Reporter Genes Are Used to Define Enhancers Other Regulatory Elements

By ligating regions of DNA suspected of harboring regulatory sequences to various reporter genes (the reporter or chimeric gene approach) (Figures 39-10 and 39-11), 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 are ligated to a suitable reporter gene and

Figure 39-9. Formation and putative structure of the enhanceosome formed on the human p-interferon gene enhancer. Diagramatically represented at the top is the distribution of the multiple cis-elements (HMG, PRDIV, PRDI-III, PRDII, NRDI) composing the p-interferon gene enhancer. The intact enhancer mediates transcriptional induction of the p-interferon gene (over 100-fold) upon virus infection of human cells. The cis-elements of this modular enhancer represent the binding sites for the trans-factors HMG I(Y), cJun-ATF-2, IRF3, IRF7, and NF-kB, respectively. The factors interact with these DNA elements in an obligatory, ordered, and highly cooperative fashion as indicated by the arrow. Initial binding of four HMG I(Y) proteins induces sharp DNA bends in the enhancer, causing the entire 70-80 bp region to assume a high level of curvature. This curvature is integral to the subsequent highly cooperative binding of the other trans-factors since this enables the DNA-bound factors to make important, direct protein-protein interactions that both contribute to the formation and stability of the enhanceosome and generate a unique three-dimensional surface that serves to recruit chromatin-modifying activities (eg, Swi/Snf and P/CAF) as well as the general transcription machinery (RNA polymerase II and GTFs). Although four of the five cis-elements (PRDIV, PRDI-III, PRDII, NRDI) independently can modestly stimulate (-tenfold) transcription of a reporter gene in transfected cells (see Figures 39-10 and 39-12), all five cis-elements, in appropriate order, are required to form an enhancer that can appropriately stimulate mRNA gene transcription (ie, > 100-fold) in response to viral infection of a human cell. This distinction indicates the strict requirement for appropriate enhanceosome architecture for efficient trans-activation. Similar enhanceosomes, involving distinct cis- and trans-factors, are proposed to form on many other mammalian genes.

HMG prdiv^.hmg/^PRDI-II!^ prdiivsmg^"nrdi' hmg^

HMG prdiv^.hmg/^PRDI-II!^ prdiivsmg^"nrdi' hmg^

introduced into a host cell (Figure 39-10). Basal expression of the reporter gene will be increased if the DNA contains an enhancer. Addition of a hormone or heavy metal to the culture medium will increase expression of the reporter gene if the DNA contains a hormone or metal response element (Figure 39-11). The location of the element can be pinpointed by using progressively shorter pieces of DNA, deletions, or point mutations (Figure 39-11).

This strategy, using transfected cells in culture and transgenic animals, has led to the identification of dozens of enhancers, repressors, 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. The challenge now is to figure out how this occurs.

Test promoter

GENE v-

ENHANCER-PROMOTER

Reporter gene

REPORTER GENE: TEST ENHANCER-PROMOTER DRIVING TRANSCRIPTION CAT GENE

Divide and re-plate

Hormones

HARVEST 24 HOURS LATER ASSAY FOR CAT ACTIVITY

Identification of control elements

TRANSFECT CELLS USING CaPO4 PRECIPITATED DNA

Divide and re-plate

Hormones

HARVEST 24 HOURS LATER ASSAY FOR CAT ACTIVITY

Identification of control elements

Figure 39-10. The use of reporter genes to define DNA regulatory elements. A DNA fragment 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 bacterial enzyme chloramphenicol transferase (CAT). The enzyme luciferase (abbreviated LUC) is another popular reporter gene. Neither LUC nor CAT is present in mammalian cells; hence, detection of these activities in a cell extract means that the cell was successfully transfected by the plasmid. An increase of CAT activity over the basal level, eg, 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 to pinpoint the response element (see Figure 39-11 for deletion mapping of the relevant HREs).

ner, but the process in most genes, especially in mammals, is much more complicated. Signals representing a number of complex environmental stimuli may converge on a single gene. The response of the gene to these signals can have several physiologic characteristics. First, the response may extend over a considerable range. This is accomplished by having additive and syn-ergistic positive responses counterbalanced by negative or repressing effects. In some cases, either the positive or the negative response can be dominant. Also required is a mechanism whereby an effector such as a hormone can activate some genes in a cell while repressing others and leaving still others unaffected. When all of these processes are coupled with tissue-specific element factors, considerable flexibility is afforded. These physiologic variables obviously require an arrangement much more complicated than an on-off switch. The array of DNA elements in a promoter specifies—with associated factors—how a given gene will respond. Some simple examples are illustrated in Figure 39-12.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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