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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 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.

Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II are necessary for DNA-damage-response (DDR) focus formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNA polymerase II preinitiation complex, MED1 and CDK9. Absence or inactivation of these factors causes a reduction in DDR foci both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins, such as 53BP1, into foci that exhibit liquid–liquid phase-separation condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis, which stimulates phase separation of DDR factors in the shape of foci. Pessina et al. report that DNA damage induces the assembly of a functional promoter at double-strand breaks and the transcribed RNAs promote phase separation of damage-response factors such as 53BP1.

TAZ promotes growth, development and tumorigenesis by regulating the expression of target genes. However, the manner in which TAZ orchestrates the transcriptional responses is poorly defined. Here we demonstrate that TAZ forms nuclear condensates through liquid–liquid phase separation to compartmentalize its DNA-binding cofactor TEAD4, coactivators BRD4 and MED1, and the transcription elongation factor CDK9 for transcription. TAZ forms phase-separated droplets in vitro and liquid-like nuclear condensates in vivo, and this ability is negatively regulated by Hippo signalling through LATS-mediated phosphorylation and is mediated by the coiled-coil (CC) domain. Deletion of the TAZ CC domain or substitution with the YAP CC domain prevents the phase separation of TAZ and its ability to induce the expression of TAZ-specific target genes. Thus, we identify a mechanism of transcriptional activation by TAZ and demonstrate that pathway-specific transcription factors also engage the phase-separation mechanism for efficient and specific transcriptional activation.Wu et al. show that TAZ can undergo phase separation and the resulting condensates locally concentrate transcriptional activators to facilitate the expression of TAZ-controlled genes.

BRD4, a major tandem-bromodomain-containing transcription regulator, has two isoforms. The long isoform (BRD4L) has an extended C terminus that binds transcription cofactors, while the short isoform (BRD4S) lacks this C-terminal extension. Unlike BRD4L, the role of BRD4S in gene transcription remains unclear. Here, we report that, in human cancer cells, BRD4S forms nuclear puncta that possess liquid-like properties and that colocalize with BRD4L, MED1 and sites of histone H3 lysine 27 acetylation. BRD4 puncta are correlated with BRD4S but not BRD4L expression levels. BRD4S knockdown reduces BRD4S condensation, and ectopic expression promotes puncta formation and target gene transcription. BRD4S nuclear condensation is mediated by its intrinsically disordered regions and binding of its bromodomains to DNA and acetylated chromatin, respectively, and BRD4S phosphorylation diminishes BRD4 condensation. Our study illuminates a previously unappreciated role of BRD4S in organizing chromatin and transcription factors through phase separation to sustain gene transcription in chromatin for cancer cell proliferation.A combination of cellular, in vitro phase separation and functional assays shows that the intrinsically disordered regions and bromodomains of the BRD4 short isoform induce formation of liquid-like condensates in cancer cell nuclei and enhance transcriptional activity.

Cancer stem cells (CSCs) play a critical role in invasive growth and metastasis of human head and neck squamous cell carcinoma (HNSCC). Although significant progress has been made in understanding the self-renewal and pro-tumorigenic potentials of CSCs, a key challenge remains on how to eliminate CSCs and halt metastasis effectively. Here we show that super-enhancers (SEs) play a critical role in the transcription of cancer stemness genes as well as pro-metastatic genes, thereby controlling their tumorigenic potential and metastasis. Mechanistically, we find that bromodomain-containing protein 4 (BRD4) recruits Mediators and NF-κB p65 to form SEs at cancer stemness genes such as TP63 , MET and FOSL1 , in addition to oncogenic transcripts. In vivo lineage tracing reveals that disrupting SEs by BET inhibitors potently inhibited CSC self-renewal and eliminated CSCs in addition to elimination of proliferating non-stem tumor cells in a mouse model of HNSCC. Moreover, disrupting SEs also inhibits the invasive growth and lymph node metastasis of human CSCs isolated from human HNSCC. Taken together, our results suggest that targeting SEs may serve as an effective therapy for HNSCC by eliminating CSCs. Little is known on the transcriptional mechanisms that control cancer stem cell (CSC) self-renewal and stemness. Here the authors show superenhancers (SEs) play a role in the transcription of cancer stemness genes as well as prometastatic genes, thereby controlling their tumorigenic potential; disrupting SEs inhibited CSC self-renewal and eliminated CSCs.

A proof of principle study shows that by exome sequencing of cell-free circulating DNA from cancer patient plasma samples, the genomic evolution of metastatic cancers and the acquisition of resistance in response to therapy can be tracked over time. Plasma screening tracks tumour evolution This proof-of-principle study shows that by sequencing cancer exomes from patient plasma samples, it is possible to track genomic evolution of metastatic cancers and acquisition of resistance in response to therapy. In six patients with advanced breast, lung and ovarian cancers, sequences were derived from two to five plasma samples taken during multiple courses of treatment. Mutations associated with the emergence of resistance to drugs including cisplatin, tamoxifen and gefitinib were identified. Cancers acquire resistance to systemic treatment as a result of clonal evolution and selection 1 , 2 . Repeat biopsies to study genomic evolution as a result of therapy are difficult, invasive and may be confounded by intra-tumour heterogeneity 3 , 4 . Recent studies have shown that genomic alterations in solid cancers can be characterized by massively parallel sequencing of circulating cell-free tumour DNA released from cancer cells into plasma, representing a non-invasive liquid biopsy 5 , 6 , 7 . Here we report sequencing of cancer exomes in serial plasma samples to track genomic evolution of metastatic cancers in response to therapy. Six patients with advanced breast, ovarian and lung cancers were followed over 1–2 years. For each case, exome sequencing was performed on 2–5 plasma samples (19 in total) spanning multiple courses of treatment, at selected time points when the allele fraction of tumour mutations in plasma was high, allowing improved sensitivity. For two cases, synchronous biopsies were also analysed, confirming genome-wide representation of the tumour genome in plasma. Quantification of allele fractions in plasma identified increased representation of mutant alleles in association with emergence of therapy resistance. These included an activating mutation in PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha) following treatment with paclitaxel 8 ; a truncating mutation in RB1 (retinoblastoma 1) following treatment with cisplatin 9 ; a truncating mutation in MED1 (mediator complex subunit 1) following treatment with tamoxifen and trastuzumab 10 , 11 , and following subsequent treatment with lapatinib 12 , 13 , a splicing mutation in GAS6 (growth arrest-specific 6) in the same patient; and a resistance-conferring mutation in EGFR (epidermal growth factor receptor; T790M) following treatment with gefitinib 14 . These results establish proof of principle that exome-wide analysis of circulating tumour DNA could complement current invasive biopsy approaches to identify mutations associated with acquired drug resistance in advanced cancers. Serial analysis of cancer genomes in plasma constitutes a new paradigm for the study of clonal evolution in human cancers.

The importance of BET protein BRD4 in gene transcription is well recognized through the study of chemical modulation of its characteristic tandem bromodomain (BrD) binding to lysine-acetylated histones and transcription factors. However, while monovalent inhibition of BRD4 by BET BrD inhibitors such as JQ1 blocks growth of hematopoietic cancers, it is much less effective generally in solid tumors. Here, we report a thienodiazepine-based bivalent BrD inhibitor, MS645, that affords spatially constrained tandem BrD inhibition and consequently sustained repression of BRD4 transcriptional activity in blocking proliferation of solid-tumor cells including a panel of triple-negative breast cancer (TNBC) cells. MS645 blocks BRD4 binding to transcription enhancer/mediator proteins MED1 and YY1 with potency superior to monovalent BET inhibitors, resulting in down-regulation of proinflammatory cytokines and genes for cell-cycle control and DNA damage repair that are largely unaffected by monovalent BrD inhibition. Our study suggests a therapeutic strategy to maximally control BRD4 activity for rapid growth of solid-tumor TNBC cells.

Renin cells are crucial for survival - they control fluid-electrolyte and blood pressure homeostasis, vascular development, regeneration, and oxygen delivery to tissues. During embryonic development, renin cells are progenitors for multiple cell types that retain the memory of the renin phenotype. When there is a threat to survival, those descendants are transformed and reenact the renin phenotype to restore homeostasis. We tested the hypothesis that the molecular memory of the renin phenotype resides in unique regions and states of these cells' chromatin. Using renin cells at various stages of stimulation, we identified regions in the genome where the chromatin is open for transcription, mapped histone modifications characteristic of active enhancers such as H3K27ac, and tracked deposition of transcriptional activators such as Med1, whose deletion results in ablation of renin expression and low blood pressure. Using the rank ordering of super-enhancers, epigenetic rewriting, and enhancer deletion analysis, we found that renin cells harbor a unique set of super-enhancers that determine their identity. The most prominent renin super-enhancer may act as a chromatin sensor of signals that convey the physiologic status of the organism, and is responsible for the transformation of renin cell descendants to the renin phenotype, a fundamental process to ensure homeostasis.

Lens epithelium-derived growth factor/p75 (LEDGF/p75, or PSIP1) is a transcriptional coactivator that tethers other proteins to gene bodies. The chromatin tethering function of LEDGF/p75 is hijacked by HIV integrase to ensure viral integration at sites of active transcription. LEDGF/p75 is also important for the development of mixed-lineage leukemia (MLL), where it tethers the MLL1 fusion complex at aberrant MLL targets, inducing malignant transformation. However, little is known about how the LEDGF/p75 protein interaction network is regulated. Here, we obtained solution structures of the complete interfaces between the LEDGF/p75 integrase binding domain (IBD) and its cellular binding partners and validated another binding partner, Mediator subunit 1 (MED1). We reveal that structurally conserved IBD-binding motifs (IBMs) on known LEDGF/p75 binding partners can be regulated by phosphorylation, permitting switching between low- and high-affinity states. Finally, we showthat elimination of IBM phosphorylation sites on MLL1 disrupts the oncogenic potential of primary MLL1-rearranged leukemic cells. Our results demonstrate that kinase-dependent phosphorylation of MLL1 represents a previously unknown oncogenic dependency that may be harnessed in the treatment of MLL-rearranged leukemia.

Background The role of Mediator complex subunit 1 (MED1), a pivotal transcriptional coactivator implicated in diverse biological pathways, remains unexplored in the context of oral squamous cell carcinoma (OSCC). This study aims to elucidate the contributory mechanisms and potential impact of MED1 on the progression of OSCC. Methods The expression and clinical significance of MED1 in OSCC tissues were evaluated through the bioinformatics analyses. The effects of MED1 on the biological behavior of OSCC cancer cells were assessed both in vitro and in vivo. Dual-luciferase reporter assay, chromatin immunoprecipitation (ChIP) assay, bioinformatic analysis, CD8 + T cell isolation experiment, coculture experiment, enzyme-linked immunosorbent assay (ELISA), and flow cytometric analysis were employed to elucidate the underlying mechanism through which MED1 operates in the progression of OSCC. Results MED1 exhibited upregulation in both OSCC tissues and multiple OSCC cell lines, which correlated with decreased overall survival in patients. In vitro experiments demonstrated that knockdown of MED1 in metastatic OSCC cell lines SCC-9 and UPCI-SCC-154 hindered cell migration and invasion, while overexpression of MED1 promoted these processes. Whereas, MED1 knockdown had no impact on proliferation of cell lines mentioned above. In vivo studies further revealed that downregulation of MED1 effectively suppressed distant metastasis in OSCC. Mechanistically, MED1 enhanced the binding of transcription factors c-Jun and c-Fos to the matrix metalloprotein 9 (MMP9) promoters, resulting in a significant upregulation of MMP9 transcription. This process contributes to the migration and invasion of SCC-9 and UPCI-SCC-154 cells. Furthermore, MED1 modulated the expression of programmed death-ligand 1 (PD-L1) through the Notch signaling pathway, consequently impacting the tumor-killing capacity of CD8 + T cells in the tumor microenvironment. Conclusions Our findings indicate that MED1 plays a pivotal role in OSCC progression through the activation of MMP9 transcription and suppression of CD8 + T cell antitumor immunity, suggesting that MED1 may serve as a novel prognostic marker and therapeutic target in OSCC.