Functional characterization of BRD4 in transcription elongation and termination

The eukaryotic transcription machinery consisting of RNA polymerase II (Pol II) and a small number of minimally required initiation, elongation and termination factors is well-characterized structurally and biochemically, while functional characterization traditionally has been hampered by the limitations of the available perturbation strategies and readouts. In addition, knowledge of accessory transcription regulators is likely incomplete. Proteins of the conserved bromodomain and extra-terminal domain (BET) family have been demonstrated to function in maintaining normal transcription genome-wide by promoting elongation. However, the underlying molecular mechanism and also the specific contribution of each of the protein family members remained elusive. Moreover, it was unclear if BET proteins serve additional direct or indirect functions in transcription or transcription-coupled processes. To address these aspects, this study combines targeted protein degradation, a new strategy to near-completely deplete a protein of interest within minutes to hours, with complementary transcriptome-, genome- and proteome-wide readouts of high temporal or spatial resolution. It provides evidence that specifically BRD4 is involved in transcription control, although BRD2 and BRD3 might have partially overlapping functions. Within 120 minutes, BRD4-selective degradation results in a global reduction of elongating Pol II from the gene body of most protein-coding and long non-coding RNA genes, as can be detected by native elongating transcript sequencing with spike-in normalization (SI-NET-seq). Concomitantly, Pol II accumulates in the promoter-proximal region, suggesting a defect in promoter-proximal pause release. Profiling elongation factors upon BRD4 depletion reveals a decrease in occupancy, suggesting an assembly defect of the active elongation complex. In particular, PAF1 binding is decreased throughout the transcribed region. Unexpectedly, BRD4-selective degradation also induces a severe transcription termination defect at a subset of genes. Particularly, Pol II continues transcribing on average three kilobases beyond its usual termination zone, which is accompanied by inefficient cleavage of the nascent transcript at the pA site. Chromatin immunoprecipitation with sequencing (ChIP-seq) reveals a significant reduction of RNA 3’ end processing factors of the CPSF and CstF modules near the 3’ gene end as well as in the promoter-proximal and the gene body region. The failure to recruit RNA 3’ end processing as well as PAF subunits, SPT5 and SPT6, but not CDK9, could be independently confirmed by quantitative mass spectrometry. Co-immunoprecipitation experiments indicate that the recruitment of 3’ processing factors either directly depends on BRD4 or could be mediated by elongation factors. Also, BRD4-selective degradation locally increases the binding of Pol II and SPT5 at transcribed enhancers, whereas enhancer accessibility appears to be not BRD4-dependent. Altogether, the data establish a role of BRD4 in coordinating the recruitment of elongation and RNA 3’ end processing factors during an early step of transcription.Die eukaryotische Transkriptionsmaschinerie — insbesondere RNA-Polymerase II (Pol II) und eine überschaubare Gruppe essentieller Initiation-, Elongations- und Terminationsfaktoren — ist strukturell als auch biochemisch gut charakterisiert, wohingegen die funktionelle Charakterisierung durch das Fehlen geeigneter Pertubationsmethoden und sensitiver experimenteller Analyseverfahren lange erschwert wurde. Auch sind viele nicht-essentielle Transkriptionsregulatoren wahrscheinlich noch unbekannt. Es konnte gezeigt werden, dass die durch zwei Bromodomänen und eine sog. extra-terminale Domäne gekennzeichneten Proteine der konservierten BET-Proteinfamilie nötig sind, die normale Transkriptionsaktivität der Zelle — vermutlich durch Regulation der Elongation — aufrecht zu erhalten. Unklar sind jedoch der molekulare Mechanismus als auch die Rolle der einzelnen Proteine der Proteinfamilie. Darüber hinaus ist denkbar, dass BET-Proteine direkt oder indirekt auch an weiteren transkriptionellen oder co-transkriptionellen Prozessen beteiligt sind. Die genannten Aspekte adressiert die vorliegende Studie einerseits durch die Verwendung eines relativ neuen Verfahrens zur gezielten und schnellen Degradierung eines Proteins — in diesem Fall BRD4 — und andererseits mit komplementären transkriptom-, genom- und proteomweiten Methoden, die eine hohe zeitliche oder räumliche Auflösung bieten. Auf diese Weise kann gezeigt werden, dass die Transkription in hohem Maße von BRD4 abhängt, obgleich nicht auszuschließen ist, dass BRD2 oder BRD3 eine ähnliche Funktion haben. So belegt SI-NET-seq, eine spike-in-gestützten Methode zur quantitativen Analyse naszenter RNA unter nativen Bedingungen, dass die gezielte Degradierung von BRD4 innerhalb von 120 Minuten zur globalen Verringerung transkribierender Pol II am Genkörper der meisten proteinkodierenden und langen nicht-kodierenden RNA-Gene führt. Gleichzeitig akkumuliert transkribierende Pol II in der promotor-proximalen Region, was auf einen Defekt am Übergang von der promotor-proximalen Pause zur produktiven Elongation hindeutet. Einen Hinweis auf eine Funktion von BRD4 bei der Assemblierung des Elongationskomplexes liefert die Beobachtung, dass die Chromatinbindung von Elongationsfaktoren nach BRD4-Degradierung verringert ist. Insbesondere PAF1 zeigt eine Abnahme in allen Genbereichen. Unerwarteterweise führt die gezielte Degradierung von BRD4 bei einer großen Gruppe von Genen auch zu einem Terminationsdefekt — im Durchschnitt erfolgt die Termination drei Kilobasen später als unter Kontrollbedingungen — und vermindert die Effizient, mit der die naszente RNA an der Polyadenylierungsstelle enzymatisch geschnitten wird. Tatsächlich binden CPSF- und CstF-Faktoren der 3'-RNA-Prozessierungsmaschinerie nach BRD4-Degradierung signifikant weniger am 3'-Ende der Gene, aber auch im promotor-proximalen Bereich und am Genkörper, wie mittels Chromatinimmunpräzipitation (ChIP) gezeigt werden kann. Eine signifikante Abnahme von 3'-Prozessierungsfaktoren sowie von PAF-Untereinheiten, SPT5 und SPT6, nicht aber von CDK9, ist auch durch quantitative Massenspektrometrie nachweisbar. Zudem legen Co-Immunpräzipitationsexperimente nahe, dass 3‘-RNA-Prozessierungsfaktoren entweder direkt durch BRD4 oder indirekt über Elongationsfaktoren rekrutiert werden können. Außerdem korreliert die BRD4-spezifische Degradierung mit der lokalen Zunahme von Pol II und SPT5 an transkribierten Enhancer-Regionen; die Zugänglichkeit des Chromatins gegenüber Tn5-Transposase erscheint hingegen nicht BRD4-abhänging zu sein. Zusammenfassend legen unsere Daten nahe, dass BRD4 zu einem relativ frühen Zeitpunkt des Transkriptionszyklus' die Rekrutierung von Elongations-, aber auch von RNA 3'-Prozessierungsfaktoren koordiniert

Zwei Reinigungssysteme im Vergleich

Das Universitätsspital Basel (USB) ist eines der fünf grössten Gesundheitszentren der Schweiz. Die Abteilung Infrastruktur ist u.a. verantwortlich für die Erbringung von bedürfnisgerechten und den neusten Erkenntnissen entsprechenden Reinigungsdienstleistungen. Im Rahmen einer Überprüfung der Reinigungsprozesse wurde das Institut für Facility Management (IFM) der Zürcher Hochschule für Angewandte Wissenschaften beauftragt, die Prozesse zu analysieren und mögliche Optimierungspotenziale aufzuzeigen

Vascular Diseases of the Spinal Cord: A Review

Opinion statement: • In acute spinal cord ischemia syndrome (ASCIS), treatment recommendations are derived from data of cerebral ischemic stroke, atherosclerotic vascular disease and acute spinal cord injury. Besides acute management, secondary prevention is of major importance. Pathologies affecting the aorta as well as underlying cerebrovascular conditions should be treated whenever possible. • ASCIS may occur after aortic surgery, less often after thoracic endovascular aortic repair (TEVAR). Protocols are proposed. • Acute spinal cord hemorrhage can be treated surgically and/or pharmacologically. • Symptomatic treatment in patients with a spinal cord lesion is of major importance. Depending on level and extension of the lesion, there is a risk for systemic and neurological complications, which may be life-threatening. • Each spinal vascular malformation is a unique lesion that needs an individualized treatment algorithm. In case of a symptomatic vascular malformation, endovascular intervention is the primary treatment option. • Spinal dural Arteriovenous fistula (AVF) may be treated endovascularly or surgically. If preoperative localization of the fistula is possible, surgery is feasible with a low complication rate. In comparison, endovascular approaches are less invasive. • Spinal AVM are rather treated endovascularly than surgically or in a stepwise multidisciplinary approach. • Symptomatic and exophytic spinal cavernous angiomas should be treated surgically. Deep spinal cavernous angiomas that are asymptomatic or only show mild symptoms can be observe

Cerebral Venous Sinus Thrombosis Associated with Vaccine-Induced Thrombotic Thrombocytopenia—A Narrative Review

In March 2021, cerebral venous sinus thrombosis and thrombocytopenia after vaccination with adenovirus-based vaccine against SARS-CoV-2 were first reported. The underlining condition has been termed vaccine-induced immune thrombocytopenia (VITT). Anti-platelet factor 4 antibodies have been proposed as a central component of the pathomechanism. Treatment recommendations entailed immunomodulation with intravenous immunoglobulins, avoidance of heparins and avoidance of platelet transfusions. Although mortality from VITT-associated cerebral venous sinus thrombosis has decreased over time, it remains high. The aim of this narrative review is to describe different aspects of this disease according to the current state of knowledge

Bleeding skin lesions in gestating sows of a piglet producing farm in Austria.

BACKGROUND Stomoxys calcitrans, the stable fly, occurs in pig producing countries worldwide. While in cattle the impact of this blood sucking insect is quite well described, its role in pig production is poorly investigated. Here we describe a case of a massive stable fly overpopulation in the gestation unit of a piglet producing farm in Austria that resulted in bleeding skin lesions in bitten sows. CASE PRESENTATION In October 2021, the responsible herd veterinarian of the case farm reported of sows in the gestation area presenting with bloody crusts on the whole skin surface of the body and of bleeding skin lesions. 33/55 sows were affected by moderate to severe skin lesions. Reproductive performance decreased during the time of massive stable fly overpopulation. Sows in the gestation unit showed defensive behaviour and at a certain time point resigned and accepted being bitten by stable flies. After controlling the fly population, reproductive performance improved and even exceeded the performance before the massive overgrowth of the stable fly population. CONCLUSIONS Stable flies are a serious harm to pigs and should be kept in mind for improved animal health and welfare. Knowledge about the determination of Stomoxys calcitrans and early recognition of an increasing stable fly population in pig farming systems followed by proper insect control measures have to be performed to reduce losses caused by this harming insect

High-sensitive nascent transcript sequencing reveals BRD4-specific control of widespread enhancer and target gene transcription

Gene transcription by RNA polymerase II (Pol II) is under control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II correlating with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcribed enhancers in human cells. A multi-omics analysis shows that genome-wide enhancer transcription depends on the BET family protein BRD4. Specifically, BRD4 co-localizes to enhancer and promoter-proximal gene regions, and is required for elongation activation at enhancers and their genes. BRD4 keeps a set of enhancers and genes in proximity through long-range contacts. From these studies BRD4 emerges as a general regulator of enhancer transcription that may link transcription at enhancers and genes

Temporal Trends and Risk Factors for Delayed Hospital Admission in Suspected Stroke Patients.

(1) Background: The benefit of acute ischemic stroke (AIS) treatment declines with any time delay until treatment. Hence, factors influencing the time from symptom onset to admission (TTA) are of utmost importance. This study aimed to assess temporal trends and risk factors for delays in TTA. (2) Methods: We included 1244 consecutive patients from 2015 to 2018 with suspected stroke presenting within 24 h after symptom onset registered in our prospective, pre-specified hospital database. Temporal trends were assessed by comparing with a cohort of a previous study in 2006. Factors associated with TTA were assessed by univariable and multivariable regression analysis. (3) Results: In 1244 patients (median [IQR] age 73 [60-82] years; 44% women), the median TTA was 96 min (IQR 66-164). The prehospital time delay reduced by 27% in the last 12 years and the rate of patients referred by Emergency medical services (EMS) increased from 17% to 51% and the TTA for admissions by General Practitioner (GP) declined from 244 to 207 min. Factors associated with a delay in TTA were stroke severity (beta-1.9; 95% CI-3.6 to -0.2 min per point NIHSS score), referral by General Practitioner (GP, beta +140 min, 95% CI 100-179), self-admission (+92 min, 95% CI 57-128) as compared to admission by emergency medical services (EMS) and symptom onset during nighttime (+57 min, 95% CI 30-85). Conclusions: Although TTA improved markedly since 2006, our data indicates that continuous efforts are mandatory to raise public awareness on the importance of fast hospital referral in patients with suspected stroke by directly informing EMS, avoiding contact of a GP, and maintaining high effort for fast transportation also in patients with milder symptoms

Clinical prediction of large vessel occlusion in anterior circulation stroke: mission impossible?

Simple clinical scores to predict large vessel occlusion (LVO) in acute ischemic stroke would be helpful to triage patients in the prehospital phase. We assessed the ability of various combinations of National Institutes of Health Stroke Scale (NIHSS) subitems and published stroke scales (i.e., RACE scale, 3I-SS, sNIHSS-8, sNIHSS-5, sNIHSS-1, mNIHSS, a-NIHSS items profiles A-E, CPSS1, CPSS2, and CPSSS) to predict LVO on CT or MR arteriography in 1085 consecutive patients (39.4 % women, mean age 67.7 years) with anterior circulation strokes within 6 h of symptom onset. 657 patients (61 %) had an occlusion of the internal carotid artery or the M1/M2 segment of the middle cerebral artery. Best cut-off value of the total NIHSS score to predict LVO was 7 (PPV 84.2 %, sensitivity 81.0 %, specificity 76.6 %, NPV 72.4 %, ACC 79.3 %). Receiver operating characteristic curves of various combinations of NIHSS subitems and published scores were equally or less predictive to show LVO than the total NIHSS score. At intersection of sensitivity and specificity curves in all scores, at least 1/5 of patients with LVO were missed. Best odds ratios for LVO among NIHSS subitems were best gaze (9.6, 95 %-CI 6.765-13.632), visual fields (7.0, 95 %-CI 3.981-12.370), motor arms (7.6, 95 %-CI 5.589-10.204), and aphasia/neglect (7.1, 95 %-CI 5.352-9.492). There is a significant correlation between clinical scores based on the NIHSS score and LVO on arteriography. However, if clinically relevant thresholds are applied to the scores, a sizable number of LVOs are missed. Therefore, clinical scores cannot replace vessel imaging

Transiente Globale Amnesie versus Transiente Ischämische Attacke – Klinik und Schlaganfallrisiko

Transient global amnesia versus transient ischaemic attack: clinical presentation and cerebral vascular accident risk Transient global amnesia is an acute, benign, isolated and temporarily limited disturbance of memory, that can occur repeatedly but shows no increased risk of cardiovascular events or stroke in particular. Therefore, patients with the typical clinical presentation and a normal brain magnetic resonance-scan require neither further diagnostic nor therapeutic interventions. Since the differential diagnosis of transient global amnesia is wide, and transient ischaemic attacks can present similarly, a careful clinical evaluation and neuroimaging is recommended. In any case of doubt further diagnostic steps according to stroke workup should be initiated. In contrast, a transient ischaemic attack represents a neurological emergency where clinical and diagnostic evaluation must be introduced fast. The rapid establishment of therapeutic and secondary preventive measures decreases the clearly elevated stroke risk and prevents disability