scholarly journals Promoter Boundaries for theluxCDABEandbetIBA-proXWVOperons inVibrio harveyiDefined by the Method Rapid Arbitrary PCR Insertion Libraries (RAIL)

2018 ◽  
Vol 200 (11) ◽  
Author(s):  
Christine M. Hustmyer ◽  
Chelsea A. Simpson ◽  
Stephen G. Olney ◽  
Douglas B. Rusch ◽  
Matthew L. Bochman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Transcriptional Regulation ◽  
Vibrio Harveyi ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Model Systems ◽  
New Method ◽  
Regulatory Sequences ◽  
Content Type

ABSTRACTExperimental studies of transcriptional regulation in bacteria require the ability to precisely measure changes in gene expression, often accomplished through the use of reporter genes. However, the boundaries of promoter sequences required for transcription are often unknown, thus complicating the construction of reporters and genetic analysis of transcriptional regulation. Here, we analyze reporter libraries to define the promoter boundaries of theluxCDABEbioluminescence operon and thebetIBA-proXWVosmotic stress operon inVibrio harveyi. We describe a new method calledrapidarbitrary PCRinsertionlibraries (RAIL) that combines the power of arbitrary PCR and isothermal DNA assembly to rapidly clone promoter fragments of various lengths upstream of reporter genes to generate large libraries. To demonstrate the versatility and efficiency of RAIL, we analyzed the promoters driving expression of theluxCDABEandbetIBA-proXWVoperons and created libraries of DNA fragments from these loci fused to fluorescent reporters. Using flow cytometry sorting and deep sequencing, we identified the DNA regions necessary and sufficient for maximum gene expression for each promoter. These analyses uncovered previously unknown regulatory sequences and validated known transcription factor binding sites. We applied this high-throughput method togfp,mCherry, andlacZreporters and multiple promoters inV. harveyi. We anticipate that the RAIL method will be easily applicable to other model systems for genetic, molecular, and cell biological applications.IMPORTANCEGene reporter constructs have long been essential tools for studying gene regulation in bacteria, particularly following the recent advent of fluorescent gene reporters. We developed a new method that enables efficient construction of promoter fusions to reporter genes to study gene regulation. We demonstrate the versatility of this technique in the model bacteriumVibrio harveyiby constructing promoter libraries for three bacterial promoters using three reporter genes. These libraries can be used to determine the DNA sequences required for gene expression, revealing regulatory elements in promoters. This method is applicable to various model systems and reporter genes for assaying gene expression.

scholarly journals Promoter boundaries for theluxCDABEandbetIBA-proXWVoperons inVibrio harveyidefined by the method RAIL: Rapid Arbitrary PCR Insertion Libraries

2017 ◽  
Author(s):  
Christine M. Hustmyer ◽  
Chelsea A. Simpson ◽  
Stephen G. Olney ◽  
Matthew L. Bochman ◽  
Julia C. van Kessel
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Transcriptional Regulation ◽  
Dna Sequences ◽  
Vibrio Harveyi ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Model Systems ◽  
New Method ◽  
Regulatory Sequences

AbstractExperimental studies of transcriptional regulation in bacteria require the ability to precisely measure changes in gene expression, often accomplished through the use of reporter genes. However, the boundaries of promoter sequences required for transcription are often unknown, thus complicating construction of reporters and genetic analysis of transcriptional regulation. Here, we analyze reporter libraries to define the promoter boundaries of theluxCDABEbioluminescence operon and thebetIBA-proXWVosmotic stress operon inVibrio harveyi. We describe a new method called RAIL (RapidArbitrary PCRInsertionLibraries) that combines the power of arbitrary PCR and isothermal DNA assembly to rapidly clone promoter fragments of various lengths upstream of reporter genes to generate large libraries. To demonstrate the versatility and efficiency of RAIL, we analyzed the promoters driving expression of theluxCDABEandbetIBA-proXWVoperons and created libraries of DNA fragments from these loci fused to fluorescent reporters. Using flow cytometry sorting and deep sequencing, we identified the DNA regions necessary and sufficient for maximum gene expression for each promoter. These analyses uncovered previously unknown regulatory sequences and validated known transcription factor binding sites. We applied this high-throughput method togfp, mCherry, andlacZreporters and multiple promoters inV. harveyi. We anticipate that the RAIL method will be easily applicable to other model systems for genetic, molecular, and cell biological applications.ImportanceGene reporter constructs have long been essential tools for studying gene regulation in bacteria, particularly following the recent advent of fluorescent gene reporters. We developed a new method that enables efficient construction of promoter fusions to reporter genes to study gene regulation. We demonstrate the versatility of this technique in the model bacteriumVibrio harveyiby constructing promoter libraries for three bacterial promoters using three reporter genes. These libraries can be used to determine the DNA sequences required for gene expression, revealing regulatory elements in promoters. This method is applicable to various model systems and reporter genes for assaying gene expression.

scholarly journals Impact and evolutionary determinants of Neanderthal introgression on transcriptional and post-transcriptional regulation

2019 ◽  
Author(s):  
Martin Silvert ◽  
Lluis Quintana-Murci ◽  
Maxime Rotival
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Transcriptional Regulation ◽  
Regulatory Elements ◽  
Strong Impact ◽  
Modern Humans ◽  
Archaic Admixture ◽  
Archaic Introgression ◽  
Post Transcriptional Regulation ◽  
The Impact

AbstractArchaic admixture is increasingly recognized as an important source of diversity in modern humans, with Neanderthal haplotypes covering 1-3% of the genome of present-day Eurasians. Recent work has shown that archaic introgression has contributed to human phenotypic diversity, mostly through the regulation of gene expression. Yet, the mechanisms through which archaic variants alter gene expression, and the forces driving the introgression landscape at regulatory regions remain elusive. Here, we explored the impact of archaic introgression on transcriptional and post-transcriptional regulation, focusing on promoters and enhancers across 127 different tissues as well as microRNA-mediated regulation. Although miRNAs themselves harbor few archaic variants, we found that some of these variants may have a strong impact on miRNA-mediated gene regulation. Enhancers were by far the regulatory elements most affected by archaic introgression, with one third of the tissues tested presenting significant enrichments. Specifically, we found strong enrichments of archaic variants in adipose-related tissues and primary T cells, even after accounting for various genomic and evolutionary confounders such as recombination rate and background selection. Interestingly, we identified signatures of adaptive introgression at enhancers of some key regulators of adipogenesis, raising the interesting hypothesis of a possible adaptation of early Eurasians to colder climates. Collectively, this study sheds new light onto the mechanisms through which archaic admixture have impacted gene regulation in Eurasians and, more generally, increases our understanding of the contribution of Neanderthals to the regulation of acquired immunity and adipose homeostasis in modern humans.

scholarly journals The regulatory genome of the malaria vector Anopheles gambiae: integrating chromatin accessibility and gene expression

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
José L Ruiz ◽  
Lisa C Ranford-Cartwright ◽  
Elena Gómez-Díaz
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Anopheles Gambiae ◽  
Mosquito Control ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Malaria Vectors ◽  
Specific Gene ◽  
Mosquito Vector ◽  
Human Malaria

Abstract Anopheles gambiae mosquitoes are primary human malaria vectors, but we know very little about their mechanisms of transcriptional regulation. We profiled chromatin accessibility by the assay for transposase-accessible chromatin by sequencing (ATAC-seq) in laboratory-reared A. gambiae mosquitoes experimentally infected with the human malaria parasite Plasmodium falciparum. By integrating ATAC-seq, RNA-seq and ChIP-seq data, we showed a positive correlation between accessibility at promoters and introns, gene expression and active histone marks. By comparing expression and chromatin structure patterns in different tissues, we were able to infer cis-regulatory elements controlling tissue-specific gene expression and to predict the in vivo binding sites of relevant transcription factors. The ATAC-seq assay also allowed the precise mapping of active regulatory regions, including novel transcription start sites and enhancers that were annotated to mosquito immune-related genes. Not only is this study important for advancing our understanding of mechanisms of transcriptional regulation in the mosquito vector of human malaria, but the information we produced also has great potential for developing new mosquito-control and anti-malaria strategies.

scholarly journals CFTR Cooperative Cis-Regulatory Elements in Intestinal Cells

2021 ◽  
Vol 22 (5) ◽  
pp. 2599
Author(s):  
Mégane Collobert ◽  
Ozvan Bocher ◽  
Anaïs Le Nabec ◽  
Emmanuelle Génin ◽  
Claude Férec ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Gene Regulation ◽  
Genetic Diseases ◽  
Regulatory Elements ◽  
Cftr Gene ◽  
Intestinal Cells ◽  
Cooperative Effects ◽  
Cis Acting ◽  
Study Techniques

About 8% of the human genome is covered with candidate cis-regulatory elements (cCREs). Disruptions of CREs, described as “cis-ruptions” have been identified as being involved in various genetic diseases. Thanks to the development of chromatin conformation study techniques, several long-range cystic fibrosis transmembrane conductance regulator (CFTR) regulatory elements were identified, but the regulatory mechanisms of the CFTR gene have yet to be fully elucidated. The aim of this work is to improve our knowledge of the CFTR gene regulation, and to identity factors that could impact the CFTR gene expression, and potentially account for the variability of the clinical presentation of cystic fibrosis as well as CFTR-related disorders. Here, we apply the robust GWAS3D score to determine which of the CFTR introns could be involved in gene regulation. This approach highlights four particular CFTR introns of interest. Using reporter gene constructs in intestinal cells, we show that two new introns display strong cooperative effects in intestinal cells. Chromatin immunoprecipitation analyses further demonstrate fixation of transcription factors network. These results provide new insights into our understanding of the CFTR gene regulation and allow us to suggest a 3D CFTR locus structure in intestinal cells. A better understand of regulation mechanisms of the CFTR gene could elucidate cases of patients where the phenotype is not yet explained by the genotype. This would thus help in better diagnosis and therefore better management. These cis-acting regions may be a therapeutic challenge that could lead to the development of specific molecules capable of modulating gene expression in the future.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

scholarly journals How the Avidity of Polymerase Binding to the -35/-10 Promoter Sites Affects Gene Expression

2019 ◽  
Author(s):  
Tal Einav ◽  
Rob Phillips
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Regulatory Elements ◽  
Physical Principle ◽  
Regulatory Sequences ◽  
Energy Matrix ◽  
Cellular Behavior ◽  
Inhibit Gene Expression ◽  
Small Set ◽  
Polymerase Binding

AbstractAlthough the key promoter elements necessary to drive transcription inEscherichia colihave long been understood, we still cannot predict the behavior of arbitrary novel promoters, hampering our ability to characterize the myriad of sequenced regulatory architectures as well as to design novel synthetic circuits. This work builds on a beautiful recent experiment by Urtechoet al.who measured the gene expression of over 10,000 promoters spanning all possible combinations of a small set of regulatory elements. Using this data, we demonstrate that a central claim in energy matrix models of gene expression – that each promoter element contributes independently and additively to gene expression – contradicts experimental measurements. We propose that a key missing ingredient from such models is the avidity between the -35 and -10 RNA polymerase binding sites and develop what we call arefined energy matrixmodel that incorporates this effect. We show that this the refined energy matrix model can characterize the full suite of gene expression data and explore several applications of this framework, namely, how multivalent binding at the -35 and -10 sites can buffer RNAP kinetics against mutations and how promoters that bind overly tightly to RNA polymerase can inhibit gene expression. The success of our approach suggests that avidity represents a key physical principle governing the interaction of RNA polymerase to its promoter.Significance StatementCellular behavior is ultimately governed by the genetic program encoded in its DNA and through the arsenal of molecular machines that actively transcribe its genes, yet we lack the ability to predict how an arbitrary DNA sequence will perform. To that end, we analyze the performance of over 10,000 regulatory sequences and develop a model that can predict the behavior of any sequence based on its composition. By considering promoters that only vary by one or two elements, we can characterize how different components interact, providing fundamental insights into the mechanisms of transcription.

scholarly journals Antibacterial Mechanism of 405-Nanometer Light-Emitting Diode against Salmonella at Refrigeration Temperature

2016 ◽  
Vol 83 (5) ◽  
Author(s):  
Min-Jeong Kim ◽  
Hyun-Gyun Yuk
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Light Emitting Diode ◽  
Dna Oxidation ◽  
Uptake System ◽  
Antibacterial Mechanism ◽  
Content Type ◽  
Light Emitting ◽  
Expression Levels ◽  
Gene Expression Levels

ABSTRACT The aim of this study was to elucidate the antibacterial mechanism of 405 ± 5-nm light-emitting diode (LED) illumination against Salmonella at 4°C in phosphate-buffered saline (PBS) by determining endogenous coproporphyrin content, DNA oxidation, damage to membrane function, and morphological change. Gene expression levels, including of oxyR, recA, rpoS, sodA, and soxR, were also examined to understand the response of Salmonella to LED illumination. The results showed that Salmonella strains responded differently to LED illumination, revealing that S. enterica serovar Enteritidis (ATCC 13076) and S. enterica subsp. enterica serovar Saintpaul (ATCC 9712) were more susceptible and resistant, respectively, than the 16 other strains tested. There was no difference in the amounts of endogenous coproporphyrin in the two strains. Compared with that in nonilluminated cells, the DNA oxidation levels in illuminated cells increased. In illuminated cells, we observed a loss of efflux pump activity, damage to the glucose uptake system, and changes in membrane potential and integrity. Transmission electron microscopy revealed a disorganization of chromosomes and ribosomes due to LED illumination. The levels of the five genes measured in the nonilluminated and illuminated S. Saintpaul cells were upregulated in PBS at a set temperature of 4°C, indicating that increased gene expression levels might be due to a temperature shift and nutrient deficiency rather than to LED illumination. In contrast, only oxyR in S. Enteritidis cells was upregulated. Thus, different sensitivities of the two strains to LED illumination were attributed to differences in gene regulation. IMPORTANCE Bacterial inactivation using visible light has recently received attention as a safe and environmentally friendly technology, in contrast with UV light, which has detrimental effects on human health and the environment. This study was designed to understand how 405 ± 5-nm light-emitting diode (LED) illumination kills Salmonella strains at refrigeration temperature. The data clearly demonstrated that the effectiveness of LED illumination on Salmonella strains depended highly on the serotype and strain. Our findings also revealed that its antibacterial mechanism was mainly attributed to DNA oxidation and a loss of membrane functions rather than membrane lipid peroxidation, which has been proposed by other researchers who studied the antibacterial effect of LED illumination by adding exogenous photosensitizers, such as chlorophyllin and hypericin. Therefore, this study suggests that the detailed antibacterial mechanisms of 405-nm LED illumination without additional photosensitizers may differ from that by exogenous photosensitizers. Furthermore, a change in stress-related gene regulation may alter the susceptibility of Salmonella cells to LED illumination at refrigeration temperature. Thus, our study provides new insights into the antibacterial mechanism of 405 ± 5-nm LED illumination on Salmonella cells.

scholarly journals Hierarchical Transcriptional Control of the LuxR Quorum-Sensing Regulon of Vibrio harveyi

2020 ◽  
Vol 202 (14) ◽  
Author(s):  
Ryan R. Chaparian ◽  
Alyssa S. Ball ◽  
Julia C. van Kessel
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Quorum Sensing ◽  
Cell Density ◽  
Regulatory Networks ◽  
Vibrio Harveyi ◽  
Sugar Transport ◽  
Content Type ◽  
Second Tier

ABSTRACT In vibrios, quorum sensing controls hundreds of genes that are required for cell density-specific behaviors including bioluminescence, biofilm formation, competence, secretion, and swarming motility. The central transcription factor in the quorum-sensing pathway is LuxR/HapR, which directly regulates ∼100 genes in the >400-gene regulon of Vibrio harveyi. Among these directly controlled genes are 15 transcription factors, which we predicted would comprise the second tier in the hierarchy of the LuxR regulon. We confirmed that LuxR binds to the promoters of these genes in vitro and quantified the extent of LuxR activation or repression of transcript levels. Transcriptome sequencing (RNA-seq) indicates that most of these transcriptional regulators control only a few genes, with the exception of MetJ, which is a global regulator. The genes regulated by these transcription factors are predicted to be involved in methionine and thiamine biosynthesis, membrane stability, RNA processing, c-di-GMP degradation, sugar transport, and other cellular processes. These data support a hierarchical model in which LuxR directly regulates 15 transcription factors that drive the second level of the gene expression cascade to influence cell density-dependent metabolic states and behaviors in V. harveyi. IMPORTANCE Quorum sensing is important for survival of bacteria in nature and influences the actions of bacterial groups. In the relatively few studied examples of quorum-sensing-controlled genes, these genes are associated with competition or cooperation in complex microbial communities and/or virulence in a host. However, quorum sensing in vibrios controls the expression of hundreds of genes, and their functions are mostly unknown or uncharacterized. In this study, we identify the regulators of the second tier of gene expression in the quorum-sensing system of the aquaculture pathogen Vibrio harveyi. Our identification of regulatory networks and metabolic pathways controlled by quorum sensing can be extended and compared to other Vibrio species to understand the physiology, ecology, and pathogenesis of these organisms.

scholarly journals Transposable elements as a potent source of diverse cis -regulatory sequences in mammalian genomes

2020 ◽  
Vol 375 (1795) ◽  
pp. 20190347 ◽  
Author(s):  
Vasavi Sundaram ◽  
Joanna Wysocka
Keyword(s):  
Gene Regulation ◽  
Transposable Elements ◽  
Regulatory Networks ◽  
Regulatory Elements ◽  
Regulatory Function ◽  
Specific Gene ◽  
Regulatory Sequences ◽  
Dependent Manner ◽  
Unique Contribution ◽  
Regulatory Potential

Eukaryotic gene regulation is mediated by cis -regulatory elements, which are embedded within the vast non-coding genomic space and recognized by the transcription factors in a sequence- and context-dependent manner. A large proportion of eukaryotic genomes, including at least half of the human genome, are composed of transposable elements (TEs), which in their ancestral form carried their own cis -regulatory sequences able to exploit the host trans environment to promote TE transcription and facilitate transposition. Although not all present-day TE copies have retained this regulatory function, the preexisting regulatory potential of TEs can provide a rich source of cis -regulatory innovation for the host. Here, we review recent evidence documenting diverse contributions of TE sequences to gene regulation by functioning as enhancers, promoters, silencers and boundary elements. We discuss how TE-derived enhancer sequences can rapidly facilitate changes in existing gene regulatory networks and mediate species- and cell-type-specific regulatory innovations, and we postulate a unique contribution of TEs to species-specific gene expression divergence in pluripotency and early embryogenesis. With advances in genome-wide technologies and analyses, systematic investigation of TEs' cis -regulatory potential is now possible and our understanding of the biological impact of genomic TEs is increasing. This article is part of a discussion meeting issue ‘Crossroads between transposons and gene regulation’.

scholarly journals Renal Anemia Model Mouse Established by Transgenic Rescue with an Erythropoietin Gene Lacking Kidney-Specific Regulatory Elements

2016 ◽  
Vol 37 (4) ◽  
Author(s):  
Ikuo Hirano ◽  
Norio Suzuki ◽  
Shun Yamazaki ◽  
Hiroki Sekine ◽  
Naoko Minegishi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mouse ◽  
Artificial Chromosome ◽  
Regulatory Elements ◽  
Renal Anemia ◽  
Content Type ◽  
Transgenic Expression ◽  
Complex Process ◽  
Model Mouse ◽  
Mouse Lines

ABSTRACT The erythropoietin (Epo) gene is under tissue-specific inducible regulation. Because the kidney is the primary EPO-producing tissue in adults, impaired EPO production in chronic kidney disorders results in serious renal anemia. The Epo gene contains a liver-specific enhancer in the 3′ region, but the kidney-specific enhancer for gene expression in renal EPO-producing (REP) cells remains elusive. Here, we examined a conserved upstream element for renal E po regulation (CURE) region that spans 17.4 kb to 3.6 kb upstream of the Epo gene and harbors several phylogenetically conserved elements. We prepared various Epo gene-reporter constructs utilizing a bacterial artificial chromosome and generated a number of transgenic-mouse lines. We observed that deletion of the CURE region (δCURE) abrogated Epo gene expression in REP cells. Although transgenic expression of the δCURE construct rescued Epo-deficient mice from embryonic lethality, the rescued mice had severe EPO-dependent anemia. These mouse lines serve as an elaborate model for the search for erythroid stimulatory activity and are referred to as AnRED (anemic model with renal EPO deficiency) mice. We also dissected the CURE region by exploiting a minigene harboring four phylogenetically conserved elements in reporter transgenic-mouse analyses. Our analyses revealed that Epo gene regulation in REP cells is a complex process that utilizes multiple regulatory influences.

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