The metazoan Mediator co-activator complex as an integrative hub for transcriptional regulation

Abstract

The Mediator is an evolutionarily conserved, multiprotein complex that is a key regulator of protein-coding genes. In metazoan cells, multiple pathways that are responsible for homeostasis, cell growth and differentiation converge on the Mediator through transcriptional activators and repressors that target one or more of the almost 30 subunits of this complex. Besides interacting directly with RNA polymerase II, Mediator has multiple functions and can interact with and coordinate the action of numerous other co-activators and co-repressors, including those acting at the level of chromatin. These interactions ultimately allow the Mediator to deliver outputs that range from maximal activation of genes to modulation of basal transcription to long-term epigenetic silencing.

Figure 1. Involvement of Mediator in multiple steps of transcription

Current models of transcriptional activation involve multiple steps. a | An idealized pathway is shown that begins with chromatin, in which nucleosomes exist in a characteristic beads-on-a-string array. This contrasts to transcriptionally inert chromatin, a highly compacted structure in which DNA is tightly packaged not just through wrapping around nucleosomes but through additional higher-order structures that entail linker histones and other proteins such as heterochromatin protein 1 (not shown). b | The activation pathway is initiated by one or more transcriptional activators that bind to their cognate sites in the regulatory region of the gene. These factors recruit a series of chromatin co-activators that can covalently modify nucleosomes at specific histone residues (not identified) and mobilize nucleosomes through ATP-requiring reactions. c | The resulting intermediate contains chromatin that is characterized by distinct covalent modifications, such as acetylation (Ac) and methylation (Me), and by a relative dearth of nucleosomes. The activators then recruit Mediator. In some cases, the intact Mediator that consists of the core and the kinase module might be recruited at this stage. d | Pre-initiation complex (PIC) assembly, entailing the various general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH) and RNA polymerase II (Pol II), and transcription initiation then ensue with concomitant restructuring of the Mediator that results from loss of the kinase module. e | After Pol II clears the promoter, there are two possible outcomes. As shown on the right, the process can proceed directly to the elongation phase at which it is associated with elongation factors that include DSIF and P-TEFb, whose entry into the transcription elongation complex (TEC) may coincide with capping (7-methyl-guanosine; MeG) of the nascent RNA. The RPB1 carboxy-terminal domain (CTD) also undergoes substantial phosphorylation at Ser2 and Ser5 (Pol IIO) through the sequential actions of TFIIH and P-TEFb. Importantly, a scaffold containing a vestigial subset of general transcription factors and Mediator remains at the promoter to potentially facilitate subsequent rounds of transcription. Alternatively, as shown on the left, at many loci, Pol II may be subject to promoter-proximal pausing (at approximately nucleotide +50). This Pol II would be associated with DSIF but phosphorylated only at Ser 5 (Pol IIA). Under appropriate conditions, the paused Pol II complex can also mature into an elongation complex. Although depicted linearly, the intermediates are not likely to be as clear-cut as shown. Indeed, as discussed in the text, Mediator couples many of the steps. Moreover, some aspects of the reaction are reversible, again this occurs under the control of Mediator.

Figure 2. Modular structure of Mediator and interactions with diverse factors

A composite depiction of the subunit structure of the human Mediator complex is shown. Note that although the relative placement of the subunits in the subcomplexes is based on published binary interaction and partial structural data^,,,, it is primarily for illustration; the precise locations have yet to be mapped. The MED1 and MED26 subunits, which are not present in all isolates, are likely to be located at the junction of the middle and tail modules. MED28 and MED30 have been provisionally placed in the middle module. Transcription factors selected for discussion in the main text and their target subunits in Mediator are also indicated. The unified nomenclature proposed by Bourbon et al. has been used here. However, very recent bioinformatic analyses strongly suggest that MED27 and MED29, which were earlier considered to be metazoan-specific, are related to yeast MED3 and MED2, respectively. Here they are referred to as MED3 (also known as MED27) and MED2 (also known as MED29). Similarly, MED5 and MED24 may be related and referred to as MED5 (also known as MED24). ER, oestrogen receptor; GR, glucocorticoid receptor; HNF4, hepatocyte nuclear factor; NHR49, nuclear hormone receptor 49; PPARγ, peroxisome proliferator-activated receptor-γ; SREBP1, sterol regulatory element-binding protein 1; TGFβ, transforming growth factor-β; VDR, vitamin D3 receptor.

Figure 3. Modulation of Mediator function by ancillary factors: coordination of chromatin remodelling and PIC formation

Although it plays a crucial part in assembling the pre-initiation complex (PIC), which is formed on nucleosome-free templates, Mediator also contributes to chromatin remodelling through functional interactions with chromatin co-factors. A potential activation pathway is shown that follows the binding of ligand-bound thyroid hormone receptor (TR) in association with its heterodimerization partner retinoic acid receptor-α (RXRα) to their cognate site embedded in chromatin. In the first step, the histone acetyltransferase p300, is recruited. On some genes, this interaction is stimulated by peroxisome proliferator-activated receptor-γ co-activator 1α (PGC1α); other contributors (for example, p160 co-activators) are not shown. As both chromatin co-activators and Mediator bind to the activation function 2 (AF2) domains of nuclear receptors, it was previously suggested that their binding would be mutually exclusive and that Mediator-driven PIC formation would provide the driving force for co-factor exchange on the receptor. More recent evidence suggests that Mediator and p300 can co-exist in a transient ternary intermediate (shown in the second step in the figure) that could facilitate transitions resulting in functional PICs (shown in the third step in the figure). Given further that PGC1α dynamically interacts with the Mediator through the Mediator complex subunit 1 (MED1) (structural rearrangements are depicted as an altered orientation of PGC1α), it might further modulate this coordination role of the Mediator^,. Any potential role of the kinase module in this particular scenario remains unaddressed and has been omitted for clarity.

Figure 4. Modulation of Mediator function by ancillary factors: epigenetic silencing

In concert with additional negatively acting factors, Mediator can contribute to long-term silencing of certain developmentally regulated loci. Starting with an active pre-initiation complex (PIC; which would likely contain the PC2 form of the Mediator), REST binding to the cognate site commits the gene to a heterochromatin fate as development proceeds. A ternary complex containing REST, the histone methyltransferase G9a and the intact Mediator is first assembled. The multipartite interactions are anchored through the MED12 subunit of the kinase module. Following G9a action and dimethylation of histone H3 lysine 9 (H3K9) to H3K9me2, heterochromatin protein 1 (HP1) and DNA methyltransferase 1 (DNMT1) are recruited to the site. Ultimately, the gene is embedded in transcriptionally inert heterochromatin marked by H3K9me2 and methylated DNA (CH₃). Other factors (for example, histone deacetylase 1 and lysine-specific demethylase 1) that contribute to silencing are not shown. The figure is based on the findings described in REF. .