Promoter Fusions to Study Gene Expression

Abstract

One important tool in molecular biology takes advantage of promoter fusions where the expression of one gene is controlled by the promoter or regulatory elements of another gene. Promoter fusions engineered in vitro can help to determine how gene expression is regulated in cells and whole organisms. Promoter fusions generated in vivo, for example, by promoter trapping, can help to identify new genes and generate useful genetic resources for dissecting important biological processes. A binary effector–reporter system can be used to direct the production of a protein of interest in specific cells of an organism for functional studies. It can also be used to direct the production of interference ribonucleic acid molecules that can inhibit the activity of their target genes in specific cells of an organism. The GAL4–UAS version of this binary system is a valuable genetic tool for investigations of mechanisms that control development.

Key Concepts:

  • The first step towards expressing a gene is transcription.

  • Gene transcription is controlled by regulatory DNA sequences (elements) that contain binding sites for regulatory proteins.

  • The regulatory DNA element of one gene can exert its effect on the expression of another gene in a chimaeric (fusion) construct.

  • Engineered fusion constructs can be introduced to cells or whole organisms.

  • Reporter genes are used in fusion constructs because their encoded proteins can be monitored or assayed easily.

  • Reporter fusion constructs can be used to analyse the functions of the regulatory DNA elements.

  • An effector–reporter system can be used to study the relationship between a regulatory DNA element and the regulatory proteins that bind to it.

  • Promoter trapping and enhancer trapping are useful techniques to generate genome‐scale resources for genetic studies.

  • A binary effector–reporter system (a version of which is commonly known as the GAL4–UAS system) is a powerful genetic tool.

Keywords: promoter; enhancer; transcription; gene fusions; activators

Figure 1.

A promoter fusion construct generated in vitro. The expression of the lacZ reporter gene in this fusion construct is controlled by the enhancer sequence located upstream. Promoter (Prom.) and polyadenylation (PolyA) sequences are required for proper expression of genes in eukaryotic cells. The line under the fusion gene construct denotes the product of transcription, messenger RNA (mRNA), which is then translated into the protein β‐galactosidase.

Figure 2.

A cotransfection assay. Two different fusion constructs, an effector (top) and a lacZ reporter (bottom), are introduced into the same cell. The effector gene encodes an activator that can stimulate the expression of the reporter gene by binding to DNA sequences within the enhancer. This assay can be used not only to delineate the specific sequences in the enhancer but also to further study the functions of the activator protein. The GAL4–UAS binary system is based on the same principle depicted in this figure. In that binary system, GAL4 is the activator and UAS is the enhancer.

Figure 3.

Enhancer trap. A reporter gene construct without an enhancer is used to integrate randomly into the genome of an organism. This gene will not be expressed unless it is inserted near an enhancer, thus ‘trapping’ that enhancer.

Figure 4.

Promoter trap. In this design of promoter trap, the lacZ reporter gene contains neither an enhancer nor a promoter but has a splice acceptor site (SA) placed upstream. This reporter gene will not be expressed unless it is inserted within an intron (sequence between the exons) of an endogenous gene in the same transcription direction. Splicing is an RNA‐processing event that joins exon RNA sequences together by removing the intron sequences. The splice acceptor located upstream of lacZ ensures that its RNA appears in the spliced product (mRNA) for translation into protein. Enh., enhancer; Prom., promoter; pA, polyA.

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References

Arimbasseri AG, Rijal K and Maraia RJ (2013) Transcription termination by the eukaryotic RNA polymerase III. Biochimica et Biophysica Acta 1829: 318–330.

Bateman JR, Lee AM and Wu CT (2006) Site‐specific transformation of Drosophila via phiC31 integrase‐mediated cassette exchange. Genetics 173: 769–777.

Bellen HJ, O'Kane CJ, WIlson C et al. (1989) P‐element‐mediated enhancer detection: a versatile method to study development in Drosophila. Genes & Development 3: 1288–1300.

Brand AH and Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415.

Brent R (2004) Building an artificial regulatory system to understand a natural one. Cell 116: S73–S74.

Casadaban MJ (1976) Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriophage lambda and Mu. Journal of Molecular Biology 104: 541–555.

Dietzl G, Chen D, Schnorrer F et al. (2007) A genome‐wide transgenic RNAi library for conditional gene inactivation in Drosophila. Nature 448: 151–156.

Fischer JA, Giniger E, Maniatis T and Ptashne M (1988) GAL4 activates transcription in Drosophila. Nature 332: 853–856.

Friedrich G and Soriano P (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes & Development 5: 1513–1523.

Grunberg S and Hahn S (2013) Structural insights into transcription initiation by RNA polymerase II. Trends in Biochemical Sciences 38(12): 603–611.

Guarente L and Ptashne M (1981) Fusion of Escherichia coli lacZ to the cytochrome c gene of Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the USA 78: 2199–2203.

Kakidani H and Ptashne M (1988) GAL4 activates gene expression in mammalian cells. Cell 52: 161–167.

Lee DJ, Minchin SD and Busby SJ (2012) Activating transcription in bacteria. Annual Review of Microbiology 66: 125–152.

Lee T, Winter C, Marticke SS, Lee A and Luo L (2000) Essential roles of Drosophila RhoA in the regulation of neuroblast proliferation and dendritic but not axonal morphogenesis. Neuron 25: 307–316.

Levine M (2011) Paused RNA polymerase II as a developmental checkpoint. Cell 145: 502–511.

Liu X, Bushnell DA and Kornberg RD (2013) RNA polymerase II transcription: structure and mechanism. Biochimica et Biophysica Acta 1829: 2–8.

Ma J (2004) Actively seeking activating sequences. Cell 116: S75–S76.

Ma J (2005) Crossing the line between activation and repression. Trends in Genetics 21: 54–59.

Ma J (2011) Transcriptional activators and activation mechanisms. Protein & Cell 2: 879–888.

Ma J, Przibilla E, Hu J, Bogorad L and Ptashne M (1988) Yeast activators stimulate plant gene expression. Nature 334: 631–633.

Nowotschin S, Eakin GS and Hadjantonakis AK (2009) Live‐imaging fluorescent proteins in mouse embryos: multi‐dimensional, multi‐spectral perspectives. Trends in Biotechnology 27: 266–276.

Ong CT and Corces VG (2011) Enhancer function: new insights into the regulation of tissue‐specific gene expression. Nature Reviews Genetics 12: 283–293.

Rougvie AE and Lis JT (1988) The RNA polymerase II molecule at the 5′‐end of the uninduced hsp70 genes of D. melanogaster is transcriptionally engaged. Cell 54: 795–804.

Sadowski I, Ma J, Triezenberg S and Ptashne M (1988) GAL4‐VP16 is an unusually potent transcriptional activator. Nature 335: 563–564.

Saecker RM, Record MT Jr and Dehaseth PL (2011) Mechanism of bacterial transcription initiation: RNA polymerase – promoter binding, isomerization to initiation‐competent open complexes, and initiation of RNA synthesis. Journal of Molecular Biology 412: 754–771.

Further Reading

Alberts B, Johnson A, Lewis J et al. (2007) Molecular Biology of the Cell, 5th edn. New York: Garland Publishing.

Krebs JE, Goldstein ES and Kilpatrick ST (2012) Lewin's GENES XI. Burlington, MA: Jones & Bartlett Learning.

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Ma, Jun(Feb 2014) Promoter Fusions to Study Gene Expression. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000976.pub3]