By ligating regions of DNA suspected of harboring regulatory sequences to various reporter genes (the reporter or chimeric gene approach) (Figures 1, 2, and 3), one can determine which regions in the vicinity of structural genes have an influence on their expression. Pieces of DNA thought to harbor regulatory elements, often identified by bioinformatic sequence alignments, are ligated to a suitable reporter gene and introduced into a host cell (see Figure 2). Expression of the reporter gene will be increased if the DNA contains a particular enhancer. For example, addition of different hormones to separate cultures will increase expression of the reporter gene if the DNA contains a particular hormone response DNA element (HRE) (see Figure 3). The location of the element can be pinpointed by using progressively shorter pieces of DNA, deletions, or and/ or point mutations (see Figure 3).
Fig1. A schematic illustrating the methods used to study the organization and action of enhancers and other cis acting regulatory elements. These model chimeric genes, all con structed by recombinant DNA techniques in vitro (see Chapter 39), consist of a reporter gene that encodes a protein that can be readily assayed, and that is not normally produced in the cells to be studied, a promoter that ensures accurate initiation of transcription, and the indicated enhancer (regulatory response) elements. In all cases, high level transcription from the indicated chimeras depends on the presence of enhancers, which stimulate transcription ≥100-fold over basal transcriptional levels (ie, transcription of the same chimeric genes containing just promoters fused to the indicated reporter genes). Examples (A) and (B) illustrate the fact that enhancers (eg, here SV40) work in either orientation and upon a heterologous promoter. Example (C) illustrates that the metallothionein (mt) regulatory element (which under the influence of cadmium or zinc induces transcription of the endogenous mt gene and hence the metal-binding mt protein) will work through the herpes simplex virus (HSV) thymidine kinase (tk) gene promoter to enhance transcription of the human growth hormone (hGH) reporter gene. In a separate experiment, this engineered genetic construct was introduced into the male pronuclei of single-cell mouse embryos and the embryos placed into the uterus of a surrogate mother to develop as transgenic animals. Offspring have been generated under these conditions, and in some the addition of zinc ions to their drinking water effects an increase in growth hormone expression in liver. In this case, these transgenic animals have responded to the high levels of growth hormone by becoming twice as large as their normal litter mates. Example (D) illustrates that a glucocorticoid response element (GRE) enhancer will work through homologous (PEPCK gene) or heterologous gene promoters (not shown; ie, HSV tk promoter, SV40 promoter, β-globin promoter, etc) to drive expression of the chloramphenicol acetyltransferase (CAT) reporter gene.
Fig2. The use of reporter genes to define DNA regulatory elements. A DNA fragment bearing regulatory cis-elements (triangles, square, circles in diagram) from the gene in question—in this example, approximately 2 kb of 5′-flanking DNA and cognate promoter—is ligated into a plasmid vector that contains a suitable reporter gene—in this case, the enzyme firefly luciferase, abbreviated LUC. As noted in Figure 1 in such experiments, the reporter cannot be present endogenously in the cells transfected. Consequently, any detection of these activities in a cell extract means that the cell was successfully transfected by the plasmid. Not shown here, but typically one cotransfects an additional reporter such as Renilla luciferase to serve as a transfection efficiency control. Assay conditions for the firefly andRenillaluciferases are different, hence the two activities can be independently sequentially assayed using the same cell extract. An increase of firefly luciferase activity over the basal level, for example, after addition of one or more hormones, means that the region of DNA inserted into the reporter gene plasmid contains functional hormone response elements (HRE). Progressively shorter pieces of DNA, regions with internal deletions, or regions with point mutations can be constructed and inserted upstream of the reporter gene to pinpoint the response element (Figure3). One caveat of this approach is that the transfected plasmid DNAs likely do not form “classical” chromatin structures.
Fig3. Mapping distinct hormone response elements (HREs) (A), (B), and (C) using the reporter gene–transfection approach. A family of reporter genes, constructed as described in Figures 1 and 2, can be transfected individually into recipient cells. By analyzing when certain hormone responses are lost in comparison to the 5′ deletion end point, specific hormone response enhancer elements can be located and defined, ultimately with nucleotide-level precision (see summary, bottom).
This strategy, typically performed using transfected cells in culture (ie, cells induced to take up exogenous DNAs), has led to the identification of hundreds of enhancers, silencers/ repressors such as tissue-specific elements, and hormone, heavy metal, and drug-response elements. The activity of a gene at any moment reflects the interaction of these numerous cis-acting DNA elements with their respective trans-acting factors. Overall, transcriptional output is determined by the balance of positive and negative signaling to the transcription machinery. The challenge now is to figure out exactly how this regulation occurs at the molecular level so that we might ultimately have the ability to modulate gene transcription therapeutically.
