Direct observation of a condensate effect on super-enhancer controlled gene bursting.

Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 µm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 µm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.

A dual role for H2A.Z.1 in modulating the dynamics of RNA polymerase II initiation and elongation.

RNA polymerase II (RNAPII) pausing immediately downstream of the transcription start site is a critical rate-limiting step for the expression of most metazoan genes. During pause release, RNAPII encounters a highly conserved +1 H2A.Z nucleosome, yet how this histone variant contributes to transcription is poorly understood. Here, using an inducible protein degron system combined with genomic approaches and live cell super-resolution microscopy, we show that H2A.Z.1 modulates RNAPII dynamics across most genes in murine embryonic stem cells. Our quantitative analysis shows that H2A.Z.1 slows the rate of RNAPII pause release and consequently impacts negative elongation factor dynamics as well as nascent transcription. Consequently, H2A.Z.1 also impacts re-loading of the pre-initiation complex components TFIIB and TBP. Altogether, this work provides a critical mechanistic link between H2A.Z.1 and the proper induction of mammalian gene expression programs through the regulation of RNAPII dynamics and pause release.

Clinical Experience with Use of Remdesivir in the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2: a Case Series.

BACKGROUND: A novel antiviral agent, remdesivir (RDV), is a promising candidate treatment for coronavirus disease 2019 (COVID-19) in the absence of any proven therapy. MATERIALS AND METHODS: This retrospective case series included 10 patients with a clinically and laboratory confirmed diagnosis of severe COVID-19 pneumonia who had received RDV for 5 days (n = 5) or 10 days (n = 5) in the Phase III clinical trial of RDV (GS-US-540-5773) conducted by Gilead Sciences. The clinical and laboratory data for these patients were extracted. RESULTS: One patient in the 10-day group received RDV for only 5 days because of nausea and elevated liver transaminases. No patient had respiratory comorbidity. Seven patients had bilateral lesions and three had unilateral lesions on imaging. All patients had received other medications for COVID-19, including lopinavir/ritonavir and hydroxychloroquine, before administration of RDV. Five patients required supplemental oxygen and one required mechanical ventilation. All patients showed clinical and laboratory evidence of improvement. Half of the patients developed elevated liver transaminases and three had nausea. There were no adverse events exceeding grade 2. CONCLUSION: Our experience indicates that RDV could be a therapeutic option for COVID-19. A well-designed randomized controlled clinical trial is now needed to confirm the efficacy of RDV in patients with COVID-19.

A CRISPR/Cas9 platform for MS2-labelling of single mRNA in live stem cells.

The MS2 system is a powerful tool for investigating transcription dynamics at the single molecule directly in live cells. In the past, insertion of the RNA-labelling cassette at specific gene loci has been a major hurdle. Here, we present a CRISPR/Cas9-based approach to insert an MS2 cassette with selectable marker at the start of the 3' untranslated region of any coding gene. We demonstrate applicability of our approach by tagging RNA of the stem cell transcription factor Esrrb in mouse embryonic stem cells. Using quantitative fluorescence microscopy we determine the number of nascent transcripts at the Esrrb locus and the fraction of cells expressing the gene. We find that upon differentiation towards epiblast-like cells, expression of Esrrb is down-regulated in an increasing fraction of cells in a binary manner.

Mediator and RNA polymerase II clusters associate in transcription-dependent condensates.

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.

Analysis of Tertiary Interactions between SART3 and U6 Small Nuclear RNA Using Modified Nanocapillaries.

We employed modified glass nanocapillaries to investigate interactions between the RNA-binding protein, known as cell carcinoma antigen recognized by T cells-3 (SART3), and the noncoding spliceosome component, U6 small nuclear RNA (snRNA), at the single-molecule level. We functionalized the nanocapillaries with U6 snRNA fragments, which were hybridized to DNA molecules and then covalently attached to the nanocapillary surface. When transported through the modified nanocapillaries, two different SART3-derived constructs, HAT-RRM1-RRM2 and RRM1-RRM2, exhibited resistive ionic current pulses with different dwell times, which represented their different binding affinities to tethered U6 snRNAs. The dissociation constants (KD), estimated from the bias voltage dependence of translocation events, were approximately 1.9 µM and 201 µM for HAT-RRM1-RRM2 and RRM1-RRM2, respectively. These values were comparable to corresponding values obtained with isothermal titration calorimetry, demonstrating that the modified glass nanocapillaries are applicable to analyses of protein-ligand interactions at the single-molecule level.

Selective Detection of Single-Stranded DNA Molecules Using a Glass Nanocapillary Functionalized with DNA.

We describe glass nanocapillaries with single-stranded DNA molecules (ssDNA) covalently attached to the capillary surface. These DNA-functionalized nanocapillaries selectively facilitate the translocation of target ssDNA that is complementary to the probe ssDNA. In addition, the complementary target ssDNA exhibits an event duration time longer than that of the noncomplementary target ssDNA. The temperature dependence measurements of translocation events show that the longer duration time is a result of an interaction between probe and target ssDNA and is dependent on the base pair binding strength. These results demonstrate that single-base mismatch transport selectivity can be achieved using the DNA-functionalized nanocapillaries.

A photoemission study of Pd ultrathin films on Pt (111).

The origin of surface core-level shift (SCLS) of Pd thin films on Pt (111) substrate is investigated. At submonolayer coverage of Pd thin films, the splitting of Pd 3d core-level peaks indicate the contribution of both initial and final states of photoionization processes while no change on valence band (VB) spectra is found. When the coverage of Pd reaches to single monolayer, the final-state relaxation effect on the Pd 3d vanishes and only the initial-state effect, a negative SCLS, is present. Also, the VB spectrum at Pd monolayer films shows a clear band narrowing, that is, the origin of the negative SCLS at monolayer coverage. As the Pd coverage is increased to more than monolayer thickness, the Pd 3d peaks start to show the surface layer contribution from second and third layers and the VB spectra show even narrower bandwidth, possibly due to the formation of surface states and strained effect of Pd adlayers on top of the first pseudomorphic layer.