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.

In the electron energy loss spectra for amorphous, atomic layer deposited (ALD) Al2O3 film, a peak at 6.4 eV was observed. First principle quantum chemical simulation shows that it relates to excitation of neutral oxygen vacancy in Al2O3. The 2.91 eV luminescence excited in a band near 6.0 eV in amorphous Al2O3 is similar to that in bulk crystals which is associated with neutral oxygen vacancy. Thus, the amorphous ALD Al2O3 film is oxygen deficient and the oxygen vacancy parameters are similar in crystalline and amorphous Al2O3.

RNAPII pausing immediately downstream of the transcription start site (TSS) is a critical rate limiting step at most metazoan genes that allows fine-tuning of gene expression in response to diverse signals [1][2][3][4][5] . During pause-release, RNA Polymerase II (RNAPII) encounters an H2A.Z.1 nucleosome [6][7][8] , yet how this variant contributes to transcription is poorly understood. Here, we use high resolution genomic approaches 2,9 (NET-seq and ChIP-nexus) along with live cell superresolution microscopy (tcPALM) 10 to investigate the role of H2A.Z.1 on RNAPII dynamics in embryonic stem cells (ESCs). Using a rapid, inducible protein degron system 11 combined with transcriptional initiation and elongation inhibitors, our quantitative analysis shows that H2A.Z.1 slows the release of RNAPII, impacting both RNAPII and NELF dynamics at a single molecule level. We also find that H2A.Z.1 loss has a dramatic impact on nascent transcription at stably paused, signal-dependent genes. Furthermore, we demonstrate that H2A.Z.1 inhibits reassembly and re-initiation of the PIC to reinforce the paused state and acts as a strong additional pause signal at stably paused genes. Together, our study suggests that H2A.Z.1 fine-tunes gene expression by regulating RNAPII kinetics in mammalian cells.1 .

No abstract

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.