ability to function over long distances ( > 1000 bp) irrespective of their position relative to the cap site

4. preferential stimulation of transcription from the most proximal of two

tandem promoters.

Enhancers like promoters are modular in nature. All the modules contain multiple, overlapping sites for different transcriptional activator proteins. Modules in turn can be subdivided into ’enhansons*. These are short D N A sequences that are the basic units of enhancer structure.

Inducible enhancers have been found conserved in the upstream regions of the

metallothionein-I (MT-I) gene from man to Drosophila melanogaster. Several

motifs exist which are involved in the transcriptional induction caused by heavy metal ions, glucocorticoid hormones and bacterial lipopolysaccharides. Disruption of the metal regulatory element (MRE) found in the promoter causes a reduction of the heavy metal response but not its total elimination. This led to the identification of four homologous copies of the MRE in a more upstream region. When synthetic copies of these sequences were placed as dimers in a heterologous

promoter (HSV tk gene) it was found that four of the five sequences could confer

heavy-metal regulation (Stuart et a l, 1985).

Tissue-specific enhancers have been detected in the Inununoglobulin (Ig) genes. During lymphoid cell differentiation, the Ig heavy chain (IgH) genes are assembled by fusion of one variable (V) gene segment to a diversity (D ) and a joining (J) segment and separated by a large intron, to a constant (C) segment.

Within the intron, a strong enhancer is present. This was the first genetic element identified to confer cell-type specificity. The IgH enhancer is required for establishing stable transcriptional complexes in the early part of B cell differentiation. Subsequently, its presence is not required for the maintenance of transcription (reviewed by, Maniatis et a l, 1987; Atchison, 1988).

A number of models have been proposed to explain the interaction of protein complexes bound at the enhancer and other transcription factors at the promoter leading to the initiation of transcription. In the looping model, the intervening D N A sequences between remote sequences and proximal promoter elements is ’looped out’ allowing contact between the two regions. In contrast, the scanning model proposes that RNA polymerase II (or a transcription factor) recognizes enhancers/upstream promoter elements, binds and then moves in either direction along the D N A backbone, until it encounters proximal promoter elements and at that point a transcription initiation complex is formed.

Evidence has accumulated in support of the looping model. Heuchel et a l

(1989) found that promoter usage is unaffected by enhancer position. If a scanning model were in existence, the promoter proximal to an enhancer would be expected to be preferentially activated. It has also been demonstrated that enhancer sequences can stimulate transcription in trans (Muller and Schaffner, 1990). Again, this is consistent with a looping model, in which enhancers or promoter elements can stimulate transcription without covalent linkage to the gene as long as enhancer and promoter are in close proximity.

A variety of reporter gene constructs with different combinations of enhancers and promoters have been compared by transient expression in mammalian cells. It has been found that the combination of heterologous elements stimulated transcription as efficiently as constructs with homologous elements in their promoter and enhancer regions. This flexibility led to the suggestion that the interaction between reporter binding sites in an enhancer position and the promoter is mediated by components of the general transcription machinery. This idea fits in with the increasing evidence that eukaryotic transcription factors usually do not bind to D N A cooperatively but nevertheless stimulate transcription synergistically.

Synergistic activation has been shown using either in vitro transcription involving a derivative of the yeast activator GAL4 and the mammalian transcription factor ATF or multiple-bound copies of GAL4 derivatives. This synergy is thought to be a consequence of interaction of each of the bound activators with some component or components of the transcriptional machinery rather than with each other. This result suggests multiple contacts with a single target or with multiple copies of a single target (Lin et aL, 1990; Carey et a l, 1990).

At least three different types of activating domains (acidic, glutamine-rich, and proline-rich) have been identified in transcription factors. These regions of 30-100 amino acids are separate from the D N A binding domain. The first defined activation regions, in GAL4 and GCN4, have significant negative charge and can form amphipathic a-helical structures. The position of these regions can be manipulated within the same protein or actually transferred to a heterologous D N A binding domain, and yet still retain the ability to activate transcription of a reporter gene carrying the relevant binding site. The glucocorticoid hormone receptor and A P-l/Jun also possesses an acidic a-helix (Ptashne, 1988). The second type of activation domain is the glutamine-rich region found in the two

most potent activation domains of Spl, as well as; the D.melanogaster gene

products of Antennapedia, Ultrabithorax and Zeste; the yeast proteins H A Pl, HAP2 and G a lll; and the mammalian proteins OCT-1, OCT-2, Jun, AP-2 and SRF. The glutamine-rich region, like the acidic domain, may actually be interchangeable since when this region from Antennapedia is linked to the Spl zinc fingers it can partially substitute for the activation domains of Spl (Courey

et a l, 1989). The third type of activation domain identified in CTF/NF-1, AP-2, Jun, OCT-2 and SRF is proline-rich. This region in CTF can also activate transcription when linked to other D N A binding domains including the zinc fingers of Spl (Mermod et a l, 1989).

These activation domains may facilitate transcription initiation by interacting with either general components of the initiation complex, such as the TATA- binding factor TFIID or with RNA polymerase II itself or its subunits. The highly conserved carboxy-terminal heptapeptide repeats of the largest subunit of RNA polymerase II have been postulated to be one such target. As enhancers and

upstream promoter sequences have similar structures, it is possible that RNA polymerase II could function through an initial interaction with either type of control region. Where RNA polymerase first recognizes transcription factors bound to a remote enhancer, it might be brought closer to the promoter by transient looping and/or short range diffusion (Mitchell and Tjian, 1989).

1,12 i Regulation at the Post-Transcriptional Level

Post-transcriptional regulation may occur at the level of: 1. RNA processing.

The roles played by the 5’ untranslated region (UTR), 3’ UTR, 5’ CAP (m^Gppp, 7-methylguanosine residues joined to mRNAs by triphosphate linkages), poly(A) tail and alternative splicing of R N A transcripts. 2. mRNA stability.