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Many components of eukaryotic transcription machinery—such as transcription factors and cofactors including BRD4, subunits of the Mediator complex, and RNA polymerase II—contain intrinsically disordered low-complexity domains. Now a conceptual framework connecting the nature and behavior of their interactions to their functions in transcription regulation is emerging (see the Perspective by Plys and Kingston). Chong et al. found that low-complexity domains of transcription factors form concentrated hubs via functionally relevant dynamic, multivalent, and sequence-specific protein-protein interaction. These hubs have the potential to phase-separate at higher concentrations. Indeed, Sabari et al. showed that at super-enhancers, BRD4 and Mediator form liquid-like condensates that compartmentalize and concentrate the transcription apparatus to maintain expression of key cell-identity genes. Cho et al. further revealed the differential sensitivity of Mediator and RNA polymerase II condensates to selective transcription inhibitors and how their dynamic interactions might initiate transcription elongation. Science , this issue p. eaar2555 , p. eaar3958 , p. 412 ; see also p. 329 Critical components of transcription machinery form stable, condensate-like, transcription-dependent clusters in cells. Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.

A class of long non-coding RNA (lncRNA) with enhancer-like activity is found to associate with the co-activator complex Mediator and promote its genomic association and enzymatic activity; together with Mediator, the lncRNAs also help to maintain the chromosomal architecture of active regulatory elements. Mediator acts with ncRNA-a in gene regulation Long non-coding RNAs (lncRNAs) can both repress and activate gene expression. Here, a class of lncRNAs with enhancer-like activity is found to associate with the translational co-activator complex Mediator. Termed ncRNA-activating (ncRNA-a), these molecules promote the genomic association and enzymatic activity of Mediator, and acting together with Mediator, they also help to maintain the chromosomal architecture of active regulatory elements. Importantly, Mediator complexes containing disease-linked mutant MED12 proteins fail to associate with ncRNA-a. The MED12 gene encodes a Mediator complex subunit, and MED12 mutations have been linked to FG syndrome, a rare genetic disorder with symptoms including intellectual disability. This work suggests that the loss of Mediator–ncRNA-a interactions might be a possible contributing factor in such developmental diseases. Recent advances in genomic research have revealed the existence of a large number of transcripts devoid of protein-coding potential in multiple organisms 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 . Although the functional role for long non-coding RNAs (lncRNAs) has been best defined in epigenetic phenomena such as X-chromosome inactivation and imprinting, different classes of lncRNAs may have varied biological functions 8 , 9 , 10 , 11 , 12 , 13 . We and others have identified a class of lncRNAs, termed ncRNA-activating (ncRNA-a), that function to activate their neighbouring genes using a cis -mediated mechanism 5 , 14 , 15 , 16 . To define the precise mode by which such enhancer-like RNAs function, we depleted factors with known roles in transcriptional activation and assessed their role in RNA-dependent activation. Here we report that depletion of the components of the co-activator complex, Mediator, specifically and potently diminished the ncRNA-induced activation of transcription in a heterologous reporter assay using human HEK293 cells. In vivo , Mediator is recruited to ncRNA-a target genes and regulates their expression. We show that ncRNA-a interact with Mediator to regulate its chromatin localization and kinase activity towards histone H3 serine 10. The Mediator complex harbouring disease- 17 , 18 displays diminished ability to associate with activating ncRNAs. Chromosome conformation capture confirmed the presence of DNA looping between the ncRNA-a loci and its targets. Importantly, depletion of Mediator subunits or ncRNA-a reduced the chromatin looping between the two loci. Our results identify the human Mediator complex as the transducer of activating ncRNAs and highlight the importance of Mediator and activating ncRNA association in human disease.

Respiratory syncytial virus (RSV), the most common cause of bronchiolitis and pneumonia in infants, elicits a remarkably weak innate immune response. This is partly due to type I interferon (IFN) antagonism by the non-structural RSV NS1 protein. It was recently suggested that NS1 could modulate host transcription via an interaction with the MED25 subunit of the Mediator complex. Previous work emphasized the role of the NS1 C-terminal helix α3 for recruitment of the MED25 ACID domain, a target of transcription factors (TFs). Here we show that the NS1 α/β core domain binds to MED25 ACID and acts cooperatively with NS1 α3 to achieve nanomolar affinity. The strong interaction is rationalized by the dual NS1 binding site on MED25 ACID predicted by AlphaFold and confirmed by NMR, which overlaps with the two canonical binding interfaces of TF transactivation domains. Single amino acid substitutions in the NS1 α/β domain, notably NS1 E110A, significantly reduced the affinity of NS1 for MED25 ACID, both in vitro and in cellula. These mutations resulted in attenuated replication of recombinant RSV (rRSV-mCherry). They did not significantly upregulate type I or III IFN levels in IFN-competent BEAS-2B cells, contrary to the NS1 α3 deletion. However, in line with attenuated replication, the NS1 E110A mutation enhanced expression of the antiviral interferon-stimulated gene ISG15, and NS1 I54A upregulated ISG15, OAS1A and IFIT1 in IFN-competent cells. In MED25-knockdown cells, rRSV-mCherry replication was further attenuated at a late post-infection timepoint. The difference between WT and NS1 mutant rRSV-mCherry was partially lost, suggesting that the NS1–MED25 ACID complex contributes to controlling antiviral responses at this timepoint. The strong interaction and the extended binding interface between NS1 and MED25 ACID provide evidence for a mechanism, where NS1 blocks access of transcription factors to MED25, and thereby MED25-mediated transcription activation.

Many components of eukaryotic transcription machinery—such as transcription factors and cofactors including BRD4, subunits of the Mediator complex, and RNA polymerase II—contain intrinsically disordered low-complexity domains. Now a conceptual framework connecting the nature and behavior of their interactions to their functions in transcription regulation is emerging (see the Perspective by Plys and Kingston). Chong et al. found that low-complexity domains of transcription factors form concentrated hubs via functionally relevant dynamic, multivalent, and sequence-specific protein-protein interaction. These hubs have the potential to phase-separate at higher concentrations. Indeed, Sabari et al. showed that at super-enhancers, BRD4 and Mediator form liquid-like condensates that compartmentalize and concentrate the transcription apparatus to maintain expression of key cell-identity genes. Cho et al. further revealed the differential sensitivity of Mediator and RNA polymerase II condensates to selective transcription inhibitors and how their dynamic interactions might initiate transcription elongation. Science , this issue p. eaar2555 , p. eaar3958 , p. 412 ; see also p. 329 Phase-separated condensates compartmentalize the transcription apparatus at super-enhancers of key cell-identity genes. Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes.

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex 1 – 4 . The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus 5 , 6 . Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain 7 – 12 . Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers 7 , 8 , and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites 9 – 12 . Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference. RNA polymerase II with a hypophosphorylated C-terminal domain preferentially incorporates into mediator condensates, and with a hyperphosphorylated C-terminal domain into splicing-factor condensates, revealing phosphorylation as a regulatory mechanism in condensate preference.

Disruption of lignin biosynthesis has been proposed as a way to improve forage and bioenergy crops, but it can result in stunted growth and developmental abnormalities; here, the undesirable features of one such manipulation are shown to depend on the transcriptional co-regulatory complex Mediator. Digestible lignin for biofuel crops Disruption of the biosynthesis of lignin — the complex biopolymer that imparts strength and rigidity to the plant cell wall — has been proposed as a means to improve forage and bioenergy crops. However, genetic perturbations of lignin biosynthesis tend to result in stunted growth and developmental abnormalities. Working in Arabidopsis , these authors show that these undesirable features depend on the transcriptional co-regulatory complex Mediator. Mutant analyses implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis. Biomass recalcitrance can be greatly reduced by blocking the synthesis of G and S lignin subunits, without necessarily sacrificing biomass yield. This finding suggests potential targets for the production of genetically modified cellulosic biofuel crops. Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall 1 , 2 . Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood 3 . Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8 , without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p -hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

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.

The 3.4 Å crystal structure of the 15-subunit core Mediator complex in yeast. How Mediator triggers transcription The multiprotein Mediator complex has an essential role in regulating RNA polymerase II (Pol II) transcription in eukaryotes. Here, Patrick Cramer and colleagues report a 3.4 Å crystal structure of the 15-subunit core Mediator complex in yeast. They combine this with a previously determined cryo-EM structure of the Pol II pre-initiation complex to obtain an atomic model of Mediator bound to the pre-initiation complex. This model allows insights into the interactions of the head and middle modules of Mediator and provides a framework for understanding how Mediator stimulates Pol II C-terminal domain phosphorylation by TFIIH, a process which triggers productive transcription. Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II 1 , 2 , 3 . The Mediator head and middle modules form the essential core Mediator (cMed) 4 , 5 , 6 , whereas the tail and kinase modules play regulatory roles 7 . The architecture of Mediator 5 , 8 , 9 , 10 and its position on the PIC 5 are known, but atomic details are limited to Mediator subcomplexes 11 , 12 . Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module 13 , 14 , 15 , and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16 ) and Med17 (ref. 17 ) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined 5 with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC) 18 . The resulting atomic model of the cPIC–cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge 19 and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC 20 . The hook also contains residues that crosslink to the CTD and reside in a previously described cradle 5 . These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.

For the initiation of transcription, RNA polymerase II (Pol II) assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here we report cryo-electron microscopy structures of the Saccharomyces cerevisiae PIC and PIC–core Mediator complex at nominal resolutions of 4.7 Å and 5.8 Å, respectively. The structures reveal transcription factor IIH (TFIIH), and suggest how the core and kinase TFIIH modules function in the opening of promoter DNA and the phosphorylation of Pol II, respectively. The TFIIH core subunit Ssl2 (a homologue of human XPB) is positioned on downstream DNA by the ‘E-bridge’ helix in TFIIE, consistent with TFIIE-stimulated DNA opening. The TFIIH kinase module subunit Tfb3 (MAT1 in human) anchors the kinase Kin28 (CDK7), which is mobile in the PIC but preferentially located between the Mediator hook and shoulder in the PIC–core Mediator complex. Open spaces between the Mediator head and middle modules may allow access of the kinase to its substrate, the C-terminal domain of Pol II. Cryo-electron microscopy structures of the yeast pre-initiation complex (PIC) and its complex with core Mediator provide insights into the opening of promoter DNA and the initiation of transcription. TFIIH in the transcription pre-initiation complex To initiate gene transcription, RNA polymerase (Pol) II assembles with general transcription factors on promoter DNA to form the pre-initiation complex (PIC). Here, Patrick Cramer and colleagues describe cryo-electron microscopy structures of the yeast PIC and the PIC bound to the core Mediator (cMed) complex. The latter structure with the general coactivator Mediator has 46 factors, including all those that are essential for transcription initiation in yeast. The structures reveal the architecture of transcription factor IIH (TFIIH) and suggest how its 'core' and 'kinase' modules might function in promoter opening and Pol II phosphorylation, respectively.

One function of Mediator complex subunit MED23 is to mediate transcriptional activation by the phosphorylated transcription factor Elk-1, in response to the Ras-MAPK signaling pathway. Using cryogenic electron microscopy, we solve a 3.0 Å structure of human MED23 complexed with the phosphorylated activation domain of Elk-1. Elk-1 binds to MED23 via a hydrophobic sequence PSIHFWSTLS P P containing one phosphorylated residue (S383 p ), which forms a tight turn around the central Phenylalanine. Binding of Elk-1 induces allosteric changes in MED23 that propagate to the opposite face of the subunit, resulting in the dynamic behavior of a 19-residue segment, which alters the molecular surface of MED23. We design a specific MED23 mutation (G382F) that disrupts Elk­-1 binding and consequently impairs Elk-1-dependent serum-induced activation of target genes in the Ras-Raf-MEK-ERK signaling pathway. The structure provides molecular details and insights into a Mediator subunit-transcription factor interface. The Mediator complex subunit MED23 contributes to transcriptional activation by the phosphorylated transcription factor Elk-1, in response to Ras-MAPK signalling. Here, the authors determine a cryo-EM structure of human MED23 with the phosphorylated activation domain of Elk-1 providing insights into the Mediator subunit-transcription factor interface.