expressed and active at the same time. The core information processing machinery consists of modular DNA sequence elements, called cis-modules, that interact with transcription factors. The cis-modules “read” the information contained in the regulatory state of the cell through transcription factor binding, “process” it, and directly or indirectly communicate with the basal transcription apparatus to determine gene expression. This endowment of each gene with the information-receiving capacity through their cis-regulatory modules is essential for the response to every possible regulatory state to which it might be exposed during all phases of the life cycle and in all cell types. We present here a set of challenges addressed by our CYRENE research project aimed at studying the cis-regulatory code of the regulatory genome. The CYRENE Project is devoted to (1) the construction of a database, the cis-Lexicon, containing comprehensive information across species about experimentally validated cis-regulatory modules; and (2) the software development of a next-generation genome browser, the cis-Browser, specialized for the regulatory genome. The presentation is anchored on three main computational challenges: the Gene Naming Problem, the Consensus Sequence Bottleneck Problem, and the Logic Function Inference Problem.

A balance of activity in brain control and reward systems predicts self-regulatory outcomes

PubMed Central

Chen, Pin-Hao A.; Huckins, Jeremy F.; Hofmann, Wilhelm; Kelley, William M.; Heatherton, Todd F.

2017-01-01

Abstract Previous neuroimaging work has shown that increased reward-related activity following exposure to food cues is predictive of self-control failure. The balance model suggests that self-regulation failures result from an imbalance in reward and executive control mechanisms. However, an open question is whether the relative balance of activity in brain systems associated with executive control (vs reward) supports self-regulatory outcomes when people encounter tempting cues in daily life. Sixty-nine chronic dieters, a population known for frequent lapses in self-control, completed a food cue-reactivity task during an fMRI scanning session, followed by a weeklong sampling of daily eating behaviors via ecological momentary assessment. We related participants’ food cue activity in brain systems associated with executive control and reward to real-world eating patterns. Specifically, a balance score representing the amount of activity in brain regions associated with self-regulatory control, relative to automatic reward-related activity, predicted dieters’ control over their eating behavior during the following week. This balance measure may reflect individual self-control capacity and be useful for examining self-regulation success in other domains and populations. PMID:28158874

A balance of activity in brain control and reward systems predicts self-regulatory outcomes.

PubMed

Lopez, Richard B; Chen, Pin-Hao A; Huckins, Jeremy F; Hofmann, Wilhelm; Kelley, William M; Heatherton, Todd F

2017-05-01

Previous neuroimaging work has shown that increased reward-related activity following exposure to food cues is predictive of self-control failure. The balance model suggests that self-regulation failures result from an imbalance in reward and executive control mechanisms. However, an open question is whether the relative balance of activity in brain systems associated with executive control (vs reward) supports self-regulatory outcomes when people encounter tempting cues in daily life. Sixty-nine chronic dieters, a population known for frequent lapses in self-control, completed a food cue-reactivity task during an fMRI scanning session, followed by a weeklong sampling of daily eating behaviors via ecological momentary assessment. We related participants' food cue activity in brain systems associated with executive control and reward to real-world eating patterns. Specifically, a balance score representing the amount of activity in brain regions associated with self-regulatory control, relative to automatic reward-related activity, predicted dieters' control over their eating behavior during the following week. This balance measure may reflect individual self-control capacity and be useful for examining self-regulation success in other domains and populations. © The Author (2017). Published by Oxford University Press.

Insights into Structural and Mechanistic Features of Viral IRES Elements

PubMed Central

Martinez-Salas, Encarnacion; Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Embarek, Azman M.

2018-01-01

Internal ribosome entry site (IRES) elements are cis-acting RNA regions that promote internal initiation of protein synthesis using cap-independent mechanisms. However, distinct types of IRES elements present in the genome of various RNA viruses perform the same function despite lacking conservation of sequence and secondary RNA structure. Likewise, IRES elements differ in host factor requirement to recruit the ribosomal subunits. In spite of this diversity, evolutionarily conserved motifs in each family of RNA viruses preserve sequences impacting on RNA structure and RNA–protein interactions important for IRES activity. Indeed, IRES elements adopting remarkable different structural organizations contain RNA structural motifs that play an essential role in recruiting ribosomes, initiation factors and/or RNA-binding proteins using different mechanisms. Therefore, given that a universal IRES motif remains elusive, it is critical to understand how diverse structural motifs deliver functions relevant for IRES activity. This will be useful for understanding the molecular mechanisms beyond cap-independent translation, as well as the evolutionary history of these regulatory elements. Moreover, it could improve the accuracy to predict IRES-like motifs hidden in genome sequences. This review summarizes recent advances on the diversity and biological relevance of RNA structural motifs for viral IRES elements. PMID:29354113

Human Epidermal Langerhans Cells Maintain Immune Homeostasis in Skin by Activating Skin Resident Regulatory T Cells

PubMed Central

Seneschal, Julien; Clark, Rachael A.; Gehad, Ahmed; Baecher-Allan, Clare M.; Kupper, Thomas S.

2013-01-01

Recent discoveries indicate that the skin of a normal individual contains 10-20 billion resident memory T cells ( which include various T helper, T cytotoxic, and T regulatory subsets, that are poised to respond to environmental antigens. Using only autologous human tissues, we report that both in vitro and in vivo, resting epidermal Langerhan cells (LC) selectively and specifically induced the activation and proliferation of skin resident regulatory T cells (Treg), a minor subset of skin resident memory T cells. In the presence of foreign pathogen, however, the same LC activated and induced proliferation of effector memory T (Tem) cells and limited Treg cells activation. These underappreciated properties of LC: namely maintenance of tolerance in normal skin, and activation of protective skin resident memory T cells upon infectious challenge, help clarify the role of LC in skin. PMID:22560445

Early Evolution of Conserved Regulatory Sequences Associated with Development in Vertebrates

PubMed Central

McEwen, Gayle K.; Goode, Debbie K.; Parker, Hugo J.; Woolfe, Adam; Callaway, Heather; Elgar, Greg

2009-01-01

Comparisons between diverse vertebrate genomes have uncovered thousands of highly conserved non-coding sequences, an increasing number of which have been shown to function as enhancers during early development. Despite their extreme conservation over 500 million years from humans to cartilaginous fish, these elements appear to be largely absent in invertebrates, and, to date, there has been little understanding of their mode of action or the evolutionary processes that have modelled them. We have now exploited emerging genomic sequence data for the sea lamprey, Petromyzon marinus, to explore the depth of conservation of this type of element in the earliest diverging extant vertebrate lineage, the jawless fish (agnathans). We searched for conserved non-coding elements (CNEs) at 13 human gene loci and identified lamprey elements associated with all but two of these gene regions. Although markedly shorter and less well conserved than within jawed vertebrates, identified lamprey CNEs are able to drive specific patterns of expression in zebrafish embryos, which are almost identical to those driven by the equivalent human elements. These CNEs are therefore a unique and defining characteristic of all vertebrates. Furthermore, alignment of lamprey and other vertebrate CNEs should permit the identification of persistent sequence signatures that are responsible for common patterns of expression and contribute to the elucidation of the regulatory language in CNEs. Identifying the core regulatory code for development, common to all vertebrates, provides a foundation upon which regulatory networks can be constructed and might also illuminate how large conserved regulatory sequence blocks evolve and become fixed in genomic DNA. PMID:20011110

Self-Regulatory Processes and Exercise Adherence in Older Adults

PubMed Central

McAuley, Edward; Mullen, Sean P.; Szabo, Amanda N.; White, Siobhan M.; Wójcicki, Thomas R.; Mailey, Emily L.; Gothe, Neha P.; Olson, Erin A.; Voss, Michelle; Erickson, Kirk; Prakash, Ruchika; Kramer, Arthur F.

2011-01-01

Background Self-efficacy and the use of self-regulatory strategies are consistently associated with physical activity behavior. Similarly, behavioral inhibition and cognitive resource allocation, indices of executive control function, have also been associated with this health behavior. Purpose The purpose of this study was to examine the hypothesis that self-efficacy mediates the relationship between self-regulatory processes, such as executive function, and sustained exercise behavior. Methods Older adults (N = 177, mean age = 66.44 years) completed measures of executive function, self-reported use of self-regulatory strategies and self-efficacy prior to and during the first month of a 12-month exercise intervention. Percentage of exercise classes attended over the following 11 months was used to represent adherence. Data were collected from 2007 to 2010 and analyzed in 2010–2011. Structural equation models were tested examining the effect of executive function and strategy use on adherence via efficacy. Results As hypothesized, results showed significant direct effects of two elements of executive function and of strategy use on self-efficacy and of efficacy on adherence. In addition, there were significant indirect effects of strategy use and executive function on adherence via self-efficacy. Conclusions Higher levels of executive function and use of self-regulatory strategies at the start of an exercise program enhance beliefs in exercise capabilities, which in turn leads to greater adherence. PMID:21855742

Conserved Noncoding Elements in the Most Distant Genera of Cephalochordates: The Goldilocks Principle

PubMed Central

Yue, Jia-Xing; Kozmikova, Iryna; Ono, Hiroki; Nossa, Carlos W.; Kozmik, Zbynek; Putnam, Nicholas H.; Yu, Jr-Kai; Holland, Linda Z.

2016-01-01

Cephalochordates, the sister group of vertebrates + tunicates, are evolving particularly slowly. Therefore, genome comparisons between two congeners of Branchiostoma revealed so many conserved noncoding elements (CNEs), that it was not clear how many are functional regulatory elements. To more effectively identify CNEs with potential regulatory functions, we compared noncoding sequences of genomes of the most phylogenetically distant cephalochordate genera, Asymmetron and Branchiostoma, which diverged approximately 120–160 million years ago. We found 113,070 noncoding elements conserved between the two species, amounting to 3.3% of the genome. The genomic distribution, target gene ontology, and enriched motifs of these CNEs all suggest that many of them are probably cis-regulatory elements. More than 90% of previously verified amphioxus regulatory elements were re-captured in this study. A search of the cephalochordate CNEs around 50 developmental genes in several vertebrate genomes revealed eight CNEs conserved between cephalochordates and vertebrates, indicating sequence conservation over >500 million years of divergence. The function of five CNEs was tested in reporter assays in zebrafish, and one was also tested in amphioxus. All five CNEs proved to be tissue-specific enhancers. Taken together, these findings indicate that even though Branchiostoma and Asymmetron are distantly related, as they are evolving slowly, comparisons between them are likely optimal for identifying most of their tissue-specific cis-regulatory elements laying the foundation for functional characterizations and a better understanding of the evolution of developmental regulation in cephalochordates. PMID:27412606

77 FR 70846 - Regulatory Guide 1.182, “Assessing and Managing Risk Before Maintenance Activities at Nuclear...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-11-27

... Risk Before Maintenance Activities at Nuclear Power Plants'' AGENCY: Nuclear Regulatory Commission... Activities at Nuclear Power Plants,'' published in May 2000. The document is redundant due to the inclusion... Risk Before Maintenance Activities at Nuclear Power Plants,'' published in May 2000. The requirements...

Viral infection upregulates myostatin promoter activity in orange-spotted grouper (Epinephelus coioides)

PubMed Central

Chen, Yi-Tien; Lin, Chao-Fen; Chen, Young-Mao; Lo, Chih-En; Chen, Wan-Erh

2017-01-01

Myostatin is a negative regulator of myogenesis and has been suggested to be an important factor in the development of muscle wasting during viral infection. The objective of this study was to characterize the main regulatory element of the grouper myostatin promoter and to study changes in promoter activity due to viral stimulation. In vitro and in vivo experiments indicated that the E-box E6 is a positive cis-and trans-regulation motif, and an essential binding site for MyoD. In contrast, the E-box E5 is a dominant negative cis-regulatory. The characteristics of grouper myostatin promoter are similar in regulation of muscle growth to that of other species, but mainly through specific regulatory elements. According to these results, we conducted a study to investigate the effect of viral infection on myostatin promoter activity and its regulation. The nervous necrosis virus (NNV) treatment significantly induced myostatin promoter activity. The present study is the first report describing that specific myostatin motifs regulate promoter activity and response to viral infection. PMID:29036192

Viral infection upregulates myostatin promoter activity in orange-spotted grouper (Epinephelus coioides).

PubMed

Chen, Yi-Tien; Lin, Chao-Fen; Chen, Young-Mao; Lo, Chih-En; Chen, Wan-Erh; Chen, Tzong-Yueh

2017-01-01

Myostatin is a negative regulator of myogenesis and has been suggested to be an important factor in the development of muscle wasting during viral infection. The objective of this study was to characterize the main regulatory element of the grouper myostatin promoter and to study changes in promoter activity due to viral stimulation. In vitro and in vivo experiments indicated that the E-box E6 is a positive cis-and trans-regulation motif, and an essential binding site for MyoD. In contrast, the E-box E5 is a dominant negative cis-regulatory. The characteristics of grouper myostatin promoter are similar in regulation of muscle growth to that of other species, but mainly through specific regulatory elements. According to these results, we conducted a study to investigate the effect of viral infection on myostatin promoter activity and its regulation. The nervous necrosis virus (NNV) treatment significantly induced myostatin promoter activity. The present study is the first report describing that specific myostatin motifs regulate promoter activity and response to viral infection.

76 FR 82334 - Self-Regulatory Organizations; Financial Industry Regulatory Authority, Inc.; Notice of Filing of...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-30

... Schedule A to the FINRA By-Laws to adjust the rate of FINRA's Trading Activity Fee (``TAF'') for... Fee (TAF). These fees are used to fund FINRA's regulatory activities, including examinations... adopted the TAF in 2002 as a replacement for an earlier regulatory fee based on trades reported to Nasdaq...

76 FR 24942 - Self-Regulatory Organizations; Financial Industry Regulatory Authority, Inc.; Notice of Filing of...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-03

... Schedule A to the FINRA By-Laws to adjust the rate of FINRA's Trading Activity Fee (``TAF'') for... Fee (TAF). These fees are used to fund FINRA's regulatory activities, including examinations... adopted the TAF in 2002 as a replacement for an earlier regulatory fee based on trades reported to Nasdaq...

Monascus-fermented red mold dioscorea protects mice against alcohol-induced liver injury, whereas its metabolites ankaflavin and monascin regulate ethanol-induced peroxisome proliferator-activated receptor-γ and sterol regulatory element-binding transcription factor-1 expression in HepG2 cells.

PubMed

Cheng, Chih-Fu; Pan, Tzu-Ming

2018-03-01

Alcoholic hepatitis is a necroinflammatory process that is associated with fibrosis and leads to cirrhosis in 40% of cases. The hepatoprotective effects of red mold dioscorea (RMD) from Monascus purpureus NTU 568 were evaluated in vivo using a mouse model of chronic alcohol-induced liver disease (ALD). ALD mice were orally administered vehicle (ALD group) or vehicle plus 307.5, 615.0 or 1537.5 mg kg -1 (1 ×, 2 × and 5 ×) RMD for 5 weeks. RMD lowered serum leptin, hepatic total cholesterol, free fatty acid and hepatic triglyceride levels and increased serum adiponectin, hepatic alcohol dehydrogenase and antioxidant enzyme levels. Furthermore, ankaflavin (AK) and monascin (MS), metabolites of RMD fermented with M. purpureus 568, induced peroxisome proliferator-activated receptor-γ expression and the concomitant suppression of ethanol-induced elevation of sterol regulatory element-binding transcription factor-1 and TG in HepG2 cells. These results indicate the hepatoprotective effect of Monascus-fermented RMD. Moreover, AK and MS were identified as the active constituents of RMD for the first time and were shown to protect against ethanol-induced liver damage. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Distal regulatory regions restrict the expression of cis-linked genes to the tapetal cells.

PubMed

Franco, Luciana O; de O Manes, Carmem Lara; Hamdi, Said; Sachetto-Martins, Gilberto; de Oliveira, Dulce E

2002-04-24

The oleosin glycine-rich protein genes Atgrp-6, Atgrp-7, and Atgrp-8 occur in clusters in the Arabidopsis genome and are expressed specifically in the tapetum cells. The cis-regulatory regions involved in the tissue-specific gene expression were investigated by fusing different segments of the gene cluster to the uidA reporter gene. Common distal regulatory regions were identified that coordinate expression of the sequential genes. At least two of these genes were regulated spatially by proximal and distal sequences. The cis-acting elements (122 bp upstream of the transcriptional start point) drive the uidA expression to floral tissues, whereas distal 5' upstream regions restrict the gene activity to tapetal cells.

Genetic and epigenetic variation in the lineage specification of regulatory T cells

PubMed Central

Arvey, Aaron; van der Veeken, Joris; Plitas, George; Rich, Stephen S; Concannon, Patrick; Rudensky, Alexander Y

2015-01-01

Regulatory T (Treg) cells, which suppress autoimmunity and other inflammatory states, are characterized by a distinct set of genetic elements controlling their gene expression. However, the extent of genetic and associated epigenetic variation in the Treg cell lineage and its possible relation to disease states in humans remain unknown. We explored evolutionary conservation of regulatory elements and natural human inter-individual epigenetic variation in Treg cells to identify the core transcriptional control program of lineage specification. Analysis of single nucleotide polymorphisms in core lineage-specific enhancers revealed disease associations, which were further corroborated by high-resolution genotyping to fine map causal polymorphisms in lineage-specific enhancers. Our findings suggest that a small set of regulatory elements specify the Treg lineage and that genetic variation in Treg cell-specific enhancers may alter Treg cell function contributing to polygenic disease. DOI: http://dx.doi.org/10.7554/eLife.07571.001 PMID:26510014

cis- and trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis.

PubMed

Wahli, W; Martinez, E; Corthésy, B; Cardinaux, J R

1989-01-01

Vitellogenin genes are expressed under strict estrogen control in the liver of female oviparous vertebrates. Gene transfer experiments using estrogen-responsive cells have shown that the 13 bp perfect palindromic element GGTCACTGTGACC found upstream of the Xenopus laevis vitellogenin gene A2 promoter mediates hormonal stimulation and thus, was called the estrogen-responsive element (ERE). In the Xenopus vitellogenin genes B1 and B2 there are two closely adjacent EREs with one or more base substitutions when compared to the consensus ERE GGTCANNNTGACC. On their own, these degenerated elements have only a low or no regulatory capacity at all but act together synergistically to form an estrogen-responsive unit (ERU) with the same strength as the perfect palindromic 13 bp element. Analysis of estrogen receptor binding to the gene B1 ERU revealed a cooperative interaction of receptor dimers to the two adjacent imperfect EREs which most likely explains the synergistic stimulation observed in vivo. Furthermore, a promoter activator element located between positions --113 and --42 of the gene B1 and functional in the human MCF-7 and the Xenopus B3.2 cells has been identified and shown to be involved in the high level of induced transcription activity when the ERE is placed at a distance from the promoter. Finally, a hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to characterize two additional novel cis-acting elements within the vitellogenin gene B1 promoter. One of them, a negative regulatory element (NRE), is responsible for repression of promoter activity in the absence of hormone. The second is related to the NF-I binding site and is required, together with the ERE, to mediate hormonal induction. Moreover, we detected three trans-acting activities in Xenopus liver nuclear extracts that interact with these regions and demonstrated that they participate in the regulation of the expression of the vitellogenin

On the role of sparseness in the evolution of modularity in gene regulatory networks

PubMed Central

2018-01-01

Modularity is a widespread property in biological systems. It implies that interactions occur mainly within groups of system elements. A modular arrangement facilitates adjustment of one module without perturbing the rest of the system. Therefore, modularity of developmental mechanisms is a major factor for evolvability, the potential to produce beneficial variation from random genetic change. Understanding how modularity evolves in gene regulatory networks, that create the distinct gene activity patterns that characterize different parts of an organism, is key to developmental and evolutionary biology. One hypothesis for the evolution of modules suggests that interactions between some sets of genes become maladaptive when selection favours additional gene activity patterns. The removal of such interactions by selection would result in the formation of modules. A second hypothesis suggests that modularity evolves in response to sparseness, the scarcity of interactions within a system. Here I simulate the evolution of gene regulatory networks and analyse diverse experimentally sustained networks to study the relationship between sparseness and modularity. My results suggest that sparseness alone is neither sufficient nor necessary to explain modularity in gene regulatory networks. However, sparseness amplifies the effects of forms of selection that, like selection for additional gene activity patterns, already produce an increase in modularity. That evolution of new gene activity patterns is frequent across evolution also supports that it is a major factor in the evolution of modularity. That sparseness is widespread across gene regulatory networks indicates that it may have facilitated the evolution of modules in a wide variety of cases. PMID:29775459

On the role of sparseness in the evolution of modularity in gene regulatory networks.

PubMed

Espinosa-Soto, Carlos

2018-05-01

Modularity is a widespread property in biological systems. It implies that interactions occur mainly within groups of system elements. A modular arrangement facilitates adjustment of one module without perturbing the rest of the system. Therefore, modularity of developmental mechanisms is a major factor for evolvability, the potential to produce beneficial variation from random genetic change. Understanding how modularity evolves in gene regulatory networks, that create the distinct gene activity patterns that characterize different parts of an organism, is key to developmental and evolutionary biology. One hypothesis for the evolution of modules suggests that interactions between some sets of genes become maladaptive when selection favours additional gene activity patterns. The removal of such interactions by selection would result in the formation of modules. A second hypothesis suggests that modularity evolves in response to sparseness, the scarcity of interactions within a system. Here I simulate the evolution of gene regulatory networks and analyse diverse experimentally sustained networks to study the relationship between sparseness and modularity. My results suggest that sparseness alone is neither sufficient nor necessary to explain modularity in gene regulatory networks. However, sparseness amplifies the effects of forms of selection that, like selection for additional gene activity patterns, already produce an increase in modularity. That evolution of new gene activity patterns is frequent across evolution also supports that it is a major factor in the evolution of modularity. That sparseness is widespread across gene regulatory networks indicates that it may have facilitated the evolution of modules in a wide variety of cases.

Integrator element as a promoter of active learning in engineering teaching

NASA Astrophysics Data System (ADS)

Oliveira, Paulo C.; Oliveira, Cristina G.

2014-03-01

In this paper, we present a teaching proposal used in an Introductory Physics course to civil engineering students from Porto's Engineering Institute/Instituto Superior de Engenharia do Porto (ISEP). The proposal was born from the need to change students' perception and motivation for learning physics. It consists in the use of an integrator element, called the physics elevator project. This integrator element allows us to use, in a single project, all the content taught in the course and uses several active learning strategies. In this paper, we analyse this project as: (i) a clarifying element of the contents covered in the course; (ii) a promoter element of motivation and active participation in class and finally and (iii) a link between the contents covered in the course and the 'real world'. The data were collected by a questionnaire and interviews to students. From the data collected, it seems that the integrator element improves students' motivation towards physics and develops several skills that they consider to be important to their professional future. It also acts as a clarifying element and makes the connection between the physics that is taught and the 'real world'.

Role of Oxygen as Surface-Active Element in Linear GTA Welding Process

NASA Astrophysics Data System (ADS)

Yadaiah, Nirsanametla; Bag, Swarup

2013-11-01

Although the surface-active elements such as oxygen and sulfur have an adverse effect on momentum transport in liquid metals during fusion welding, such elements can be used beneficially up to a certain limit to increase the weld penetration in the gas tungsten arc (GTA) welding process. The fluid flow pattern and consequently the weld penetration and width change due to a change in coefficient of surface tension from a negative value to a positive value. The present work is focused on the analysis of possible effects of surface-active elements to change the weld pool dimensions in linear GTA welding. A 3D finite element-based heat transfer and fluid flow model is developed to study the effect of surface-active elements on stainless steel plates. A velocity in the order of 180 mm/s due to surface tension force is estimated at an optimum concentration of surface-active elements. Further, the differential evolution-based global optimization algorithm is integrated with the numerical model to estimate uncertain model parameters such as arc efficiency, effective arc radius, and effective values of material properties at high temperatures. The effective values of thermal conductivity and viscosity are estimated to be enhanced nine and seven times, respectively, over corresponding room temperature values. An error analysis is also performed to find out the overall reliability of the computed results, and a maximum reliability of 0.94 is achieved.

Regulatory behavior and frontal activity: Considering the role of revised-BIS in relative right frontal asymmetry.

PubMed

Gable, Philip A; Neal, Lauren B; Threadgill, A Hunter

2018-01-01

Essential to human behavior are three core personality systems: approach, avoidance, and a regulatory system governing the two motivational systems. Decades of research has linked approach motivation with greater relative left frontal-cortical asymmetry. Other research has linked avoidance motivation with greater relative right frontal-cortical asymmetry. However, past work linking withdrawal motivation with greater relative right frontal asymmetry has been mixed. The current article reviews evidence suggesting that activation of the regulatory system (revised Behavioral Inhibition System [r-BIS]) may be more strongly related to greater relative right frontal asymmetry than withdrawal motivation. Specifically, research suggests that greater activation of the r-BIS is associated with greater relative right frontal activity, and reduced r-BIS activation is associated with reduced right frontal activity (greater relative left frontal activity). We review evidence examining trait and state frontal activity using EEG, source localization, lesion studies, neuronal stimulation, and fMRI supporting the idea that r-BIS may be the core personality system related to greater relative right frontal activity. In addition, the current review seeks to disentangle avoidance motivation and r-BIS as substrates of relative right frontal asymmetry. © 2017 Society for Psychophysiological Research.

Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus

PubMed Central

Smith, Emily M.; Lajoie, Bryan R.; Jain, Gaurav; Dekker, Job

2016-01-01

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation. PMID:26748519

APG: an Active Protein-Gene network model to quantify regulatory signals in complex biological systems.

PubMed

Wang, Jiguang; Sun, Yidan; Zheng, Si; Zhang, Xiang-Sun; Zhou, Huarong; Chen, Luonan

2013-01-01

Synergistic interactions among transcription factors (TFs) and their cofactors collectively determine gene expression in complex biological systems. In this work, we develop a novel graphical model, called Active Protein-Gene (APG) network model, to quantify regulatory signals of transcription in complex biomolecular networks through integrating both TF upstream-regulation and downstream-regulation high-throughput data. Firstly, we theoretically and computationally demonstrate the effectiveness of APG by comparing with the traditional strategy based only on TF downstream-regulation information. We then apply this model to study spontaneous type 2 diabetic Goto-Kakizaki (GK) and Wistar control rats. Our biological experiments validate the theoretical results. In particular, SP1 is found to be a hidden TF with changed regulatory activity, and the loss of SP1 activity contributes to the increased glucose production during diabetes development. APG model provides theoretical basis to quantitatively elucidate transcriptional regulation by modelling TF combinatorial interactions and exploiting multilevel high-throughput information.

APG: an Active Protein-Gene Network Model to Quantify Regulatory Signals in Complex Biological Systems

PubMed Central

Wang, Jiguang; Sun, Yidan; Zheng, Si; Zhang, Xiang-Sun; Zhou, Huarong; Chen, Luonan

2013-01-01

Synergistic interactions among transcription factors (TFs) and their cofactors collectively determine gene expression in complex biological systems. In this work, we develop a novel graphical model, called Active Protein-Gene (APG) network model, to quantify regulatory signals of transcription in complex biomolecular networks through integrating both TF upstream-regulation and downstream-regulation high-throughput data. Firstly, we theoretically and computationally demonstrate the effectiveness of APG by comparing with the traditional strategy based only on TF downstream-regulation information. We then apply this model to study spontaneous type 2 diabetic Goto-Kakizaki (GK) and Wistar control rats. Our biological experiments validate the theoretical results. In particular, SP1 is found to be a hidden TF with changed regulatory activity, and the loss of SP1 activity contributes to the increased glucose production during diabetes development. APG model provides theoretical basis to quantitatively elucidate transcriptional regulation by modelling TF combinatorial interactions and exploiting multilevel high-throughput information. PMID:23346354

The Yersinia pestis gcvB gene encodes two small regulatory RNA molecules

PubMed Central

McArthur, Sarah D; Pulvermacher, Sarah C; Stauffer, George V

2006-01-01

Background In recent years it has become clear that small non-coding RNAs function as regulatory elements in bacterial virulence and bacterial stress responses. We tested for the presence of the small non-coding GcvB RNAs in Y. pestis as possible regulators of gene expression in this organism. Results In this study, we report that the Yersinia pestis KIM6 gcvB gene encodes two small RNAs. Transcription of gcvB is activated by the GcvA protein and repressed by the GcvR protein. The gcvB-encoded RNAs are required for repression of the Y. pestis dppA gene, encoding the periplasmic-binding protein component of the dipeptide transport system, showing that the GcvB RNAs have regulatory activity. A deletion of the gcvB gene from the Y. pestis KIM6 chromosome results in a decrease in the generation time of the organism as well as a change in colony morphology. Conclusion The results of this study indicate that the Y. pestis gcvB gene encodes two small non-coding regulatory RNAs that repress dppA expression. A gcvB deletion is pleiotropic, suggesting that the sRNAs are likely involved in controlling genes in addition to dppA. PMID:16768793

Cooperative action of multiple cis-acting elements is required for N-myc expression in branchial arches: specific contribution of GATA3.

PubMed

Potvin, Eric; Beuret, Laurent; Cadrin-Girard, Jean-François; Carter, Marcelle; Roy, Sophie; Tremblay, Michel; Charron, Jean

2010-11-01

The precise expression of the N-myc proto-oncogene is essential for normal mammalian development, whereas altered N-myc gene regulation is known to be a determinant factor in tumor formation. Using transgenic mouse embryos, we show that N-myc sequences from kb -8.7 to kb +7.2 are sufficient to reproduce the N-myc embryonic expression profile in developing branchial arches and limb buds. These sequences encompass several regulatory elements dispersed throughout the N-myc locus, including an upstream limb bud enhancer, a downstream somite enhancer, a branchial arch enhancer in the second intron, and a negative regulatory element in the first intron. N-myc expression in the limb buds is under the dominant control of the limb bud enhancer. The expression in the branchial arches necessitates the interplay of three regulatory domains. The branchial arch enhancer cooperates with the somite enhancer region to prevent an inhibitory activity contained in the first intron. The characterization of the branchial arch enhancer has revealed a specific role of the transcription factor GATA3 in the regulation of N-myc expression. Together, these data demonstrate that correct N-myc developmental expression is achieved via cooperation of multiple positive and negative regulatory elements.

Sequence and functional characterization of MIRNA164 promoters from Brassica shows copy number dependent regulatory diversification among homeologs.

PubMed

Jain, Aditi; Anand, Saurabh; Singh, Neer K; Das, Sandip

2018-03-12

The impact of polyploidy on functional diversification of cis-regulatory elements is poorly understood. This is primarily on account of lack of well-defined structure of cis-elements and a universal regulatory code. To the best of our knowledge, this is the first report on characterization of sequence and functional diversification of paralogous and homeologous promoter elements associated with MIR164 from Brassica. The availability of whole genome sequence allowed us to identify and isolate a total of 42 homologous copies of MIR164 from diploid species-Brassica rapa (A-genome), Brassica nigra (B-genome), Brassica oleracea (C-genome), and allopolyploids-Brassica juncea (AB-genome), Brassica carinata (BC-genome) and Brassica napus (AC-genome). Additionally, we retrieved homologous sequences based on comparative genomics from Arabidopsis lyrata, Capsella rubella, and Thellungiella halophila, spanning ca. 45 million years of evolutionary history of Brassicaceae. Sequence comparison across Brassicaceae revealed lineage-, karyotype, species-, and sub-genome specific changes providing a snapshot of evolutionary dynamics of miRNA promoters in polyploids. Tree topology of cis-elements associated with MIR164 was found to re-capitulate the species and family evolutionary history. Phylogenetic shadowing identified transcription factor binding sites (TFBS) conserved across Brassicaceae, of which, some are already known as regulators of MIR164 expression. Some of the TFBS were found to be distributed in a sub-genome specific (e.g., SOX specific to promoter of MIR164c from MF2 sub-genome), lineage-specific (YABBY binding motif, specific to C. rubella in MIR164b), or species-specific (e.g., VOZ in A. thaliana MIR164a) manner which might contribute towards genetic and adaptive variation. Reporter activity driven by promoters associated with MIR164 paralogs and homeologs was majorly in agreement with known role of miR164 in leaf shaping, regulation of lateral root development and

cis-Regulatory control of the initial neurogenic pattern of onecut gene expression in the sea urchin embryo.

PubMed

Barsi, Julius C; Davidson, Eric H

2016-01-01

Specification of the ciliated band (CB) of echinoid embryos executes three spatial functions essential for postgastrular organization. These are establishment of a band about 5 cells wide which delimits and bounds other embryonic territories; definition of a neurogenic domain within this band; and generation within it of arrays of ciliary cells that bear the special long cilia from which the structure derives its name. In Strongylocentrotus purpuratus the spatial coordinates of the future ciliated band are initially and exactly determined by the disposition of a ring of cells that transcriptionally activate the onecut homeodomain regulatory gene, beginning in blastula stage, long before the appearance of the CB per se. Thus the cis-regulatory apparatus that governs onecut expression in the blastula directly reveals the genomic sequence code by which these aspects of the spatial organization of the embryo are initially determined. We screened the entire onecut locus and its flanking region for transcriptionally active cis-regulatory elements, and by means of BAC recombineered deletions identified three separated and required cis-regulatory modules that execute different functions. The operating logic of the crucial spatial control module accounting for the spectacularly precise and beautiful early onecut expression domain depends on spatial repression. Previously predicted oral ectoderm and aboral ectoderm repressors were identified by cis-regulatory mutation as the products of goosecoid and irxa genes respectively, while the pan-ectodermal activator SoxB1 supplies a transcriptional driver function. Copyright © 2015. Published by Elsevier Inc.

Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor 5 expression

PubMed Central

Wei, Zhiquan; Yan, Li; Chen, Yixin; Bao, Chuanhong; Deng, Jing; Deng, Jiagang

2016-01-01

Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP-1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP-1 cells were differentiated to macrophages by phorbol 12-myristate 13-acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon-γ (IFN-γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription-quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme-linked immunosorbent assay. IRF5 protein and nuclei co-localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN-γ stimulation-induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels. PMID:27277156

Characterization of the evolution of the pharmaceutical regulatory environment.

PubMed

Shafiei, Nader; Ford, James L; Morecroft, Charles W; Lisboa, Paulo J; Taylor, Mark J

2013-01-01

This paper is part of a research study that is intended to identify pharmaceutical quality risks induced by the ongoing transformation in the industry. This study establishes the current regulatory context by characterizing the development of the pharmaceutical regulatory environment. The regulatory environment is one of the most important external factors that affects a company's organization, processes, and technological strategy. This is especially the case with the pharmaceutical industry, where its products affect the quality of life of the consumers. The quantitative analysis of regulatory events since 1813 and review of the associated literature resulted in identification of six factors influencing the regulatory environment, namely public health protection, public health promotion, crisis management, harmonization, innovation, and modernization. From 1813 to the 1970s the focus of regulators was centered on crisis management and public health protection-a basic mission that has remained consistent over the years. Since the 1980s a gradual move in the regulatory environment towards a greater focus on public health promotion, international harmonization, innovation, and agency modernization may be seen. The pharmaceutical industry is currently going through changes that affect the way it performs its research, manufacturing, and regulatory activities. The impact of these changes on the approaches to quality risk management requires more understanding. The authors are engaged in research to identify elements of the changes that influence pharmaceutical quality. As quality requirements are an integral part of the pharmaceutical regulations, a comprehensive understanding of these regulations is seen as the first step. The results of this study show that (i) public health protection, public health promotion, crisis management, harmonization, innovation, and modernization are factors that affect regulations in the pharmaceutical industry; (ii) the regulators' main

Prediction of Geomagnetic Activity and Key Parameters in High-Latitude Ionosphere-Basic Elements

NASA Technical Reports Server (NTRS)

Lyatsky, W.; Khazanov, G. V.

2007-01-01

Prediction of geomagnetic activity and related events in the Earth's magnetosphere and ionosphere is an important task of the Space Weather program. Prediction reliability is dependent on the prediction method and elements included in the prediction scheme. Two main elements are a suitable geomagnetic activity index and coupling function -- the combination of solar wind parameters providing the best correlation between upstream solar wind data and geomagnetic activity. The appropriate choice of these two elements is imperative for any reliable prediction model. The purpose of this work was to elaborate on these two elements -- the appropriate geomagnetic activity index and the coupling function -- and investigate the opportunity to improve the reliability of the prediction of geomagnetic activity and other events in the Earth's magnetosphere. The new polar magnetic index of geomagnetic activity and the new version of the coupling function lead to a significant increase in the reliability of predicting the geomagnetic activity and some key parameters, such as cross-polar cap voltage and total Joule heating in high-latitude ionosphere, which play a very important role in the development of geomagnetic and other activity in the Earth s magnetosphere, and are widely used as key input parameters in modeling magnetospheric, ionospheric, and thermospheric processes.

Regulatory T cells in the control of host-microorganism interactions (*).

PubMed

Belkaid, Yasmine; Tarbell, Kristin

2009-01-01

Each microenvironment requires a specific set of regulatory elements that are finely and constantly tuned to maintain local homeostasis. Various populations of regulatory T cells contribute to the maintenance of this equilibrium and establishment of controlled immune responses. In particular, regulatory T cells limit the magnitude of effector responses, which may result in failure to adequately control infection. However, regulatory T cells also help limit collateral tissue damage caused by vigorous antimicrobial immune responses against pathogenic microbes as well as commensals. In this review, we describe various situations in which the balance between regulatory T cells and effector immune functions influence the outcome of host-microorganism coexistence and discuss current hypotheses and points of polemic associated with the origin, target, and antigen specificity of both endogenous and induced regulatory T cells during these interactions.

The BAFF receptor TACI controls IL-10 production by regulatory B cells and CLL B cells.

PubMed

Saulep-Easton, D; Vincent, F B; Quah, P S; Wei, A; Ting, S B; Croce, C M; Tam, C; Mackay, F

2016-01-01

Interleukin (IL)-10-producing B cells (B10 cells) have emerged as important regulatory elements with immunosuppressive roles. Chronic lymphocytic leukemia (CLL) B cells also secrete IL-10 and share features of B10 cells, suggesting a possible contribution of CLL B cells to immunosuppression in CLL patients. Factors controlling the emergence of B10 cells are not known. B-cell-activating factor of the tumor necrosis factor (TNF) family (BAFF) is critical for B-cell maturation and survival, and is implicated in the development and progression of CLL. We sought to investigate the role of BAFF in the emergence of IL-10-producing regulatory B cells in healthy donors and CLL patients. Here, we report that BAFF signaling promotes IL-10 production by CLL B cells in a mouse model of CLL and in CLL patients. Moreover, BAFF-mediated IL-10 production by normal and CLL B cells is mediated via its receptor transmembrane activator and cyclophilin ligand interactor. Our work uncovered a major targetable pathway important for the generation of regulatory B cells that is detrimental to immunity in CLL.

Cyclosporin A and FK-506 both affect DNA binding of regulatory nuclear proteins to the human interleukin-2 promoter.

PubMed

Baumann, G; Geisse, S; Sullivan, M

1991-03-01

The structurally unrelated immunosuppressive drugs cyclosporin A (Sandimmun) and FK-506 both interfere with the process of T-cell proliferation by blocking the transcription of the T-cell growth factor interleukin-2 (IL-2). Here we demonstrate that the transcriptional activation of this gene requires the binding of regulatory nuclear proteins to a promoter element with sequence similarity to the consensus binding site for NF-kappa B-related transcription factors. We present evidence that the binding by regulatory nuclear proteins to the kappa B element of the IL-2 promoter is affected negatively by cyclosporin A and FK-506 at concentrations paralleling their immunosuppressive activity in vivo. The decrease in DNA-protein complex formation induced by the immunosuppressive drugs correlates with a decrease in IL-2 production. FK-506 is 10 to 100 times more potent than cyclosporin A in its ability to inhibit sequence-specific DNA binding and IL-2 production. Our findings suggest that the actions of both drugs converge at the level of DNA-protein interaction.

EGRINs (Environmental Gene Regulatory Influence Networks) in Rice That Function in the Response to Water Deficit, High Temperature, and Agricultural Environments[OPEN

PubMed Central

Hafemeister, Christoph; Nicotra, Adrienne B.; Jagadish, S.V. Krishna; Bonneau, Richard; Purugganan, Michael

2016-01-01

Environmental gene regulatory influence networks (EGRINs) coordinate the timing and rate of gene expression in response to environmental signals. EGRINs encompass many layers of regulation, which culminate in changes in accumulated transcript levels. Here, we inferred EGRINs for the response of five tropical Asian rice (Oryza sativa) cultivars to high temperatures, water deficit, and agricultural field conditions by systematically integrating time-series transcriptome data, patterns of nucleosome-free chromatin, and the occurrence of known cis-regulatory elements. First, we identified 5447 putative target genes for 445 transcription factors (TFs) by connecting TFs with genes harboring known cis-regulatory motifs in nucleosome-free regions proximal to their transcriptional start sites. We then used network component analysis to estimate the regulatory activity for each TF based on the expression of its putative target genes. Finally, we inferred an EGRIN using the estimated transcription factor activity (TFA) as the regulator. The EGRINs include regulatory interactions between 4052 target genes regulated by 113 TFs. We resolved distinct regulatory roles for members of the heat shock factor family, including a putative regulatory connection between abiotic stress and the circadian clock. TFA estimation using network component analysis is an effective way of incorporating multiple genome-scale measurements into network inference. PMID:27655842

Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp.

PubMed Central

Montgomery, J; Pollard, V; Deikman, J; Fischer, R L

1993-01-01

The tomato fruit consists of a thick, fleshy pericarp composed predominantly of highly vacuolated parenchymatous cells, which surrounds the seeds. During ripening, the activation of gene expression results in dramatic biochemical and physiological changes in the pericarp. The polygalacturonase (PG) gene, unlike many fruit ripening-induced genes, is not activated by the increase in ethylene hormone concentration associated with the onset of ripening. To investigate ethylene concentration-independent gene transcription in ripe tomato fruit, we analyzed the expression of chimeric PG promoter-beta-glucuronidase (GUS) reporter gene fusions in transgenic tomato plants. We determined that a 1.4-kb PG promoter directs ripening-regulated transcription in outer pericarp but not in inner pericarp cells, with a sharp boundary of PG promoter activity located midway through the pericarp. Promoter deletion analysis indicated that a minimum of three promoter regions influence the spatial regulation of PG transcription. A positive regulatory region from -231 to -134 promotes gene transcription in the outer pericarp of ripe fruit. A second positive regulatory region from -806 to -443 extends gene activity to the inner pericarp. However, a negative regulatory region from -1411 to -1150 inhibits gene transcription in the inner pericarp. DNase I footprint analysis showed that nuclear proteins in unripe and ripe fruit interact with DNA sequences within each of these three regulatory regions. Thus, temporal and spatial control of PG transcription is mediated by the interaction of negative and positive regulatory promoter elements, resulting in gene activity in the outer pericarp but not the inner pericarp of ripe tomato fruit. The expression pattern of PG suggests that, although they are morphologically similar, there is a fundamental difference between the parenchymatous cells within the inner and outer pericarp. PMID:8400876

Observation of New Spontaneous Fission Activities from Elements 100 TO 105.

NASA Astrophysics Data System (ADS)

Somerville, Lawrence Patrick

Several new Spontaneous Fission (SF) activities have been found. Their half-lives and production cross sections in several reactions have been measured by collecting and transporting recoils at known speed past mica track detectors. No definite identification could be made for any of the new SF activities; however, half-lives and possible assignments to element-104 isotopes consistent with several cross bombardments include ('257)Rf(3.8 s, 14% SF), ('258)Rf(13 ms), ('259)Rf((TURN)3 s, 8% SF), ('260)Rf((TURN)20 ms), and ('262)Rf((TURN)50ms). The 80-ms SF activity claimed by the Dubna group for the discovery of element 104 (('260)104) was not observed. A difficulty exists in the interpretation that ('260)Rf is a (TURN)20-ms SF activity: in order to be correct, for example, the SF activities with half-lives between 14 and 24 ms produced in the reactions 109- to 119-MeV ('18)O + ('248)Cm, 88- to 100-MeV ('15)N + ('249)Bk, and 96-MeV ('18)O + ('249)Cf must be other nuclides due to their large production cross sections, or the cross sections for production of ('260)Rf must be enhanced by unknown mechanisms. Based on calculated total production cross sections a possible (TURN)1% electron-capture branch in ('258)Lr(4.5 s) to the SF emitter ('258)No(1.2 ms) and an upper limit of 0.05% for SF branching in ('254)No(55 s) were determined. Other measured half-lives from unknown nuclides produced in respective reactions include (TURN)1.6 s (('18)O + ('248)Cm), indications of a (TURN)47-s SF activity (75-MeV ('12)C + ('249)Cf), and two or more SF activities with 3 s (LESSTHEQ) T(, 1/2) (LESSTHEQ) 60 s (('18)O + ('249)Bk). The most exciting conclusion of this work is that if the tentative assignments to even-even element -104 isotopes are correct, there would be a sudden change in the SF half-life systematics at element 104 which has been predicted theoretically by Randrup et al. and Baran et al. and attributed to the disappearance of the second hump of the double-humped fission

DREAM (Downstream Regulatory Element Antagonist Modulator) contributes to synaptic depression and contextual fear memory

PubMed Central

2010-01-01

The downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, binds specifically to DNA and several nucleoproteins regulating gene expression and with proteins outside the nucleus to regulate membrane excitability or calcium homeostasis. DREAM is highly expressed in the central nervous system including the hippocampus and cortex; however, the roles of DREAM in hippocampal synaptic transmission and plasticity have not been investigated. Taking advantage of transgenic mice overexpressing a Ca2+-insensitive DREAM mutant (TgDREAM), we used integrative methods including electrophysiology, biochemistry, immunostaining, and behavior tests to study the function of DREAM in synaptic transmission, long-term plasticity and fear memory in hippocampal CA1 region. We found that NMDA receptor but not AMPA receptor-mediated current was decreased in TgDREAM mice. Moreover, synaptic plasticity, such as long-term depression (LTD) but not long-term potentiation (LTP), was impaired in TgDREAM mice. Biochemical experiments found that DREAM interacts with PSD-95 and may inhibit NMDA receptor function through this interaction. Contextual fear memory was significantly impaired in TgDREAM mice. By contrast, sensory responses to noxious stimuli were not affected. Our results demonstrate that DREAM plays a novel role in postsynaptic modulation of the NMDA receptor, and contributes to synaptic plasticity and behavioral memory. PMID:20205763

TU-AB-204-00: CDRH/FDA Regulatory Processes and Device Science Activities

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

The responsibilities of the Food and Drug Administration (FDA) have increased since the inception of the Food and Drugs Act in 1906. Medical devices first came under comprehensive regulation with the passage of the 1938 Food, Drug, and Cosmetic Act. In 1971 FDA also took on the responsibility for consumer protection against unnecessary exposure to radiation-emitting devices for home and occupational use. However it was not until 1976, under the Medical Device Regulation Act, that the FDA was responsible for the safety and effectiveness of medical devices. This session will be presented by the Division of Radiological Health (DRH) andmore » the Division of Imaging, Diagnostics, and Software Reliability (DIDSR) from the Center for Devices and Radiological Health (CDRH) at the FDA. The symposium will discuss on how we protect and promote public health with a focus on medical physics applications organized into four areas: pre-market device review, post-market surveillance, device compliance, current regulatory research efforts and partnerships with other organizations. The pre-market session will summarize the pathways FDA uses to regulate the investigational use and commercialization of diagnostic imaging and radiation therapy medical devices in the US, highlighting resources available to assist investigators and manufacturers. The post-market session will explain the post-market surveillance and compliance activities FDA performs to monitor the safety and effectiveness of devices on the market. The third session will describe research efforts that support the regulatory mission of the Agency. An overview of our regulatory research portfolio to advance our understanding of medical physics and imaging technologies and approaches to their evaluation will be discussed. Lastly, mechanisms that FDA uses to seek public input and promote collaborations with professional, government, and international organizations, such as AAPM, International Electrotechnical Commission

Activation of interferon regulatory factor-3 via toll-like receptor 3 and immunomodulatory functions detected in A549 lung epithelial cells exposed to misplaced U1-snRNA.

PubMed

Sadik, Christian D; Bachmann, Malte; Pfeilschifter, Josef; Mühl, Heiko

2009-08-01

U1-snRNA is an integral part of the U1 ribonucleoprotein pivotal for pre-mRNA splicing. Toll-like receptor (TLR) signaling has recently been associated with immunoregulatory capacities of U1-snRNA. Using lung A549 epithelial/carcinoma cells, we report for the first time on interferon regulatory factor (IRF)-3 activation initiated by endosomally delivered U1-snRNA. This was associated with expression of the IRF3-inducible genes interferon-beta (IFN-beta), CXCL10/IP-10 and indoleamine 2,3-dioxygenase. Mutational analysis of the U1-snRNA-activated IFN-beta promoter confirmed the crucial role of the PRDIII element, previously proven pivotal for promoter activation by IRF3. Notably, expression of these parameters was suppressed by bafilomycin A(1), an inhibitor of endosomal acidification, implicating endosomal TLR activation. Since resiquimod, an agonist of TLR7/8, failed to stimulate A549 cells, data suggest TLR3 to be of prime relevance for cellular activation. To assess the overall regulatory potential of U1-snRNA-activated epithelial cells on cytokine production, co-cultivation with peripheral blood mononuclear cells (PBMC) was performed. Interestingly, A549 cells activated by U1-snRNA reinforced phytohemagglutinin-induced interleukin-10 release by PBMC but suppressed that of tumor necrosis factor-alpha, indicating an anti-inflammatory potential of U1-snRNA. Since U1-snRNA is enriched in apoptotic bodies and epithelial cells are capable of performing efferocytosis, the present data in particular connect to immunobiological aspects of apoptosis at host/environment interfaces.

Effects of Four Different Regulatory Mechanisms on the Dynamics of Gene Regulatory Cascades

NASA Astrophysics Data System (ADS)

Hansen, Sabine; Krishna, Sandeep; Semsey, Szabolcs; Lo Svenningsen, Sine

2015-07-01

Gene regulatory cascades (GRCs) are common motifs in cellular molecular networks. A given logical function in these cascades, such as the repression of the activity of a transcription factor, can be implemented by a number of different regulatory mechanisms. The potential consequences for the dynamic performance of the GRC of choosing one mechanism over another have not been analysed systematically. Here, we report the construction of a synthetic GRC in Escherichia coli, which allows us for the first time to directly compare and contrast the dynamics of four different regulatory mechanisms, affecting the transcription, translation, stability, or activity of a transcriptional repressor. We developed a biologically motivated mathematical model which is sufficient to reproduce the response dynamics determined by experimental measurements. Using the model, we explored the potential response dynamics that the constructed GRC can perform. We conclude that dynamic differences between regulatory mechanisms at an individual step in a GRC are often concealed in the overall performance of the GRC, and suggest that the presence of a given regulatory mechanism in a certain network environment does not necessarily mean that it represents a single optimal evolutionary solution.

A Hox regulatory network of hindbrain segmentation is conserved to the base of vertebrates.

PubMed

Parker, Hugo J; Bronner, Marianne E; Krumlauf, Robb

2014-10-23

A defining feature governing head patterning of jawed vertebrates is a highly conserved gene regulatory network that integrates hindbrain segmentation with segmentally restricted domains of Hox gene expression. Although non-vertebrate chordates display nested domains of axial Hox expression, they lack hindbrain segmentation. The sea lamprey, a jawless fish, can provide unique insights into vertebrate origins owing to its phylogenetic position at the base of the vertebrate tree. It has been suggested that lamprey may represent an intermediate state where nested Hox expression has not been coupled to the process of hindbrain segmentation. However, little is known about the regulatory network underlying Hox expression in lamprey or its relationship to hindbrain segmentation. Here, using a novel tool that allows cross-species comparisons of regulatory elements between jawed and jawless vertebrates, we report deep conservation of both upstream regulators and segmental activity of enhancer elements across these distant species. Regulatory regions from diverse gnathostomes drive segmental reporter expression in the lamprey hindbrain and require the same transcriptional inputs (for example, Kreisler (also known as Mafba), Krox20 (also known as Egr2a)) in both lamprey and zebrafish. We find that lamprey hox genes display dynamic segmentally restricted domains of expression; we also isolated a conserved exonic hox2 enhancer from lamprey that drives segmental expression in rhombomeres 2 and 4. Our results show that coupling of Hox gene expression to segmentation of the hindbrain is an ancient trait with origin at the base of vertebrates that probably led to the formation of rhombomeric compartments with an underlying Hox code.

Active pixel sensors with substantially planarized color filtering elements

NASA Technical Reports Server (NTRS)

Fossum, Eric R. (Inventor); Kemeny, Sabrina E. (Inventor)

1999-01-01

A semiconductor imaging system preferably having an active pixel sensor array compatible with a CMOS fabrication process. Color-filtering elements such as polymer filters and wavelength-converting phosphors can be integrated with the image sensor.

Gluconacetobacter diazotrophicus PAL5 possesses an active quorum sensing regulatory system.

PubMed

Bertini, Elisa V; Nieto Peñalver, Carlos G; Leguina, Ana C; Irazusta, Verónica P; de Figueroa, Lucía I C

2014-09-01

The endophytic bacterium Gluconacetobacter diazotrophicus colonizes a broad range of host plants. Its plant growth-promoting capability is related to the capacity to perform biological nitrogen fixation, the biosynthesis of siderophores, antimicrobial substances and the solubilization of mineral nutrients. Colonization of and survival in these endophytic niche requires a complex regulatory network. Among these, quorum sensing systems (QS) are signaling mechanisms involved in the control of several genes related to microbial interactions, host colonization and stress survival. G. diazotrophicus PAL5 possesses a QS composed of a luxR and a luxI homolog, and produces eight molecules from the AHL family as QS signals. In this report data are provided showing that glucose concentration modifies the relative levels of these signal molecules. The activity of G. diazotrophicus PAL5 QS is also altered in presence of other carbon sources and under saline stress conditions. Inactivation of the QS system of G. diazotrophicus PAL5 by means of a quorum quenching strategy allowed the identification of extracellular and intracellular proteins under the control of this regulatory mechanism.

Regulatory effects of fisetin on microglial activation.

PubMed

Chuang, Jing-Yuan; Chang, Pei-Chun; Shen, Yi-Chun; Lin, Chingju; Tsai, Cheng-Fang; Chen, Jia-Hong; Yeh, Wei-Lan; Wu, Ling-Hsuan; Lin, Hsiao-Yun; Liu, Yu-Shu; Lu, Dah-Yuu

2014-06-26

Increasing evidence suggests that inflammatory processes in the central nervous system that are mediated by microglial activation play a key role in neurodegeneration. Fisetin, a plant flavonol commonly found in fruits and vegetables, is frequently added to nutritional supplements due to its antioxidant properties. In the present study, treatment with fisetin inhibited microglial cell migration and ROS (reactive oxygen species) production. Treatment with fisetin also effectively inhibited LPS plus IFN-γ-induced nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) expression in microglial cells. Furthermore, fisetin also reduced expressions of iNOS and NO by stimulation of peptidoglycan, the major component of the Gram-positive bacterium cell wall. Fisetin also inhibited the enhancement of LPS/IFN-γ- or peptidoglycan-induced inflammatory mediator IL (interlukin)-1 β expression. Besides the antioxidative and anti-inflammatory effects of fisetin, our study also elucidates the manner in fisetin-induced an endogenous anti-oxidative enzyme HO (heme oxygenase)-1 expression. Moreover, the regulatory molecular mechanism of fisetin-induced HO-1 expression operates through the PI-3 kinase/AKT and p38 signaling pathways in microglia. Notably, fisetin also significantly attenuated inflammation-related microglial activation and coordination deficit in mice in vivo. These findings suggest that fisetin may be a candidate agent for the development of therapies for inflammation-related neurodegenerative diseases.

Phylum-Level Conservation of Regulatory Information in Nematodes despite Extensive Non-coding Sequence Divergence

PubMed Central

Gordon, Kacy L.; Arthur, Robert K.; Ruvinsky, Ilya

2015-01-01

Gene regulatory information guides development and shapes the course of evolution. To test conservation of gene regulation within the phylum Nematoda, we compared the functions of putative cis-regulatory sequences of four sets of orthologs (unc-47, unc-25, mec-3 and elt-2) from distantly-related nematode species. These species, Caenorhabditis elegans, its congeneric C. briggsae, and three parasitic species Meloidogyne hapla, Brugia malayi, and Trichinella spiralis, represent four of the five major clades in the phylum Nematoda. Despite the great phylogenetic distances sampled and the extensive sequence divergence of nematode genomes, all but one of the regulatory elements we tested are able to drive at least a subset of the expected gene expression patterns. We show that functionally conserved cis-regulatory elements have no more extended sequence similarity to their C. elegans orthologs than would be expected by chance, but they do harbor motifs that are important for proper expression of the C. elegans genes. These motifs are too short to be distinguished from the background level of sequence similarity, and while identical in sequence they are not conserved in orientation or position. Functional tests reveal that some of these motifs contribute to proper expression. Our results suggest that conserved regulatory circuitry can persist despite considerable turnover within cis elements. PMID:26020930

Official Methods for the Determination of Minerals and Trace Elements in Infant Formula and Milk Products: A Review.

PubMed

Poitevin, Eric

2016-01-01

The minerals and trace elements that account for about 4% of total human body mass serve as materials and regulators in numerous biological activities in body structure building. Infant formula and milk products are important sources of endogenic and added minerals and trace elements and hence, must comply with regulatory as well as nutritional and safety requirements. In addition, reliable analytical data are necessary to support product content and innovation, health claims, or declaration and specific safety issues. Adequate analytical platforms and methods must be implemented to demonstrate both the compliance and safety assessment of all declared and regulated minerals and trace elements, especially trace-element contaminant surveillance. The first part of this paper presents general information on the mineral composition of infant formula and milk products and their regulatory status. In the second part, a survey describes the main techniques and related current official methods determining minerals and trace elements in infant formula and milk products applied for by various international organizations (AOAC INTERNATIONAL, the International Organization for Standardization, the International Dairy Federation, and the European Committe for Standardization). The third part summarizes method officialization activities by Stakeholder Panels on Infant Formula and Adult Nutritionals and Stakeholder Panel on Strategic Food Analytical Methods. The final part covers a general discussion focusing on analytical gaps and future trends in inorganic analysis that have been applied for in infant formula and milk-based products.

Functional Assessment of Disease-Associated Regulatory Variants In Vivo Using a Versatile Dual Colour Transgenesis Strategy in Zebrafish

PubMed Central

Bhatia, Shipra; Gordon, Christopher T.; Foster, Robert G.; Melin, Lucie; Abadie, Véronique; Baujat, Geneviève; Vazquez, Marie-Paule; Amiel, Jeanne; Lyonnet, Stanislas; van Heyningen, Veronica; Kleinjan, Dirk A.

2015-01-01

Disruption of gene regulation by sequence variation in non-coding regions of the genome is now recognised as a significant cause of human disease and disease susceptibility. Sequence variants in cis-regulatory elements (CREs), the primary determinants of spatio-temporal gene regulation, can alter transcription factor binding sites. While technological advances have led to easy identification of disease-associated CRE variants, robust methods for discerning functional CRE variants from background variation are lacking. Here we describe an efficient dual-colour reporter transgenesis approach in zebrafish, simultaneously allowing detailed in vivo comparison of spatio-temporal differences in regulatory activity between putative CRE variants and assessment of altered transcription factor binding potential of the variant. We validate the method on known disease-associated elements regulating SHH, PAX6 and IRF6 and subsequently characterise novel, ultra-long-range SOX9 enhancers implicated in the craniofacial abnormality Pierre Robin Sequence. The method provides a highly cost-effective, fast and robust approach for simultaneously unravelling in a single assay whether, where and when in embryonic development a disease-associated CRE-variant is affecting its regulatory function. PMID:26030420

Regulatory physiology discipline science plan

NASA Technical Reports Server (NTRS)

1991-01-01

The focus of the Regulatory Physiology discipline of the Space Physiology and Countermeasures Program is twofold. First, to determine and study how microgravity and associated factors of space flight affect the regulatory mechanisms by which humans adapt and achieve homeostasis and thereby regulate their ability to respond to internal and external signals; and, second, to study selected physiological systems that have been demonstrated to be influenced by gravity. The Regulatory Physiology discipline, as defined here, is composed of seven subdisciplines: (1) Circadian Rhythms, (2) Endocrinology, (3) Fluid and Electrolyte Regulation, (4) Hematology, (5) Immunology, (6) Metabolism and Nutrition, and (7) Temperature Regulation. The purpose of this Discipline Science Plan is to provide a conceptual strategy for NASA's Life Sciences Division research and development activities in the area of regulatory physiology. It covers the research areas critical to NASA's programmatic requirements for the Extended-Duration Orbiter, Space Station Freedom, and exploration mission science activities. These science activities include ground-based and flight; basic, applied, and operational; and animal and human research and development. This document summarizes the current status of the program, outlines available knowledge, establishes goals and objectives, identifies science priorities, and defines critical questions in regulatory physiology. It contains a general plan that will be used by both NASA Headquarters Program Offices and the field centers to review and plan basic, applied, and operational intramural and extramural research and development activities in this area.

76 FR 3825 - Regulatory Compliance

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-21

... Compliance Memorandum for the Heads of Executive Departments and Agencies My Administration is committed to... regulatory compliance and enforcement activities, such as information with respect to administrative... compliance information fosters fair and consistent enforcement of important regulatory obligations. Such...

Transterm: a database to aid the analysis of regulatory sequences in mRNAs

PubMed Central

Jacobs, Grant H.; Chen, Augustine; Stevens, Stewart G.; Stockwell, Peter A.; Black, Michael A.; Tate, Warren P.; Brown, Chris M.

2009-01-01

Messenger RNAs, in addition to coding for proteins, may contain regulatory elements that affect how the protein is translated. These include protein and microRNA-binding sites. Transterm (http://mRNA.otago.ac.nz/Transterm.html) is a database of regions and elements that affect translation with two major unique components. The first is integrated results of analysis of general features that affect translation (initiation, elongation, termination) for species or strains in Genbank, processed through a standard pipeline. The second is curated descriptions of experimentally determined regulatory elements that function as translational control elements in mRNAs. Transterm focuses on protein binding sites, particularly those in 3′-untranslated regions (3′-UTR). For this release the interface has been extensively updated based on user feedback. The data is now accessible by strain rather than species, for example there are 10 Escherichia coli strains (genomes) analysed separately. In addition to providing a repository of data, the database also provides tools for users to query their own mRNA sequences. Users can search sequences for Transterm or user defined regulatory elements, including protein or miRNA targets. Transterm also provides a central core of links to related resources for complementary analyses. PMID:18984623

Self-discrepancy and regulatory fit in avatar-based exergames.

PubMed

Jin, Seung-A Annie

2012-12-01

Drawing from Higgins's self-discrepancy theory and regulatory focus theory, this study examined the use of activated selves and regulatory foci in health games. Utilizing the Wii's avatar-creating and exergaming features, a 2 (activated self: actual self versus ideal self) x 2 (regulatory focus: promotion versus prevention) x 2 (efficacy appeals: self-efficacy versus response-efficacy) between-subjects experiment tested the interactions of activated selves, regulatory foci, and efficacy appeals on low-calorie dieting intentions after health game playing. Results from an experiment with 156 participants demonstrated that a fit between regulatory focus and efficacy appeals induced greater dieting intentions when the actual self was activated while the opposite effect occurred when the ideal self was activated. Theoretical contributions to basic and applied social psychology as well as managerial implications for consumer behavior research are considered.

Invariant TAD Boundaries Constrain Cell-Type-Specific Looping Interactions between Promoters and Distal Elements around the CFTR Locus.

PubMed

Smith, Emily M; Lajoie, Bryan R; Jain, Gaurav; Dekker, Job

2016-01-07

Three-dimensional genome structure plays an important role in gene regulation. Globally, chromosomes are organized into active and inactive compartments while, at the gene level, looping interactions connect promoters to regulatory elements. Topologically associating domains (TADs), typically several hundred kilobases in size, form an intermediate level of organization. Major questions include how TADs are formed and how they are related to looping interactions between genes and regulatory elements. Here we performed a focused 5C analysis of a 2.8 Mb chromosome 7 region surrounding CFTR in a panel of cell types. We find that the same TAD boundaries are present in all cell types, indicating that TADs represent a universal chromosome architecture. Furthermore, we find that these TAD boundaries are present irrespective of the expression and looping of genes located between them. In contrast, looping interactions between promoters and regulatory elements are cell-type specific and occur mostly within TADs. This is exemplified by the CFTR promoter that in different cell types interacts with distinct sets of distal cell-type-specific regulatory elements that are all located within the same TAD. Finally, we find that long-range associations between loci located in different TADs are also detected, but these display much lower interaction frequencies than looping interactions within TADs. Interestingly, interactions between TADs are also highly cell-type-specific and often involve loci clustered around TAD boundaries. These data point to key roles of invariant TAD boundaries in constraining as well as mediating cell-type-specific long-range interactions and gene regulation. Copyright © 2016 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Functional analysis of the Arabidopsis PLDZ2 promoter reveals an evolutionarily conserved low-Pi-responsive transcriptional enhancer element

PubMed Central

Oropeza-Aburto, Araceli; Cruz-Ramírez, Alfredo; Acevedo-Hernández, Gustavo J.; Pérez-Torres, Claudia-Anahí; Caballero-Pérez, Juan; Herrera-Estrella, Luis

2012-01-01

Plants have evolved a plethora of responses to cope with phosphate (Pi) deficiency, including the transcriptional activation of a large set of genes. Among Pi-responsive genes, the expression of the Arabidopsis phospholipase DZ2 (PLDZ2) is activated to participate in the degradation of phospholipids in roots in order to release Pi to support other cellular activities. A deletion analysis was performed to identify the regions determining the strength, tissue-specific expression, and Pi responsiveness of this regulatory region. This study also reports the identification and characterization of a transcriptional enhancer element that is present in the PLDZ2 promoter and able to confer Pi responsiveness to a minimal, inactive 35S promoter. This enhancer also shares the cytokinin and sucrose responsive properties observed for the intact PLDZ2 promoter. The EZ2 element contains two P1BS motifs, each of which is the DNA binding site of transcription factor PHR1. Mutation analysis showed that the P1BS motifs present in EZ2 are necessary but not sufficient for the enhancer function, revealing the importance of adjacent sequences. The structural organization of EZ2 is conserved in the orthologous genes of at least eight families of rosids, suggesting that architectural features such as the distance between the two P1BS motifs are also important for the regulatory properties of this enhancer element. PMID:22210906

Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gaur, Naseem Akhtar; Manoharlal, Raman; Saini, Preeti

2005-06-24

Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild typemore » CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.« less

RSAT 2015: Regulatory Sequence Analysis Tools

PubMed Central

Medina-Rivera, Alejandra; Defrance, Matthieu; Sand, Olivier; Herrmann, Carl; Castro-Mondragon, Jaime A.; Delerce, Jeremy; Jaeger, Sébastien; Blanchet, Christophe; Vincens, Pierre; Caron, Christophe; Staines, Daniel M.; Contreras-Moreira, Bruno; Artufel, Marie; Charbonnier-Khamvongsa, Lucie; Hernandez, Céline; Thieffry, Denis; Thomas-Chollier, Morgane; van Helden, Jacques

2015-01-01

RSAT (Regulatory Sequence Analysis Tools) is a modular software suite for the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, appropriate to genome-wide data sets like ChIP-seq, (ii) transcription factor binding motif analysis (quality assessment, comparisons and clustering), (iii) comparative genomics and (iv) analysis of regulatory variations. Nine new programs have been added to the 43 described in the 2011 NAR Web Software Issue, including a tool to extract sequences from a list of coordinates (fetch-sequences from UCSC), novel programs dedicated to the analysis of regulatory variants from GWAS or population genomics (retrieve-variation-seq and variation-scan), a program to cluster motifs and visualize the similarities as trees (matrix-clustering). To deal with the drastic increase of sequenced genomes, RSAT public sites have been reorganized into taxon-specific servers. The suite is well-documented with tutorials and published protocols. The software suite is available through Web sites, SOAP/WSDL Web services, virtual machines and stand-alone programs at http://www.rsat.eu/. PMID:25904632

77 FR 8072 - Semiannual Regulatory Flexibility Agenda

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-13

... six months. ADDRESSES: Comments should be addressed to Jennifer J. Johnson, Secretary of the Board... Regulatory and Deregulatory Actions, which is coordinated by the Office of Management and Budget under... pledged; and certain other elements including a strategic analysis of the company's plans for maintaining...

Physical Activity in the Transition to University: The Role of Past Behavior and Concurrent Self-Regulatory Efficacy

ERIC Educational Resources Information Center

Crozier, Alyson J.; Gierc, Madelaine S. H.; Locke, Sean R.; Brawley, Lawrence R.

2015-01-01

Objective: Two studies were conducted to examine the relationship between past physical activity, concurrent self-regulatory efficacy (CSRE), and current physical activity during the transition to university. Participants: Study 1 included 110 first-year undergraduate students recruited during October/November of 2012. Study 2 involved 86…

Parainfluenza virus chimeric mini-replicons indicate a novel regulatory element in the leader promoter.

PubMed

Matsumoto, Yusuke; Ohta, Keisuke; Goto, Hideo; Nishio, Machiko

2016-07-01

Gene expression of paramyxoviruses is regulated by genome-encoded cis-acting elements; however, whether all the required elements for viral growth have been identified is not clear. Using a mini-replicon system, it has been shown that human parainfluenza virus type 2 (hPIV2) polymerase can recognize the promoter elements of parainfluenza virus type 5 (PIV5), but reporter activity is lower in this case. We constructed a series of luciferase-encoding chimeric PIV2/5 mini-genomes that are basically hPIV2, but whose leader (le), mRNA start signal and trailer sequence are partially replaced with those of PIV5. Studies of the chimeric PIV2/5 mini-replicons demonstrated that replacement of hPIV2 le with PIV5 le results in remarkably weak luciferase expression. Further mutagenesis identified the responsible region as positions 25-30 of the PIV5 le. Using recombinant hPIV2, the impact of this region on viral life cycles was assessed. Insertion of the mutation at this region facilitated viral growth, genomic replication and mRNA transcription at the early stage of infection, which elicited severe cell damage. In contrast, at the late infection stage it caused a reduction in viral transcription. Here, we identify a novel cis-acting element in the internal region of an le sequence that is involved in the regulation of polymerase, and which contributes to maintaining a balance between viral growth and cytotoxicity.

Trace elements and antibacterial activity in amniotic fluid.

PubMed

Honkonen, E; Näntö, V; Hyörä, H; Vuorinen, K; Erkkola, R

1986-01-01

Antibacterial activity and trace element concentrations in amniotic fluid (AF) were determined in a population of 39 pregnant women in the second half of gestation. Antibacterial activity in each AF was measured by a spectrophotometric micromethod after 18 h incubation at 37 degrees C using Escherichia coli K 12 as a reference bacterium. Concentrations of zinc, iron, copper, calcium, potassium and bromine were measured by particle-induced X-ray emission method and the zinc concentration was also measured by atomic absorption spectrophotometry. Phosphate concentration was determined by direct albumin adding method. In AFs with good antibacterial activity significantly lower concentrations of potassium and bromine were found when compared to AFs with lower antibacterial activity. Concentrations of zinc, iron, copper, calcium or phosphate did not correlate with antibacterial activity in AF.

Upstream regulatory elements are necessary and sufficient for transcription of a U6 RNA gene by RNA polymerase III.

PubMed Central

Das, G; Henning, D; Wright, D; Reddy, R

1988-01-01

Whereas the genes coding for trimethyl guanosine-capped snRNAs are transcribed by RNA polymerase II, the U6 RNA genes are transcribed by RNA polymerase III. In this study, we have analyzed the cis-regulatory elements involved in the transcription of a mouse U6 snRNA gene in vitro and in frog oocytes. Transcriptional analysis of mutant U6 gene constructs showed that, unlike most known cases of polymerase III transcription, intragenic sequences except the initiation nucleotide are dispensable for efficient and accurate transcription of U6 gene in vitro. Transcription of 5' deletion mutants in vitro and in frog oocytes showed that the upstream region, within 79 bp from the initiation nucleotide, contains elements necessary for U6 gene transcription. Transcription studies were carried out in frog oocytes with U6 genes containing 5' distal sequence; these studies revealed that the distal element acts as an orientation-dependent enhancer when present upstream to the gene, while it is orientation-independent but distance-dependent enhancer when placed down-stream to the U6 gene. Analysis of 3' deletion mutants showed that the transcription termination of U6 RNA is dependent on a T cluster present on the 3' end of the gene, thus providing further support to other lines of evidence that U6 genes are transcribed by RNA polymerase III. These observations suggest the involvement of a composite of components of RNA polymerase II and III transcription machineries in the transcription of U6 genes by RNA polymerase III. Images PMID:3366121

A role for circadian evening elements in cold-regulated gene expression in Arabidopsis.

PubMed

Mikkelsen, Michael D; Thomashow, Michael F

2009-10-01

The plant transcriptome is dramatically altered in response to low temperature. The cis-acting DNA regulatory elements and trans-acting factors that regulate the majority of cold-regulated genes are unknown. Previous bioinformatic analysis has indicated that the promoters of cold-induced genes are enriched in the Evening Element (EE), AAAATATCT, a DNA regulatory element that has a role in circadian-regulated gene expression. Here we tested the role of EE and EE-like (EEL) elements in cold-induced expression of two Arabidopsis genes, CONSTANS-like 1 (COL1; At5g54470) and a gene encoding a 27-kDa protein of unknown function that we designated COLD-REGULATED GENE 27 (COR27; At5g42900). Mutational analysis indicated that the EE/EEL elements were required for cold induction of COL1 and COR27, and that their action was amplified through coupling with ABA response element (ABRE)-like (ABREL) motifs. An artificial promoter consisting solely of four EE motifs interspersed with three ABREL motifs was sufficient to impart cold-induced gene expression. Both COL1 and COR27 were found to be regulated by the circadian clock at warm growth temperatures and cold-induction of COR27 was gated by the clock. These results suggest that cold- and clock-regulated gene expression are integrated through regulatory proteins that bind to EE and EEL elements supported by transcription factors acting at ABREL sequences. Bioinformatic analysis indicated that the coupling of EE and EEL motifs with ABREL motifs is highly enriched in cold-induced genes and thus may constitute a DNA regulatory element pair with a significant role in configuring the low-temperature transcriptome.

ABO alleles are linked with haplotypes of an erythroid cell-specific regulatory element in intron 1 with a few exceptions attributable to genetic recombination.

PubMed

Nakajima, T; Sano, R; Takahashi, Y; Watanabe, K; Kubo, R; Kobayashi, M; Takahashi, K; Takeshita, H; Kominato, Y

2016-01-01

Recent investigation of transcriptional regulation of the ABO genes has identified a candidate erythroid cell-specific regulatory element, named the +5·8-kb site, in the first intron of ABO. Six haplotypes of the site have been reported previously. The present genetic population study demonstrated that each haplotype was mostly linked with specific ABO alleles with a few exceptions, possibly as a result of hybrid formation between common ABO alleles. Thus, investigation of these haplotypes could provide a clue to further elucidation of ABO alleles. © 2015 International Society of Blood Transfusion.

Genome-Scale Architecture of Small Molecule Regulatory Networks and the Fundamental Trade-Off between Regulation and Enzymatic Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reznik, Ed; Christodoulou, Dimitris; Goldford, Joshua E.

Metabolic flux is in part regulated by endogenous small molecules that modulate the catalytic activity of an enzyme, e.g., allosteric inhibition. In contrast to transcriptional regulation of enzymes, technical limitations have hindered the production of a genome-scale atlas of small molecule-enzyme regulatory interactions. Here, we develop a framework leveraging the vast, but fragmented, biochemical literature to reconstruct and analyze the small molecule regulatory network (SMRN) of the model organism Escherichia coli, including the primary metabolite regulators and enzyme targets. Using metabolic control analysis, we prove a fundamental trade-off between regulation and enzymatic activity, and we combine it with metabolomic measurementsmore » and the SMRN to make inferences on the sensitivity of enzymes to their regulators. By generalizing the analysis to other organisms, we identify highly conserved regulatory interactions across evolutionarily divergent species, further emphasizing a critical role for small molecule interactions in the maintenance of metabolic homeostasis.« less

Genome-Scale Architecture of Small Molecule Regulatory Networks and the Fundamental Trade-Off between Regulation and Enzymatic Activity

DOE PAGES

Reznik, Ed; Christodoulou, Dimitris; Goldford, Joshua E.; ...

2017-09-12

Metabolic flux is in part regulated by endogenous small molecules that modulate the catalytic activity of an enzyme, e.g., allosteric inhibition. In contrast to transcriptional regulation of enzymes, technical limitations have hindered the production of a genome-scale atlas of small molecule-enzyme regulatory interactions. Here, we develop a framework leveraging the vast, but fragmented, biochemical literature to reconstruct and analyze the small molecule regulatory network (SMRN) of the model organism Escherichia coli, including the primary metabolite regulators and enzyme targets. Using metabolic control analysis, we prove a fundamental trade-off between regulation and enzymatic activity, and we combine it with metabolomic measurementsmore » and the SMRN to make inferences on the sensitivity of enzymes to their regulators. By generalizing the analysis to other organisms, we identify highly conserved regulatory interactions across evolutionarily divergent species, further emphasizing a critical role for small molecule interactions in the maintenance of metabolic homeostasis.« less

Hepatic carcinoma-associated fibroblasts induce IDO-producing regulatory dendritic cells through IL-6-mediated STAT3 activation

PubMed Central

Cheng, J-t; Deng, Y-n; Yi, H-m; Wang, G-y; Fu, B-s; Chen, W-j; Liu, W; Tai, Y; Peng, Y-w; Zhang, Q

2016-01-01

Although carcinoma-associated fibroblasts (CAFs) in tumor microenvironments have a critical role in immune cell modulation, their effects on the generation of regulatory dendritic cells (DCs) are still unclear. In this study, we initially show that CAFs derived from hepatocellular carcinoma (HCC) tumors facilitate the generation of regulatory DCs, which are characterized by low expression of costimulatory molecules, high suppressive cytokines production and enhanced regulation of immune responses, including T-cell proliferation impairment and promotion of regulatory T-cell (Treg) expansion via indoleamine 2,3-dioxygenase (IDO) upregulation. Our findings also indicate that STAT3 activation in DCs, as mediated by CAF-derived interleukin (IL)-6, is essential to IDO production. Moreover, IDO inhibitor, STAT3 and IL-6 blocking antibodies can reverse this hepatic CAF-DC regulatory function. Therefore, our results provide new insights into the mechanisms by which CAFs induce tumor immune escape as well as a novel cancer immunotherapeutic approach (for example, targeting CAFs, IDO or IL-6). PMID:26900950

Genome-wide comparative analysis reveals human-mouse regulatory landscape and evolution.

PubMed

Denas, Olgert; Sandstrom, Richard; Cheng, Yong; Beal, Kathryn; Herrero, Javier; Hardison, Ross C; Taylor, James

2015-02-14

Because species-specific gene expression is driven by species-specific regulation, understanding the relationship between sequence and function of the regulatory regions in different species will help elucidate how differences among species arise. Despite active experimental and computational research, relationships among sequence, conservation, and function are still poorly understood. We compared transcription factor occupied segments (TFos) for 116 human and 35 mouse TFs in 546 human and 125 mouse cell types and tissues from the Human and the Mouse ENCODE projects. We based the map between human and mouse TFos on a one-to-one nucleotide cross-species mapper, bnMapper, that utilizes whole genome alignments (WGA). Our analysis shows that TFos are under evolutionary constraint, but a substantial portion (25.1% of mouse and 25.85% of human on average) of the TFos does not have a homologous sequence on the other species; this portion varies among cell types and TFs. Furthermore, 47.67% and 57.01% of the homologous TFos sequence shows binding activity on the other species for human and mouse respectively. However, 79.87% and 69.22% is repurposed such that it binds the same TF in different cells or different TFs in the same cells. Remarkably, within the set of repurposed TFos, the corresponding genome regions in the other species are preferred locations of novel TFos. These events suggest exaptation of some functional regulatory sequences into new function. Despite TFos repurposing, we did not find substantial changes in their predicted target genes, suggesting that CRMs buffer evolutionary events allowing little or no change in the TFos - target gene associations. Thus, the small portion of TFos with strictly conserved occupancy underestimates the degree of conservation of regulatory interactions. We mapped regulatory sequences from an extensive number of TFs and cell types between human and mouse using WGA. A comparative analysis of this correspondence unveiled the

Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing-activated transfer of a mobile genetic element.

PubMed

Ramsay, Joshua P; Tester, Laura G L; Major, Anthony S; Sullivan, John T; Edgar, Christina D; Kleffmann, Torsten; Patterson-House, Jackson R; Hall, Drew A; Tate, Warren P; Hynes, Michael F; Ronson, Clive W

2015-03-31

Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSym(R7A) is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNA(phe) from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSym(R7A), suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSym(R7A)-encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSym(R7A) excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSym(R7A) transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSym(R7A) transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels.

Ribosomal frameshifting and dual-target antiactivation restrict quorum-sensing–activated transfer of a mobile genetic element

PubMed Central

Ramsay, Joshua P.; Tester, Laura G. L.; Major, Anthony S.; Sullivan, John T.; Edgar, Christina D.; Kleffmann, Torsten; Patterson-House, Jackson R.; Hall, Drew A.; Tate, Warren P.; Hynes, Michael F.; Ronson, Clive W.

2015-01-01

Symbiosis islands are integrative and conjugative mobile genetic elements that convert nonsymbiotic rhizobia into nitrogen-fixing symbionts of leguminous plants. Excision of the Mesorhizobium loti symbiosis island ICEMlSymR7A is indirectly activated by quorum sensing through TraR-dependent activation of the excisionase gene rdfS. Here we show that a +1 programmed ribosomal frameshift (PRF) fuses the coding sequences of two TraR-activated genes, msi172 and msi171, producing an activator of rdfS expression named Frameshifted excision activator (FseA). Mass-spectrometry and mutational analyses indicated that the PRF occurred through +1 slippage of the tRNAphe from UUU to UUC within a conserved msi172-encoded motif. FseA activated rdfS expression in the absence of ICEMlSymR7A, suggesting that it directly activated rdfS transcription, despite being unrelated to any characterized DNA-binding proteins. Bacterial two-hybrid and gene-reporter assays demonstrated that FseA was also bound and inhibited by the ICEMlSymR7A-encoded quorum-sensing antiactivator QseM. Thus, activation of ICEMlSymR7A excision is counteracted by TraR antiactivation, ribosomal frameshifting, and FseA antiactivation. This robust suppression likely dampens the inherent biological noise present in the quorum-sensing autoinduction circuit and ensures that ICEMlSymR7A transfer only occurs in a subpopulation of cells in which both qseM expression is repressed and FseA is translated. The architecture of the ICEMlSymR7A transfer regulatory system provides an example of how a set of modular components have assembled through evolution to form a robust genetic toggle that regulates gene transcription and translation at both single-cell and cell-population levels. PMID:25787256

[Bacteriophage λ: electrostatic properties of the genome and its elements].

PubMed

Krutinina, G G; Krutinin, E A; Kamzolova, S G; Osypov, A A

2015-01-01

Bacteriophage λ is a classical model object in molecular biology, but little is still known on the physical properties of its DNA and regulatory elements. A study was made of the electrostatic properties of phage λ DNA and regulatory elements. A global electrostatic potential distribution along the phage genome was found to be nonuniform with main regulatory elements being located in a limited region with a high potential. The RNA polymerase binding frequency on the linearized phage chromosome directly correlates with its local potential. Strong promoters of the phage and its host Escherichia coli have distinct electrostatic upstream elements, which differ in nucleotide sequence. Attachment and recombination sites of phage λ and its host have a higher potential, which possibly facilitates their recognition by integrase. Phage λ and host Rho-independent terminators have a symmetrical M-shaped potential profile, which only slightly depends on the annotated terminator palindrome length, and occur in a region with a substantially higher potential, which may cause polymerase retention, facilitating the formation of a terminator hairpin in RNA. It was concluded that virtually all elements of phage λ genome have potential distribution specifics, which are related to their structural properties and may play a role in their biological function. The global potential distribution along the phage genome reflects the architecture of the regulation of its transcription and integration in the host genome.

Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor 5 expression.

PubMed

Wei, Zhiquan; Yan, Li; Chen, Yixin; Bao, Chuanhong; Deng, Jing; Deng, Jiagang

2016-08-01

Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP‑1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP‑1 cells were differentiated to macrophages by phorbol 12‑myristate 13‑acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon‑γ (IFN‑γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription‑quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme‑linked immunosorbent assay. IRF5 protein and nuclei co‑localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN‑γ stimulation‑induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels.

Binding among select episodic elements is altered via active short-term retrieval.

PubMed

Bridge, Donna J; Voss, Joel L

2015-08-01

Of the many elements that comprise an episode, are any disproportionately bound to the others? We tested whether active short-term retrieval selectively increases binding. Individual objects from multiobject displays were retrieved after brief delays. Memory was later tested for the other objects. Cueing with actively retrieved objects facilitated memory of associated objects, which was associated with unique patterns of viewing behavior during study and enhanced ERP correlates of retrieval during test, relative to other reminder cues that were not actively retrieved. Active short-term retrieval therefore enhanced binding of retrieved elements with others, thus creating powerful memory cues for entire episodes. © 2015 Bridge and Voss; Published by Cold Spring Harbor Laboratory Press.

The diabetes type 1 locus Idd6 modulates activity of CD4+CD25+ regulatory T-cells.

PubMed

Rogner, Ute Christine; Lepault, Françoise; Gagnerault, Marie-Claude; Vallois, David; Morin, Joëlle; Avner, Philip; Boitard, Christian

2006-01-01

The genetic locus Idd6 confers susceptibility to the spontaneous development of type 1 diabetes in the NOD mouse. Our studies on disease resistance of the congenic mouse strain NOD.C3H 6.VIII showed that Idd6 influences T-cell activities in the peripheral immune system and suggest that a major mechanism by which the Idd6 locus modifies diabetes development is via modulation of regulatory T-cell activities. Our transfer experiments using total splenocytes and purified T-cells demonstrated that the locus specifically controls the efficiency of disease protection mediated by the regulatory CD4(+)CD25(+) T-cell subset. Our data also implicate the Idd6 locus in controlling the balance between infiltrating lymphocytes and antigen-presenting cells within the pancreatic islet.

Mechanistic and regulatory aspects of intestinal iron absorption

PubMed Central

Gulec, Sukru; Anderson, Gregory J.

2014-01-01

Iron is an essential trace mineral that plays a number of important physiological roles in humans, including oxygen transport, energy metabolism, and neurotransmitter synthesis. Iron absorption by the proximal small bowel is a critical checkpoint in the maintenance of whole-body iron levels since, unlike most other essential nutrients, no regulated excretory systems exist for iron in humans. Maintaining proper iron levels is critical to avoid the adverse physiological consequences of either low or high tissue iron concentrations, as commonly occurs in iron-deficiency anemia and hereditary hemochromatosis, respectively. Exquisite regulatory mechanisms have thus evolved to modulate how much iron is acquired from the diet. Systemic sensing of iron levels is accomplished by a network of molecules that regulate transcription of the HAMP gene in hepatocytes, thus modulating levels of the serum-borne, iron-regulatory hormone hepcidin. Hepcidin decreases intestinal iron absorption by binding to the iron exporter ferroportin 1 on the basolateral surface of duodenal enterocytes, causing its internalization and degradation. Mucosal regulation of iron transport also occurs during low-iron states, via transcriptional (by hypoxia-inducible factor 2α) and posttranscriptional (by the iron-sensing iron-regulatory protein/iron-responsive element system) mechanisms. Recent studies demonstrated that these regulatory loops function in tandem to control expression or activity of key modulators of iron homeostasis. In health, body iron levels are maintained at appropriate levels; however, in several inherited disorders and in other pathophysiological states, iron sensing is perturbed and intestinal iron absorption is dysregulated. The iron-related phenotypes of these diseases exemplify the necessity of precisely regulating iron absorption to meet body demands. PMID:24994858

2012 Global Summit on Regulatory Science (GSRS-2012)--modernizing toxicology.

PubMed

Miller, Margaret A; Tong, Weida; Fan, Xiaohui; Slikker, William

2013-01-01

Regulatory science encompasses the tools, models, techniques, and studies needed to assess and evaluate product safety, efficacy, quality, and performance. Several recent publications have emphasized the role of regulatory science in improving global health, supporting economic development and fostering innovation. As for other scientific disciplines, research in regulatory science is the critical element underpinning the development and advancement of regulatory science as a modern scientific discipline. As a regulatory agency in the 21st century, the Food and Drug Administration (FDA) has an international component that underpins its domestic mission; foods, drugs, and devices are developed and imported to the United States from across the world. The Global Summit on Regulatory Science, an international conference for discussing innovative technologies, approaches, and partnerships that enhance the translation of basic science into regulatory applications, is providing leadership for the advancement of regulatory sciences within the global context. Held annually, this international conference provides a platform where regulators, policy makers, and bench scientists from various countries can exchange views on how to develop, apply, and implement innovative methodologies into regulatory assessments in their respective countries, as well as developing a harmonized strategy to improve global public health through global collaboration.

The twilight zone of cis element alignments.

PubMed

Sebastian, Alvaro; Contreras-Moreira, Bruno

2013-02-01

Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein-DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein-DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments.

Direct activation of a notochord cis-regulatory module by Brachyury and FoxA in the ascidian Ciona intestinalis.

PubMed

Passamaneck, Yale J; Katikala, Lavanya; Perrone, Lorena; Dunn, Matthew P; Oda-Ishii, Izumi; Di Gregorio, Anna

2009-11-01

The notochord is a defining feature of the chordate body plan. Experiments in ascidian, frog and mouse embryos have shown that co-expression of Brachyury and FoxA class transcription factors is required for notochord development. However, studies on the cis-regulatory sequences mediating the synergistic effects of these transcription factors are complicated by the limited knowledge of notochord genes and cis-regulatory modules (CRMs) that are directly targeted by both. We have identified an easily testable model for such investigations in a 155-bp notochord-specific CRM from the ascidian Ciona intestinalis. This CRM contains functional binding sites for both Ciona Brachyury (Ci-Bra) and FoxA (Ci-FoxA-a). By combining point mutation analysis and misexpression experiments, we demonstrate that binding of both transcription factors to this CRM is necessary and sufficient to activate transcription. To gain insights into the cis-regulatory criteria controlling its activity, we investigated the organization of the transcription factor binding sites within the 155-bp CRM. The 155-bp sequence contains two Ci-Bra binding sites with identical core sequences but opposite orientations, only one of which is required for enhancer activity. Changes in both orientation and spacing of these sites substantially affect the activity of the CRM, as clusters of identical sites found in the Ciona genome with different arrangements are unable to activate transcription in notochord cells. This work presents the first evidence of a synergistic interaction between Brachyury and FoxA in the activation of an individual notochord CRM, and highlights the importance of transcription factor binding site arrangement for its function.

Regulatory affairs and biotechnology in Europe. I. Introduction into good regulatory practice.

PubMed

Tryzelaar, B

1988-01-01

The high cost and risks associated with the research and development of new drugs demand an alert as well as realistic legislative policy at both national and international levels. Registration of a new drug required before a marketing license is granted, is important for all branches of the pharmaceutical industry but is crucial for success in the innovative biotechnological sector. Innovation as such is no guarantee to be profitable. Increasing government demands have introduced uncertainty on whether new products will secure registration and have led to a disproportionate increase in the economical risks for innovative industry. Preparation and submission of an application for registration should be undertaken seriously and professionally since it has significantly more consequences than simply obtaining a marketing licence. It will influence marketing strategies and results. It is proposed--since dealing with regulatory affairs can be considered as an essential specialism--to apply a Quality Assurance approach. Activities in this context should comply with the same performance standards as developed for GMP, GLP and GCP leading to Good Regulatory Practice (GRP). By acknowledging regulatory affairs as a quality assurance means one can define a set of standard procedures within an organization to ensure that decisions are made on current and future regulations. In such a setup regulatory affairs becomes a marketing tool. This paper illustrates the complex problems found in registration activities. It underlines the necessity of introducing a GRP-approach of performance resulting in substantive evidence of regulatory efficacy.

A minimal murine Msx-1 gene promoter. Organization of its cis-regulatory motifs and their role in transcriptional activation in cells in culture and in transgenic mice.

PubMed

Takahashi, T; Guron, C; Shetty, S; Matsui, H; Raghow, R

1997-09-05

To dissect the cis-regulatory elements of the murine Msx-1 promoter, which lacks a conventional TATA element, a putative Msx-1 promoter DNA fragment (from -1282 to +106 base pairs (bp)) or its congeners containing site-specific alterations were fused to luciferase reporter and introduced into NIH3T3 and C2C12 cells, and the expression of luciferase was assessed in transient expression assays. The functional consequences of the sequential 5' deletions of the promotor revealed that multiple positive and negative regulatory elements participate in regulating transcription of the Msx-1 gene. Surprisingly, however, the optimal expression of Msx-1 promoter in either NIH3T3 or C2C12 cells required only 165 bp of the upstream sequence to warrant detailed examination of its structure. Therefore, the functional consequences of site-specific deletions and point mutations of the cis-acting elements of the minimal Msx-1 promoter were systematically examined. Concomitantly, potential transcriptional factor(s) interacting with the cis-acting elements of the minimal promoter were also studied by gel electrophoretic mobility shift assays and DNase I footprinting. Combined analyses of the minimal promoter by DNase I footprinting, electrophoretic mobility shift assays, and super shift assays with specific antibodies revealed that 5'-flanking regions from -161 to -154 and from -26 to -13 of the Msx-1 promoter contains an authentic E box (proximal E box), capable of binding a protein immunologically related to the upstream stimulating factor 1 (USF-1) and a GC-rich sequence motif which can bind to Sp1 (proximal Sp1), respectively. Additionally, we observed that the promoter activation was seriously hampered if the proximal E box was removed or mutated, and the promoter activity was eliminated completely if the proximal Sp1 site was similarly altered. Absolute dependence of the Msx-1 minimal promoter on Sp1 could be demonstrated by transient expression assays in the Sp1-deficient

Targeted Deletion of the Antisilencer/Enhancer (ASE) Element from Intron 1 of the Myelin Proteolipid Protein Gene (Plp1) in Mouse Reveals that the Element Is Dispensable for Plp1 Expression in Brain during Development and Remyelination

PubMed Central

Pereira, Glauber B.; Meng, Fanxue; Kockara, Neriman T.; Yang, Baoli; Wight, Patricia A.

2012-01-01

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. While removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is nonfunctional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene. PMID:23157328

Targeted deletion of the antisilencer/enhancer (ASE) element from intron 1 of the myelin proteolipid protein gene (Plp1) in mouse reveals that the element is dispensable for Plp1 expression in brain during development and remyelination.

PubMed

Pereira, Glauber B; Meng, Fanxue; Kockara, Neriman T; Yang, Baoli; Wight, Patricia A

2013-02-01

Myelin proteolipid protein gene (Plp1) expression is temporally regulated in brain, which peaks during the active myelination period of CNS development. Previous studies with Plp1-lacZ transgenic mice demonstrated that (mouse) Plp1 intron 1 DNA is required for high levels of expression in oligodendrocytes. Deletion-transfection analysis revealed the intron contains a single positive regulatory element operative in the N20.1 oligodendroglial cell line, which was named ASE (antisilencer/enhancer) based on its functional properties in these cells. To investigate the role of the ASE in vivo, the element was deleted from the native gene in mouse using a Cre/lox strategy. Although removal of the ASE from Plp1-lacZ constructs profoundly decreased expression in transfected oligodendroglial cell lines (N20.1 and Oli-neu), the element was dispensable to achieve normal levels of Plp1 gene expression in mouse during development (except perhaps at postnatal day 15) and throughout the remyelination period following cuprizone-induced (acute) demyelination. Thus, it is possible that the ASE is non-functional in vivo, or that loss of the ASE from the native gene in mouse can be compensated for by the presence of other regulatory elements within the Plp1 gene. © 2012 International Society for Neurochemistry.

An Organismal Model for Gene Regulatory Networks in the Gut-Associated Immune Response

PubMed Central

Buckley, Katherine M.; Rast, Jonathan P.

2017-01-01

The gut epithelium is an ancient site of complex communication between the animal immune system and the microbial world. While elements of self-non-self receptors and effector mechanisms differ greatly among animal phyla, some aspects of recognition, regulation, and response are broadly conserved. A gene regulatory network (GRN) approach provides a means to investigate the nature of this conservation and divergence even as more peripheral functional details remain incompletely understood. The sea urchin embryo is an unparalleled experimental model for detangling the GRNs that govern embryonic development. By applying this theoretical framework to the free swimming, feeding larval stage of the purple sea urchin, it is possible to delineate the conserved regulatory circuitry that regulates the gut-associated immune response. This model provides a morphologically simple system in which to efficiently unravel regulatory connections that are phylogenetically relevant to immunity in vertebrates. Here, we review the organism-wide cellular and transcriptional immune response of the sea urchin larva. A large set of transcription factors and signal systems, including epithelial expression of interleukin 17 (IL17), are important mediators in the activation of the early gut-associated response. Many of these have homologs that are active in vertebrate immunity, while others are ancient in animals but absent in vertebrates or specific to echinoderms. This larval model provides a means to experimentally characterize immune function encoded in the sea urchin genome and the regulatory interconnections that control immune response and resolution across the tissues of the organism. PMID:29109720

EFFECT OF MOISTURE ON ADSORPTION OF ELEMENTAL MERCURY BY ACTIVATED CARBON

EPA Science Inventory

The paper discusses experiments using activated carbon to capture elemental mercury (Hgo), and a bench-scale dixed-bed reactor and a flow reactor to determine the role of surface moisture in Hgo adsorption. Three activated-carbon samples, with different pore structure and ash co...

RSAT 2015: Regulatory Sequence Analysis Tools.

PubMed

Medina-Rivera, Alejandra; Defrance, Matthieu; Sand, Olivier; Herrmann, Carl; Castro-Mondragon, Jaime A; Delerce, Jeremy; Jaeger, Sébastien; Blanchet, Christophe; Vincens, Pierre; Caron, Christophe; Staines, Daniel M; Contreras-Moreira, Bruno; Artufel, Marie; Charbonnier-Khamvongsa, Lucie; Hernandez, Céline; Thieffry, Denis; Thomas-Chollier, Morgane; van Helden, Jacques

2015-07-01

RSAT (Regulatory Sequence Analysis Tools) is a modular software suite for the analysis of cis-regulatory elements in genome sequences. Its main applications are (i) motif discovery, appropriate to genome-wide data sets like ChIP-seq, (ii) transcription factor binding motif analysis (quality assessment, comparisons and clustering), (iii) comparative genomics and (iv) analysis of regulatory variations. Nine new programs have been added to the 43 described in the 2011 NAR Web Software Issue, including a tool to extract sequences from a list of coordinates (fetch-sequences from UCSC), novel programs dedicated to the analysis of regulatory variants from GWAS or population genomics (retrieve-variation-seq and variation-scan), a program to cluster motifs and visualize the similarities as trees (matrix-clustering). To deal with the drastic increase of sequenced genomes, RSAT public sites have been reorganized into taxon-specific servers. The suite is well-documented with tutorials and published protocols. The software suite is available through Web sites, SOAP/WSDL Web services, virtual machines and stand-alone programs at http://www.rsat.eu/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Soils element activities for the period October 1973--September 1974

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fowler, E.B.; Essington, E.H.; White, M.G.

Soils Element activities were conducted on behalf of the U. S. Atomic Energy Commission's Nevada Applied Ecology Group (NAEG) program to provide source term information for the other program elements and maintain continuous cognizance of program requirements for sampling, sample preparation, and analysis. Activities included presentation of papers; participation in workshops; analysis of soil, vegetation, and animal tissue samples for $sup 238$Pu, $sup 239-240$Pu, $sup 241$Am, $sup 137$Cs, $sup 60$Co, and gamma scan for routine and laboratory quality control purposes; preparation and analysis of animal tissue samples for NAEG laboratory certification; studies on a number of analytical, sample preparation, andmore » sample collection procedures; and contributions to the evaluation of procedures for calculation of specialized counting statistics. (auth)« less

An ensemble of regulatory elements controls Runx3 spatiotemporal expression in subsets of dorsal root ganglia proprioceptive neurons.

PubMed

Appel, Elena; Weissmann, Sarit; Salzberg, Yehuda; Orlovsky, Kira; Negreanu, Varda; Tsoory, Michael; Raanan, Calanit; Feldmesser, Ester; Bernstein, Yael; Wolstein, Orit; Levanon, Ditsa; Groner, Yoram

2016-12-01

The Runx3 transcription factor is essential for development and diversification of the dorsal root ganglia (DRGs) TrkC sensory neurons. In Runx3-deficient mice, developing TrkC neurons fail to extend central and peripheral afferents, leading to cell death and disruption of the stretch reflex circuit, resulting in severe limb ataxia. Despite its central role, the mechanisms underlying the spatiotemporal expression specificities of Runx3 in TrkC neurons were largely unknown. Here we first defined the genomic transcription unit encompassing regulatory elements (REs) that mediate the tissue-specific expression of Runx3. Using transgenic mice expressing BAC reporters spanning the Runx3 locus, we discovered three REs-dubbed R1, R2, and R3-that cross-talk with promoter-2 (P2) to drive TrkC neuron-specific Runx3 transcription. Deletion of single or multiple elements either in the BAC transgenics or by CRISPR/Cas9-mediated endogenous ablation established the REs' ability to promote and/or repress Runx3 expression in developing sensory neurons. Our analysis reveals that an intricate combinatorial interplay among the three REs governs Runx3 expression in distinct subtypes of TrkC neurons while concomitantly extinguishing its expression in non-TrkC neurons. These findings provide insights into the mechanism regulating cell type-specific expression and subtype diversification of TrkC neurons in developing DRGs. © 2016 Appel et al.; Published by Cold Spring Harbor Laboratory Press.

The Regulatory and Kinase Domains but Not the Interdomain Linker Determine Human Double-stranded RNA-activated Kinase (PKR) Sensitivity to Inhibition by Viral Non-coding RNAs.

PubMed

Sunita, S; Schwartz, Samantha L; Conn, Graeme L

2015-11-20

Double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an important component of the innate immune system that presents a crucial first line of defense against viral infection. PKR has a modular architecture comprising a regulatory N-terminal dsRNA binding domain and a C-terminal kinase domain interposed by an unstructured ∼80-residue interdomain linker (IDL). Guided by sequence alignment, we created IDL deletions in human PKR (hPKR) and regulatory/kinase domain swap human-rat chimeric PKRs to assess the contributions of each domain and the IDL to regulation of the kinase activity by RNA. Using circular dichroism spectroscopy, limited proteolysis, kinase assays, and isothermal titration calorimetry, we show that each PKR protein is properly folded with similar domain boundaries and that each exhibits comparable polyinosinic-cytidylic (poly(rI:rC)) dsRNA activation profiles and binding affinities for adenoviral virus-associated RNA I (VA RNAI) and HIV-1 trans-activation response (TAR) RNA. From these results we conclude that the IDL of PKR is not required for RNA binding or mediating changes in protein conformation or domain interactions necessary for PKR regulation by RNA. In contrast, inhibition of rat PKR by VA RNAI and TAR RNA was found to be weaker than for hPKR by 7- and >300-fold, respectively, and each human-rat chimeric domain-swapped protein showed intermediate levels of inhibition. These findings indicate that PKR sequence or structural elements in the kinase domain, present in hPKR but absent in rat PKR, are exploited by viral non-coding RNAs to accomplish efficient inhibition of PKR. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Development and validation of an ICP-MS method for the determination of elemental impurities in TP-6076 active pharmaceutical ingredient (API) according to USP 〈232〉/〈233〉.

PubMed

Chahrour, Osama; Malone, John; Collins, Mark; Salmon, Vrushali; Greenan, Catherine; Bombardier, Amy; Ma, Zhongze; Dunwoody, Nick

2017-10-25

The new guidelines of the United States pharmacopeia (USP), European pharmacopeia (EP) and international conference on harmonization (ICH) regulating elemental impurities limits in pharmaceuticals signify the end of unspecific analysis of metals as outlined in USP 〈231〉. The new guidelines specify both daily doses and concentration/limits of elemental impurities in pharmaceutical final products, active pharmaceutical ingredients (API) and excipients. In chapter USP 〈233〉 method implementation, validation and quality control during the analytical process are described. We herein report the use of a stabilising matrix that overcomes low spike recovery problem encountered with Os and allows the determination of all USP required elemental impurities (As, Cd, Hg, Pb, V, Cr, Ni, Mo, Cu, Pt, Pd, Ru, Rh, Os and Ir) in a single analysis. The matrix was used in the validation of a method to determine elemental impurities in TP-6076 active pharmaceutical ingredient (API) by ICP-MS according to the procedures defined in USP〈233〉 and to GMP requirements. This validation will support the regulatory submission of TP-6076 which is a novel tetracycline analogue effective against the most urgent multidrug-resistant gram-negative bacteria. Evaluation of TP-6076 in IND-enabling toxicology studies has led to the initiation of a phase 1 clinical trial. Copyright © 2017 Elsevier B.V. All rights reserved.

76 FR 40092 - Department Regulatory Agenda; Semiannual Summary

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-07

... Department. The agenda provides the public with information about the Department of Transportation's... allow it to more effectively participate in the Department's regulatory activity. The public is also... of Transportation's regulatory activities online, go to http://regs.dot.gov . Among other things...

ERK-MAPK drives lamellipodia protrusion by activating the WAVE2 regulatory complex.

PubMed

Mendoza, Michelle C; Er, E Emrah; Zhang, Wenjuan; Ballif, Bryan A; Elliott, Hunter L; Danuser, Gaudenz; Blenis, John

2011-03-18

Cell movement begins with a leading edge protrusion, which is stabilized by nascent adhesions and retracted by mature adhesions. The ERK-MAPK (extracellular signal-regulated kinase-mitogen-activated protein kinase) localizes to protrusions and adhesions, but how it regulates motility is not understood. We demonstrate that ERK controls protrusion initiation and protrusion speed. Lamellipodial protrusions are generated via the WRC (WAVE2 regulatory complex), which activates the Arp2/3 actin nucleator for actin assembly. The WRC must be phosphorylated to be activated, but the sites and kinases that regulate its intermolecular changes and membrane recruitment are unknown. We show that ERK colocalizes with the WRC at lamellipodial leading edges and directly phosphorylates two WRC components: WAVE2 and Abi1. The phosphorylations are required for functional WRC interaction with Arp2/3 and actin during cell protrusion. Thus, ERK coordinates adhesion disassembly with WRC activation and actin polymerization to promote productive leading edge advancement during cell migration. Copyright © 2011 Elsevier Inc. All rights reserved.

ERK-MAPK Drives Lamellipodia Protrusion by Activating the WAVE2 Regulatory Complex

PubMed Central

Mendoza, Michelle C.; Emrah, E.; Zhang, Wenjuan; Ballif, Bryan A.; Elliott, Hunter L.; Danuser, Gaudenz; Blenis, John

2011-01-01

Summary Cell movement begins with a leading edge protrusion, which is stabilized by nascent adhesions and retracted by mature adhesions. The ERK-MAPK (extracellular signal regulated kinasemitogen-activated protein kinase) localizes to protrusions and adhesions, but how it regulates motility is not understood. We demonstrate ERK controls protrusion initiation and protrusion speed. Lamellipodial protrusions are generated via the WRC (WAVE2 Regulatory Complex), which activates the Arp2/3 actin nucleator for actin assembly. The WRC must be phosphorylated to be activated, but the sites and kinases that regulate its intermolecular changes and membrane recruitment are unknown. We show ERK co-localizes with the WRC at lamellipodial leading edges and directly phosphorylates two WRC components: WAVE2 and Abi1. The phosphorylations are required for functional WRC interaction with Arp2/3 and actin during cell protrusion. Thus, ERK coordinates adhesion disassembly with WRC activation and actin polymerization to promote productive leading edge advancement during cell migration. PMID:21419341

Transcriptional activation of short interspersed elements by DNA-damaging agents.

PubMed

Rudin, C M; Thompson, C B

2001-01-01

Short interspersed elements (SINEs), typified by the human Alu repeat, are RNA polymerase III (pol III)-transcribed sequences that replicate within the genome through an RNA intermediate. Replication of SINEs has been extensive in mammalian evolution: an estimated 5% of the human genome consists of Alu repeats. The mechanisms regulating transcription, reverse transcription, and reinsertion of SINE elements in genomic DNA are poorly understood. Here we report that expression of murine SINE transcripts of both the B1 and B2 classes is strongly upregulated after prolonged exposure to cisplatin, etoposide, or gamma radiation. A similar induction of Alu transcripts in human cells occurs under these conditions. This induction is not due to a general upregulation of pol III activity in either species. Genotoxic treatment of murine cells containing an exogenous human Alu element induced Alu transcription. Concomitant with the increased expression of SINEs, an increase in cellular reverse transcriptase was observed after exposure to these same DNA-damaging agents. These findings suggest that genomic damage may be an important activator of SINEs, and that SINE mobility may contribute to secondary malignancy after exposure to DNA-damaging chemotherapy.

A system-level model for the microbial regulatory genome.

PubMed

Brooks, Aaron N; Reiss, David J; Allard, Antoine; Wu, Wei-Ju; Salvanha, Diego M; Plaisier, Christopher L; Chandrasekaran, Sriram; Pan, Min; Kaur, Amardeep; Baliga, Nitin S

2014-07-15

Microbes can tailor transcriptional responses to diverse environmental challenges despite having streamlined genomes and a limited number of regulators. Here, we present data-driven models that capture the dynamic interplay of the environment and genome-encoded regulatory programs of two types of prokaryotes: Escherichia coli (a bacterium) and Halobacterium salinarum (an archaeon). The models reveal how the genome-wide distributions of cis-acting gene regulatory elements and the conditional influences of transcription factors at each of those elements encode programs for eliciting a wide array of environment-specific responses. We demonstrate how these programs partition transcriptional regulation of genes within regulons and operons to re-organize gene-gene functional associations in each environment. The models capture fitness-relevant co-regulation by different transcriptional control mechanisms acting across the entire genome, to define a generalized, system-level organizing principle for prokaryotic gene regulatory networks that goes well beyond existing paradigms of gene regulation. An online resource (http://egrin2.systemsbiology.net) has been developed to facilitate multiscale exploration of conditional gene regulation in the two prokaryotes. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Microarray and comparative genomics-based identification of genes and gene regulatory regions of the mouse immune system

PubMed Central

Hutton, John J; Jegga, Anil G; Kong, Sue; Gupta, Ashima; Ebert, Catherine; Williams, Sarah; Katz, Jonathan D; Aronow, Bruce J

2004-01-01

Background In this study we have built and mined a gene expression database composed of 65 diverse mouse tissues for genes preferentially expressed in immune tissues and cell types. Using expression pattern criteria, we identified 360 genes with preferential expression in thymus, spleen, peripheral blood mononuclear cells, lymph nodes (unstimulated or stimulated), or in vitro activated T-cells. Results Gene clusters, formed based on similarity of expression-pattern across either all tissues or the immune tissues only, had highly significant associations both with immunological processes such as chemokine-mediated response, antigen processing, receptor-related signal transduction, and transcriptional regulation, and also with more general processes such as replication and cell cycle control. Within-cluster gene correlations implicated known associations of known genes, as well as immune process-related roles for poorly described genes. To characterize regulatory mechanisms and cis-elements of genes with similar patterns of expression, we used a new version of a comparative genomics-based cis-element analysis tool to identify clusters of cis-elements with compositional similarity among multiple genes. Several clusters contained genes that shared 5–6 cis-elements that included ETS and zinc-finger binding sites. cis-Elements AP2 EGRF ETSF MAZF SP1F ZF5F and AREB ETSF MZF1 PAX5 STAT were shared in a thymus-expressed set; AP4R E2FF EBOX ETSF MAZF SP1F ZF5F and CREB E2FF MAZF PCAT SP1F STAT cis-clusters occurred in activated T-cells; CEBP CREB NFKB SORY and GATA NKXH OCT1 RBIT occurred in stimulated lymph nodes. Conclusion This study demonstrates a series of analytic approaches that have allowed the implication of genes and regulatory elements that participate in the differentiation, maintenance, and function of the immune system. Polymorphism or mutation of these could adversely impact immune system functions. PMID:15504237

Strengthening the Canadian alcohol advertising regulatory system.

PubMed

Heung, Carly M; Rempel, Benjamin; Krank, Marvin

2012-05-24

Research evidence points to harmful effects from alcohol advertising among children and youth. In particular, exposure to alcohol advertising has been associated with adolescents drinking both earlier and heavier. Although current federal and provincial guidelines have addressed advertising practices to prevent underage drinking, practice has not been supported by existing policy. While protective measures such as social marketing campaigns have the potential for counteracting the effects from alcohol advertising, the effectiveness of such measures can be easily drowned out with increasing advertising activities from the alcohol industry, especially without effective regulation. Research reviewed by the European Focus on Alcohol Safe Environment (FASE) Project has identified a set of key elements that are necessary to make alcohol advertising policy measures effective at protecting children and youth from the harmful effects of alcohol marketing. Using these key elements as an evaluation framework, there are critical components in the Canadian alcohol advertising regulatory system that clearly require strengthening. To protect impressionable children and youth against the harmful effects of alcohol advertising, 13 recommendations to strengthen current alcohol advertising regulations in Canada are provided for Canadian policy-makers, advertising standard agencies, and public health groups.

77 FR 7980 - Department Regulatory Agenda; Semiannual Summary

Federal Register 2010, 2011, 2012, 2013, 2014

2012-02-13

...The Regulatory Agenda is a semiannual summary of all current and projected rulemakings, reviews of existing regulations, and completed actions of the Department. The Agenda provides the public with information about the Department of Transportation's regulatory activity. It is expected that this information will enable the public to be more aware of and allow it to more effectively participate in the Department's regulatory activity. The public is also invited to submit comments on any aspect of this Agenda.

75 FR 79811 - Department Regulatory Agenda; Semiannual Summary

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-20

...The regulatory agenda is a semiannual summary of all current and projected rulemakings, reviews of existing regulations, and completed actions of the Department. The agenda provides the public with information about the Department of Transportation's regulatory activity. It is expected that this information will enable the public to be more aware of and allow it to more effectively participate in the Department's regulatory activity. The public is also invited to submit comments on any aspect of this agenda.

75 FR 21839 - Department Regulatory Agenda; Semiannual Summary

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-26

...The regulatory agenda is a semiannual summary of all current and projected rulemakings, reviews of existing regulations, and completed actions of the Department. The agenda provides the public with information about the Department of Transportation's regulatory activity. It is expected that this information will enable the public to be more aware of and allow it to more effectively participate in the Department's regulatory activity. The public is also invited to submit comments on any aspect of this agenda.

RSAT: regulatory sequence analysis tools.

PubMed

Thomas-Chollier, Morgane; Sand, Olivier; Turatsinze, Jean-Valéry; Janky, Rekin's; Defrance, Matthieu; Vervisch, Eric; Brohée, Sylvain; van Helden, Jacques

2008-07-01

The regulatory sequence analysis tools (RSAT, http://rsat.ulb.ac.be/rsat/) is a software suite that integrates a wide collection of modular tools for the detection of cis-regulatory elements in genome sequences. The suite includes programs for sequence retrieval, pattern discovery, phylogenetic footprint detection, pattern matching, genome scanning and feature map drawing. Random controls can be performed with random gene selections or by generating random sequences according to a variety of background models (Bernoulli, Markov). Beyond the original word-based pattern-discovery tools (oligo-analysis and dyad-analysis), we recently added a battery of tools for matrix-based detection of cis-acting elements, with some original features (adaptive background models, Markov-chain estimation of P-values) that do not exist in other matrix-based scanning tools. The web server offers an intuitive interface, where each program can be accessed either separately or connected to the other tools. In addition, the tools are now available as web services, enabling their integration in programmatic workflows. Genomes are regularly updated from various genome repositories (NCBI and EnsEMBL) and 682 organisms are currently supported. Since 1998, the tools have been used by several hundreds of researchers from all over the world. Several predictions made with RSAT were validated experimentally and published.

The Regulatory Framework for Privacy and Security

NASA Astrophysics Data System (ADS)

Hiller, Janine S.

The internet enables the easy collection of massive amounts of personally identifiable information. Unregulated data collection causes distrust and conflicts with widely accepted principles of privacy. The regulatory framework in the United States for ensuring privacy and security in the online environment consists of federal, state, and self-regulatory elements. New laws have been passed to address technological and internet practices that conflict with privacy protecting policies. The United States and the European Union approaches to privacy differ significantly, and the global internet environment will likely cause regulators to face the challenge of balancing privacy interests with data collection for many years to come.

Inference of cancer-specific gene regulatory networks using soft computing rules.

PubMed

Wang, Xiaosheng; Gotoh, Osamu

2010-03-24

Perturbations of gene regulatory networks are essentially responsible for oncogenesis. Therefore, inferring the gene regulatory networks is a key step to overcoming cancer. In this work, we propose a method for inferring directed gene regulatory networks based on soft computing rules, which can identify important cause-effect regulatory relations of gene expression. First, we identify important genes associated with a specific cancer (colon cancer) using a supervised learning approach. Next, we reconstruct the gene regulatory networks by inferring the regulatory relations among the identified genes, and their regulated relations by other genes within the genome. We obtain two meaningful findings. One is that upregulated genes are regulated by more genes than downregulated ones, while downregulated genes regulate more genes than upregulated ones. The other one is that tumor suppressors suppress tumor activators and activate other tumor suppressors strongly, while tumor activators activate other tumor activators and suppress tumor suppressors weakly, indicating the robustness of biological systems. These findings provide valuable insights into the pathogenesis of cancer.

FARME DB: a functional antibiotic resistance element database

PubMed Central

Wallace, James C.; Port, Jesse A.; Smith, Marissa N.; Faustman, Elaine M.

2017-01-01

Antibiotic resistance (AR) is a major global public health threat but few resources exist that catalog AR genes outside of a clinical context. Current AR sequence databases are assembled almost exclusively from genomic sequences derived from clinical bacterial isolates and thus do not include many microbial sequences derived from environmental samples that confer resistance in functional metagenomic studies. These environmental metagenomic sequences often show little or no similarity to AR sequences from clinical isolates using standard classification criteria. In addition, existing AR databases provide no information about flanking sequences containing regulatory or mobile genetic elements. To help address this issue, we created an annotated database of DNA and protein sequences derived exclusively from environmental metagenomic sequences showing AR in laboratory experiments. Our Functional Antibiotic Resistant Metagenomic Element (FARME) database is a compilation of publically available DNA sequences and predicted protein sequences conferring AR as well as regulatory elements, mobile genetic elements and predicted proteins flanking antibiotic resistant genes. FARME is the first database to focus on functional metagenomic AR gene elements and provides a resource to better understand AR in the 99% of bacteria which cannot be cultured and the relationship between environmental AR sequences and antibiotic resistant genes derived from cultured isolates. Database URL: http://staff.washington.edu/jwallace/farme PMID:28077567

Developmental gene regulatory network architecture across 500 million years of echinoderm evolution

NASA Technical Reports Server (NTRS)

Hinman, Veronica F.; Nguyen, Albert T.; Cameron, R. Andrew; Davidson, Eric H.

2003-01-01

Evolutionary change in morphological features must depend on architectural reorganization of developmental gene regulatory networks (GRNs), just as true conservation of morphological features must imply retention of ancestral developmental GRN features. Key elements of the provisional GRN for embryonic endomesoderm development in the sea urchin are here compared with those operating in embryos of a distantly related echinoderm, a starfish. These animals diverged from their common ancestor 520-480 million years ago. Their endomesodermal fate maps are similar, except that sea urchins generate a skeletogenic cell lineage that produces a prominent skeleton lacking entirely in starfish larvae. A relevant set of regulatory genes was isolated from the starfish Asterina miniata, their expression patterns determined, and effects on the other genes of perturbing the expression of each were demonstrated. A three-gene feedback loop that is a fundamental feature of the sea urchin GRN for endoderm specification is found in almost identical form in the starfish: a detailed element of GRN architecture has been retained since the Cambrian Period in both echinoderm lineages. The significance of this retention is highlighted by the observation of numerous specific differences in the GRN connections as well. A regulatory gene used to drive skeletogenesis in the sea urchin is used entirely differently in the starfish, where it responds to endomesodermal inputs that do not affect it in the sea urchin embryo. Evolutionary changes in the GRNs since divergence are limited sharply to certain cis-regulatory elements, whereas others have persisted unaltered.

Finite element predictions of active buckling control of stiffened panels

NASA Astrophysics Data System (ADS)

Thompson, Danniella M.; Griffin, O. H., Jr.

1993-04-01

Materials systems and structures that can respond 'intelligently' to their environment are currently being proposed and investigated. A series of finite element analyses was performed to investigate the potential for active buckling control of two different stiffened panels by embedded shape memory alloy (SMA) rods. Changes in the predicted buckling load increased with the magnitude of the actuation level for a given structural concept. Increasing the number of actuators for a given concept yielded greater predicted increases in buckling load. Considerable control authority was generated with a small number of actuators, with greater authority demonstrated for those structural concepts where the activated SMA rods could develop greater forces and moments on the structure. Relatively simple and inexpensive analyses were performed with standard finite elements to determine such information, indicating the viability of these types of models for design purposes.

Telomeric P elements associated with cytotype regulation of the P transposon family in Drosophila melanogaster.

PubMed Central

Stuart, Jeremy R; Haley, Kevin J; Swedzinski, Douglas; Lockner, Samuel; Kocian, Paul E; Merriman, Peter J; Simmons, Michael J

2002-01-01

P elements inserted at the left end of the Drosophila X chromosome were isolated genetically from wild-type P strains. Stocks carrying these elements were tested for repression of P-strain-induced gonadal dysgenesis in females and for repression of transposase-catalyzed P-element excision in males and females. Both traits were repressed by stocks carrying either complete or incomplete P elements inserted near the telomere of the X chromosome in cytological region 1A, but not by stocks carrying only nontelomeric X-linked P elements. All three of the telomeric P elements that were analyzed at the molecular level were inserted in one of the 1.8-kb telomere-associated sequence (TAS) repeats near the end of the X chromosome. Stocks with these telomeric P elements strongly repressed P-element excision induced in the male germline by a P strain or by the transposase-producing transgenes H(hsp/CP)2, H(hsp/CP)3, a combination of these two transgenes, and P(ry(+), delta2-3)99B. For H(hsp/CP)2 and P(ry(+), delta2-3)99B, the repression was also effective when the flies were subjected to heat-shock treatments. However, these stocks did not repress the somatic transposase activity of P(ry(+), delta2-3)99B. Repression of transposase activity in the germline required maternal transmission of the telomeric P elements themselves. Paternal transmission of these elements, or maternal transmission of the cytoplasm from carriers, both were insufficient to repress transposase activity. Collectively, these findings indicate that the regulatory abilities of telomeric P elements are similar to those of the P cytotype. PMID:12524339

Gene regulatory network of unfolded protein response genes in endoplasmic reticulum stress.

PubMed

Takayanagi, Sayuri; Fukuda, Riga; Takeuchi, Yuuki; Tsukada, Sakiko; Yoshida, Kenichi

2013-01-01

In the endoplasmic reticulum (ER), secretory and membrane proteins are properly folded and modified, and the failure of these processes leads to ER stress. At the same time, unfolded protein response (UPR) genes are activated to maintain homeostasis. Despite the thorough characterization of the individual gene regulation of UPR genes to date, further investigation of the mutual regulation among UPR genes is required to understand the complex mechanism underlying the ER stress response. In this study, we aimed to reveal a gene regulatory network formed by UPR genes, including immunoglobulin heavy chain-binding protein (BiP), X-box binding protein 1 (XBP1), C/EBP [CCAAT/enhancer-binding protein]-homologous protein (CHOP), PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring 1 (IRE1), activating transcription factor 6 (ATF6), and ATF4. For this purpose, we focused on promoter-luciferase reporters for BiP, XBP1, and CHOP genes, which bear an ER stress response element (ERSE), and p5 × ATF6-GL3, which bears an unfolded protein response element (UPRE). We demonstrated that the luciferase activities of the BiP and CHOP promoters were upregulated by all the UPR genes, whereas those of the XBP1 promoter and p5 × ATF6-GL3 were upregulated by all the UPR genes except for BiP, CHOP, and ATF4 in HeLa cells. Therefore, an ERSE- and UPRE-centered gene regulatory network of UPR genes could be responsible for the robustness of the ER stress response. Finally, we revealed that BiP protein was degraded when cells were treated with DNA-damaging reagents, such as etoposide and doxorubicin; this finding suggests that the expression level of BiP is tightly regulated at the post-translational level, rather than at the transcriptional level, in the presence of DNA damage.

Reconstructing gene regulatory networks from knock-out data using Gaussian Noise Model and Pearson Correlation Coefficient.

PubMed

Mohamed Salleh, Faridah Hani; Arif, Shereena Mohd; Zainudin, Suhaila; Firdaus-Raih, Mohd

2015-12-01

A gene regulatory network (GRN) is a large and complex network consisting of interacting elements that, over time, affect each other's state. The dynamics of complex gene regulatory processes are difficult to understand using intuitive approaches alone. To overcome this problem, we propose an algorithm for inferring the regulatory interactions from knock-out data using a Gaussian model combines with Pearson Correlation Coefficient (PCC). There are several problems relating to GRN construction that have been outlined in this paper. We demonstrated the ability of our proposed method to (1) predict the presence of regulatory interactions between genes, (2) their directionality and (3) their states (activation or suppression). The algorithm was applied to network sizes of 10 and 50 genes from DREAM3 datasets and network sizes of 10 from DREAM4 datasets. The predicted networks were evaluated based on AUROC and AUPR. We discovered that high false positive values were generated by our GRN prediction methods because the indirect regulations have been wrongly predicted as true relationships. We achieved satisfactory results as the majority of sub-networks achieved AUROC values above 0.5. Copyright © 2015 Elsevier Ltd. All rights reserved.

The twilight zone of cis element alignments

PubMed Central

Sebastian, Alvaro; Contreras-Moreira, Bruno

2013-01-01

Sequence alignment of proteins and nucleic acids is a routine task in bioinformatics. Although the comparison of complete peptides, genes or genomes can be undertaken with a great variety of tools, the alignment of short DNA sequences and motifs entails pitfalls that have not been fully addressed yet. Here we confront the structural superposition of transcription factors with the sequence alignment of their recognized cis elements. Our goals are (i) to test TFcompare (http://floresta.eead.csic.es/tfcompare), a structural alignment method for protein–DNA complexes; (ii) to benchmark the pairwise alignment of regulatory elements; (iii) to define the confidence limits and the twilight zone of such alignments and (iv) to evaluate the relevance of these thresholds with elements obtained experimentally. We find that the structure of cis elements and protein–DNA interfaces is significantly more conserved than their sequence and measures how this correlates with alignment errors when only sequence information is considered. Our results confirm that DNA motifs in the form of matrices produce better alignments than individual sequences. Finally, we report that empirical and theoretically derived twilight thresholds are useful for estimating the natural plasticity of regulatory sequences, and hence for filtering out unreliable alignments. PMID:23268451