scholarly journals Expanded FLP toolbox for spatiotemporal protein degradation and transcriptomic profiling in C. elegans

2021 ◽  
Author(s):  
Adrian Fragoso-Luna ◽  
Cristina Ayuso ◽  
Michael Eibl ◽  
Celia Munoz-Jimenez ◽  
Vladimir Benes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Degradation ◽  
Gene Function ◽  
Gene Knockout ◽  
Added Value ◽  
Nuclear Pore ◽  
Specific Gene ◽  
Transcriptomic Profiling ◽  
Control Of Gene Expression ◽  
C Elegans

Control of gene expression in specific tissues and/or at certain stages of development allows the study and manipulation of gene function with high precision. Site-specific genome recombination by the Flippase (FLP) and Cre enzymes has proven particularly relevant. Joint efforts of many research groups have led to the creation of efficient FLP and Cre drivers to regulate gene expression in a variety of tissues in Caenorhabditis elegans. Here, we extend this toolkit by the addition of FLP lines that drive recombination specifically in distal tip cells, the somatic gonad, coelomocytes and the epithelial P lineage. In some cases, recombination-mediated gene knockouts do not completely deplete protein levels due to persistence of long-lived proteins. To overcome this, we developed a spatiotemporally regulated degradation system for GFP fusion proteins (GFPdeg) based on FLP-mediated recombination. Using two stable nuclear pore proteins, MEL-28/ELYS and NPP-2/NUP85 as examples, we report the benefit of combining tissue-specific gene knockout and protein degradation to achieve complete protein depletion. We also demonstrate that FLP-mediated recombination can be utilized to identify nascent transcripts in a tissue of interest. We have adapted thiol(SH)-linked alkylation for the metabolic sequencing of RNA in tissue (SLAM-ITseq) for C. elegans. By focusing on a well-characterized tissue, the hypodermis, we show that the vast majority of genes identified by SLAM-ITseq are known to be expressed in this tissue, but with the added value of temporal resolution. These tools allow combining FLP activity for simultaneous gene inactivation and transcriptomic profiling, thus enabling the inquiry of gene function in various complex biological processes.

scholarly journals Attenuation of Glucocorticoid Signaling through Targeted Degradation of p300 via the 26S Proteasome Pathway

2002 ◽  
Vol 16 (12) ◽  
pp. 2819-2827 ◽  
Author(s):  
Qiao Li ◽  
Anna Su ◽  
Jihong Chen ◽  
Yvonne A. Lefebvre ◽  
Robert J. G. Haché
Keyword(s):  
Gene Expression ◽  
Glucocorticoid Receptor ◽  
Sodium Butyrate ◽  
Steroid Receptor ◽  
26S Proteasome ◽  
Regulatory Control ◽  
Specific Gene ◽  
Control Of Gene Expression ◽  
Steroid Receptor Coactivator ◽  
Proteasome Pathway

Abstract The effects of acetylation on gene expression are complex, with changes in chromatin accessibility intermingled with direct effects on transcriptional regulators. For the nuclear receptors, both positive and negative effects of acetylation on specific gene transcription have been observed. We report that p300 and steroid receptor coactivator 1 interact transiently with the glucocorticoid receptor and that the acetyltransferase activity of p300 makes an important contribution to glucocorticoid receptor-mediated transcription. Treatment of cells with the deacetylase inhibitor, sodium butyrate, inhibited steroid-induced transcription and altered the transient association of glucocorticoid receptor with p300 and steroid receptor coactivator 1. Additionally, sustained sodium butyrate treatment induced the degradation of p300 through the 26S proteasome pathway. Treatment with the proteasome inhibitor MG132 restored both the level of p300 protein and the transcriptional response to steroid over 20 h of treatment. These results reveal new levels for the regulatory control of gene expression by acetylation and suggest feedback control on p300 activity.

scholarly journals A Floxed exon (Flexon) approach to Cre-mediated conditional gene expression

2021 ◽  
Author(s):  
Justin M Shaffer ◽  
Iva Greenwald
Keyword(s):  
Gene Expression ◽  
Specific Protein ◽  
Specific Gene ◽  
Coding Region ◽  
Specific Expression ◽  
Control Of Gene Expression ◽  
Tissue Specific ◽  
Nonsense Mediated Decay ◽  
Conditional Gene Expression ◽  
Stop Cassette

Conditional gene expression allows for genes to be manipulated and lineages to be marked during development. In the established "lox-stop-lox" approach, Cre-mediated tissue-specific gene expression is achieved by excising the stop cassette, a lox-flanked translational stop that is inserted into the 5' untranslated region of a gene to halt its expression. Although lox-stop-lox has been successfully used in many experimental systems, the design of traditional stop cassettes also has common issues and limitations. Here, we describe the Floxed exon (Flexon), a stop cassette within an artificial exon that can be inserted flexibly into the coding region of any gene to cause premature termination of translation and nonsense-mediated decay of the mRNA. We demonstrate its efficacy in C. elegans by showing that, when promoters that cause weak and/or transient cell-specific expression are used to drive Cre in combination with a gfp(flexon) transgene, strong and sustained expression is obtained in specific lineages. We also describe several potential additional applications for using Flexon for developmental studies, including more precise control of gene expression using intersectional methods, tissue-specific protein degradation or RNAi, and generation of genetic mosaics. The Flexon approach should be feasible in any system where any site-specific recombination-based method may be applied.

scholarly journals Maternal and zygotic gene regulatory effects of endogenous RNAi pathways

2018 ◽  
Author(s):  
Miguel Vasconcelos Almeida ◽  
António Miguel de Jesus Domingues ◽  
René F. Ketting
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Gene Silencing ◽  
Small Rnas ◽  
Self Regulation ◽  
Fine Tuning ◽  
Specific Gene ◽  
Next Generation ◽  
C Elegans ◽  
Argonaute Proteins

AbstractEndogenous small RNAs (sRNAs) and Argonaute proteins are ubiquitous regulators of gene expression in germline and somatic tissues. sRNA-Argonaute complexes are often expressed in gametes and are consequently inherited by the next generation upon fertilization. In Caenorhabditis elegans, 26G-RNAs are primary endogenous sRNAs that trigger the expression of downstream secondary sRNAs. Two subpopulations of 26G-RNAs exist, each of which displaying strongly compartmentalized expression: one is expressed in the spermatogenic gonad and associates with the Argonautes ALG-3/4; plus another expressed in oocytes and in embryos, which associates with the Argonaute ERGO-1. The determinants and dynamics of gene silencing elicited by 26G-RNAs are largely unknown. Here, we provide diverse new insights into these endogenous sRNA pathways of C. elegans. Using genetics and deep sequencing, we dissect a maternal effect of the ERGO-1 branch sRNA pathway. We find that maternal primary sRNAs can trigger the production of zygotic secondary sRNAs that are able to silence targets, even in the absence of zygotic primary triggers. Thus, the interaction of maternal and zygotic sRNA populations, assures target gene silencing throughout animal development. Furthermore, we find that sRNA abundance, the pattern of origin of sRNA and 3’ UTR length are predictors of the regulatory outcome by the Argonautes ALG-3/4. Lastly, we discovered that ALG-3- and ALG-4-bound 26G-RNAs are dampening the expression of their own mRNAs, revealing a negative feedback loop. Altogether, we provide several new regulatory insights on the dynamics, target regulation and self-regulation of the endogenous RNAi pathways of C. elegans.Author SummarySmall RNAs (sRNAs) and their partner Argonaute proteins regulate the expression of target RNAs. When sperm and egg meet upon fertilization, a diverse set of proteins and RNA, including sRNA-Argonaute complexes, is passed on to the developing progeny. Thus, these two players are important to initiate specific gene expression programs in the next generation. The nematode Caenorhabditis elegans expresses several classes of sRNAs. 26G-RNAs are a particular class of sRNAs that are divided into two subpopulations: one expressed in the spermatogenic gonad and another expressed in oocytes and in embryos. In this work, we describe the dynamics whereby oogenic 26G-RNAs setup gene silencing in the next generation. We also show several ways that spermatogenic 26G-RNAs and their partner Argonautes, ALG-3 and ALG-4, use to regulate their targets. Finally, we show that ALG-3 and ALG-4 are fine-tuning their own expression, a rare role of Argonaute proteins. Overall, we provide new insights into how sRNAs and Argonautes are regulating gene expression.

scholarly journals Precise optical control of gene expression in C. elegans using improved genetic code expansion and Cre recombinase

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Lloyd Davis ◽  
Inja Radman ◽  
Angeliki Goutou ◽  
Ailish Tynan ◽  
Kieran Baxter ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genetic Code ◽  
Cre Recombinase ◽  
Symmetric Pair ◽  
Control Of Gene Expression ◽  
C Elegans ◽  
Individual Contributions ◽  
Touch Response ◽  
Genetic Code Expansion ◽  
The Individual

Synthetic strategies for optically controlling gene expression may enable the precise spatiotemporal control of genes in any combination of cells that cannot be targeted with specific promoters. We develop an improved genetic code expansion system in C. elegans and use it to create a photo-activatable Cre recombinase. We laser-activate Cre in single neurons within a bilaterally symmetric pair to selectively switch on expression of a loxP controlled optogenetic channel in the targeted neuron. We use the system to dissect, in freely moving animals, the individual contributions of the mechanosensory neurons PLML/PLMR to the C. elegans touch response circuit, revealing distinct and synergistic roles for these neurons. We thus demonstrate how genetic code expansion and optical targeting can be combined to break the symmetry of neuron pairs and dissect behavioural outputs of individual neurons that cannot be genetically targeted.

scholarly journals Intronic enhancers regulate the expression of genes involved in tissue-specific functions and homeostasis

2020 ◽  
Author(s):  
Beatrice Borsari ◽  
Pablo Villegas-Mirón ◽  
Hafid Laayouni ◽  
Alba Segarra-Casas ◽  
Jaume Bertranpetit ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Stem ◽  
Gene Expression Pattern ◽  
Specific Gene ◽  
Control Of Gene Expression ◽  
Tissue Specific ◽  
Tissue Specific Gene ◽  
Specific Gene Expression ◽  
Expression Of Genes ◽  
Genomic Location

AbstractTissue function and homeostasis reflect the gene expression signature by which the combination of ubiquitous and tissue-specific genes contribute to the tissue maintenance and stimuli-responsive function. Enhancers are central to control this tissue-specific gene expression pattern. Here, we explore the correlation between the genomic location of enhancers and their role in tissue-specific gene expression. We found that enhancers showing tissue-specific activity are highly enriched in intronic regions and regulate the expression of genes involved in tissue-specific functions, while housekeeping genes are more often controlled by intergenic enhancers. Notably, an intergenic-to-intronic active enhancers continuum is observed in the transition from developmental to adult stages: the most differentiated tissues present higher rates of intronic enhancers, while the lowest rates are observed in embryonic stem cells. Altogether, our results suggest that the genomic location of active enhancers is key for the tissue-specific control of gene expression.

scholarly journals Sexually dimorphic control of gene expression in sensory neurons regulates decision-making behavior in C. elegans

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Zoë A Hilbert ◽  
Dennis H Kim
Keyword(s):  
Gene Expression ◽  
Decision Making ◽  
Sensory Neurons ◽  
Regulation Of Gene Expression ◽  
Sensory Information ◽  
Specific Expression ◽  
Control Of Gene Expression ◽  
Neuron Pair ◽  
C Elegans ◽  
Male Specific

Animal behavior is directed by the integration of sensory information from internal states and the environment. Neuroendocrine regulation of diverse behaviors of Caenorhabditis elegans is under the control of the DAF-7/TGF-β ligand that is secreted from sensory neurons. Here, we show that C. elegans males exhibit an altered, male-specific expression pattern of daf-7 in the ASJ sensory neuron pair with the onset of reproductive maturity, which functions to promote male-specific mate-searching behavior. Molecular genetic analysis of the switch-like regulation of daf-7 expression in the ASJ neuron pair reveals a hierarchy of regulation among multiple inputs—sex, age, nutritional status, and microbial environment—which function in the modulation of behavior. Our results suggest that regulation of gene expression in sensory neurons can function in the integration of a wide array of sensory information and facilitate decision-making behaviors in C. elegans.

p130Cas-dependent actin remodelling regulates myogenic differentiation

2012 ◽  
Vol 445 (3) ◽  
pp. 323-332 ◽  
Author(s):  
Keiko Kawauchi ◽  
Wee Wee Tan ◽  
Keigo Araki ◽  
Farhana Binte Abu Bakar ◽  
Minsoo Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Localization ◽  
Myogenic Differentiation ◽  
Specific Gene ◽  
Control Of Gene Expression ◽  
Cytoskeletal Organization ◽  
Specific Gene Expression ◽  
Myotube Formation ◽  
Integrin Β3 ◽  
Actin Remodelling

Actin dynamics are implicated in various cellular processes, not only through the regulation of cytoskeletal organization, but also via the control of gene expression. In the present study we show that the Src family kinase substrate p130Cas (Cas is Crk-associated substrate) influences actin remodelling and concomitant muscle-specific gene expression, thereby regulating myogenic differentiation. In C2C12 myoblasts, silencing of p130Cas expression by RNA interference impaired F-actin (filamentous actin) formation and nuclear localization of the SRF (serum-response factor) co-activator MAL (megakaryocytic acute leukaemia) following the induction of myogenic differentiation. Consequently, formation of multinucleated myotubes was abolished. Re-introduction of wild-type p130Cas, but not its phosphorylation-defective mutant, into p130Cas-knockdown myoblasts restored F-actin assembly, MAL nuclear localization and myotube formation. Depletion of the adhesion molecule integrin β3, a key regulator of myogenic differentiation as well as actin cytoskeletal organization, attenuated p130Cas phosphorylation and MAL nuclear localization during C2C12 differentiation. Moreover, knockdown of p130Cas led to the activation of the F-actin-severing protein cofilin. The introduction of a dominant-negative mutant of cofilin into p130Cas-knockdown myoblasts restored muscle-specific gene expression and myotube formation. The results of the present study suggest that p130Cas phosphorylation, mediated by integrin β3, facilitates cofilin inactivation and promotes myogenic differentiation through modulating actin cytoskeleton remodelling.

Genome-wide analysis of temporally regulated and compartment-specific gene expression in sporulating cells of Bacillus subtilis

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 399-420 ◽  
Author(s):  
Leif Steil ◽  
Mónica Serrano ◽  
Adriano O. Henriques ◽  
Uwe Völker
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Mother Cell ◽  
Molecular Mechanisms ◽  
Sigma Factors ◽  
Specific Gene ◽  
Temporal Expression ◽  
Control Of Gene Expression ◽  
Genome Wide ◽  
Specific Control

Temporal and compartment-specific control of gene expression during sporulation in Bacillus subtilis is governed by a cascade of four RNA polymerase subunits. σ F in the prespore and σ E in the mother cell control early stages of development, and are replaced at later stages by σ G and σ K, respectively. Ultimately, a comprehensive description of the molecular mechanisms underlying spore morphogenesis requires the knowledge of all the intervening genes and their assignment to specific regulons. Here, in an extension of earlier work, DNA macroarrays have been used, and members of the four compartment-specific sporulation regulons have been identified. Genes were identified and grouped based on: i) their temporal expression profile and ii) the use of mutants for each of the four sigma factors and a bofA allele, which allows σ K activation in the absence of σ G. As a further test, artificial production of active alleles of the sigma factors in non-sporulating cells was employed. A total of 439 genes were found, including previously characterized genes whose transcription is induced during sporulation: 55 in the σ F regulon, 154 σ E-governed genes, 113 σ G-dependent genes, and 132 genes under σ K control. The results strengthen the view that the activities of σ F, σ E, σ G and σ K are largely compartmentalized, both temporally as well as spatially, and that the major vegetative sigma factor (σ A) is active throughout sporulation. The results provide a dynamic picture of the changes in the overall pattern of gene expression in the two compartments of the sporulating cell, and offer insight into the roles of the prespore and the mother cell at different times of spore morphogenesis.

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