Promoter Fusions to Study Gene Expression


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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Further Reading

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