XB-ART-54787Chromosoma January 1, 2018; 127 (3): 361-374.
Imaging the dynamics of transcription loops in living chromosomes.
When in the lampbrush configuration, chromosomes display thousands of visible DNA loops that are transcribed at exceptionally high rates by RNA polymerase II (pol II). These transcription loops provide unique opportunities to investigate not only the detailed architecture of pol II transcription sites but also the structural dynamics of chromosome looping, which is receiving fresh attention as the organizational principle underpinning the higher-order structure of all chromosome states. The approach described here allows for extended imaging of individual transcription loops and transcription units under conditions in which loop RNA synthesis continues. In intact nuclei from lampbrush-stage Xenopus oocytes isolated under mineral oil, highly specific targeting of fluorescent fusions of the RNA-binding protein CELF1 to nascent transcripts allowed functional transcription loops to be observed and their longevity assessed over time. Some individual loops remained extended and essentially static structures over time courses of up to an hour. However, others were less stable and shrank markedly over periods of 30-60 min in a manner that suggested that loop extension requires continued dense coverage with nascent transcripts. In stable loops and loop-derived structures, the molecular dynamics of the visible nascent RNP component were addressed using photokinetic approaches. The results suggested that CELF1 exchanges freely between the accumulated nascent RNP and the surrounding nucleoplasm, and that it exits RNP with similar kinetics to its entrance. Overall, it appears that on transcription loops, nascent transcripts contribute to a dynamic self-organizing structure that exemplifies a phase-separated nuclear compartment.
PubMed ID: 29610944
PMC ID: PMC6096578
Article link: Chromosoma
Species referenced: Xenopus
Genes referenced: akap13 celf1 kidins220 mtor
Article Images: [+] show captions
|Fig. 1. Distinctive chromosomal targeting of fluorescently tagged RNA-binding proteins in unfixed nuclear spread preparations. Fluorescence and phase contrast images showing a U1C.mCherry targeting to loops of a lampbrush bivalent and to a type of nuclear body, the B snurposome (arrow). The large, highly refractile nuclear bodies in the phase contrast image are extrachromosomal nucleoli. b Specific CELF1.GFP targeting to four lateral loops, which correspond to the four chromatids comprising each locus in the 4C lampbrush bivalent; two of these morphologically unremarkable loops at homologous sites are arrowed in the phase contrast image (note that one of the lower pairs of sister loops is collapsed onto the chromosome axis and is viewed “end-on”). c A set of loops with a distinctive contorted morphology are targeted by CELF1.GFP, contorted loops at homologous loci (arrows) on LBC 7 in an unfixed spread preparation. Note that only the phase-dark regions of the loops appear highly fluorescent (lower arrow). In d, co-expression and co-targeting of U1C.mCherry (pseudocolored green in merge) and CELF1.GFP (pseudocolored red) to contorted loops are shown. One of the two homologous contorted loop loci is arrowed in each panel|
|Fig. 2. Retention of pol II transcriptional activity by transcription loops in oil-isolated nuclei. a Pol II immunostaining of lampbrush loops in a fixed spread prepared from an oocyte nucleus that had been isolated into oil and kept for about 3 h prior to spread preparation. The α-pol II monoclonal antibody that stains the loop axes recognizes a CTD phosphoisomer associated with transcriptionally active pol II. The brightly immunostained objects are B snurposomes, which contain epitopes that also cross-react with this antibody (Doyle et al. 2002). b Continued loop RNA synthesis in oil-isolated nuclei detected by Br-U incorporation followed by immunostaining. As summarized in the diagram, nuclei were isolated into oil and pre-incubated for about 2 h prior to injection of Br-UTP. Following incubation in oil for a further 3 h, each nucleus was transferred into a drop of oil under an aqueous solution into which it was then pushed, allowing the production of a fixed nuclear spread preparation. Immunostaining with an α-BrU antibody demonstrates that RNA synthesis was occurring on lampbrush loops at least 2 h after nuclear isolation (left-hand panels). As controls, a nuclear spread was prepared directly from an oocyte that had been injected 3 h previously with Br-UTP (center panels) and from an oil-isolated nucleus that was not injected with Br-UTP prior to incubation (right panels). Images are reproduced using the same contrast function|
|Fig. 3. Transcription loops targeted by fluorescent CELF1 are detectable in intact, oil-isolated nuclei. a Survey view of part of an oil-isolated nucleus taken from an oocyte expressing CELF1.GFP. Brightly fluorescent structures (arrowhead) are detectable against a lower nucleoplasmic background fluorescence. Dotted line indicates position of oil/nuclear envelope interface. b–f Higher-magnification, wide-field images of lampbrush loops targeted by CELF1.mCherry in oil-isolated nuclei. Loops with either a typical, “thin-thick” morphology (b, c) or one characteristic of contorted loops (d–f) are shown. Arrows in b, c indicate the predicted direction in which pol II is tracking along these loops. Images b–f to same scale|
|Fig. 4. Stability of transcription loops over time in oil-isolated nuclei. a–d show images at regular time points of four transcription loops each targeted by CELF1-GFP and exhibiting a range of RNP compartment morphologies. a Survey view of a CELF1-targeted loop (arrow) extending from a lampbrush bivalent that is detectable via faint background labeling (location of a chiasma is indicted by a large arrowhead and approximate positions of the ends of the homologous chromosome arms are indicated by small arrowheads). The neighboring bright sister loop is viewed end-on and may be collapsed; the homologous locus in the homolog to the right is detectable in a different focal plane. The indicated loop appeared essentially unchanged over the time course. b Example of a loop that initially exhibits a convoluted/kinked morphology and which over time produces internal “sub-loops” (arrowhead). Although an essentially looped track with two definable insertions at its base is maintained over the time course, there also appears some contraction in overall loop length. c Example of a contorted loop locus that shows an orientation change (curved arrow) during the time course, although the complex morphology of the GFP-labeled RNP compartment appears stable over time. d Example of a loop that initially exhibits a typical “thin-thick” asymmetry in RNP distribution along its length. A marked reduction in loop axial length has begun by 8 min and by 30 min no longer are an overt loop-like form nor a clearly asymmetric distribution of RNP apparent. This time-lapse series was obtained from the same nucleus, and completed prior to, the one shown in a. All scale bars = 10 μm|
|Fig. 5. FRAP of CELF1.mCherry in contorted loop loci of oil-isolated nuclei. a Selected images from two confocal FRAP time courses. Photobleaching was performed on individual contorted loop loci using a bleach region that completely enclosed each structure. The recovery of 80 to 100% CELF1.mCherry fluorescence in single optical sections of 2 μm over time is shown. b FRAP recovery curves based on three contorted loop loci of varying size and appearance. Because of their morphological differences, individual FRAP curves for the different loci were not averaged but all predict similar half-times for recovery of 1.5 to 2 min|
|Fig. 6. Photoactivation of CELF1.PA-GFP in contorted loop loci and fluorescence loss over time. a Contorted loops from an oil-isolated nucleus that contain both CELF1.mCherry, which is detected at 561 nm in single optical sections, and unactivated CELF1.PA-GFP, which is not initially detectable at 488 nm. After photoactivation at 405 nm within an ROI encompassing the whole locus (dotted circle), bright fluorescence at 488 nm is detectable. The intensity of fluorescence detected at 488 nm in the immediately post-activation image becomes reduced over time until it is undetectable. The overall fluorescence of CELF1.mCherry in the same loops appears unaltered, although the locus undergoes conformational changes over the time course. b Experiment as in a except using a spread preparation in which the contorted loops are suspended in saline rather than nucleoplasm. Again, robust photoactivation of CELF1.PA-GFP was detected at 488 nm in contorted loop loci but, although imaged under the same conditions used for a, there was no appreciable loss of fluorescence relative to immediately post-activation levels. c Fluorescence decay curves plotted separately from quantitative data obtained for four different contorted loop loci. These show that 50% of the initial fluorescence intensity of CELF1.PA-GFP are lost within 2.5–6 min of photoactivation. Three of the curves (red and black symbols) were obtained from loop loci that were photoactivated in their entirety, while one (green symbols) is derived from the experiment shown in d, in which only a sub-region of a contorted loop locus was photoactivated. d Regional photoactivation of CELF1.PA-GFP in a contorted loop locus was obtained by confining the 405-nm laser beam to a diffraction limited spot targeted by reference to the co-localized CELF1.mCherry image (dotted circle). Loss of fluorescence at 488 nm from the photoactivated region occurred over time at similar rates to the experiment shown in a|
References [+] :
Anderson, Patterns of synthesis and accumulation of heterogeneous RNA in lampbrush stage oocytes of Xenopus laevis (Daudin). 1979, Pubmed, Xenbase