# An intrinsically disordered region in MED13 turns
Mediator on/off on cue

Vincent Villeret, Didier Monté, Alexis Verger

► To cite this version:

Vincent Villeret, Didier Monté, Alexis Verger. An intrinsically disordered region in MED13 turns Mediator on/off on cue. Molecular Cell, 2024, 84 (16), pp.3005-3007. 10.1016/j.molcel.2024.07.027. hal-04676270

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Submitted on 27 Oct 2024

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# An intrinsically disordered region in MED13 turns Mediator on/off on cue

Vincent Villeret¹, Didier Monté¹ and Alexis Verger¹, *

¹CNRS EMR 9002 Integrative Structural Biology, Inserm U 1167 – RID-AGE, Univ. Lille,
CHU Lille, Institut Pasteur de Lille, F-59000 Lille, France

*Lead author ([Alexis.Verger@univ-lille.fr](mailto:Alexis.Verger@univ-lille.fr))

Complementary studies by Zhao et al.¹ and Chen et al.² reveal how an intrinsically disordered region in MED13 controls mutually exclusive binding of RNA Polymerase II and CDK8 kinase module to Mediator, switching on and off Mediator and transcription activation.

Since its discovery over three decades ago in yeast, distinct compositional forms of the
Mediator complex (MED) have been isolated as stable entities. The most common form is a 21-
subunit core complex (26 subunits in mammals) organized into three topological modules
named Head, Middle and Tail, and a 25-subunit CDK8-Mediator complex (29 subunits in
mammals) (Figure 1A). CDK8 represents a separable four-subunit kinase module (CKM)
consisting of MED12, MED13, Cyclin C and CDK8. In mammals, core Mediator contains five
additional specific subunits, one of which (MED26) dissociates upon CKM binding.
Vertebrates also evolved subunit paralogs, called MED12L, MED13L, and CDK19, which
expand the functional diversity of the kinase module.³ Mediator plays a critical role for
transcription factor (TF)-dependent transcription activation by connecting enhancer-bound TFs
and the RNA Polymerase II (Pol II) machinery at the promoter. Mediator directly influences
preinitiation complex (PIC) assembly and stabilization, and stimulates TFIIH-dependent
phosphorylation of the Pol II C-terminal domain (CTD).⁴ These functions are disrupted upon
kinase module binding, which blocks PIC assembly. Many studies have documented reversible
“on/off” binding of CKM to Mediator but the mechanistic details remain elusive.³,⁵ Recent
years have seen an explosion of structural studies of yeast and mammalian Mediators
complexed with the Pol II transcription initiation machinery.⁴ However, revealing the structure
of the dissociable CDK8 kinase module bound to Mediator remains one of the greatest
challenges in the field. Additionally, although CKM influences PIC formation, questions
remain regarding transcriptional regulator recruitment of CKM-bound Mediator and how
Mediator relays activating signals to initiate PIC assembly. Studies from Zhao et al.¹ in a recent
issue of *Molecular Cell* and Chen et al.² in a preprint uncover the architecture of mammalian
Mediator in complex with the kinase module, assigning a function to a structureless central region of the MED13 subunit. Using a combination of structural, biochemical, and functional methods, they collectively elucidate the molecular basis of CKM repression and Mediator activation. Competition between an intrinsically disordered region in MED13, Pol II CTD and MED26 for binding to Mediator represents a key regulatory control that activates Mediator upon transcription factor binding.

Building on previous low-resolution EM studies of the CKM-MED complex⁵,⁶ and the yeast CKM in isolation⁷, Tsai and colleagues resolved the cryo-EM architecture of the human CKM-MED complex. Their composite CKM-MED map shows human CKM is anchored principally via MED12 to a protrusion of the Mediator hook region, with other CKM regions extending away from core Mediator (Figure 1B). The structure also reveals MED12/MED13 organization and contacts between MED12 and CDK8/Cyc C essential for kinase activity. Overall, the architecture of human and yeast CKM is similar, with MED12 mainly scaffolded with HEAT-repeat domains and MED13 organized around Argonaute-like domains. Nevertheless, the human CKM-MED binding mode appears distinct from that observed in yeast⁶,⁷ where the entire CKM extends along the Middle and Head modules and sterically blocks the association of MED with Pol II. Initially, this human CKM-MED structure failed to explain why MED13 was originally thought to be instrumental for Mediator binding.⁵,⁶ The Tsai and Asturias labs, like detectives in a police investigation, pieced together the clues and solved this enigma. Combining structural analyses, cross-linking mass spectrometry (XL-MS) and biochemical analyses, they identified and apprehended the culprit: a large intrinsically disordered region in MED13 (MED13 IDR) which is involved in core MED binding (Figure 1B). One might wonder what MED13 IDR could do other than stabilizing the CKM-MED interface? The evidence gathered by Zhao et al.¹ and Chen et al.² not only incriminates MED13 IDR but also uncovers its underlying motive. (i) XL-MS analyses identified several unique residue pairs between MED13 IDR and Mediator subunits that bind either MED26 or Pol II CTD. (ii) Nearly all Mediator subunits associate with MED13 IDR, but not MED26 nor Pol II. (iii) Using RNA-seq, ChiP-seq and CUT&RUN experiments in MED26⁻/⁻ cells, Zhao et al.¹ validated MED26's *in vivo* involvement in facilitating Pol II interaction with Mediator at gene promoters. (iv) Analyses of the CKM-MED Cryo-EM maps show several extra densities (attributed to the MED13 IDR) at locations where Pol II CTD and MED26 normally interact with Mediator in the absence of CKM. Collectively, the findings of Zhao et al.¹ and Chen et al.² suggest a model where MED13 IDR blocks MED26 and CTD interaction to repress Mediator and regulates the transition from CKM-MED to MED-PIC (Figure 1B).

But then, do transcription factors play a role in MED13 IDR regulation? Asturias and colleagues found that nuclear receptors (TR and VDR) prefer to interact with CKM-MED. Their TR-MED Cryo-EM data show that the densities of MED13 IDR observed near Pol II CTD and MED26 binding sites in the CKM-MED structure are absent, suggesting TR can revert MED inhibition by CKM (Figure 1B).

These two studies reveal a fundamental mechanism of transcriptional regulation, showing how a structureless domain facilitates the transition between the inactive and active states of Mediator. Given that many transcription factors form condensates with Mediator to activate transcription⁸ and that IDRs promote liquid-liquid phase separation (LLPS), transcriptional condensates might also facilitate conformational transitions of CKM-MED. CDK8 phosphorylates many TFs and Mediator subunits, including MED12, MED13 and MED26.⁹
Notably, the phosphorylation site identified in MED13 (S749) is located in its IDR. Exploring its potential impact on MED13 function is an intriguing question for future research. Furthermore, given MED1's location at the Tail-Middle junction, the question arises whether the effects on CKM-MED are specific to nuclear receptors or whether other TFs targeting different subunits could exert the same influence. The functional interplay recently identified between the Tail subunits and the kinase module¹⁰ suggests that the proposed mechanism for NR may be general. The molecular events that trigger release of CKM from the enhancer-bound Mediator and the subsequent transition to a fully functional PIC (with MED26) on promoters are questions for future research, along with a better understanding of the positive role of CKM in post-initiation events. Finally, Zhao et al.¹ and Chen et al.² provide an invaluable structural framework for interpreting disease causing mutations reported in the CKM subunits.

<figure><img src="image_2.png" /><figcaption>**Figure 1: Switching Mediator on and off.** (A) Composition of Mediator and CKM. Mammalian subunits with no yeast homologs are in bold. Mediator modules and specific subunits are color-coded. TR: Thyroid hormone receptor; VDR: Vitamin D nuclear receptor (B) Dynamic association of Pol II CTD and Mediator subunits (CKM and MED26) with Mediator. Extended interaction of MED13 IDR with MED (CKM-MED “OFF”) blocks Pol II CTD and MED26. Enhancer-bound Nuclear Receptor (NR) interaction with CKM-MED through MED1 repositions MED13 IDR (CKM-MED “primed”), facilitating recruitment of Pol II CTD and MED26 (MED “ON”). Head and Middle modules are also repositioned (not shown) and CTD binding diminishes MED25 interaction with the Tail. This ON/OFF switch promotes</figcaption></figure>
MED-PIC assembly and initiates transcription. Only Pol II is shown for clarity. TSS:
Transcription start site.

**Declaration of interests**

The authors declare no competing interests.

**References**

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6. Tsai, K.-L., Sato, S., Tomomori-Sato, C., Conaway, R.C., Conaway, J.W., and Asturias, F.J. (2013). A conserved Mediator-CDK8 kinase module association regulates Mediator-RNA polymerase II interaction. Nat Struct Mol Biol 20, 611–619. https://doi.org/10.1038/nsmb.2549.

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