The RNA polymerase II (Pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator - a large, conformationally flexible protein complex with a variable subunit composition (for example, a four-subunit cyclin-dependent kinase 8 module can reversibly associate with it) These biochemical characteristics are fundamentally important for Mediator's ability to control various processes that are important for transcription, including the organization of chromatin architecture and the regulation of Pol II pre-initiation, initiation, re-initiation, pausing and elongation Although Mediator exists in all eukaryotes, a variety of Mediator functions seem to be specific to metazoans, which is indicative of more diverse regulatory requirements

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The Mediator complex: a central integrator of transcription

Dynamic regulation of pol II transcription by the mammalian Mediator complex.

Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

Transcription regulation by the Mediator complex.

Mammalian euchromatic gene silencing results from the combined repressive effects of histone and DNA methyltransferases. Little is known of the mechanism by which these enzymes cooperate to induce silencing. Here we show that mammalian HP1 family members mediate communication between histone and DNA methyltransferases. In vitro, methylation of histone 3 Lys 9 by G9a creates a binding platform for HP1alpha, beta, and gamma. DNMT1 interacts with HP1 resulting in increased DNA methylation on DNA and chromatin templates in vitro. The functional and physical interaction can be recapitulated in vivo. Binding of GAL4-HP1 to a reporter construct is sufficient to induce repression and DNA methylation in DNMT1 wild-type but not DNMT1-null cells. Additionally, silencing of the Survivin gene coincides with recruitment of G9a and HP1 in DNMT1 wild-type but not null cells. We conclude that direct interactions between HP1 and DNMT1 mediate silencing of euchromatic genes.

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Functional cooperation between HP1 and DNMT1 mediates gene silencing

The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module.

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The Mediator complex and transcription regulation.

Activator-mediated transcription complex assembly on templates lacking chromatin requires the interaction of activators with two major coactivator complexes: TFIID and mediator. Here we employed immobilized template assays to correlate transcriptional activation with mediator and TFIID recruitment. In reactions reconstituted with purified proteins, we found that activator, TFIID, and mediator engage in reciprocal cooperative interactions to form a complex on promoter DNA. Preassembly of the coactivator complex accelerates the rate of transcription in a cell-free system depleted of TFIID and mediator. Our data argue that this coactivator complex is an intermediate in the assembly of an active transcription complex. Furthermore, the reciprocity of the interactions demonstrates that the complex could in principle be nucleated with either TFIID or mediator, implying that alternative pathways could be utilized to generate diversity in the way activators function in vivo.

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TFIID and human mediator coactivator complexes assemble cooperatively on promoter DNA

Assembly of a mediator/TFIID/TFIIA complex bypasses the need for an activator.

The immobilized template assay for measuring cooperativity in eukaryotic transcription complex assembly.

https://www.cell.com/current-biology/pdf/S0960-9822(01)00306-2.pdf

Kristina M. Johnson

Papers

TFIID and human mediator coactivator complexes assemble cooperatively on promoter DNA

Assembly of a mediator/TFIID/TFIIA complex bypasses the need for an activator.

The immobilized template assay for measuring cooperativity in eukaryotic transcription complex assembly.

Eukaryotic transcription: the core of eukaryotic gene activation.