Live images of RNA polymerase II transcription units

Patel, Snehal; Novikova, Natalya; Beenders, Brent; Austin, Christopher M.; Bellini, Michel

doi:10.1007/s10577-007-1189-z

Cited by 14 publications

(17 citation statements)

References 17 publications

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“…We think that LBCs, and the newt LBCs in particular, provide an invaluable biological system for settling this issue of a general importance for unravelling the relationships and functional role of the Ro proteins and RNPs. New cellular imaging methods have been developed for the study of LBCs, such as the visualisation of LBCs in oil-isolated GVs under in vivo-like conditions (Patel et al 2008) or the analysis of the distribution of proteins on specific loops by superresolution imaging (Kaufmann et al 2012). These should provide powerful tools for studying the localisation and dynamics of the different protein partners of the Ro RNPs directly at the level of the loop matrix.…”

Section: Discussionmentioning

confidence: 99%

Precocious detection on amphibian oocyte lampbrush chromosomes of subtle changes in the cellular localisation of the Ro52 protein induced by in vitro culture

2012

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Subterminal lampbrush loops of one of the 12 bivalents of the oocyte karyotype of Pleurodeles waltl (Amphibian, Urodele) underwent prominent morphological changes upon in vitro culture. These loops exhibited a fine ribonucleoprotein (RNP) granular matrix, which evolved during culture into huge structures that we have named 'chaussons' (slippers). This phenomenon involved progressive accumulation of proteins in the RNP matrix without protein neosynthesis. One of these proteins, which translocated into the nucleus during the culture, was identified as a homolog of the human Ro52 E3 ubiquitin ligase. RNA polymerase III was also found to accumulate on the same loops. These results suggest that the subterminal loops of bivalent XII act as a storage site for the components of a nuclear machinery involved in the quality control of RNA synthesis and maturation in response to cellular stress. They also emphasise the considerable value of the lampbrush chromosome system for a direct visualisation of modifications in gene expression and open the question of a nuclear accumulation of Ro52 in human or animal oocytes cultured in vitro for assisted reproductive technologies (ART).

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Section: Discussionmentioning

confidence: 99%

Precocious detection on amphibian oocyte lampbrush chromosomes of subtle changes in the cellular localisation of the Ro52 protein induced by in vitro culture

2012

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“…We present two of them here, namely the de novo formation of lampbrush chromatids (Gall and Murphy 1998) and the visualization of organelles (Handwerger et al 2003), as well as LBCs in oil-isolated nuclei (Patel et al 2008). The remarkable demonstration that sperm heads can yield individual lampbrush chromatids within a few hours after nuclear injection provides new opportunities of characterizing the dynamic molecular events that contribute to the formation and maintenance of LBCs.…”

Section: Future Perspectivesmentioning

confidence: 99%

“…We also find that cohesin staining is very robust at the axes, whereas little or no cohesin is found on loops. Finally, we utilized our novel under oil method of characterizing chromosomes of the Xenopus oocyte (Patel et al 2008) to study cohesin dynamics by fluorescence recovery after photobleaching (FRAP) of YFP-hRAD21. These data are among the first direct evidence (see also McNairn and Gerton, this issue) for cohesin dynamics on a small and defined region of a single chromosome.…”

Section: Cohesin and Meiosismentioning

confidence: 99%

“…Oil-isolation of nuclei and FRAP Non-defolliculated oocytes were injected with RNA coding for the YFP-hRAD21-3HA proteins, and the isolation of nuclei in mineral oil was performed 24 h later as described in Patel et al (2008). In some cases, oocytes were also injected with DAPI (20 nl of a 5 ng/μl solution of DAPI in water) 5 min prior nuclei isolation to confirm the chromosomal distribution of YFP-hRAD21-3HA.…”

Section: Sperm Nuclei Preparation and Injectionmentioning

confidence: 99%

“…We recently optimized the procedure for imaging LBCs in oil-isolated nuclei by light microscopy (Patel et al 2008), and we used this technique here to begin analyzing the dynamic properties of a newly expressed hRAD21-3HA fused to the yellow fluorescent (YFP) protein. Capped, sense strand transcripts coding for the YFP-hRAD21-3HA protein were injected into stage IV-V oocytes.…”

Section: Newly Expressed Hrad21 Is Recruited To Lbcs Where It Co-locamentioning

confidence: 99%

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Lampbrush chromosomes enable study of cohesin dynamics

et al. 2009

Self Cite

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The lampbrush chromosomes present in the nuclei of amphibian oocytes offer unique biological approaches for study of the mechanisms that regulate chromatin structure with high spatial resolution. We discuss fundamental aspects of the remarkable organization and plasticity exhibited by lampbrush chromosomes. We then utilize lampbrush chromosomes to characterize the chromosomal distribution and dynamics of cohesin, the four-protein complex (RAD21/MCD1/ SCC1, SMC1, SMC3, SCC3/SA2) responsible for sister chromatid cohesion. We find that endogenous SMC3 and newly expressed hRAD21 co-localize on chromosomal axes, sites where sister chromatids are tightly paired. We present evidence suggesting that hRAD21 recruitment to lampbrush chromosomes is modulated by chromosomal SMC1 and SMC3. Notably, using a technique for de novo chromosome assembly, we demonstrate that both SMC3 and hRAD21 are recruited to single, unreplicated lampbrush chromatids. Finally, we used our novel method of analyzing the oocyte nucleus under oil combined with fluorescence recovery after photobleaching, to provide direct evidence that cohesin is highly dynamic at discrete, condensed chromosomal regions. Collectively, these data demonstrate that lampbrush chromosomes provide a unique and powerful tool for combining biochemical and cytological analyses for dissection of complex chromosomal processes.

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Imaging the dynamics of transcription loops in living chromosomes

Morgan

2018

Chromosoma

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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.Electronic supplementary materialThe online version of this article (10.1007/s00412-018-0667-8) contains supplementary material, which is available to authorized users.

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Live images of RNA polymerase II transcription units

Cited by 14 publications

References 17 publications

Precocious detection on amphibian oocyte lampbrush chromosomes of subtle changes in the cellular localisation of the Ro52 protein induced by in vitro culture

Precocious detection on amphibian oocyte lampbrush chromosomes of subtle changes in the cellular localisation of the Ro52 protein induced by in vitro culture

Lampbrush chromosomes enable study of cohesin dynamics

Imaging the dynamics of transcription loops in living chromosomes

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