GAL4/UAS system

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An example GAL4-UAS system, with GAL4 lines and UAS reporter lines. Gal4UAS-System.png
An example GAL4-UAS system, with GAL4 lines and UAS reporter lines.

The GAL4-UAS system is a biochemical method used to study gene expression and function in organisms such as the fruit fly. It is based on the finding by Hitoshi Kakidani and Mark Ptashne, [1] and Nicholas Webster and Pierre Chambon [2] in 1988 that Gal4 binding to UAS sequences activates gene expression. The method was introduced into flies by Andrea Brand and Norbert Perrimon in 1993 [3] and is considered a powerful technique for studying the expression of genes. [4] The system has two parts: the Gal4 gene, encoding the yeast transcription activator protein Gal4, and the UAS (Upstream Activation Sequence), an enhancer to which GAL4 specifically binds to activate gene transcription.

Contents

Overview

The Gal4 system allows separation of the problems of defining which cells express a gene or protein and what the experimenter wants to do with this knowledge. Geneticists have created genetic variants of model organisms (typically fruit flies), called GAL4 lines, each of which expresses GAL4 in some subset of the animal's tissues. For example, some lines might express GAL4 only in muscle cells, or only in nerves, or only in the antennae, and so on. For fruit flies in particular, there are tens of thousands of such lines, with the most useful expressing GAL4 in only a very specific subset of the animal—perhaps, for example, only those neurons that connect two specific compartments of the fly's brain. The presence of GAL4, by itself, in these cells has little or no effect, since GAL4's main effect is to bind to a UAS region, and most cells have no (or innocuous) UAS regions.

Since Gal4 by itself is not visible, and has little effect on cells, the other necessary part of this system are the "reporter lines". These are strains of flies with the special UAS region next to a desired gene. These genetic instructions occur in every cell of the animal, but in most cells nothing happens since that cell is not producing GAL4. In the cells that are producing GAL4, however, the UAS is activated, the gene next to it is turned on, and it starts producing its resulting protein. This may report to the investigator which cells are expressing GAL4, hence the term "reporter line", but genes intended to manipulate the cell behavior are often used as well.

Typical reporter genes include:

For example, scientists can first visualize a class of neurons by choosing a fly from a GAL4 line that expresses GAL4 in the desired set of neurons, and crossing it with a reporter line that express GFP. In the offspring, the desired subset of cells will make GAL4, and in these cells the GAL4 will bind to the UAS, and enable the production of GFP. So the desired subset of cells will now fluoresce green and can be followed with a fluorescence microscope. Next, to figure out what these cells might do, the experimenter might express channelrhodopsin in each of these cells, by crossing the same GAL4 line with a channelrhodopsin reporter line. In the offspring the selected cells, and only those cells, will contain channelrhodopsin and can be triggered by a bright light. Now the scientist can trigger these particular cells at will, and examine the resulting behavior to see what these cells might do.

Operation

Gal4 is a modular protein consisting broadly of a DNA-binding domain and an activation domain. The UAS to which GAL4 binds is CGG-N11-CCG, where N can be any base. [6] Although GAL4 is a yeast protein not normally present in other organisms it has been shown to work as a transcription activator in a variety of organisms such as Drosophila, [7] and human cells, highlighting that the same mechanisms for gene expression have been conserved over the course of evolution. [2]

For study in Drosophila, the GAL4 gene is placed under the control of a native gene promoter, or driver gene, while the UAS controls expression of a target gene. GAL4 is then only expressed in cells where the driver gene is usually active. In turn, GAL4 should only activate gene transcription where a UAS has been introduced. For example, by fusing a gene encoding a visible marker like GFP (Green Fluorescent Protein) the expression pattern of the driver genes can be determined. GAL4 and the UAS are very useful for studying gene expression in Drosophila as they are not normally present and their expression does not interfere with other processes in the cell. For example, GAL4/UAS-regulated transgenes in Drosophila have been used to alter glial expression to produce arrhythmic behavior in a known rhythmic circadian output called pigment dispersing factor (PDF). [8] However, some research has indicated that over-expression of GAL4 in Drosophila can have side-effects, probably relating to immune and stress responses to what is essentially an alien protein. [9]

The GAL4-UAS system has also been employed to study gene expression in organisms besides Drosophila such as the African clawed frog Xenopus [10] and zebrafish. [11]

The GAL4/UAS system is also utilized in Two-Hybrid Screening, a method of identifying interactions between two proteins or a protein with DNA.

Extensions

Gal4 expression can be made even more specific by means of "intersectional strategies". These can combine two different GAL4 lines—say, A and B—in a way that GAL4 is only expressed in the cells that are in line A but not line B, or those that are in both lines A and B. When combined with intrinsically sparse GAL4 lines, this offers very specific selection, often limited to a single cell type. The disadvantage is that at least three independent insertion sites are required, so the lines must use different and independent insertion sites, and creating the desired final organisms needs more than a single cross. This is a very active field of research, and there are many such intersectional strategies, of which two are discussed below.

One way to create GAL4 expression in the cells that are in line A but not line B, requires line A to be made to express GAL4, and line B made to express Gal80 , which is a GAL4 inhibitor. Therefore, only the cells that are in A but not B will have active GAL4, which can then drive the reporter gene. [12] [13]

To express GAL4 in only the cells contained in both A and B, a technique called "split-GAL4" can be used. Line A is made to express half of the GAL4 protein, which is inactive by itself. Similarly, line B is made to express the other half of GAL4, also inactive by itself. Only the cells that are in both lines make both halves, which self-assemble by leucine zipper into GAL4 and activate the reporter gene. [14]

Related Research Articles

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Ectopic is a word used with a prefix, ecto, meaning “out of place.” Ectopic expression is an abnormal gene expression in a cell type, tissue type, or developmental stage in which the gene is not usually expressed. The term ectopic expression is predominantly used in studies using metazoans, especially in Drosophila melanogaster for research purposes.

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An upstream activating sequence or upstream activation sequence (UAS) is a cis-acting regulatory sequence. It is distinct from the promoter and increases the expression of a neighbouring gene. Due to its essential role in activating transcription, the upstream activating sequence is often considered to be analogous to the function of the enhancer in multicellular eukaryotes. Upstream activation sequences are a crucial part of induction, enhancing the expression of the protein of interest through increased transcriptional activity. The upstream activation sequence is found adjacently upstream to a minimal promoter and serves as a binding site for transactivators. If the transcriptional transactivator does not bind to the UAS in the proper orientation then transcription cannot begin. To further understand the function of an upstream activation sequence, it is beneficial to see its role in the cascade of events that lead to transcription activation. The pathway begins when activators bind to their target at the UAS recruiting a mediator. A TATA-binding protein subunit of a transcription factor then binds to the TATA box, recruiting additional transcription factors. The mediator then recruits RNA polymerase II to the pre-initiation complex. Once initiated, RNA polymerase II is released from the complex and transcription begins.

Andrea Hilary Brand is the Herchel Smith Professor of Molecular Biology and a Fellow of Jesus College, Cambridge. She heads a lab investigating nervous system development at the Gurdon Institute and the Department of Physiology, Development and Neuroscience. She developed the GAL4/UAS system with Norbert Perrimon which has been described as “a fly geneticist's Swiss army knife”.

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<span class="mw-page-title-main">Enhancer-FACS-seq</span>

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<span class="mw-page-title-main">Roger Brent</span> American biologist

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The Gal4 transcription factor is a positive regulator of gene expression of galactose-induced genes. This protein represents a large fungal family of transcription factors, Gal4 family, which includes over 50 members in the yeast Saccharomyces cerevisiae e.g. Oaf1, Pip2, Pdr1, Pdr3, Leu3.

Ravi Allada is an Indian-American chronobiologist studying the circadian and homeostatic regulation of sleep primarily in the fruit fly Drosophila. He is the Edward C. Stuntz Distinguished Professor of Neuroscience and Chair of the Department of Neurobiology at Northwestern University. Working with Michael Rosbash, he positionally cloned the Drosophila Clock gene. In his laboratory at Northwestern, he discovered a conserved mechanism for circadian control of sleep-wake cycle, as well as circuit mechanisms that manage levels of sleep.

References

  1. ↑ Kakidani, Hitoshi; Ptashne, Mark (1988). "GAL4 activates gene expression in mammalian cells". Cell. 52 (2): 161–167. doi:10.1016/0092-8674(88)90504-1. PMID   2830021. S2CID   9524012.
  2. 1 2 Webster, N.; Jin, J. R.; Green, S.; Hollis, M.; Chambon, P. (1988). "The yeast UASG is a transcription enhancer in human HeLa cells in the presence of the GAL4 trans-activator". Cell. 52 (2): 169–78. doi:10.1016/0092-8674(88)90505-3. PMID   2830022. S2CID   26819676.
  3. ↑ Brand, A. H.; Perrimon, N. (1993). "Targeted gene expression as a means of altering cell fates and generating dominant phenotypes". Development. 118 (2): 401–415. doi:10.1242/dev.118.2.401. PMID   8223268.
  4. ↑ Duffy, J. B. (2002). "GAL4 system in Drosophila: A fly geneticist's Swiss army knife". Genesis. 34 (1–2): 1–15. doi: 10.1002/gene.10150 . PMID   12324939. S2CID   5073328.
  5. ↑ Kitamoto, Toshihiro (2001). "Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons". Journal of Neurobiology. 47 (2): 81–92. doi:10.1002/neu.1018. PMID   11291099.
  6. ↑ Campbell, R. N.; Leverentz, M. K.; Ryan, L. A.; Reece, R. J. (2008). "Metabolic control of transcription: Paradigms and lessons from Saccharomyces cerevsiae". Biochemical Journal. 414 (2): 177–87. doi:10.1042/BJ20080923. PMID   18687061. S2CID   19379977.
  7. ↑ Janice A. Fischer; Edward Giniger; Tom Maniatis; Mark Ptashne (1988). "GAL4 activates transcription in Drosophila". Nature . 332 (6167): 853–6. Bibcode:1988Natur.332..853F. doi:10.1038/332853a0. PMID   3128741. S2CID   31793830.
  8. ↑ Ng, F. S.; Tangredi, M. M.; Jackson, F. R. (April 2011). "Glial cells physiologically modulate clock neurons and circadian behavior in a calcium-dependent manner". Current Biology. 21 (8): 625–34. doi:10.1016/j.cub.2011.03.027. PMC   3081987 . PMID   21497088.
  9. ↑ Liu Y, Lehman M (2008). "A genomic response to the yeast transcription factor GAL4 in Drosophila". Fly (Austin). 2 (2): 92–8. doi:10.4161/fly.6311. PMC   2803330 . PMID   18820459.
  10. ↑ Katharine O. Hartley; Stephen L. Nutt; Enrique Amaya (2002). "Targeted gene expression in transgenic Xenopus using the binary Gal4-UAS system". Proceedings of the National Academy of Sciences . 99 (3): 1377–82. Bibcode:2002PNAS...99.1377H. doi: 10.1073/pnas.022646899 . PMC   122198 . PMID   11818539.
  11. ↑ Davison, J. M.; Akitake CM; Goll M. G.; Rhee, J. M.; Gosse, N.; Baier, H.; Halpern, M. E.; Leach, S. D.; Parsons, M. J. (2007). "Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish". Developmental Biology. 304 (2): 811–24. doi:10.1016/j.ydbio.2007.01.033. PMC   3470427 . PMID   17335798.
  12. ↑ Suster, Maximiliano L.; Seugnet, Laurent; Bate, Michael; Sokolowski, Marla B. (August 2004). "Refining GAL4-driven transgene expression in Drosophila with a GAL80 enhancer-trap". Genesis. 39 (4): 240–245. doi:10.1002/gene.20051. PMID   15286996. S2CID   43478668.
  13. ↑ Fujimoto, Esther; Gaynes, Brooke; Brimley, Cameron; J. Chien, Chi-Bin & Bonkowsky, Joshua L. (2011). "Gal80 intersectional regulation of cell-type specific expression in vertebrates". Developmental Dynamics. 240 (10): 2324–2334. doi:10.1002/dvdy.22734. PMC   3178006 . PMID   21905164.
  14. ↑ Luan, Haojiang; Peabody, Nathan C.; Vinson, Charles R. & White, Benjamin H. (2006). "Refined spatial manipulation of neuronal function by combinatorial restriction of transgene expression". Neuron. 52 (3): 425–436. doi:10.1016/j.neuron.2006.08.028. PMC   1713190 . PMID   17088209.
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