RNA functions at enhancers remain mysterious. Here we show that the 7SK small nuclear RNA (snRNA) inhibits enhancer transcription by modulating nucleosome position. 7SK occupies enhancers and super enhancers genome-wide in mouse and human cells, and 7SK is required to limit eRNA initiation and synthesis in a manner distinct from promoter pausing. Clustered elements at super enhancers uniquely require 7SK to prevent convergent transcription and DNA damage signaling. 7SK physically interacts with the BAF chromatin remodeling complex, recruit BAF to enhancers, and inhibits enhancer transcription by modulating chromatin structure. In turn, 7SK occupancy at enhancers coincides with Brd4 and is exquisitely sensitive to the bromodomain inhibitor JQ1. Thus, 7SK employs distinct mechanisms to counteract diverse consequences of pervasive transcription that distinguish super enhancers, enhancers, and promoters.
To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the γ-tubulin ring complex (γTuRC). Although γTuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved γ-tubulin nucleation activator (γTuNA) domain has been reported to increase the number of microtubules (MTs) generated by γTuRCs. However, previously we and others observed that γTuNA had a minimal effect on the activity of antibody-purified Xenopus γTuRCs in vitro (Thawani et al., eLife, 2020; Liu et al., 2020). Here we instead report, based on improved versions of γTuRC, γTuNA, and our TIRF assay, the first real-time observation that γTuNA directly increases γTuRC activity in vitro, which is thus a bona fide γTuRC activator. We further validate this effect in Xenopus egg extract. Via mutation analysis, we find that γTuNA is an obligate dimer. Moreover, efficient dimerization as well as γTuNA's L70, F75, and L77 residues are required for binding to and activation of γTuRC. Finally, we find that γTuNA's activating effect opposes inhibitory regulation by stathmin. In sum, our improved assays prove that direct γTuNA binding strongly activates γTuRCs, explaining previously observed effects of γTuNA expression in cells and illuminating how γTuRC-mediated microtubule nucleation is regulated.
12Determining how microtubules (MTs) are nucleated is essential for understanding how the 13 cytoskeleton assembles. Yet, half a century after the discovery of MTs and ab-tubulin subunits 14 and decades after the identification of the γ-tubulin ring complex (γ-TuRC) as the universal MT 15 nucleator, the underlying mechanism largely remains a mystery. Using single molecule studies, 16we uncover that γ-TuRC nucleates a MT more efficiently than spontaneous assembly. The laterally 17 interacting array of γ-tubulins on γ-TuRC facilitates the lateral association of αβ-tubulins, while 18 longitudinal affinity between γ/αβ-tubulin is surprisingly weak. During nucleation, 3-4 αβ-tubulin 19 dimers bind stochastically to γ-TuRC on average until two of them create a lateral contact and 20 overcome the nucleation barrier. Although γ-TuRC defines the nucleus, XMAP215 significantly 21 increases reaction efficiency by facilitating ab-tubulin incorporation. In sum, we elucidate how 22 MT initiation occurs from γ-TuRC and determine how it is regulated. 23Results 49 50 Reconstituting and visualizing microtubule nucleation from γ-TuRC 51To study how γ-TuRC nucleates MTs (Fig. 1A), we purified endogenous γ-TuRC from Xenopus 52 egg extracts and biotinylated the complexes to immobilize them on functionalized glass ( Fig. S1A-53 C). Upon perfusing fluorescent αβ-tubulin, we visualized MT nucleation live with total internal 54 reflection fluorescence microscopy (TIRFM). Strikingly, MT nucleation events occurred 55 specifically from single γ-TuRC molecules ( Fig. 1B; Fig. S1D and Movie S1-2). Kymographs 56 revealed that attached γ-TuRC assembled ab-tubulin into a MT de novo starting from zero length 57 within the diffraction limit of light microscopy (Fig. 1C), ruling out an alternative model where 58MTs first spontaneously nucleate and then become stabilized via γ-TuRC. By observing the 59 fiduciary marks on the MT lattice ( Fig. 1C) and generating polarity-marked MTs from attached g-60 TuRC (Fig. S1E), we showed that γ-TuRC caps the MT minus-end, while only the plus-end 61 polymerizes. Altogether, our results show that γ-TuRC directly nucleates MTs. 62 63 Defining the microtubule nucleus on γ-TuRC 64To determine how γ-TuRC nucleates MTs, we measured the kinetics of MT nucleation for a 65 constant density of γ-TuRC and increasing αβ-tubulin concentration ( Fig. 1D and Movie S3). 66 Surprisingly, γ-TuRC nucleated MTs starting from 7 µM tubulin (Fig. 1D), which is higher than 67 the minimum tubulin concentration (C*) needed for growth at pre-formed MT plus-ends (C* = 1.4 68 µM, Fig. 1E). Furthermore, the number of MTs nucleated from γ-TuRC increased non-linearly 69 with tubulin concentration as opposed to the linear increase in MT's growth-speed with tubulin 70 concentration (Fig. 1E). By measuring the number of MTs nucleated over time with varying ab-71 interferometry, we compared the interaction of ab-tubulin dimers with themselves versus with g-118 tubulin. Specific interactions between probe-bound αβ-tubulin and increasing concentratio...
Centrosomes are self-assembling, micron-scale, nonmembrane bound organelles that nucleate microtubules (MTs) and organize the microtubule cytoskeleton of the cell. They orchestrate critical cellular processes such as ciliary-based motility, vesicle trafficking, and cell division. Much is known about the role of the centrosome in these contexts, but we have a less comprehensive understanding of how the centrosome assembles and generates microtubules. Studies over the past 10 years have fundamentally shifted our view of these processes. Subdiffraction imaging has probed the amorphous haze of material surrounding the core of the centrosome revealing a complex, hierarchically organized structure whose composition and size changes profoundly during the transition from interphase to mitosis. New biophysical insights into protein phase transitions, where a diffuse protein spontaneously separates into a locally concentrated, nonmembrane bounded compartment, have provided a fresh perspective into how the centrosome might rapidly condense from diffuse cytoplasmic components. In this Perspective, we focus on recent findings that identify several centrosomal proteins that undergo phase transitions. We discuss how to reconcile these results with the current model of the underlying organization of proteins in the centrosome. Furthermore, we reflect on how these findings impact our understanding of how the centrosome undergoes self-assembly and promotes MT nucleation.
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