Boese QF et al. (1998) East Coast Worm Meeting "Mapping and Cloning the mut-2 Mutator"

Collins JJ, Pelliccia JG, Alcazar R, Boese QF

[East Coast Worm Meeting, 1998]

We are interested in the function of mut-2, a gene that regulates the movement of all known transposons in the genome of Caenorhabditis elegans. Our goal is to map, clone and characterize this interesting and elusive gene. We have mapped mut-2 to a 0.24 map unit region of Linkage Group 1 flanked by dpy-14 and sem-4 . This interval corresponds to a physical distance of about 200-300 kilobases that has complete cosmid/YAC coverage and for which complete sequence is available. However, the phenotypes conferred by mut-2(r459) -- enhanced germ-line transposition (Mut) and a high incidence of males (Him) -- preclude straightforward rescue approaches for identifying a molecular clone of the gene. To circumvent this obstacle, we are searching the mut-2 interval for Bristol/Bergerac polymorphisms to use in higher resolution mapping of the gene. We are also searching the finished sequence of the region for candidate genes to examine. Understanding how mut-2 functions will enhance our understanding of the mechanisms that control transposition in eukaryotic genomes. It should also provide better ways to manipulate transposon activity in C. elegans, facilitating reverse genetic approaches in this important model organism. This work is supported by several Grants-in-aid of Research from Sigma Xi, the Scientific Research Society and the Graduate School of UNH.

Boese QF et al. (1997) International C. elegans Meeting "MAPPING AND CLONING THE MUT-2 MUTATOR"

Collins JJ, Boese QF, Pelliccia JG

[International C. elegans Meeting, 1997]

We are interested in mapping, cloning and characterizing the mut-2 gene in C. elegans in order to understand its role as a global regulator of transposon activity within the genome of our favorite nematode. Information regarding the role of mut-2 will certainly enhance the utility of transposons as molecular tools in reverse genetic approaches. More importantly, characterization of the mut-2 gene will elucidate the mechanism by which a host genome regulates transposon activity, and provide evidence as to whether similar genes regulate transposon activity in other hosts. The mut-2 gene was first identified in a screen of EMS-induced mutations that elevated 100-fold the reversion frequency of an unc54::Tc1 mutant. Further studies of strains carrying the mut-2(r459) mutation revealed the presence of three new families of transposons: Tc3, Tc4, and Tc5. In fact, these three families are known only to be active in the presence of the mut-2 mutation. These features suggest that mut-2 plays an interesting global role in maintaining the integrity of the worm genome. Mapping studies in our lab confirmed and refined the position of mut-2 to a 200 to 300 kilobase region to the left of sem-4 on Linkage Group I. We have obtained candidate cosmids from an ordered array spanning this region. The cosmids are currently being tested for mut-2 function by microinjection into the appropriate strains. Due to the nature of the mut-2 phenotype (which requires screening large populations for frequencies of him and TcX activation), rescue experiments of the mutant phenotype may give ambiguous results. Therefore we are using PCR and the available sequence information to amplify candidate genes from the mut-2 mutant strain. Genetic transformation assays using these DNA fragments will be used to confirm the identity of the mut-2 gene.

Boese QF et al. (1996) East Coast Worm Meeting "Mapping the mut-2 mutator II"

Boese QF, Collins JJ, Beinhorn J, Pelliccia JG

[East Coast Worm Meeting, 1996]

We are characterizing the mut-2 gene in C. elegans. We want to understand its role in regulating transposons in the worm genome. These studies should augment transposon-based reverse genetic approaches in this organism and may reveal features of a novel process for regulating transposon activity in eukaryotic genomes. How the host genome regulates transposon activity is not well understood. The mut-2 gene was identified in a screen for EMS-induced mutations that elevated reversion frequency of an unc54::Tc1 mutant. Subsequent studies showed that this mutation, mut-2 (r459), also increased transposition of Tcl and activated transposition and excision of at least three other families of transposons in the C. elegans genome, Tc3, Tc4 and Tc5. For these reasons, this mutant allele of mut-2 has been termed the mut-2 mutator. We would like to know the function of the wild type mut-2 gene and its role in transposon control and genome integrity in C. elegans, and how these are altered by the mut-2 mutator. To this end we want to map and clone mut-2. Early studies mapped the mut-2 mutator activity to the unc-13/unc-15 region of LG1 (Finney and Horvitz, pers comm). We have refined this map position, using the high incidence of males (Him) phenotype conferred by mut-2(r459) as our genetic assay. We crossed a dpy-5;mut-2 strain by a sem-4;unc-13 strain, picked Dpy,Sem,Unc and Dpy,non-Sem,Unc recombinants and scored each for spontaneous male offspring to determine mut-2 mutator status for each. Results place mut-2 in the 1.65 map unit interval between dpy-5 and sem-4 and suggest it is near sem-4. This corresponds to a physical distance of approximately 200-300 kb, which is covered by a single YAC. Experiments are underway to clone mut-2. We will attempt to rescue the mut-2 mutator phenotype with cosmids from the region. We are also making a genomic library from a mut-2 mutant strain. We will use the existing cosmids and YAC from this region to identify candidate clones from the mut-2 mutant library and test them by microinjection for ability to donate mut-2 mutator activity.

QF Boese et al. (1999) International C. elegans Meeting "The mut-2 mutator of C. elegans"

J Collins, QF Boese

[International C. elegans Meeting, 1999]

The mut-2 mutator plays multiple regulatory roles in the C. elegans germline. In addition to regulating activity of at least four distinct transposon families it is implicated in chromosome segregation, gametogenesis, germline gene silencing and DNA double-strand break repair. Elucidating the function of mut-2 will enhance the utility of transposons for functional genomic studies in this organism and shed light on mechanisms that maintain structural and functional integrity of eukaryotic genomes. Using the Him phenotype conferred by mut-2(r459), we mapped the gene between dpy-14 and sem-4 on LGI. However, efforts to identify a molecular clone of the gene were hampered because the available phenotypes were unsuitable for standard transformation rescue approaches. This roadblock led us to examine the temperature-sensitive (ts) behavior of mut-2: the original mut-2 isolates TR674 and TR679 are inviable at 25 o C. Analysis of ancestors and recombinants derived from these isolates demonstrated that the ts phenotype cosegregates with mut-2 and is unrelated to the Bergerac ancestry of these strains. Closer inspection (with assistance from Eric Lambie) revealed that sterility results from a defect in gametogenesis. Gonads of mut-2 animals raised at 25 o C appear normal but have a dearth of sperm which exhibit variable morphology. Oocytes show no gross abnormalities but may be affected as well. DAPI staining reveals no obvious chromosomal defects. We are testing if wild-type sperm can rescue sterility and determining the temperature-critical stage of sperm (and oocyte?) development. The ts sterile phenotype is rescued by sDp2, a large free duplication that covers the dpy-14 sem-4 interval. This suggests it may be a suitable phenotype for identifying a molecular clone of mut-2. Two smaller free duplications that bisect this interval, hDp62 and hDp65, were tested; hDp65 rescues, hDp62 does not. This positions mut-2 between +1.57 and +1.66, a region covered by three cosmids, containing ~20 ORFs. Work is in progress to clone mut-2 by rescuing sterility. Recent evidence suggests mut-2 strains may be resistant to RNAi (1). We are exploring the use of this phenotype in our cloning efforts as well. 1) Tabara, H and Mello, C. 1999. (this meeting)

Wood WB et al. (2000) West Coast Worm Meeting "Does CEH-20, an Exd/Pbx homolog in C. elegans, play a role in worm embryogenesis?"

Boese QF, Wood WB

[West Coast Worm Meeting, 2000]

Several studies have shown that the TALE (three-amino-acid-loop-extension) homeodomain (HD) proteins, Exd and Hth in Drosophila and their mammalian homologs, Pbx1-3 and Meis, complex with Hox proteins to enhance binding specificity for target promoter sites. Studies have further shown that Hth/Meis interacts with Exd/Pbx to promote nuclear localization facilitating their roles as co-factors in the nucleus. Recent work in our lab revealed that loss-of-function (lf) mutations in the C. elegans Hth/Meis homolog, unc-62, result in pleiotropic defects including defects in embryonic development. To investigate whether the Exd/Pbx homolog, CEH-20 might also be involved in embryonic patterning, we have begun characterizing the defects of two strong ceh-20(lf) alleles (gift from M. Stern) with respect to their potential interactions with unc-62 and other embryonically required genes (Hox or non-Hox). Both ceh-20 alleles result in low-penetrance larval lethality and no apparent embryonic defects suggesting that ceh-20 is not normally required for embryogenesis. Injection of double-stranded ceh-20 RNA into wild-type hermaphrodites [ceh-20 (RNAi)] results in an Unc Egl phenotype but no embryonic defects. To test for an interaction with the embryonically required posterior Hox paralog gene nob-1 (1), ceh-20 (RNAi) was performed in the background of a weak allele, nob-1(ct230). No enhancement of the mutant phenotype was observed. Whether this reflects compensatory functions of two other Exd/Pbx homologs, ceh-40 and F22A3.x, in the absence of ceh-20, is under investigation. To test for interactions with unc-62, ceh-20 RNAi was performed in the background of the weak allele unc-62 (e644). The low penetrance e644 lethal phenotype was enhanced over the uninjected control, consistent with an interaction between these genes during embryogenesis. To test for interactions with other non-Hox HD proteins important for embryogenesis, ceh-20 RNAi was performed in the background of a weak pal-1 allele (e2091). The e2091 mutant phenotype was also enhanced suggesting a previously undescribed interaction between Exd/Pbx and Caudal/CDX homologs. Further tests for ceh-20 interactions with other embryonically required genes are in progress. (1) Van Auken, K. et al., 2000, PNAS 91: 4499-503

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.