Wei X et al. (2012) Nat Methods "Controlling gene expression with the Q repressible binary expression system in Caenorhabditis elegans."

Shen K, Wei X, Luo L, Potter CJ

[Nat Methods, 2012]

We established a transcription-based binary gene expression system in Caenorhabditis elegans using the recently developed Q system. This system, derived from genes in Neurospora crassa, uses the transcriptional activator QF to induce the expression of target genes. Activation can be efficiently suppressed by the transcriptional repressor QS, and suppression can be relieved by the nontoxic small molecule quinic acid. We used QF, QS and quinic acid to achieve temporal and spatial control of transgene expression in various tissues in C. elegans. We also developed a split Q system, in which we separated QF into two parts encoding its DNA-binding and transcription-activation domains. Each domain showed negligible transcriptional activity when expressed alone, but expression of both reconstituted QF activity, providing additional combinatorial power to control gene expression.

Nonet, Michael (2021) MicroPubl Biol

Nonet, Michael

[MicroPubl Biol, 2021]

The first generation of C. elegans GAL4 drivers for bipartite expression function less well than C. elegans tet ON/OFF, QF and LexA drivers. The main difference between the GAL4 drivers and the others is the absence of a flexible linker between the DNA binding and activation domain in the GAL4 construct. Addition of a linker to a GAL4-QF construct increased driver potency, while adding linkers to a GAL4-VP64 driver was much less effective. Extending the linker region of the tetR-L-QF driver also increased activity of that driver. The new GAL4 driver makes GAL4/UAS bipartite system activity comparable to the other worm bipartite expression systems.

Crittenden, Sarah L. et al. (2013) International Worm Meeting "Progress on developing the Q system to study GSCs and their control."

Crittenden, Sarah L., Kimble, Judith, Mohanty, Ipsita

[International Worm Meeting, 2013]

The Q system (Potter et al., 2010) permits inducible gene expression in C. elegans (Wei et al., 2012) and is similar in principal to the widely used GAL4-UAS system. The QF transcriptional activator directs transcription via an upstream activating sequence (QUAS). But in addition, another protein, QS, can inhibit QF activation and QS repression can be relieved by a small molecule, quinic acid or QA. QA is non-toxic and can be fed to worms (Wei et al., 2012). This inducible system has the potential to control target genes in both space and time. A chemically inducible method to control gene expression in specific cells at specific times would be tremendously useful for analyses of germline stem cells (GSCs) and their niche, the somatic distal tip cell (DTC). To this end, we are generating mosSCI insertion transgenes that rely on DTC-specific and GSC-specific regulatory sequences to drive QF expression. We are also generating a QUAS driven nuclear GFP (fused to H2B). Once we have the QF/QUAS pair working well for spatial regulation, we will add the QS/QA pair for temporal regulation. Preliminary experiments are promising and results will be shared at the meeting.

Wei, Xing et al. (2011) International Worm Meeting "Controlling Gene expression with the Q Repressible Binary Expression System In C.elegans."

Shen, Kang, Wei, Xing, Potter, Christopher J., Luo, Liqun

[International Worm Meeting, 2011]

The capability to regulate the expression of engineered transgenes has revolutionized the study of biology in multi-cellular genetic model organisms. One popular and powerful strategy is using a binary expression system such as the tetracycline-regulated tTA/TRE system in mammals and the GAL4/UAS system in Drosophila. However, so far there has not been any transcription-based binary expression system reported in nematode C.elegans. Recently, a novel repressible binary expression system, the Q system, was established in Drosophila and mammalian cells based on the regulatory genes from the Neurospora crassa qa gene cluster. The transcriptional activator QF binds to a 16bp sequence (named as QUAS) and activates expression of target genes under the control of QUAS sites; the expression can be efficiently suppressed by its transcriptional repressor QS; the transcriptional suppression can be relieved by feeding animals quinic acid, a non-toxic small molecule. So far, we have successfully adapted the Q system into C.elegans and proven its high specificity and sensitivity in nervous system. We created transgenic lines that co-express QF in a specific subset of neurons together with the QUAS::GFP, and as expected, GFP was only expressed in these neurons. When QS was expressed in these neurons with QF and QUAS::GFP, the expression of GFP was efficiently suppressed. And the suppression can be relieved in 6 hours if feeding these transgenic animals on NGM plates containing quinic acid, suggesting that the Q repressible binary system is as effective in nematode C.elegans as in mammalian cells and Drosophila. Besides precise temporal control of expression, the Q system can also be utilized to refine spatial control of transgene expression by using combinatorial promoters. Using different promoters to express QF and QS, we can label more specific subset of neuron. Furthermore, we split QF into two halves, binding half and activation half, and we fused a heterodimerizing leucine zipper fragment with each half to enhance the reconstitution efficiency of active QF. When the two halves were expressed using different promoters, the transcriptional activity could be reconstituted within the intersectional subset of two promoters. The newly introduced "split Q system" with intersectional promoters can afford even higher degree of control and achieve expression at the single cell resolution.

Chen X et al. (2015) Mol Neurodegener "Erratum to: Ethosuximide ameliorates neurodegenerative disease phenotypes by modulating DAF-16/FOXO target gene expression."

McCue HV, Chen X, Morgan A, Kashyap SS, Barclay JW, Kraemer BC, Burgoyne RD, Wong SQ

[Mol Neurodegener, 2015]

The original version of this article [1] unfortunately contained a mistake. The author list contained a spelling error for the author Hannah V. McCue. The original article has been corrected for this error. The corrected author list is given below:Xi Chen, Hannah V. McCue, Shi Quan Wong, Sudhanva S. Kashyap, Brian C. Kraemer, Jeff W. Barclay, Robert D. Burgoyne and Alan Morgan

She, X. et al. (2017) International Worm Meeting "Applying the Q system for spatiotemporal gene expression in adult C. elegans."

Hansen, M., She, X.

[International Worm Meeting, 2017]

While C. elegans has long been on the leading edge of aging research, a spatial-temporal controlled gene expression system is currently lacking to fully understand tissue- and adult-specific functions of genes modulating lifespan and healthspan in this organism. The Shen lab recently adapted the so-called Q-system for inducible gene expression in a subset of C. elegans neurons (Wei et al., Nature Methods, 2013). Originally discovered in the fungus Neurospora crassa, this binary system is comprised of the transcription factor QF and its repressor QS. In the presence of quinic acid, the QS repressor is released from binding with QF, allowing QF to activate the expression of transgenes. Temporal control is achieved through quinic-acid supplementation to the media plates, and spatial control by use of tissue-specific promoters that drive the expression of QS and QF. Here, we further developed the Q-system and tested it for its usability in C. elegans aging studies. First, we drove the elements of the Q-system with tissue-specific promoters expressing in several major tissue types to show the inducibility of the system in adult animals. Our results indicate that the Q-system can be used to induce expression in such tissues. Second, we tested the effects of quinic acid and Q-system components on C. elegans lifespan and stress resistance and detected no apparent effects on longevity or fitness, suggesting that the Q-system can be used for lifespan studies. We will present current progress towards applying the Q-system as a powerful system for spatial- and temporal analysis of gene function in adult C. elegans.

Wei X et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Application of a Novel Repressible Binary Expression System (Q System) in C.elegans"

Shen K, Luo L, Wei X

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Spatial and temporal control of gene expression is an important tool for modern genetics studies in multi-cellular model organisms. One powerful genetic strategy is to use binary expression systems such as GAL4/UAS system in Drosophila. However the GAL4/UAS does not work very well in C.elegans. Alternative method is to use DNA recombinases such as FLP or Cre, but the expression level decreases very quickly in extrachromosomal array except being integrated and the expression cannot be suppressed. Recently, A novel repressible binary expression system, Q system, based on the regulatory genes from the Neurospora crassa qa gene cluster has been well established in Drosophila and mammalian cells. The transcriptional activator QA-1F (QF) can effectively activate expression of target genes under the control of the QF binding sites (Q-UAS); this expression can be repressed by transcriptional repressor QA-1S (QS) efficiently; the transcriptional suppression can be relieved by feeding animals with quinic acid. The Q system can provide precise spatial and temporal control of gene expression in Drosophila. We have successfully adjusted the Q system into C.elegans and proven its high specificity and sensitivity in backwards locomotion circuit. unc-4 is expressed in A-type neurons (DA neurons as well as VA neurons) in the ventral nerve cord. Under the control of unc-4 promoter, QF is expressed in DA and VA neurons and can activate the expression of Q-UAS::GFP in these neurons. The expression of GFP in A-type neurons can be suppressed by using unc-4 promoter to express suppressor QS . And the suppression can be relieved in 6 hours if feeding these transgenic animals on NGM plates containing quinic acid. Furthermore, using DA neurons specific promoter unc-4c to express QS to suppress the expression of GFP in DA neurons, we can label VA neurons specifically. It means that by using different combinations of tissue specific promoters driving expression of QF and QS, we can express a target gene in a more specific subset of cells. Moreover with the development of Mos1-induced single copy insertion technique to introduce FRT sites into genome, we can develop MARCM (Mosaic analysis with a repressible cell marker) in C.elegans to analyze single mutant cell in wild type animals.

Liu Y et al. (2012) J Pharmacol Sci "Activated O2() and H2O2 mediated cell survival in SU11274-treated non-small-cell lung cancer A549 cells via c-Met-PI3K-Akt and c-Met-Grb2/SOS-Ras-p38 pathways."

Liu Y, Ikejima T, Ye YC, Onodera S, Shi QF, Tashiro SI

[J Pharmacol Sci, 2012]

The pharmacological activity of SU11274 is primarily due to its inhibition of hepotocyte growth factor receptor (c-Met) kinase overexpression. In this study, we demonstrated that the pathway involved in SU11274-induced autophagy was presumably through inhibition of c-Met and its down-stream pathways, including phosphatidylinositol 3-kinases Akt (PI3KAkt) and the growth factor receptor bound protein-2 / son of sevenless Ras p38 MAPK (Grb2/SOSRasp38) pathway. SU11274 time-dependently induced the generation of superoxide anion (O2()) and hydrogen peroxide (H2O2). There is a negative feedback loop between reactive oxygen species (ROS) induction and SU11274. Then, we investigated the role of ROS in protecting cells against SU11274-induced autophagic cell death in A549 cells. O2() and H2O2 generation activated c-MetPI3KAkt and c-MetGrb2/SOSRasp38 signaling pathways, which were suppressed by O2() scavenger superoxide dismutase (SOD) and H2O2 scavenger catalase. In conclusion, O2() and H2O2 evoked cell resistance to SU11274 via activating c-MetPI3KAkt and c-MetGrb2/SOSRasp38 pathways in A549 cells. SU11274 also induced ROS generation in Caenorhabditis elegans.

She, Xingyu et al. (2015) International Worm Meeting "Applying a Spatial-Temporal Controlled Gene Expression System to Aging Research."

She, Xingyu, Hansen, Malene

[International Worm Meeting, 2015]

C. elegans has been an effective model organism for identifying genes with important physiological functions, including in aging. However, to fully dissect gene function an effective spatial-temporal controlled gene expression system is needed. In C. elegans, several protocols have been put forward for this purpose, including driving temporal gene expression using heat shock inducible promoters (Bacaj et al. 2007), and achieving tissue-specific gene expression using site-specific recombination methods (Cre-loxP, FLP-FRT). However, none of these systems have been used for simultaneous temporal- and spatial control and it is uncertain how applicable they would be for aging studies (see Xu & Kim, 2011). While several chemicals (e.g., tetracycline, RU486) are utilized to induce gene expression in other systems, such protocols are not available in C. elegans. However, the Shen lab recently adapted the Q system to C. elegans, a system in which inducible gene expression is obtained by the chemical quinic acid (Wei et al. 2013). This binary system, originally discovered in the fungus Neurospora crassa, is comprised of the transcription factor QF and its repressor QS. In the presence of quinic acid, the QS repressor is released from binding with QF, allowing QF to activate the expression of transgenes. Temporal control is achieved through the timing of quinic acid supplementation, and spatial control by use of tissue-specific promoters that drive the expression of QS and QF (Wei et al. 2013). We are interested in applying the Q system for our research on genes with roles in aging. Towards this goal, we are focusing on three main aspects. First, we have tested the effects of quinic acid and the Q system on C. elegans lifespan and stress resistance and detected no apparent alterations on longevity or fitness, suggesting that the Q system is suitable for aging studies. Second, we are driving the elements of the Q system with tissue-specific promoters to test inducibility in major tissue types of adult animals. Preliminary results indicate that the Q system can be used to induce expression in selected tissues, including neurons and muscle. Third, we are creating a library of Gateway-compatible plasmids that contain the various elements of the Q system and tissue-specific promoters to expedite the generation of Q system transgenic strains for future gene-specific projects. We will present our current progress towards applying the Q system as a powerful tool for spatial- and temporal analysis of gene function in adult C. elegans. This work will also be discussed at the "Spatial and temporal analysis of gene function in C. elegans" workshop.

Driesschaert, Brecht et al. (2021) International Worm Meeting "Applying the Q system in Caenorhabditis elegans: observed issues and challenges"

Driesschaert, Brecht, Temmerman, Liesbet, Dens, Elisabeth, Mergan, Lucas

[International Worm Meeting, 2021]

Gaining control over spatiotemporal features of gene expression is useful to improve our understanding of biological regulation. A common approach is to (ir)reversibly switch genes on or off via conditional expression systems. One such genetic toolkit is the Q system, a binary conditional expression system that was originally developed for mammalian cells and D. melanogaster, but has quickly been adapted for use in C. elegans as well. The system consists of three components: QUAS, QF and QS. Expression of a sequence of interest can be controlled by placing it downstream of an enhancer sequence (QUAS) that can be recognized by a transcriptional activator (QF). Advantageous over the canonical Gal4UAS system, the Q system permits to temporally control gene expression in a temperature-independent manner through the addition of quinic acid, which (reversibly) sequesters the transcriptional suppressor QS. Because of this substantial benefit, we turned to the Q system to build a reporter strain to visualize the endocytic capacity of the C. elegans coelomocytes. The objective is to gain reversible temporal control (on and off) over the expression of a fluorescent reporter, mNeonGreen, to generate temporally resolved mNeonGreen secretion by source cells, of which the subsequent degradation by the coelomocytes could then be observed. However, while the Q system has successfully been used to spatially restrict gene expression, there is little support for its performance in terms of temporal control. In an ongoing effort to validate the Q system for the research purpose described above, we encountered several points of attention which we here wish to share with the community. Especially at the level of the QF/QS ratio, there appears to be but a narrow window of opportunity that avoids leaky expression on one hand, vs inefficient de-repression of transcription by quinic acid on the other. We hope these results may engage others using conditional expression systems in a discussion balancing practical challenges and opportunities of these tools.