The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression.

Nuclear pore complexes (NPCs) influence gene expression besides their established function in nuclear transport. The TREX-2 complex localizes to the NPC basket and affects gene-NPC interactions, transcription, and mRNA export. How TREX-2 regulates the gene expression machinery is unknown. Here, we show that TREX-2 interacts with the Mediator complex, an essential regulator of RNA Polymerase (Pol) II. Structural and biochemical studies identify a conserved region on TREX-2, which directly binds the Mediator Med31/Med7N submodule. TREX-2 regulates assembly of Mediator with the Cdk8 kinase and is required for recruitment and site-specific phosphorylation of Pol II. Transcriptome and phenotypic profiling confirm that TREX-2 and Med31 are functionally interdependent at specific genes. TREX-2 additionally uses its Mediator-interacting surface to regulate mRNA export suggesting a mechanism for coupling transcription initiation and early steps of mRNA processing. Our data provide mechanistic insight into how an NPC-associated adaptor complex accesses the core transcription machinery.

Phase separation directs ubiquitination of gene-body nucleosomes.

The conserved yeast E3 ubiquitin ligase Bre1 and its partner, the E2 ubiquitin-conjugating enzyme Rad6, monoubiquitinate histone H2B across gene bodies during the transcription cycle1. Although processive ubiquitination might-in principle-arise from Bre1 and Rad6 travelling with RNA polymerase II2, the mechanism of H2B ubiquitination across genic nucleosomes remains unclear. Here we implicate liquid-liquid phase separation3 as the underlying mechanism. Biochemical reconstitution shows that Bre1 binds the scaffold protein Lge1, which possesses an intrinsically disordered region that phase-separates via multivalent interactions. The resulting condensates comprise a core of Lge1 encapsulated by an outer catalytic shell of Bre1. This layered liquid recruits Rad6 and the nucleosomal substrate, which accelerates the ubiquitination of H2B. In vivo, the condensate-forming region of Lge1 is required to ubiquitinate H2B in gene bodies beyond the +1 nucleosome. Our data suggest that layered condensates of histone-modifying enzymes generate chromatin-associated 'reaction chambers', with augmented catalytic activity along gene bodies. Equivalent processes may occur in human cells, and cause neurological disease when impaired.

An Ultra-Fast TSP on a CNT Heating Layer for Unsteady Temperature and Heat Flux Measurements in Subsonic Flows.

In this paper, the authors demonstrate the application of a modified Ru(phen)-based temperature-sensitive paint which was originally developed for the evaluation of unsteady aero-thermodynamic phenomena in high Mach number but short duration experiments. In the present work, the modified TSP with a temperature sensitivity of up to -5.6%/K was applied in a low Mach number long-duration test case in a low-pressure environment. For the demonstration of the paint's performance, a flat plate with a mounted cylinder was set up in the High-Speed Cascade Wind Tunnel (HGK). The test case was designed to generate vortex shedding frequencies up to 4300 Hz which were sampled using a high-speed camera at 40 kHz frame rate to resolve unsteady surface temperature fields for potential heat-transfer estimations. The experiments were carried out at reduced ambient pressure of p∞ = 13.8 kPa for three inflow Mach numbers being Ma∞=[0.3;0.5;0.7]. In order to enable the resolution of very low temperature fluctuations down to the noise floor of 10-5 K with high spatial and temporal resolution, the flat plate model was equipped with a sprayable carbon nanotube (CNT) heating layer. This constellation, together with the thermal sensors incorporated in the model, allowed for the calculation of a quasi-heat-transfer coefficient from the surface temperature fields. Besides the results of the experiments, the paper highlights the properties of the modified TSP as well as the methodology.

Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1.

Cotranscriptional ubiquitination of histone H2B is key to gene regulation. The yeast E3 ubiquitin ligase Bre1 (human RNF20/40) pairs with the E2 ubiquitin conjugating enzyme Rad6 to monoubiquitinate H2B at Lys123. How this single lysine residue on the nucleosome core particle (NCP) is targeted by the Rad6-Bre1 machinery is unknown. Using chemical cross-linking and mass spectrometry, we identified the functional interfaces of Rad6, Bre1, and NCPs in a defined in vitro system. The Bre1 RING domain cross-links exclusively with distinct regions of histone H2B and H2A, indicating a spatial alignment of Bre1 with the NCP acidic patch. By docking onto the NCP surface in this distinct orientation, Bre1 positions the Rad6 active site directly over H2B Lys123. The Spt-Ada-Gcn5 acetyltransferase (SAGA) H2B deubiquitinase module competes with Bre1 for binding to the NCP acidic patch, indicating regulatory control. Our study reveals a mechanism that ensures site-specific NCP ubiquitination and fine-tuning of opposing enzymatic activities.

SPOC1-mediated antiviral host cell response is antagonized early in human adenovirus type 5 infection.

Little is known about immediate phases after viral infection and how an incoming viral genome complex counteracts host cell defenses, before the start of viral gene expression. Adenovirus (Ad) serves as an ideal model, since entry and onset of gene expression are rapid and highly efficient, and mechanisms used 24-48 hours post infection to counteract host antiviral and DNA repair factors (e.g. p53, Mre11, Daxx) are well studied. Here, we identify an even earlier host cell target for Ad, the chromatin-associated factor and epigenetic reader, SPOC1, recently found recruited to double strand breaks, and playing a role in DNA damage response. SPOC1 co-localized with viral replication centers in the host cell nucleus, interacted with Ad DNA, and repressed viral gene expression at the transcriptional level. We discovered that this SPOC1-mediated restriction imposed upon Ad growth is relieved by its functional association with the Ad major core protein pVII that enters with the viral genome, followed by E1B-55K/E4orf6-dependent proteasomal degradation of SPOC1. Mimicking removal of SPOC1 in the cell, knock down of this cellular restriction factor using RNAi techniques resulted in significantly increased Ad replication, including enhanced viral gene expression. However, depletion of SPOC1 also reduced the efficiency of E1B-55K transcriptional repression of cellular promoters, with possible implications for viral transformation. Intriguingly, not exclusive to Ad infection, other human pathogenic viruses (HSV-1, HSV-2, HIV-1, and HCV) also depleted SPOC1 in infected cells. Our findings provide a general model for how pathogenic human viruses antagonize intrinsic SPOC1-mediated antiviral responses in their host cells. A better understanding of viral entry and early restrictive functions in host cells should provide new perspectives for developing antiviral agents and therapies. Conversely, for Ad vectors used in gene therapy, counteracting mechanisms eradicating incoming viral DNA would increase Ad vector efficacy and safety for the patient.

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response.

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.

Treatment resistance analysis reveals GLUT-1-mediated glucose uptake as a major target of synthetic rocaglates in cancer cells.

Rocaglates are natural compounds that have been extensively studied for their ability to inhibit translation initiation. Rocaglates represent promising drug candidates for tumor treatment due to their growth-inhibitory effects on neoplastic cells. In contrast to natural rocaglates, synthetic analogues of rocaglates have been less comprehensively characterized, but were also shown to have similar effects on the process of protein translation. Here, we demonstrate an enhanced growth-inhibitory effect of synthetic rocaglates when combined with glucose anti-metabolite 2-deoxy-D-glucose (2DG) in different cancer cell lines. Moreover, we unravel a new aspect in the mechanism of action of synthetic rocaglates involving reduction of glucose uptake mediated by downregulation or abrogation of glucose transporter GLUT-1 expression. Importantly, cells with genetically induced resistance to synthetic rocaglates showed substantially less pronounced treatment effect on glucose metabolism and did not demonstrate GLUT-1 downregulation, pointing at the crucial role of this mechanism for the anti-tumor activity of the synthetic rocaglates. Transcriptome profiling revealed glycolysis as one of the major pathways differentially regulated in sensitive and resistant cells. Analysis of synthetic rocaglate efficacy in a 3D tissue context with a co-culture of tumor and normal cells demonstrated a selective effect on tumor cells and substantiated the mechanistic observations obtained in cancer cell lines. Increased glucose uptake and metabolism is a universal feature across different tumor types. Therefore, targeting this feature by synthetic rocaglates could represent a promising direction for exploitation of rocaglates in novel anti-tumor therapies.

Correlations between Motor Symptoms across Different Motor Tasks, Quantified via Random Forest Feature Classification in Parkinson's Disease.

BACKGROUND: Objective assessments of Parkinson's disease (PD) patients' motor state using motion capture techniques are still rarely used in clinical practice, even though they may improve clinical management. One major obstacle relates to the large dimensionality of motor abnormalities in PD. We aimed to extract global motor performance measures covering different everyday motor tasks, as a function of a clinical intervention, i.e., deep brain stimulation (DBS) of the subthalamic nucleus. METHODS: We followed a data-driven, machine-learning approach and propose performance measures that employ Random Forests with probability distributions. We applied this method to 14 PD patients with DBS switched-off or -on, and 26 healthy control subjects performing the Timed Up and Go Test (TUG), the Functional Reach Test (FRT), a hand coordination task, walking 10-m straight, and a 90° curve. RESULTS: For each motor task, a Random Forest identified a specific set of metrics that optimally separated PD off DBS from healthy subjects. We noted the highest accuracy (94.6%) for standing up. This corresponded to a sensitivity of 91.5% to detect a PD patient off DBS, and a specificity of 97.2% representing the rate of correctly identified healthy subjects. We then calculated performance measures based on these sets of metrics and applied those results to characterize symptom severity in different motor tasks. Task-specific symptom severity measures correlated significantly with each other and with the Unified Parkinson's Disease Rating Scale (UPDRS, part III, correlation of r2 = 0.79). Agreement rates between different measures ranged from 79.8 to 89.3%. CONCLUSION: The close correlation of PD patients' various motor abnormalities quantified by different, task-specific severity measures suggests that these abnormalities are only facets of the underlying one-dimensional severity of motor deficits. The identification and characterization of this underlying motor deficit may help to optimize therapeutic interventions, e.g., to "automatically" adapt DBS settings in PD patients.

Mediator and TREX-2: Emerging links between transcription initiation and mRNA export.

Nuclear pore proteins interact dynamically with chromatin to regulate gene activities. A key question is how nucleoporin interactions mechanistically alter a gene's intranuclear position and transcriptional output. We reported recently on a direct interaction between the nuclear pore-associated TREX-2 complex and promoter-bound Mediator. This highlights how nuclear-pore associated adaptors gain regulatory access to the core transcription machinery. In this Extra View, we discuss an additional implication that arises from our work and the recent literature: how promoter elements may regulate mRNA metabolism beyond transcription initiation.

PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3.

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.