Transcriptional activation: A complex puzzle with few easy pieces

Cell, Vol. 77, 5-8, April 8, 1994, Copyright Q 1994 by Cell Press

Transcrbtional Activation: A Complex Puzzle with Few Easy Pieces Robert T/Ian* and Tom Maniatist ‘Howard Hughes Medical Institute Department of Molecular and Ceil Biology University of California Berkeley, California 947203202 tDepartment of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02133

Transcriptional activation of eukaryotic genes during development or in response to extracellular signals involves the regulated assembly of multiprotein complexes on enhancers and promoters. The complex nature of these processes provides virtually unlimited possibilities for regulation and results in an elaborate fail-safe mechanism for controlling gene expression. Central players in this process are sequence-specific transcription factors that select genes to be activated and orchestrate the assembly of a transcription “machine” at the start site of mRNA synthesis by RNA polymerase II (pol II) (for review see McKnight and Yamamoto, 1992). A key issue in understanding this process is how a relatively small number of different transcription factors can be used to achieve the high level of specificity required to regulate the complex patterns of gene expression in higher eukaryotes. At least part of the answer is that transcription factors and enhancers are composed of modular components. For example, a typical transcription factor contains a specific DNA-binding domain that directly contact8 DNA, a multimerizationdomain that allows theformation of home- or heteromultimers, and a transcriptional activation domain. Importantly, these domain8 can be combined in a modular fashion to generate novel and fully functional transcription factors (for review see Ptashne, 1992). Similarly, enhancers contain distinct sets of transcription factor-binding sites, and variations in the arrangement of the binding site8 provide the potential to create unique nucleoprotein complexes by forming heterodimers within and among families of transcription factors, Synergistic interactions between the proteins within the complex result in specificity, a potential for multiple regulatory controls, and a high level of transcription. In this minireview, we will discuss an additional mechanism for achieving a high level of specificity and gene activation: the assembly of a stereospecific nucleoprotein complex. Significantly, this process require8 proteins that bind to DNA in a sequence-specific manner, but function as architectural components. In addition, we will review recent advances in the identification of specific interactions between transcription factors and components of the basal transcription complex. The Assembly of Stereospeclfic Enhancer Complexes An interesting property of many transcription factor-binding sites is that they can function as regulated enhancer elements when multimerized and placed upstream from a test promoter. These synthetic enhancers often mimic

Minireview

the activities of the natural enhancers from which they were derived. Thus, the relative positions and orientations of individual elements within the natural enhancer were not thought to be essential for normal enhancer function. However, recent studies have shown that the activity of at least some natural enhancers requires proteins that function as architectural components, that interactions between proteins within the enhancer are crucial, and that changes in the relative positions or orientations of proteinbinding sites within the enhancer lead to the inactivation of the enhancer. Thus, activation of at least some enhancers appears to require the assembly of a highly specific threedimensional nucieoprotein complex (the stereospecific complex). Precedents for this model are provided by earlier studies of recombination in bacteria (for review see Crothers, 1993). For example, site-specific recombination in bacteriophage Ir. involves the assembly of a highly specific nucleoprotein complex called the intasome. The assembly of the intasome requires integration host factor, which binds to the minor groove of DNA and induces a DNA bend required to promote interactions between other proteins in the complex. The first indication that a similar mechanism may be used for transcriptional activation in higher eukaryotes was provided by a study of the mouse T cell receptor a gene enhancer (TCRa; Giese et al., 1992). The minimal enhancer element was shown to contain binding sites for at least three distinct factors and all three sites are required for enhancer function. One of these sites is recognized by a T cell-specific protein designated lymphoid enhancer-binding factor 1 (LEF-1) or T cell factor la (the human counterpart of LEF-1). A series of experiment8 revealed that the LEF-1 -binding site functions only in its natural context, and mutations that alter the relative positions of the three sites inactivate the enhancer. Moreover, it appeared that the LEF-1 protein cannot activate transcription on its own, but must act in concert with factors that bind to the other two sites in the enhancer. An important clue to the function of LEF-1 was the discovery that the protein contains a high mobility group (HMG) domain, a sequence motif first recognized as the DNA-binding domain of the protein HMG-1 (for review see Landsman and Bustin, 1993). In vitro binding studies revealed that the HMG domain of LEF-1 binds to the minor groove of DNA and induces a sharp bend in DNA (Giese et al., 1992). This observation and additional studies of the LEF-1 protein led to the proposal that LEF-1 acts as an architectural component in the assembly of the T cell enhancer complex (Figure 1A). According to this model, LEF-1 induces a bend in DNA so that transcription factors bound to recognition sequences flanking the LEF-lbinding site can interact with each other. In addition, another region of LEF-1 is thought to mediate interactions between other proteins within the enhancer complex (GieseandGrosschedl, 1993; Carlssonetat., 1993). Thus, LEF-1 contains a novel transcriptional activation domain that is enhancer context specific and promotes interac-

“Acidic”aclivalion

Gin-rich

Figure 1. Stereospecific Enhancer Complexes (A) A model of the transcription complex formed on the TCRa gene. The LEF-1 protein binds to a site within the enhancer and bends DNA to facilitate interactions between the Ets and CAMP reponse eiementbinding protein (WEB) family proteins. in addition, a second domain wlthin LEF-1 mediates interactions between Ets and CREB through direct protein-protein interactions. The entire enhancer complex then interacts with targets within the basal transcrtption complex. (B) A model of the 1FN-p gene enhancer complex. In this model the HMG i(Y) proteins bend DNA and facilitate interactions among the ATF-2-cJun heterodimer, the p5fI and pS5 subunits of NF-KB, and IRF family proteins. Once assembled, the entlre enhancer complex interacts with targets within the nearby basal transcription complex.

tions between sequence-specific transcription factors. By contrast, most activation domains (discussed below) interact directly or indirectly with components of the basal transcription complex. The virus-inducible enhancer of the human interferon B (IFN-9) gene provides a particularly well-characterized example of combinatorial interactions among distinct regulatory elements (Thanos and Maniatis, 1992; Du et al., 1993). This enhancer consists of overlapping regulatory elements recognized by the transcription factors NF-~6, RF-l, and ATF-P-cJun. None of these elements function on their own, but two or more copies of any one of them can act as a virus-inducible enhancer. However, the synthetic enhancers display unusually high levels of basal activity and are less inducible than the intact enhancer. Moreover, the synthetic enhancers are also activated by many other inducers. In striking contrast, the intact enhancer is inducible only by virus. Thus, the activity of the intact enhancer is distinct from that of the individual elements. The highly specific activation of the enhancer appears to result from the precise arrangement of transcription factor binding sites and the ability of HMO l(Y) to function as an architectural component. The HMG l(Y) protein does not contain an HMG box, but does contain a highly charged basic repeat sequence that is required for its interaction wlth AT-rich duplex DNA through contacts in the minor groove. In vitro binding studies indicate that HMO I(Y) binds a sequence within the NF-K&binding site and to two regions that immediately flank the ATF-Pbinding site. HMO I(Y) binding apparently alters the structure of DNA, thereby increasing the affinity of NF-KB and ATF-2 for their respective recognition sequences. Remarkably, NF-KB directly contacts ATF-2 and RF-l, and HMG l(Y) interacts with all of these transcription factors in the absenceof DNA. Thus, direct protein-protein interactions may be involved in the transcriptional synergism between the distinct elements of the IFN-3 promoter. Moreover, HMG l(Y) may mediate both protein-DNA and protein-protein interactions in the assembly of an IFN enhancer complex (Figure 1B). A number of observations

domain

activation

domain

Figure 2. A Schematic Diagram Depicting Potential Cohesive interfaces for Directing Contacts Between Activation Domains and Target Proteins Two classes of activation domains, acidic (top) and glutaminsrich (bottom), are depicted interacting with compkmentary surfaces via pofymerk units consktkg of alternating hydrophobic residues (circles with dkgonai lines) whh acidic restdues (cirdes with horixontai lines) Eor)hydrogen bonding (three vertkai dots) vta giutamlne restdues m.

indicate that the relative phasing of the NF-KB-, IRF-l-, ATF-2-, and HMG lo-binding sites cannot be altered without adversely affecting enhancer function (D. Thanos and T. M., unpublished data). Thus, the activation of the 1FN-g gene promoter by virus infection appears to require the assembly of a stereospecific enhancer complex. The assembly of this stereospecific complex requires extensive protein-DNA and protein-protein interactions. The negative control of c-fos gene expression by the zinc finger protein Wl provides another example of protein-induced DNA bending in the control of gene expression (Natesan and Gilman, 1993). Although the Wl protein acts as a repressor of the c&s promoter, it can function as an activator in other promoter contexts. In fact, the ability of Wl to act as a repressor depends on the orientation and position of its binding site relative to other sequences in the c&s promoter. Thus, at least in the c-fos promoter, the primary function of Wl may be to organize the topology of the DNA in such a way that the interaction between a transcription activator and the baeal complex is prevented.

Tranrcrlptfonal Activation Dom8ins Following the assembly of enhancer complexes, the activation domains of some sequence-specific transcription factors within the complex must interact directly or indirectly wfth components of the basal transcription apparatus. The overall potency of a transcriptional actiiator is determined by a number of factors: the affinity for its site on the DNA and the subunit interactions necessary to assembfe a functional activator, as well as the strength of the interaction between an actfvatlon domain and its “target.” Several different types of activation domains have been identified and classified as acidic, glutamine rich, and proline rich. Thus far, the structural relationships and mechanisms of specificity of these different activation domains remain obscure. Indeed, recent studies (Gill et al., 1994) reveal that not all activation domains of a given class interact with the same target, and therefore several functionally distinct types of glutamine-rich and acidic activators may exist. Moreover, detailed mutagenesis studies suggest

Minireview 7

that the most important amino acid residues for activation are not necessarily the predominant residues such as glutamines or acidics (Cress and Triezenberg, 1991; Gill et al., 1994). Instead, in some cases, bulky hydrophobic residues that are interspersed with glutamine or acidic amino acids appear to be important elements for activation. It is likely that the cohesion of activation domains with their targets is driven by hydrophobic forces, as in protein folding, and that the specificity is achieved by the periodicityof the cohesive elements (Figure 2). Simple polymeric units such as those commonly associated with activation domains could make initial contact with multiple targets via weak interactions during different steps in the assembly of the initiation complex. These weak contacts may then subsequently “fit” into a more stable complex. The primary function of activation domains is to interact specifically with other components of the transcription apparatus. However, nuclear magnetic resonance and circular dichroism spectral studies of several isolated activation domains have revealed no evidence of secondary structure. Instead, it is envisioned that these domains might assume a specific three-dimensional structure only upon adhering to a partner or target, thus undergoing an induced fit. A conformational restructuring of activation domains affords some potential advantages. First, nonproductive interactions would be minimized if activation domains are capable of interacting with their targets in the basal complex only after binding to the enhancer and interacting with other proteins in the enhancer complex. Second, an unstructured activation domain might provide a mechanism for achieving both specificity and flexibility in protein-protein recognition. Activator-Basal Factor Interactions The ability to reconstitute activator dependent transcription in vitro with biochemically defined components has enabled a search for partners that interact with specific activation domains. Recent studies suggest that some activators are dependent on auxiliary factors that can significantly influence activation functions. Some of these components act in a positive manner and may be subunits of composite activators (Hahn, 1993) while others appear to function as negative factors that repress basal transcription (Drapkin et al., 1993). Thus, although activators may participate in multiple protein-protein interactions with various factors during activation, in this minireview we limit our discussion primarily to activator-transcriptional machinery interactions. One popular and convenient strategy to identify targets of activators has been to generate various types of protein affinity resins containing an activation domain and then to test potential binding partners for selective interactions. The first of these studies detected an interaction between the acidic activation domain of VP16 and TBP, the TATAbinding subunit of the basal transcription factor TFIID (Stringer et al., 1990). TBP was a reasonable target for specific interactions with activators, since TBP binding is thought to be the first step in the assembly of the preinitiation complex. Subsequently, many other activators have been reported to bind TBP. However, it seems clear that TBP rarely if ever exists as a single, unobstructed subunit in vivo. A major challenge, therefore, is to determine which

Figure 3. Activators Sound to DNA Interact with Multiple Targets within the Transcriptional Apparatus to Transmit Regulatory Signals

of these many TBP interactions detected in vitro are functionally significant. It is reassuring that single amino acid substitutions that inactivate VP1 6 in vivo also prevent interactions with TBP in vitro (Ingles et al., 1991) and that some activators have been shown to stabilize the binding of TBP to the TATA box in vitro. Other components of the basal transcription complex such as TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH also present potential regulatory targets for activators. One report of such interactions found that the same activation domain of VP16 that interacts with TBP also targeted TFIIB (Lin and Green, 1991). By examining proteins that associate with transcription complexes assembled on immobilized DNA, acidic activators were reported to recruit TFIIB to the initiation complex. Paradoxically, the same mutations in VP16 that disrupt VP16-TBP interactions also prevent the recruitment of TFIIB by VP16 More recently, TFIIB mutations that retain basal functions but are defective for activation were shown to be defective for VP16 interactions(Robertset al., 1993). These observations in conjunction with kinetic assays showing that TFIIB binding could be accelerated by some activators implicate TFIIB as a likely target for transcriptional activation. Activator-TBP-Associated Factor Interactions Although interactions between activators and general transcription factors may contribute to the process of activation, they are clearly not sufficient. This conclusion was based initially on the finding that the stimulation of in vitro transcription by sequence-specific activators can be detected with partially purified TFIID, but not with purified TBP (Pugh and Tjian, 1990). Subsequently, TFIID was found to exist as a remarkably stable complex composed of TBP and at least eight TBP-associated factors (TAFs). The identification of TAFs (250, 150, 110,60,60,40,30a, 306) and the demonstration that they are required to reconstitute activation in vitro led to the coactivator hypothesis (Pugh and Tjian, 1990; Dynlacht et al., 1991) which poses that at least some of the subunits of TFIID serve functionally to link transcription activation domains with the basal transcription complex. Since there are at least eight TAFs, with the possibility of additional substoichiometric and perhaps tissue-specific TAFs, it was proposed that different classes of activators might interact directly with distinct TAFs (Figure 3). To date, several activator-TAF interactions have been documented. Using three complementary protein interaction assays, the glutamine-rich activation domains of Spl were found to bind selectively to a glutamine-rich domain ofTAFll1 lO(Hoeyetal., 1993; Gill et al., 1994). Analysisof

Cdl 0

mutants in activation domains revealed a tight correlation between TAF binding and transcriptional activity. In addition, in vitro reconstitution of partial TFIID complexes supports the notion that Spl -TAFIIl 10 interactions help mediate activation. In a second case, the C-terminal activation domain (residues 452-490) of VP1 6 was shown to interact with TAFll40; antibodies that disrupt this interaction impede transcriptional activation without affecting basal transcription (Goodrich et al., 1993). These studies suggest that some of the TAFs may serve as coactivators to mediate transcriptional regulation. Moreover, the few examples that have been studied support the idea that different classes of activators (i.e., acidic or glutamine rich) may in fact interact with different subunits of the TBP-TAF complex. Indeed, all activators that have been tested thus far appear to require the TFIID complex for activation in vitro, suggesting that TAFs play a central role in mediating activator functions. Furthermore, analysis of a cell line with a temperature-sensitive TAF mutant confirms the importance of TBP-TAF complexes for mediating activation in vivo (Wang and Tjian, 1994). By contrast, in vitro reconstitution experiments have established that TAFs are not necessary or even stimulatory for basal transcription and that stable preinitiation complexes are efficiently formed in the absence of TAFs. It is important to note that the pattern of protein-protein interactions and the function of most TAFs have yet to be determined. It is therefore likely that additional interactions between TAFs and other components of the basal apparatus will be identified. Analysis of complexes isolated by gel filtration suggest that at least some TAFs may also act subsequent to activator-TFIIB interactions and that TAFs may mediate interactions between TFIID and other components of the basal complex (Choy and Green, 1993). Indeed, TAFll40 is capable of interacting selectively with TFIIB, and since TAFll40 and TFIIB each interact with distinct regions of VP1 6, a three-way complex is possible (Goodrich et al., 1993). These studies reaffirm that activators in conjunction with TAFs are required for the assembly of active basal initiation complexes. Moreover, observations made to date suggest that complex activation domains most likely contact multiple targets, including basal factors such as TBP and TFIIB as well as TAFs. It is also likely that in some promoter contexts, TAFs perform essential functions in addition to interacting with activators or basal factors. For example, the TFIID complex supports transcription from TATA-less promoters while TBP fails to do so (Pugh and Tjian, 1990). It is conceivable that TAFs contribute to the formation of stable initiation complexes by interacting directly with DNA. We know that TFIID protects an extended region overlapping the promoter and start site, whereas TBP binds to a limited 20 bp region over the TATA box. We may eventually find that TAFs play a role during several different stages of the transcription cycle, including initiation, promoter clearance, elongation, and termination. Therefore, activators, TAFs, and basal factors may mediate activation by participating at different times and steps during the dynamic process of initiation complex assembly and transcription. Taken at face value, these studies suggest that activa-

tors may contact several distinct components within the basal initiation complex and that no specific component is the exclusive target for activation domains. However, it may be premature to conclude that all activator-target interactions detected in vitro are important for mediating transcriptional regulation. The in vitro conditions for performing transcription reactions and protein binding assays can profoundly influence the outcome. Also, one limitation of protein binding assays is their bias for detecting strong interactions between single partners, whereas the potentially more relevant weak binding involving multiple partners may not be detected. In summary, rapid and significant progress has been made in charting the steps required for transcriptional activation and in characterizing the interactions among various components of the transcriptional apparatus. These studies have provided significant mechanistic insights into the problem of how specific genes are selected for activation. The challenge now is to understand the mechanism by which the assembly of a nucleoprotein complex on the promoter results in transcriptional activation. Rrferenceo Cartsson, P., Waterman, 7, 2416-2430.

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