Regulation of the RNA polymerase II pre-initiation complex by its associated coactivators

Abstract

The RNA polymerase II (Pol II) pre-initiation complex (PIC) is a critical node in eukaryotic transcription regulation, and its formation is the major rate-limiting step in transcriptional activation. Diverse cellular signals borne by transcriptional activators converge on this large, multiprotein assembly and are transduced via intermediary factors termed coactivators. Cryogenic electron microscopy, multi-omics and single-molecule approaches have recently offered unprecedented insights into both the structure and cellular functions of the PIC and two key PIC-associated coactivators, Mediator and TFIID. Here, we review advances in our understanding of how Mediator and TFIID interact with activators and affect PIC formation and function. We also discuss how their functions are influenced by their chromatin environment and selected cofactors. We consider how, through its multifarious interactions and functionalities, a Mediator-containing and TFIID-containing PIC can yield an integrated signal processing system with the flexibility to determine the unique temporal and spatial expression pattern of a given gene.

Fig. 1 |. A paradigmatic TBP-nucleated PIC assembly pathway.

A composite TATA binding protein (TBP)-nucleated pre-initiation complex (PIC) assembly pathway on a TATA element-containing promoter, based on biochemical and structural studies of the minimal PIC (see main text and references therein). In the first step, TBP binding to the minor groove of the TATA element (red box) is stabilized by TFIIA, which makes direct contacts with TBP and with upstream DNA backbone phosphates (T.A). Additional stabilization comes from TFIIB (T.A.B), both through interactions with the T.A subcomplex as well as through interactions with TFIIB recognition element (BRE) sequences in the promoter. The amino-terminal regions of TFIIB, consisting of the zinc ribbon, B-reader and B-linker domains, recruit RNA polymerase II (Pol II) and support transcription initiation through interactions, respectively, with Pol II and the template and non-template DNA strands. T.A.B is a landing pad for Pol II in a step that is facilitated by heterodimeric TFIIF, which binds close to the Pol II cleft and causes the Pol II clamp to partially open, resulting in stabilization of the double-stranded template DNA in the cleft (T.A.B.F.II). Finally, TFIIE and TFIIH enter to yield a functional PIC (T.A.B.F.II.E.H). TFIIE makes multiple contacts with Pol II, including sites at the RPB4/7 stalk. A cluster of TFIIE winged helix domains extend out over the cleft and together with TFIIF form a protein bridge that further stabilizes the template. TFIIE is also critical in recruiting TFIIH into the PIC. Formation of the first phosphodiester bond by the Pol II active site is preceded by melting of double-stranded DNA around the transcription start site (TSS) by the TFIIH XPB translocase^,,. Multiple conformational changes (such as clamp closure, stalk movements and TFIIB linker transitions) in the PIC also ensue, further stabilizing the resulting ‘open complex’. The nascent RNA chain faces a steric clash with the stabilizing interactions of N-terminal regions of TFIIB, which forces Pol II to undergo reiterative abortive synthesis of short oligomeric RNAs. Pol II promoter escape and extension of the RNA chain past a length of about 12 nucleotides entails TFIIB release from the PIC. TFIIE and TFIIF are also released, both to relinquish the open state’s stabilizing contacts and to enable entry of elongation factors (Fig. 2). Although it fully supports basal transcription, this minimal PIC is not responsive to transcriptional activators. CAK, CDK-activating kinase.

Fig. 2 |. General principles of coactivator-dependent PIC recruitment and function.

Multi-step pre-initiation complex (PIC) assembly on an idealized template (step 1) containing diverse regulatory sequence motifs that are not typically all found in a given transcription locus. Signal-bearing transcription activators can bind to cognate sites located in distal enhancers, including super-enhancers, or to sites located proximal to core promoter elements (step 2). Core promoter elements collectively specify the transcription start site (TSS). In addition to the TATA box, these may include, among others, motif ten elements (MTEs) and downstream promoter elements (DPEs). Activator binding sets off a cascade of events that include recruitment of one or more intermediary factors, depicted here as a generic ‘coactivator’ (step 3). Some coactivators play critical roles at the level of chromatin; given the focus here on the PIC, this step is not highlighted. Coactivators in turn facilitate the formation of the PIC through interactions with one or more general transcription factors (GTFs), as well as RNA polymerase II (Pol II) (step 4). Despite their historical origins as factors that promote PIC formation and function, the two coactivators discussed here, Mediator and TFIID TATA binding protein (TBP)-associated factors (TAFs), are so intimately associated with the PIC that they may justifiably be regarded as components of an expanded PIC. The PIC is tasked with ensuring precise site-specific initiation of the nascent RNA transcript and promotion of the Pol II to an elongation-competent form (step 5) following promoter escape. GTFs are also released, both to relinquish contacts that stabilize the open state and to enable entry of transcription elongation factors (TEFs), which in conjunction with other factors not discussed here have roles in promoter-proximal pausing that occurs on many genes prior to acquisition of full elongation processivity by Pol II. PIC coactivators have the potential to regulate some of these downstream events as well.

Fig. 3 |. Modular structure of metazoan Mediator.

a, Human Mediator structural modules with conventional subunit assignment to the head, middle and tail modules (right). However, cryogenic electron microscopy (cryo-EM) and cross-linking–mass spectrometry studies^– reveal that several key subunits straddle more than one module. In particular, MED14 forms a structural backbone around which the head, middle and tail modules are organized. Similarly, whereas the bulk of MED17 resides in the head, its amino terminus is embedded in the middle. Metazoan-specific subunits MED27, MED28 and MED30 also straddle the head and tail. Also note the location of MED1 in the middle. This large subunit has not yet been visualized in its entirety, but the tail-proximal location of this activator target hot spot in Mediator structure is intriguing for models of how activation signals may be processed. The kinase module, which reversibly associates with the Mediator, is also depicted (left). As shown, three of its four constituent subunits have paralogues that can give rise to multiple permutations. b, Left: a model of the Mediator complex showing the relative location of the head, middle and tail modules. The modules can move relative to each other, resulting in several conformers of the complex (not shown). The model is based on ref. , which produced high-resolution structures showing detailed subunit architecture of the complex. Right: the Mediator complex in association with the kinase module. Note that this form of the Mediator can interact with activators but not with RNA polymerase II (Pol II). In metazoans, association of the kinase module with the complex is also mutually exclusive with association of the metazoan-specific MED26 middle subunit. Precisely how the two forms of the Mediator interchange is not yet known (see also Fig. 6). The kinase module is modelled after the structure in ref. . CDK8, cyclin-dependent kinase 8.

Fig. 4 |. TFIID structure and dynamics.

a, Subunit composition of the trilobular human TFIID structure. Several TATA binding protein (TBP)-associated factors (TAFs) exist in two copies; some TAFs also heterodimerize via histone fold-containing domains. Lobe A contains TBP as well as numerous TAFs that are also found in lobe B. Lobe C includes TAFs that have been implicated in recognition of core promoter motifs. b, As visualized by cryogenic electron microscopy (cryo-EM), TFIID can exist in multiple conformations. Two extreme conformations are depicted: ‘canonical’ (left), which is a relatively compact form of the TFIID complex; and promoter-bound (right), which is in one of the various ‘extended’ forms of the complex. The promoter-bound form shown is based on a cryo-EM structure that included TFIIA, which both stabilizes the promoter complex and helps neutralize the inhibitory action of TAF1 and TAF11–TAF13. Note the dramatic relocation of TBP as a result of TFIID lobe movements. Lobe A position in the promoter-bound state is variable; it is shown here in an intermediate location (dashed outlines) based on the structure of a TFIID-containing pre-initiation complex (PIC). DPE, downstream promoter element; MTE, motif ten element.

Fig. 5 |. TFIID recruitment to promoters in the context of a nucleosome-depleted region.

A composite model showing multiple potential interactions of TFIID just upstream of a positioned +1 nucleosome. In addition to the promoter DNA interactions (Fig. 4), TATA binding protein (TBP)-associated factors (TAFs) in TFIID can have stabilizing interactions with an activator (such as TAF4 with an E protein) bound to a proximal element or with post-translationally modified histone tails in the +1 nucleosome. Documented tail interactions include recognition of acetylated histone H4 by TAF1 and of histone H3 trimethylated at lysine 4 (H3K4me3) by TAF3. A location for the highly mobile lobe A is not specified (see Fig. 4 legend).

Fig. 6 |. Pathways for delivery of Mediator from distal enhancers to the PIC.

Two general models of how Mediator that has been recruited to a distal enhancer (state 1) can be delivered to the pre-initiation complex (PIC). Multiple kinase module-associated Mediator complexes, illustrative of Mediator clustering at a super-enhancer, are depicted bound to an array of activators. In one pathway (state 2), one of these complexes participates in bridging the enhancer and promoter where it facilitates PIC assembly after having ejected the kinase module. This long-range interaction may be transient and facilitated by architectural factors that generate and stabilize DNA loops. Alternatively, an enhancer-recruited Mediator complex may detach from the chromatin (state 3) after undergoing some form of ‘activation’ following activator interaction (see main text). Such an altered Mediator would be capable of interaction with RNA polymerase II (Pol II) (via its RPB1 carboxy-terminal domain (CTD)), whose Mediator association is mutually exclusive with that of the kinase module. This complex would be able to readily diffuse to a nearby nascent PIC (state 4) within the confines of a chromatin topological structure generated by as yet uncharacterized architectural factors. Among other possibilities, an interesting variant of this pathway imagines that an activator-bound Mediator dissociates from the activator’s cognate site in the enhancer and goes on to facilitate PIC assembly. Not highlighted is the likely scenario that in contrast to the enhancer-bound Mediator, which is enriched in the kinase module, the PIC-associated Mediator might be preferentially associated with MED26. In all these models, but especially in the diffusion model (state 3), it is not clear how promiscuous enhancer–promoter interactions are minimized. In addition to chromatin topology and related enhancer condensates (see main text), activity gradients emanating from the enhancer as a point source and regulated by cycles of post-translational modifications and reversals could ensure that functionally active diffusing coactivators do not stray too far away from the cognate promoter. See main text for how Mediator might also contribute to chromatin remodelling at the promoter if it is occluded by a nucleosome.

Fig. 7 |. A TFIID-containing and Mediator-containing PIC.

A model for a TFIID-containing and Mediator-containing pre-initiation complex (PIC) adapted from ref. . Note the additional stabilizing interactions relative to a TATA binding protein (TBP)-nucleated minimal PIC that does not contain Mediator (Fig. 1). Highlighted here is the stabilization of the RPB1 carboxy-terminal domain (CTD), but numerous other subtle, and perhaps dynamic, interactions between the coactivators, RNA polymerase II (Pol II) and general transcription factors (GTFs) also take place, as discussed and referenced in the main text. Additional stabilizing interactions of the PIC might come from promoter proximally bound activators and the downstream +1 nucleosome via TFIID (Fig. 5) and Mediator (not shown; see text). Other general cofactors that may be fulfilling architectural and other roles may also contribute but are not shown (Box 2). CAK, CDK-activating kinase.