Neuron, Vol. 7, 177-181, August, 1991, Copyright 0 1991 by Cell Press Mechanisms for Diversity Review in Gene Expression Patterns Kevin Struhl tional machinery is qualitatively different from pro- Department of Biological Chemistry karyotic RNA polymerase holoenzymes that are suffi- and Molecular Pharmacology cient for efficient transcriptional initiation. Harvard Medical School In eukaryotic organisms, gene expression requires Boston, Massachusetts 02115 activator proteins that bind to specific promoter sequences and stimulate the basic transcriptional The human central nervous system contains about machinery (Mitchell and Tjian, 1989; Ptashne, 1988; 1012 cells whose actions define the world as we know Struhl, 1989). Thus, a first-order description of a partic- it. Although the number of classically defined cell ulartranscriptional regulatory pattern is simply a mat- types is rather small, the regulatory complexity dis- ter of which specific activator proteins can interact played by individual genes indicates that many, and at the promoter. In this view, a set of genes can be perhaps nearly all, of the cells in the central nervous coordinately regulated if their promoters contain re- system are distinct with respect to which genes are lated DNA sequence elements that can interact with expressed. In addition to this cellular specificity, gene a common activator protein. In general, the related regulatory patterns are constantly changing through- promoter elements are not identical, but strongly re- out development and in response to extracellular sig- semble a consensus sequence, which is often func- nals. As a result, transcriptional regulatory patterns in tionallyoptimal. This abilityto interact efficientlywith the central nervous system are extraordinarily com- a range of related sequences allows for regulatory plex. As a rough estimate, probably between IO4 and and evolutionary flexibility. However, the number of IO5 genes are expressed, many of them in unique and distinct DNA-binding specificities is far too limited to unexpectedly complicated cellular patterns (Bier et account for the diversity of transcriptional regulatory al., 1989; McKay and Hockfield, 1982; Sutcliffe, 1988). patterns. More importantly, generating complex ex- This enormous diversity and flexibility in gene ex- pression patterns would be impossible if a single pression patterns is accomplished with a relatively activator protein were sufficient to enhance transcrip- small number of transcription factors. There are un- tion, because all genes containing a common pro- doubtedly hundreds of transcription factors and pos- moter element would be coordinately expressed in a sibly as many as a few thousand, but more than this given cell type. seems very unlikely. It is evident, therefore, that a The fundamental aspect of the RNA polymerase II “one regulatory protein per gene” model, such as fre- machinery that addresses the diversity problem is that quently applies to prokaryotic organisms, is grossly efficient transcription requires the combinatorial ac- inadequate. Instead, combinatorial action of tran- tion of activator proteins. A single activator protein scriptional regulatory proteins is necessary for multi- bound at one site in the promoter typically confers a cellular organisms to generate the requisite diversity very low level of gene expression. In contrast, tran- in gene expression patterns. This review will discuss scription is stimulated much more efficiently (factors the molecular mechanisms involved in generating di- of 5-1000) by the combination of multiple activator versity. proteins bound at distinct promoter sites. Most im- portantly, such transcriptional synergy is frequently Combinatorial Activation of Transcription observed even when the multiple binding sites are recognized by distinct, and even evolutionarily dis- The most important mechanism for achieving diver- tant, proteins. As an example of such promiscuity, the sity, combinatorial activation, relieson the basic prop- combination of the mammalian glucocorticoid recep- erties of theeukaryotic transcriptional machinery. For tor and the yeast GAL4 protein is much more effective protein-coding genes, this machinery consists of RNA than either protein alone. Although the mechanism(s) polymerase II and several auxiliary factors including of synergistic and promiscuous activation remains to TFIID, which binds to the conserved TATA element be elucidated, the requirement for multiple activator found in most eukaryotic promoters (Sawadogo and proteins at a promoter permits a very large number Sentenac, 1990). By itself, RNA polymerase II is tran- of possible combinations, each of which might be scriptionally inactive on normal DNA templates. How- biologically distinct. ever, after binding of TFIID to the TATA element and The regulatory flexibility due to transcriptional syn- subsequent assembly of the other factors into an ac- ergy is greatly enhanced by the ability of activator tive transcription complex, RNA polymerase I I can ini- proteins to function bidirectionally at long and vari- tiate synthesis at a site 25-30 bp downstream of the able distances either upstream or downstream from TATA element. However, this “basic transcriptional the mRNA initiation site. Such action at a distance machinery” is not sufficient to promote transcription is believed to reflect interactions between distantly in vivo because promoters containing only the TATA bound proteins that are brought into close proximity element and initiation region are essentially inactive. by looping out of the intervening DNA. In general, an Thus, the eukaryotic RNA polymerase II transcrip- activator protein becomes less efficient when bound Figure 1. Generation of a Complex Expres- sion Pattern for a Hypothetical Gene Con- taining Seven Enhancer Elements Up- stream of the TATA Element and mRNA initiation Site Each of the eight cells (A-H) contains a par- ticular array of activator proteins (I-7) bound directly to the promoter region (closed boxes, with distinctive shadings in- dicating individual members of a multipro- tein family as in 2 and 7) or indirectly via a protein-protein interaction (between X and 7 in cell G); protein 6 is cell type spe- cific, whereas protein 5 is nearly ubiqui- tous. As a simple arbitrary rule, any three activator proteins can stimulate the basic transcriptional machinery unless a repres- sor protein is also bound to the promoter (at site 7 in cell F). Four activator proteins permit higher expression levels (cell E), and two activator proteins are insufficient (ceil C) unless there is a synergistic protein-pro- tein interaction (cell H). at increasing distances from the initiation site, but overall pattern. Finally, the principle of combinatorial individual proteinsdisplayconsiderablevariability(in activation results in regulatory networks in which sets this regard, the common distinction between “pro- of genes are coordinately controlled by specific envi- moter” and “enhancer” binding proteins is artificial). ronmental or developmental signals, yet the individ- Whatever the precise molecular mechanisms in- ual genes can be members of many different sets. volved, the important principle is that a promoter can be subject to the action of numerous proteins whose Families of Transcription Factors target sequences can be spread out over a large chro- mosomal region. Indeed, there are already examples Although the combinatorial activation process clearly in flies and mammals in which sequences 30-50 kb generates an impressive amount of diversity, it is lim- from the initiation site play an important regulatory ited bythe number of distinct DNA-binding specificit- role (Grosveld et al., 1987; Karch et al., 1990). Protein- ies of the activator proteins. The number of possible binding sites are often tightly clustered into en- recognition sequences is limited bythe small number hancers that can be moved as a functionally autono- of base pairs (typically 6-8) that are involved in high mous unit, but such a genomic organization is not affinity protein-DNA interactions. Moreover, it is very essential. likelythat the inherent chemistries of nucleotides and Given these properties, an enormous diversity of amino acids severely restrict which DNA sequences transcriptional regulatory patterns can be generated can serve as protein-binding sites. This restriction is (see Figure 1). In simple cases, dedicated promot- compounded by structural and evolutionary con- ers responding to a single activator protein can be straints on the number of DNA-binding motifs (e.g., arranged by having multiple copies of a common helix-turn-helix, zinc finger, bZIP, and helix-loop- binding site. More typically, genes whose promoters helix). contain multiple distinct sites could be efficiently ex- Multiprotein families of transcription factors that pressed only when certain developmental or environ- recognize related DNA sequences constitute an im- mental conditions are met simultaneously. Redun- portant diversity mechanism for overcoming some of dant promoters that contain more elements than the above constraints. Examples of such families are necessary can permit expression under several differ- homeodomain proteins that control key develop- ent, but specific, circumstances. For the most com- mental decisions (Levine and Hoey, 1988); steroid hor- plex expression patterns, such as observed for genes mone receptors (Evans and Hollenberg, 1988); the that determine cell fate or that are responsible for the AP-1 and ATF/CREB proteins,