SummaryNuclear 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.

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In order to facilitate automated reasoning about large Boolean combinations of nonlinear arithmetic constraints involving transcendental functions, we provide a tight integration of recent SAT solving techniques with interval-based arithmetic constraint solving. Our approach deviates substantially from lazy theorem proving approaches in that it directly controls arithmetic constraint propagation from the SAT solver rather than delegating arithmetic decisions to a subordinate solver. Through this tight integration, all the algorithmic enhancements that were instrumental to the enormous performance gains recently achieved in propositional SAT solving carry over smoothly to the rich domain of non-linear arithmetic constraints. As a consequence, our approach is able to handle large constraint systems with extremely complex Boolean structure, involving Boolean combinations of multiple thousand arithmetic constraints over some thousands of variables.

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.

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.nucleosome | ubiquitin | Bre1-Rad6 | cross-linking mass spectrometry | RING E3 ligase E ukaryotes organize their DNA into chromatin, which packages DNA while allowing coordinated gene expression. The basic unit of the chromatin polymer is the nucleosome core particle (NCP), formed by an octameric complex of core histones (two copies each of H2A, H2B, H3, and H4) that is encircled by 145-147 bp of DNA (1). A large set of chromatin factors "write," "read," or "erase" histone posttranslational modifications and thereby alter the transcriptional properties of chromatin (2). The ∼200 kDa NCP provides a varied interaction surface for chromatin factors through the flexible histone N-and C-terminal tails, the rigid disk faces of the histone octamer, and the nucleosomal DNA (3). The recognition of unstructured histone tails by numerous protein domains is well studied, but far less is known about how chromatin factors recognize the disk face of the NCP. Monoubiquitination of histone H2B occurs at lysine 123 (H2B Lys123∼Ub) in yeast (equivalent to mammalian Lys120). The enzymatic reaction is carried out by the E3 ligase Bre1 (human RNF20/40) together with the E2 enzyme Rad6 (4-7). This highly site-specific ubiquitination of H2B controls various aspects of gene expression, which include transcription initiation and elongation (8), DNA replication (9) and repair (10), and kinetochore function (11). H2B Lys123∼Ub is thought to exert its effects through altering chromatin compaction and by promoting specific histone H3 methylations (12, 13). H2B ubiquitination is reversed by a heterotetrameric deubiquitinase module, which is part of the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex (14, 15). Aberrant H2B Lys123∼Ub levels are observed in various disease states (16), suggesting that the finetuning of opposing ubiquitin ligase and deubiquitinase activities is critical for normal cell function.Ubiquitination involves a three-step enzymatic reaction requiring ubiquitin-activating (E1), ubiquitin-conjugating (E2...

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.

-This paper describes the multithreaded MiraXT SAT Solver which was designed to take advantage of current and future shared memory multiprocessor systems. The paper highlights design and implementation details that allow the multiple threads to run and cooperate efficiently. Results show that in single threaded mode, MiraXT compares well to other state of the art solvers on Industrial problems. In threaded mode, it provides cutting edge performance, as speedup is obtained on both SAT and UNSAT instances.

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.DOI: http://dx.doi.org/10.7554/eLife.10607.001