Interplay between the transcription preinitiation complex and the +1 nucleosome.

Eukaryotic transcription starts with the assembly of a preinitiation complex (PIC) on core promoters. Flanking this region is the +1 nucleosome, the first nucleosome downstream of the core promoter. While this nucleosome is rich in epigenetic marks and plays a key role in transcription regulation, how the +1 nucleosome interacts with the transcription machinery has been a long-standing question. Here, we summarize recent structural and functional studies of the +1 nucleosome in complex with the PIC. We specifically focus on how differently organized promoter-nucleosome templates affect the assembly of the PIC and PIC-Mediator on chromatin and result in distinct transcription initiation.

Structural visualization of transcription initiation in action.

Transcription initiation is a complex process, and its mechanism is incompletely understood. We determined the structures of de novo transcribing complexes TC2 to TC17 with RNA polymerase II halted on G-less promoters when nascent RNAs reach 2 to 17 nucleotides in length, respectively. Connecting these structures generated a movie and a working model. As initially synthesized RNA grows, general transcription factors (GTFs) remain bound to the promoter and the transcription bubble expands. Nucleoside triphosphate (NTP)-driven RNA-DNA translocation and template-strand accumulation in a nearly sealed channel may promote the transition from initially transcribing complexes (ITCs) (TC2 to TC9) to early elongation complexes (EECs) (TC10 to TC17). Our study shows dynamic processes of transcription initiation and reveals why ITCs require GTFs and bubble expansion for initial RNA synthesis, whereas EECs need GTF dissociation from the promoter and bubble collapse for promoter escape.

Tubular Polypyrrole with Chloride Ion Dopants as an Ultrafast Organic Anode for High-Power Lithium-Ion Batteries.

Polypyrrole (PPy), as a representative p-type conductive polymer, attracts wide attention for energy storage materials. However, the sluggish reaction kinetics and low specific capacity of PPy impede its application in high-power lithium-ion batteries (LIBs). Herein, tubular PPy with chloride and methyl orange (MO) anionic dopants is synthesized and investigated as an anode for LIBs. The Cl- and MO anionic dopants can increase the ordered aggregation and the conjugation length of pyrrolic chains, forming plentiful conductive domains and affecting the conduction channel inside the pyrrolic matrix, thereby achieving fast charge transfer and Li+ ion diffusion, low ion transfer energy barriers, and rapid reaction kinetics. On account of the above synergistic effect, PPy electrodes deliver a high specific capacity of 2067.8 mAh g-1 at 200 mA g-1 and a remarkable rate capacity of 1026 mAh g-1 at 10 A g-1 , realizing high energy density (724 Wh kg-1 ) and power density (7237 W kg-1 ) simultaneously.

Structures of +1 nucleosome-bound PIC-Mediator complex.

RNA polymerase II-mediated eukaryotic transcription starts with the assembly of the preinitiation complex (PIC) on core promoters. The +1 nucleosome is well positioned about 40 base pairs downstream of the transcription start site (TSS) and is commonly known as a barrier of transcription. The +1 nucleosome-bound PIC-Mediator structures show that PIC-Mediator prefers binding to T40N nucleosome located 40 base pairs downstream of TSS and contacts T50N but not the T70N nucleosome. The nucleosome facilitates the organization of PIC-Mediator on the promoter by binding TFIIH subunit p52 and Mediator subunits MED19 and MED26 and may contribute to transcription initiation. PIC-Mediator exhibits multiple nucleosome-binding patterns, supporting a structural role of the +1 nucleosome in the coordination of PIC-Mediator assembly. Our study reveals the molecular mechanism of PIC-Mediator organization on chromatin and underscores the significance of the +1 nucleosome in regulating transcription initiation.

The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors.

RNA polymerase II (Pol II) apparatuses are compartmentalized into transcriptional clusters. Whether protein factors control these clusters remains unknown. In this study, we find that the ATPase-associated with diverse cellular activities (AAA + ) ATPase RUVBL2 co-occupies promoters with Pol II and various transcription factors. RUVBL2 interacts with unphosphorylated Pol II in chromatin to promote RPB1 carboxy-terminal domain (CTD) clustering and transcription initiation. Rapid depletion of RUVBL2 leads to a decrease in the number of Pol II clusters and inhibits nascent RNA synthesis, and tethering RUVBL2 to an active promoter enhances Pol II clustering at the promoter. We also identify target genes that are directly linked to the RUVBL2-Pol II axis. Many of these genes are hallmarks of cancers and encode proteins with diverse cellular functions. Our results demonstrate an emerging activity for RUVBL2 in regulating Pol II cluster formation in the nucleus.

Structural insights into assembly of transcription preinitiation complex.

RNA polymerase II (Pol II)-mediated transcription in eukaryotic cells starts with assembly of preinitiation complex (PIC) on core promoter, a DNA sequence of ∼100 base pairs. The transcription PIC consists of Pol II and general transcription factors TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH. Previous structural studies focused on PIC assembled on TATA box promoters with TFIID replaced by its subunit, TATA box-binding protein (TBP). However, the megadalton TFIID complex is essential for promoter recognition, TBP loading onto promoter, and PIC assembly for almost all Pol II-mediated transcription, especially on the TATA-less promoters, which account for ∼85% of core promoters of human coding genes. The functions of TFIID could not be replaced by TBP. The recent breakthrough in structure determination of TFIID-based PIC complexes in different assembly stages revealed mechanistic insights into PIC assembly on TATA box and TATA-less promotes and provided a framework for further investigation of transcription initiation.

Coordinated regulation of RNA polymerase II pausing and elongation progression by PAF1.

Pleiotropic transcription regulator RNA polymerase II (Pol II)-associated factor 1 (PAF1) governs multiple transcriptional steps and the deposition of several epigenetic marks. However, it remains unclear how ultimate transcriptional outcome is determined by PAF1 and whether it relates to PAF1-controlled epigenetic marks. We use rapid degradation systems and reveal direct PAF1 functions in governing pausing partially by recruiting Integrator-PP2A (INTAC), in addition to ensuring elongation. Following acute PAF1 degradation, most destabilized polymerase undergoes effective release, which presumably relies on skewed balance between INTAC and P-TEFb, resulting in hyperphosphorylated substrates including SPT5. Impaired Pol II progression during elongation, along with altered pause release frequency, determines the final transcriptional outputs. Moreover, PAF1 degradation causes a cumulative decline in histone modifications. These epigenetic alterations in chromatin likely further influence the production of transcripts from PAF1 target genes.

RPAP2 regulates a transcription initiation checkpoint by inhibiting assembly of pre-initiation complex.

RNA polymerase II (Pol II)-mediated transcription in metazoans requires precise regulation. RNA Pol II-associated protein 2 (RPAP2) was previously identified to transport Pol II from cytoplasm to nucleus and dephosphorylates Pol II C-terminal domain (CTD). Here, we show that RPAP2 binds hypo-/hyper-phosphorylated Pol II with undetectable phosphatase activity. The structure of RPAP2-Pol II shows mutually exclusive assembly of RPAP2-Pol II and pre-initiation complex (PIC) due to three steric clashes. RPAP2 prevents and disrupts Pol II-TFIIF interaction and impairs in vitro transcription initiation, suggesting a function in inhibiting PIC assembly. Loss of RPAP2 in cells leads to global accumulation of TFIIF and Pol II at promoters, indicating a critical role of RPAP2 in inhibiting PIC assembly independent of its putative phosphatase activity. Our study indicates that RPAP2 functions as a gatekeeper to inhibit PIC assembly and transcription initiation and suggests a transcription checkpoint.

Combined angiogenesis and PD-1 inhibition for immunomodulatory TNBC: concept exploration and biomarker analysis in the FUTURE-C-Plus trial.

BACKGROUND: Immune checkpoint inhibitors had a great effect in triple-negative breast cancer (TNBC); however, they benefited only a subset of patients, underscoring the need to co-target alternative pathways and select optimal patients. Herein, we investigated patient subpopulations more likely to benefit from immunotherapy and inform more effective combination regimens for TNBC patients. METHODS: We conducted exploratory analyses in the FUSCC cohort to characterize a novel patient selection method and actionable targets for TNBC immunotherapy. We investigated this in vivo and launched a phase 2 trial to assess the clinical value of such criteria and combination regimen. Furthermore, we collected clinicopathological and next-generation sequencing data to illustrate biomarkers for patient outcomes. RESULTS: CD8-positivity could identify an immunomodulatory subpopulation of TNBCs with higher possibilities to benefit from immunotherapy, and angiogenesis was an actionable target to facilitate checkpoint blockade. We conducted the phase II FUTURE-C-Plus trial to assess the feasibility of combining famitinib (an angiogenesis inhibitor), camrelizumab (a PD-1 monoclonal antibody) and chemotherapy in advanced immunomodulatory TNBC patients. Within 48 enrolled patients, the objective response rate was 81.3% (95% CI, 70.2-92.3), and the median progression-free survival was 13.6 months (95% CI, 8.4-18.8). No treatment-related deaths were reported. Patients with CD8- and/or PD-L1- positive tumors benefit more from this regimen. PKD1 somatic mutation indicates worse progression-free and overall survival. CONCLUSION: This study confirms the efficacy and safety of the triplet regimen in immunomodulatory TNBC and reveals the potential of combining CD8, PD-L1 and somatic mutations to guide clinical decision-making and treatments. TRIAL REGISTRATION: ClinicalTrials.gov: NCT04129996 . Registered 11 October 2019.

Famitinib with Camrelizumab and Nab-Paclitaxel for Advanced Immunomodulatory Triple-Negative Breast Cancer (FUTURE-C-Plus): An Open-Label, Single-Arm, Phase II Trial.

PURPOSE: Camrelizumab, an mAb against programmed cell death protein 1 (PD-1), plus nab-paclitaxel exhibited promising antitumor activity in refractory metastatic immunomodulatory triple-negative breast cancer (TNBC). Famitinib is a tyrosine kinase inhibitor targeting VEGFR2, PDGFR, and c-kit. We aimed to assess the efficacy and safety of a novel combination of famitinib, camrelizumab, and nab-paclitaxel in advanced immunomodulatory TNBC. PATIENTS AND METHODS: This open-label, single-arm, phase II study enrolled patients with previously untreated, advanced, immunomodulatory TNBC (CD8 IHC staining ≥10%). Eligible patients received 20 mg of oral famitinib on days 1 to 28, 200 mg of i.v. camrelizumab on days 1 and 15, and i.v. nab-paclitaxel 100 mg/m2 on days 1, 8, and 15 in 4-week cycles. The primary endpoint was objective response rate (ORR), as assessed by investigators per RECIST v1.1. Key secondary endpoints were progression-free survival (PFS), overall survival (OS), duration of response (DOR), safety, and exploratory biomarkers. RESULTS: Forty-eight patients were enrolled and treated. Median follow-up was 17.0 months (range, 8.7-24.3). Confirmed ORR was 81.3% [95% confidence interval (CI), 70.2-92.3], with five complete and 34 partial responses. Median PFS was 13.6 months (95% CI, 8.4-18.8), and median DOR was 14.9 months [95% CI, not estimable (NE)-NE]. Median OS was not reached. No treatment-related deaths were reported. Among 30 patients with IHC, 13 (43.3%) were programmed death-ligand 1 (PD-L1)-negative, and PD-L1 was associated with favorable response. PKD1 and KAT6A somatic mutations were associated with therapy response. CONCLUSIONS: The triplet regimen was efficacious and well tolerated in previously untreated, advanced, immunomodulatory TNBC. The randomized controlled FUTURE-SUPER trial is under way to validate our findings. See related commentary by Salgado and Loi, p. 2728.

Structures of the human Mediator and Mediator-bound preinitiation complex.

The 1.3-megadalton transcription factor IID (TFIID) is required for preinitiation complex (PIC) assembly and RNA polymerase II (Pol II)-mediated transcription initiation on almost all genes. The 26-subunit Mediator stimulates transcription and cyclin-dependent kinase 7 (CDK7)-mediated phosphorylation of the Pol II C-terminal domain (CTD). We determined the structures of human Mediator in the Tail module-extended (at near-atomic resolution) and Tail-bent conformations and structures of TFIID-based PIC-Mediator (76 polypeptides, ~4.1 megadaltons) in four distinct conformations. PIC-Mediator assembly induces concerted reorganization (Head-tilting and Middle-down) of Mediator and creates a Head-Middle sandwich, which stabilizes two CTD segments and brings CTD to CDK7 for phosphorylation; this suggests a CTD-gating mechanism favorable for phosphorylation. The TFIID-based PIC architecture modulates Mediator organization and TFIIH stabilization, underscoring the importance of TFIID in orchestrating PIC-Mediator assembly.

Structural insights into preinitiation complex assembly on core promoters.

Transcription factor IID (TFIID) recognizes core promoters and supports preinitiation complex (PIC) assembly for RNA polymerase II (Pol II)-mediated eukaryotic transcription. We determined the structures of human TFIID-based PIC in three stepwise assembly states and revealed two-track PIC assembly: stepwise promoter deposition to Pol II and extensive modular reorganization on track I (on TATA-TFIID-binding element promoters) versus direct promoter deposition on track II (on TATA-only and TATA-less promoters). The two tracks converge at an ~50-subunit holo PIC in identical conformation, whereby TFIID stabilizes PIC organization and supports loading of cyclin-dependent kinase (CDK)-activating kinase (CAK) onto Pol II and CAK-mediated phosphorylation of the Pol II carboxyl-terminal domain. Unexpectedly, TBP of TFIID similarly bends TATA box and TATA-less promoters in PIC. Our study provides structural visualization of stepwise PIC assembly on highly diversified promoters.

Structural insights into TSC complex assembly and GAP activity on Rheb.

Tuberous sclerosis complex (TSC) integrates upstream stimuli and regulates cell growth by controlling the activity of mTORC1. TSC complex functions as a GTPase-activating protein (GAP) towards small GTPase Rheb and inhibits Rheb-mediated activation of mTORC1. Mutations in TSC genes cause tuberous sclerosis. In this study, the near-atomic resolution structure of human TSC complex reveals an arch-shaped architecture, with a 2:2:1 stoichiometry of TSC1, TSC2, and TBC1D7. This asymmetric complex consists of two interweaved TSC1 coiled-coil and one TBC1D7 that spans over the tail-to-tail TSC2 dimer. The two TSC2 GAP domains are symmetrically cradled within the core module formed by TSC2 dimerization domain and central coiled-coil of TSC1. Structural and biochemical analyses reveal TSC2 GAP-Rheb complimentary interactions and suggest a catalytic mechanism, by which an asparagine thumb (N1643) stabilizes γ-phosphate of GTP and accelerate GTP hydrolysis of Rheb. Our study reveals mechanisms of TSC complex assembly and GAP activity.

Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase.

The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and protein phosphatase 2A core enzyme (PP2A-AC), termed INTAC. The 3.5-angstrom-resolution structure reveals that nine human Integrator subunits and PP2A-AC assemble into a cruciform-shaped central scaffold formed by the backbone and shoulder modules, with the phosphatase and endonuclease modules flanking the opposite sides. As a noncanonical PP2A holoenzyme, the INTAC complex dephosphorylates the carboxy-terminal repeat domain of RNA polymerase II at serine-2, -5, and -7 and thus regulates transcription. Our study extends the function of PP2A to transcriptional regulation and reveals how dual enzymatic activities-RNA cleavage and RNA polymerase II dephosphorylation-are structurally and functionally integrated into the INTAC complex.

Cryo-EM structure of human mTOR complex 2.

Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) plays an essential role in regulating cell proliferation through phosphorylating AGC protein kinase family members, including AKT, PKC and SGK1. The functional core complex consists of mTOR, mLST8, and two mTORC2-specific components, Rictor and mSin1. Here we investigated the intermolecular interactions within mTORC2 complex and determined its cryo-electron microscopy structure at 4.9 Å resolution. The structure reveals a hollow rhombohedral fold with a 2-fold symmetry. The dimerized mTOR serves as a scaffold for the complex assembly. The N-terminal half of Rictor is composed of helical repeat clusters and binds to mTOR through multiple contacts. mSin1 is located close to the FRB domain and catalytic cavity of mTOR. Rictor and mSin1 together generate steric hindrance to inhibit binding of FKBP12-rapamycin to mTOR, revealing the mechanism for rapamycin insensitivity of mTORC2. The mTOR dimer in mTORC2 shows more compact conformation than that of mTORC1 (rapamycin sensitive), which might result from the interaction between mTOR and Rictor-mSin1. Structural comparison shows that binding of Rictor and Raptor (mTORC1-specific component) to mTOR is mutually exclusive. Our study provides a basis for understanding the assembly of mTORC2 and a framework to further characterize the regulatory mechanism of mTORC2 pathway.

High Permittivity Nanocomposites Embedded with Ag/TiO2 Core⁻Shell Nanoparticles Modified by Phosphonic Acid.

In this paper, nanocomposites that contain core-shell Ag/TiO2 particles as the filler and polytetrafluoroethylene (PTFE) as the matrix were investigated. Two surfactants, namely octyl phosphonic acid (OPA) and pentafluorobenzyl phosphonic acid (PFBPA), were applied to modify Ag/TiO2 fillers for uniform dispersion in the matrix. Fourier transform infrared spectroscopy analysis of bonds between the TiO2 shells and the phosphonic modifiers shows Ti⁻O⁻P chemical bonding between the Ag/TiO2 fillers and the modifiers. Thermogravimetric analysis results show a superior adsorption effect of PFBPA over OPA on the Ag/TiO2 filler surface at the same weight percentage. For nanocomposites that contain modified Ag/TiO2 nanoparticles, the loss was reduced despite the high permittivity at the same loading. The permittivity of the nanocomposites by PFBPA is larger than that of OPA, because the more uniform dispersion of inorganic particles in the PTFE matrix enhances the interfacial polarization effect. The mechanism of enhanced dielectric performance was studied and discussed.

4.4 Å Resolution Cryo-EM structure of human mTOR Complex 1.

Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) integrates signals from growth factors, cellular energy levels, stress and amino acids to control cell growth and proliferation through regulating translation, autophagy and metabolism. Here we determined the cryo-electron microscopy structure of human mTORC1 at 4.4 Å resolution. The mTORC1 comprises a dimer of heterotrimer (mTOR-Raptor-mLST8) mediated by the mTOR protein. The complex adopts a hollow rhomboid shape with 2-fold symmetry. Notably, mTORC1 shows intrinsic conformational dynamics. Within the complex, the conserved N-terminal caspase-like domain of Raptor faces toward the catalytic cavity of the kinase domain of mTOR. Raptor shows no caspase activity and therefore may bind to TOS motif for substrate recognition. Structural analysis indicates that FKBP12-Rapamycin may generate steric hindrance for substrate entry to the catalytic cavity of mTORC1. The structure provides a basis to understand the assembly of mTORC1 and a framework to characterize the regulatory mechanism of mTORC1 pathway.

One-step Preparation of Nanoarchitectured TiO2 on Porous Al as Integrated Anode for High-performance Lithium-ion Batteries.

Titanium dioxide (TiO2) is an attractive anode material for energy storage devices due to its low-volume-change and high safety. However, TiO2 anodes usually suffer from poor electrical and ionic conductivity, thus causing dramatic degradation of electrochemical performance at rapid charge/discharge rates, which has hindered its use in energy storage devices. Here, we present a novel strategy to address this main obstacle via using nanoarchitectured TiO2 anode consisting of mesoporous TiO2 wrapped in carbon on a tunnel-like etched aluminum substrate prepared by a simple one-step approach. As a result of this nanoarchitecture arrangement, the anode exhibits excellent rate performance and superior cyclability. A rate up to 100 C is achieved with a high specific capacity of about 95 mA h g(-1), and without apparent decay after 8,000 cycles.