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.

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.

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.

Martin Victor et al. (2001) International C. elegans Meeting "The HAT activity of CBP-1 is indispensable for its biological function during C. elegans embryogenesis"

Craig C Mello, Martin Victor, Yang Shi

[International C. elegans Meeting, 2001]

CBP/p300 represent a conserved family of transcriptional cofactors possessing histone acetyltransferase (HAT) activity. This group of proteins has been shown to play critical roles in differentiation and cell proliferation in C. elegans , Drosophila , mouse and human. Despite their importance, the mechanisms by which they regulate transcription and thus exert their biological functions are poorly understood. A central question is whether the HAT activity is essential for the biological functions of these proteins. In this study, we investigated the importance of the HAT activity for CBP-1 in C. elegans . We show that a truncated CBP-1 protein lacking the HAT domain fails to promote differentiation, consistent with the notion that the HAT activity of CBP-1 is essential for its biological functions. To further address this question, we used a chemical that has been shown to be a specific inhibitor of the enzymatic activity of CBP/p300 HAT. Injection of this chemical into the gonads of the hermaphrodite mothers resulted in dead embryos arrested at different stages of development. Significantly, some of these embryos appear identical to cbp-1(RNAi) embryos in every aspect, as judged by the excess cell number, lack of endoderm and mesoderm tissues but excess neuronal differentiation (Shi and Mello, 1998). Taken together, our findings provide in vivo evidence that the HAT activity is not only critical, but also may be the only biochemical activity that is necessary for the biological functions of CBP-1. Shi, Y. and Mello,C. (1998), 'A CBP/p300 homolog specifies multiple differentiation pathways in Caenorhabditis elegans .' Genes Dev. 12(7), 943-55.

Howard Chang et al. (2003) International Worm Meeting "Functional analysis of AMPA-type glutamate receptor sequences and their contribution to receptor localization at synapses"

Christopher Rongo, Howard Chang

[International Worm Meeting, 2003]

The modulation of AMPA-type glutamate receptor localization to central nervous system synapses is an important component of synaptic plasticity, and can be triggered by LTP (Long Term Potentiation) and CaMKII (Type II calcium-calmodulin-dependent protein kinase) activity. (1, 2). In mammalian hippocampal neurons, the different AMPA receptor subunits GluR1, GluR2, GluR3, and GluR4 are proposed to form heteromultimers, and individual subunits can confer localization specificity to the multimeric channels that they comprise (2,3). One likely explanation for such subunit specificity is that the targeting of AMPA receptors to the synapses is probably due to the interaction of PDZ domain-containing proteins and the receptor subunit cytosolic tail sequences (4). In C. elegans, there are four AMPA receptor subunits that are expressed in the command interneurons, including, GLR-1, GLR-2, GLR-4 and GLR-5 (5,6). GLR-1 has C-terminal sequence that contributes to channel turnover (7). We have identified a glutamate receptor subunit domain that appears to be instructive for channel localization. This new finding may shed a new light on the localization and regulation of AMPA-type glutamate receptors in response to plasticity at synapses. (1) Rongo et al. Nature (1999), vol. 402, p. 195 (2) Shi et al. Science (1999), vol. 284, p.1811 (3) Shi et al. Cell (2001), vol. 105, p. 331 (4) Rongo et al. Cell (1998), vol. 94, p. 751 (5) Brockie et al. J. Neuroscience (2001), vol. 21(5), p. 1510 (6) Mellem et al. Neuron 2002 Dec 5; 36:933-944 (7) Burbea M et al. Neuron 2002 Jul 3;35(1):107-20

David Katz et al. (2006) Development & Evolution Meeting "Histone H3 Lysine 4 Guards The Immortality Of The Germline"

William Kelly, David Katz, Thomas Edwards

[Development & Evolution Meeting, 2006]

In the C. elegans early embryo, the chromatin in all blastomeres initially contain the euchromatic mark, di-methylation of histone H3 on lysine 4 (H3K4me2). However, upon their birth, the primordial germ cells Z2 and Z3 uniquely lose this mark. This appears to be a conserved characteristic of primordial germ cells as Drosophila pole cells also initially lack this modification. The loss of H3K4me2 has been proposed to be necessary for protecting the totipotency of the germline through chromatin-based transcriptional repression, but the mechanism of its removal is not understood. Recently, Shi et al. demonstrated that the mammalian amine oxidase LSD1 can specifically demethylate H3K4me2 (Shi et al. 2004). C. elegans has three lsd1 homologs : spr-5, amx-1 and T08D10.2. In order to ask if these genes play a role in the chromatin remodeling of Z2 and Z3, we inactivated them individually and in combination. RNAi or genetic mutation of any of these genes individually has no affect on H3K4me2 levels in Z2 and Z3. To test for redundant roles in the chromatin remodeling of Z2 and Z3, we generated a spr-5;amx-1 double mutant from two existing putative null mutations. Although the double mutants display multiple pleiotropic phenotypes, we observed no immediate effects on H3meK4 levels in Z2 and Z3. However, after multiple generations the double mutant exhibits a germline mortality phenotype, in which an increasing fraction of progeny in the population grow up sterile(black sterile phenotype). Strikingly, in these later generations, the double mutants exhibit retention of H3K4me2 in Z2 and Z3, further correlating repressive chromatin with maintenance of the germ cell lineage. In addition, the multiple generations that are required for this phenotype suggest that there can be stable inheritance of epigenetic defects through the germline and that the H3K4 demethylases may play a role in resetting H3K4 methylation at each generation.

Wang, Lin et al. (2017) International Worm Meeting "Dissecting the roles of the ADAM10 metalloprotease SUP-17 in the BMP signaling pathway in Caenorhabditis elegans."

Liu, Jun, Liu, Zhiyu, Shi, Herong, Wang, Lin

[International Worm Meeting, 2017]

BMPs (Bone Morphogenetic Proteins) belong to the TGFbeta superfamily of ligands, and mediate a highly conserved signal transduction cascade. Upon ligand binding, type II receptors phosphorylate type I receptors, which in turns phosphorylate R-Smads (receptor regulated Smads). Phosphorylated R-Smads then complex with co-Smad (common mediator smad) and enter the nucleus to regulate gene transcription with different co-factors. BMPs play important roles in developmental and physiological processes. Malfunction of the pathway in humans can cause various disorders, such as skeleton diseases, heart diseases and cancers. So it is critical to strictly regulate the level of BMP signaling spatiotemporally. Using a highly specific genetic screen, we have identified several modulators of the BMP pathway in C. elegans. These include the Neogenin homolog UNC-40 [1], two paralogous tetraspanin proteins, TSP-12 and TSP-14, and an ADAM10 metalloproteinase (A Disintegrin and Metalloproteinase 10) SUP-17 [2]. We use the CRISPR/Cas9 system and tagged the endogenous TSP-12, SUP-17 and UNC-40 proteins with different fluorescence tags. We found that TSP-12 is localized to the cell surface and intracellular vesicles, and that TSP-12 can bind to SUP-17 and promote its cell surface localization. We have genetic evidence and preliminary biochemical evidence showing that UNC-40 is one, but not the only, substrate of SUP-17 in BMP signaling. We are currently identifying additional substrate(s) of SUP-17 in BMP signaling. Our work highlights the importance of intracellular signaling and protease processing in the regulation of BMP signaling. [1] Tian C, Shi H, Xiong S, Hu F, Xiong WC, Liu J. The neogenin/DCC homolog UNC-40 promotes BMP signaling via the RGM protein DRAG-1 in C. elegans. Development. 2013;140(19):4070-80. doi: 10.1242/dev.099838. PubMed PMID: 24004951; PubMed Central PMCID: PMCPMC3775419. [2] Wang L, Liu Z, Shi H, Liu J. Two Paralogous Tetraspanins TSP-12 and TSP-14 Function with the ADAM10 Metalloprotease SUP-17 to Promote BMP Signaling in Caenorhabditis elegans. PLoS Genet. 2017;13(1):e1006568. doi: 10.1371/journal.pgen.1006568. PubMed PMID: 28068334; PubMed Central PMCID: PMCPMC5261805.