How Are Transcriptional Regulatory Elements Defined?

  Both enhancers and promoters have been assigned specific roles through the development of functional assay systems, in which cloned DNA sequences can be re-introduced into cell cultures on special plasmids carrying test or "reporter" genes, and can be tested for their ability to activate reporter gene expression. These are relatively simple and reproducible assays, and allow researchers to analysis regulatory elements in detail by deleting, adding or changing DNA sequences within the element and testing the effects of these engineered changes. With a specific cell type in hand, it is now possible to demonstrate relatively quickly with an expression assay that a specific set of nucleotides functions as a transcriptional regulatory element. Knowing which DNA sequences are important for function also provides a means for isolating the protein factors that bind to them. Although the protein-DNA interactions that lead to RNA transcription from the gene promoter are well characterized, less is known about how the regulatory proteins that bind to enhancers affect transcription from such great distances (see below). One possibility is that enhancers form landing pads for regulatory proteins which are attracted to the general vicinity of a gene by their affinity for its associated enhancer sequence. These proteins may then participate in assembling the protein complex at the promoter, by bending the DNA in a loop (see Figure 2). This is only one of many possible scenarios by which DNA regulatory elements operate, and their function is currently an area of active research.

Gene Expression Assays

  Regulatory elements associated with particular genes have been assigned specific roles through the development of functional assay systems, in which cloned DNA sequences can be re-introduced into cell cultures or into live animals. Several standard molecular approaches have been developed to study gene regulation. First, if a cloned gene is small enough, it can be introduced into cells or animals as a genomic fragment containing coding and flanking sequences in an appropriate vector, and the cells subsequently harvested and tested for expression of the introduced gene by mRNA analysis. With subsequent deletions and mutations in the original gene fragment, regulatory sequences can be identified. This straightforward approach is not always feasible, however. For example, if the host cell or species is identical or closely related to that from which the cloned gene was derived, transcripts from the introduced gene cannot be distinguished from the cognate endogenous gene product. Modifying the introduced gene product, for example by deleting a portion of the coding region to produce a "minigene", or by inserting a synthetic marker sequence into the gene body, can overcome this problem.

  Alternatively, candidate regulatory sequences from tissue-specific genes can be linked to an unrelated reporter gene. These artificial transcription units can then be tested for the ability of the regulatory element to activate reporter gene expression when introduced into the appropriate cell type. Several popular examples of reporter genes used in basic research include those encoding bacterial enzymes, such as bacterial chloramphenical acetyltransferase (CAT) or beta-galactosidase (lacZ), and the human placental alkaline phosphatase (HAP) which afford quick and quantitative assays of reporter gene expression that can also be visualized by immunocytochemical or colorimetric protocols.

  An initial survey of putative regulatory elements from a gene locus usually involves the transient or stable introduction of cloned DNA into primary or immortalized cell lines derived from the tissue of interest. Cell culture-based assays are relatively simple and reproducible, provided that the appropriate cell type is available. They allow researchers to analysis regulatory elements in detail by deleting, adding or changing DNA sequences within the element and subsequently testing the effects of these engineered changes. This tactic has been successfully used to delineate the functionally important sequence motifs of many tissue-restricted genes, and is a prerequisite to the identification of the proteins responsible for cell type-specific transcription.