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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.

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.

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.

Human genetic variation affects the gut microbiota through a complex combination of environmental and host factors. Here we characterize genetic variations associated with microbial abundances in a single large-scale population-based cohort of 5,959 genotyped individuals with matched gut microbial metagenomes, and dietary and health records (prevalent and follow-up). We identified 567 independent SNP–taxon associations. Variants at the LCT locus associated with Bifidobacterium and other taxa, but they differed according to dairy intake. Furthermore, levels of Faecalicatena lactaris associated with ABO , and suggested preferential utilization of secreted blood antigens as energy source in the gut. Enterococcus faecalis levels associated with variants in the MED13L locus, which has been linked to colorectal cancer. Mendelian randomization analysis indicated a potential causal effect of Morganella on major depressive disorder, consistent with observational incident disease analysis. Overall, we identify and characterize the intricate nature of host–microbiota interactions and their association with disease. Genome-wide association analysis of gut microbial taxa in a single homogenous population-based cohort of 5,959 Finnish individuals identifies 567 independent SNP–taxon associations, including strong associations with LCT , ABO and MED13L .

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.

Key Points A basic function of the Mediator complex is to communicate regulatory signals from DNA-binding transcription factors (TFs) directly to RNA polymerase II (Pol II). Different TFs, and the signalling pathways that regulate these TFs, often interact with different Mediator subunits to regulate expression of their target genes. Mediator is composed of a large number of subunits, some of which can reversibly associate with Mediator or are expressed at variable levels in different cell types. Mediator binding to various proteins and protein complexes, such as TFs, Pol II and the cyclin-dependent kinase 8 (CDK8) module, results in large-scale structural changes. These structural changes, in turn, appear to modulate the function of Mediator and may affect its ability to bind to other proteins. Because of its direct and extensive interactions with Pol II, Mediator regulates multiple stages of Pol II transcription (for example, initiation and re-initiation). Mediator interactions with the super elongation complex (SEC) also seem to be important for its regulation of Pol II elongation. The interactions between TFs, Mediator, cohesin and the pre-initiation complex (PIC) correlate with the formation of enhancer–promoter DNA loops, which are an important regulatory mechanism. Interactions between Mediator and non-coding RNA also correlate with DNA looping. RNA polymerase II (Pol II) is globally regulated by Mediator, a large, conformationally flexible protein complex with a variable subunit composition. These biochemical characteristics are fundamental for the ability of Mediator to control processes involved in transcription, including the organization of chromatin architecture and the regulation of Pol II pre-initiation, initiation, re-initiation, pausing and elongation. 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.

The senescence response to oncogenes is believed to be a barrier to oncogenic transformation in premalignant lesions, and describing the mechanisms by which tumor cells evade this response is important for early diagnosis and treatment. The male germ cell-associated protein SSX2 is ectopically expressed in many types of cancer and is functionally involved in regulating chromatin structure and supporting cell proliferation. Similar to many well-characterized oncogenes, SSX2 has the ability to induce senescence in cells. In this study, we performed a functional genetic screen to identify proteins implicated in SSX2-induced senescence and identified several subunits of the Mediator complex, which is central in regulating RNA polymerase-mediated transcription. Further experiments showed that reduced levels of MED1, MED4, and MED14 perturbed the development of senescence in SSX2 -expressing cells. In contrast, knockdown of MED1 did not prevent development of B-Raf- and Epirubicin-induced senescence, suggesting that Mediator may be specifically linked to the cellular functions of SSX2 that may lead to development of senescence or be central in a SSX2-specific senescence response. Indeed, immunostaining of melanoma tumors, which often express SSX proteins, exhibited altered levels of MED1 compared to benign nevi. Similarly, RNA-seq analysis suggested that MED1, MED4, and MED14 were downregulated in some tumors, while upregulated in others. In conclusion, our study reveals the Mediator complex as essential for SSX2-induced senescence and suggests that changes in Mediator activity could be instrumental for tumorigenesis.

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.

The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.

Bin Tean Teh and colleagues report the genomic characterization of 100 breast fibroepithelial tumors, including benign fibroadenomas and benign, borderline and malignant phyllodes tumors. They identify mutations specific to phyllodes tumors and find somatic mutation patterns that distinguish borderline and malignant phyllodes tumors from the other tumor types. Breast fibroepithelial tumors comprise a heterogeneous spectrum of pathological entities, from benign fibroadenomas to malignant phyllodes tumors 1 . Although MED12 mutations have been frequently found in fibroadenomas and phyllodes tumors 2 , 3 , 4 , 5 , 6 , 7 , the landscapes of genetic alterations across the fibroepithelial tumor spectrum remain unclear. Here, by performing exome sequencing of 22 phyllodes tumors followed by targeted sequencing of 100 breast fibroepithelial tumors, we observed three distinct somatic mutation patterns. First, we frequently observed MED12 and RARA mutations in both fibroadenomas and phyllodes tumors, emphasizing the importance of these mutations in fibroepithelial tumorigenesis. Second, phyllodes tumors exhibited mutations in FLNA , SETD2 and KMT2D , suggesting a role in driving phyllodes tumor development. Third, borderline and malignant phyllodes tumors harbored additional mutations in cancer-associated genes. RARA mutations exhibited clustering in the portion of the gene encoding the ligand-binding domain, functionally suppressed RARA-mediated transcriptional activation and enhanced RARA interactions with transcriptional co-repressors. This study provides insights into the molecular pathogenesis of breast fibroepithelial tumors, with potential clinical implications.