Refined LexA transactivators and their use in combination with the Drosophila Gal4 system.
ABSTRACT: The use of binary transcriptional systems offers many advantages for experimentally manipulating gene activity, as exemplified by the success of the Gal4/UAS system in Drosophila. To expand the number of applications, a second independent transactivator (TA) is desirable. Here, we present the optimization of an additional system based on LexA and show how it can be applied. We developed a series of LexA TAs, selectively suppressible via Gal80, that exhibit high transcriptional activity and low detrimental effects when expressed in vivo. In combination with Gal4, an appropriately selected LexA TA permits to program cells with a distinct balance and independent outputs of the two TAs. We demonstrate how the two systems can be combined for manipulating communicating cell populations, converting transient tissue-specific expression patterns into heritable, constitutive activities, and defining cell territories by intersecting TA expression domains. Finally, we describe a versatile enhancer trap system that allows swapping TA and generating mosaics composed of Gal4 and LexA TA-expressing cells. The optimized LexA system facilitates precise analyses of complex biological phenomena and signaling pathways in Drosophila.
Project description:Tissue-specific gene expression using the upstream activating sequence (UAS)–GAL4 binary system has facilitated genetic dissection of many biological processes in Drosophila melanogaster. Refining GAL4 expression patterns or independently manipulating multiple cell populations using additional binary systems are common experimental goals. To simplify these processes, we developed a convertible genetic platform, the integrase swappable in vivo targeting element (InSITE) system. This approach allows GAL4 to be replaced with any other sequence, placing different genetic effectors under the control of the same regulatory elements. Using InSITE, GAL4 can be replaced with LexA or QF, allowing an expression pattern to be repurposed. GAL4 can also be replaced with GAL80 or split-GAL4 hemi-drivers, allowing intersectional approaches to refine expression patterns. The exchanges occur through efficient in vivo manipulations, making it possible to generate many swaps in parallel. This system is modular, allowing future genetic tools to be easily incorporated into the existing framework.
Project description:Gal4-mediated activation of GAL gene transcription in Saccharomyces cerevisiae requires the interaction of Gal3 with Gal80, the Gal4 inhibitor protein. While it is known that galactose and ATP activates Gal3 interaction with Gal80, neither the mechanism of activation nor the surface that binds to Gal80 is known. We addressed this through intragenic suppression of GAL3C alleles that cause galactose-independent Gal3-Gal80 interaction. We created a new allele, GAL3SOC, and showed that it suppressed a new GAL3C allele. We tested the effect of GAL3SOC on several newly isolated and existing GAL3C alleles that map throughout the gene. All except one GAL3C allele, D368V, were suppressible by GAL3SOC. GAL3SOC and all GAL3C alleles were localized on a Gal3 homology model that is based on the structure of the highly related Gal1 protein. These results provide evidence for allosterism in the galactose- and ATP-activation of Gal3 binding to Gal80. In addition, because D368V and residues corresponding to Gal80-nonbinder mutations colocalized to a domain that is absent in homologous proteins that do not bind to Gal80, we suggest that D368 is a part of the Gal80-binding surface.
Project description:Studies on the formation of connections in the developing nervous system are greatly aided by methods that permit the differential visualisation and manipulation of pre- and postsynaptic partner neurons. This has been facilitated by the advent of the LexA-based, GAL4/UAS-independent, binary expression system. On the molecular side, the introduction of DNA sequences into expression vectors has been simplified by the Invitrogen Gateway cloning technology. We have developed cloning vectors that combine the Gateway cloning technology with the LexA-based genetic expression system. These vectors facilitate the creation of driver and reporter constructs for the generation of Drosophila transgenic lines for the new LexA-based binary transcriptional system. We further report a new LexA::GAD sensory neuron driver and a red fluorescent membrane targeted lexAop reporter designed to complement the existing GFP-based lexAop reporter. Using these transgenic lines we have been able to differentially label motor and sensory neuron projections in the ventral nerve cord of Drosophila larvae.
Project description:Under non-inducing conditions (absence of galactose), yeast structural genes of the GAL regulon are repressed by Gal80, preventing interaction of Gal4 bound to UAS<sub>GAL</sub> promoter motifs with general factors of the transcriptional machinery. In this work, we show that Gal80 is also able to interact with histone deacetylase-recruiting corepressor proteins Cyc8 and Tup1, indicating an additional mechanism of gene repression. This is supported by our demonstration that a lexA-Gal80 fusion efficiently mediates repression of a reporter gene with an upstream lexA operator sequence. Corepressor interaction and in vivo gene repression could be mapped to a Gal80 minimal domain of 65 amino acids (aa 81-145). Site-directed mutagenesis of selected residues within this domain showed that a cluster of aromatic-hydrophobic amino acids (YLFV, aa 118-121) is important, although not solely responsible, for gene repression. Using chromatin immunoprecipitation, Cyc8 and Tup1 were shown to be present at the GAL1 promoter in a wild-type strain but not in a gal80 mutant strain under non-inducing (derepressing) growth conditions. Expression of a GAL1-lacZ fusion was elevated in a tup1 mutant (but not in a cyc8 mutant) grown in derepressing medium, indicating that Tup1 may be mainly responsible for this second mechanism of Gal80-dependent gene repression.
Project description:Characterization and functional manipulation of specific groups of neurons in the vertebrate central nervous system (CNS) remains a major hurdle for understanding complex circuitry and functions. In zebrafish, the Gal4/UAS system has permitted expression of transgenes and enhancer trap screens, but is often limited by broad expression domains. We have developed a method for cell-type specific expression using Gal80 inhibition of Gal4-dependent expression. We show that native Gal4 is able to drive strong expression, that Gal80 can inhibit this expression, and that overlapping Gal4 and Gal80 expression can achieve "intersectional" expression in spatially and genetically defined subsets of neurons. We also optimize Gal80 for expression in vertebrates, track Gal80 expression with a co-expressed fluorescent marker, and use a temperature-sensitive allele of Gal80 to temporally regulate its function. These data demonstrate that Gal80 is a powerful addition to the genetic techniques available to map and manipulate neural circuits in zebrafish.
Project description:Gal4 is a prototypical eukaryotic transcriptional activator whose recruitment function is inhibited in the absence of galactose by the Gal80 protein through masking of its transcriptional activation domain (AD). A long-standing nondissociation model posits that galactose-activated Gal3 interacts with Gal4-bound Gal80 at the promoter, yielding a tripartite Gal3-Gal80-Gal4 complex with altered Gal80-Gal4 conformation to enable Gal4 AD activity. Some recent data challenge this model, whereas other recent data support the model. To address this controversy, we imaged fluorescent-protein-tagged Gal80, Gal4, and Gal3 in live cells containing a novel GAL gene array. We find that Gal80 rapidly dissociates from Gal4 in response to galactose. Importantly, this dissociation is Gal3 dependent and concurrent with Gal4-activated GAL gene expression. When galactose-triggered dissociation is followed by galactose depletion, preexisting Gal80 reassociates with Gal4, indicating that sequestration of Gal80 by Gal3 contributes to the observed Gal80-Gal4 dissociation. Moreover, the ratio of nuclear Gal80 to cytoplasmic Gal80 decreases in response to Gal80-Gal3 interaction. Taken together, these and other results provide strong support for a GAL gene switch model wherein Gal80 rapidly dissociates from Gal4 through a mechanism that involves sequestration of Gal80 by galactose-activated Gal3.
Project description:The UAS/GAL4 system is the most used method in Drosophila melanogaster for directing the expression of a gene of interest to a specific tissue. However, the ability to control the temporal activity of GAL4 with this system is very limited. This study constructed and characterized Tet-off GAL80 transgenes designed to allow temporal control of GAL4 activity in aging adult muscles. By placing GAL80 under the control of a Tet-off promoter, GAL4 activity is regulated by the presence or absence of tetracycline in the diet. Almost complete inhibition of the expression of UAS transgenes during the pre-adult stages of the life cycle is obtained by using four copies and two types of Tet-off GAL80 transgenes. Upon treatment of newly emerged adults with tetracycline, induction of GAL4 activity is observed but the level of induction is influenced by the concentration of the inducer, the age, the sex and the anatomical location of the expression. The inhibition of GAL4 activity and the maintenance of induced expression are altered in old animals. This study reveals that the repressive ability of GAL80 is affected by the age and sex of the animal which is a major limitation to regulate gene expression with GAL80 in aged Drosophila.
Project description:Transgenic manipulation of subsets of brain cells is increasingly used for studying behaviors and their underlying neural circuits. In Drosophila, the GAL4-upstream activating sequence (UAS) binary system is powerful for gene manipulation, but GAL4 expression is often too broad for fine mapping of neural circuits. Here, we describe the development of unique molecular genetic tools to restrict GAL4 expression patterns. Building on the GAL4-UAS system, our method adds two components: a collection of enhancer-trap recombinase, Flippase (ET-FLP), transgenic lines that provide inheritable, reproducible, and tissue-specific FLP and an FRT-dependent GAL80 "flip-in" construct that converts FLP expression into tissue-specific repression of GAL4 by GAL80. By including a UAS-encoded fluorescent protein, circuit morphology can be simultaneously marked while the circuit function is assessed using another UAS transgene. In a proof-of-principle analysis, we applied this ET-FLP-induced intersectional GAL80/GAL4 repression (FINGR) method to map the neural circuitry underlying fly wing inflation. The FINGR system is versatile and powerful in combination with the vast collection of GAL4 lines for neural circuit mapping as well as for clonal analysis based on the infusion of the yeast-derived FRT/FLP system of mitotic recombination into Drosophila. The strategies and tactics underlying our FINGR system are also applicable to other genetically amenable organisms in which transgenes including the GAL4, UAS, GAL80, and FLP factors can be applied.
Project description:The DNA-binding transcriptional activator Gal4 and its regulators Gal80 and Gal3 constitute a galactose-responsive switch for the GAL genes of Saccharomyces cerevisiae. Gal4 binds to GAL gene UASGAL (upstream activation sequence in GAL gene promoter) sites as a dimer via its N-terminal domain and activates transcription via a C-terminal transcription activation domain (AD). In the absence of galactose, a Gal80 dimer binds to a dimer of Gal4, masking the Gal4AD. Galactose triggers Gal3-Gal80 interaction to rapidly initiate Gal4-mediated transcription activation. Just how Gal3 alters Gal80 to relieve Gal80 inhibition of Gal4 has been unknown, but previous analyses of Gal80 mutants suggested a possible competition between Gal3-Gal80 and Gal80 self-association interactions. Here we assayed Gal80-Gal80 interactions and tested for effects of Gal3. Immunoprecipitation, cross-linking, and denaturing and native PAGE analyses of Gal80 in vitro and fluorescence imaging of Gal80 in live cells show that Gal3-Gal80 interaction occurs concomitantly with a decrease in Gal80 multimers. Consistent with this, we find that newly discovered nuclear clusters of Gal80 dissipate in response to galactose-triggered Gal3-Gal80 interaction. We discuss the effect of Gal3 on the quaternary structure of Gal80 in light of the evidence pointing to multimeric Gal80 as the form required to inhibit Gal4.
Project description:We describe a two-hybrid strategy for detection of interactions with transactivator proteins. This repressed transactivator (RTA) system employs the N-terminal repression domain of the yeast general repressor TUP1. TUP1-GAL80 fusion proteins, when coexpressed with GAL4, are shown to inhibit transcription of GAL4-dependent reporter genes. This effect requires the C-terminal 30 residues of GAL4, which are required for interaction with GAL80 in vitro. Furthermore, repression of GAL transcription by TUP1-GAL80 requires SRB10, demonstrating that the TUP1 repression domain, in the context of a two-hybrid interaction, functions by the same mechanism as endogenous TUP1. Using this strategy, we demonstrate interactions between the mammalian basic helix-loop-helix proteins MyoD and E12, and between c-Myc and Bin-1. We have also identified interacting clones from a TUP1-cDNA fusion expression library by using GAL4-VP16 as a bait fusion. These results demonstrate that RTA is generally applicable for identifying and characterizing interactions with transactivator proteins in vivo.