Imaging the dynamics of transcription loops in living chromosomes

Abstract

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.

Super-resolution in situ analysis of active ribosomal DNA chromatin organization in the nucleolus

“…Still, while the looping of the entire rDNA cluster on chromosome XII of budding yeast has been observed with different methodologies [39][40][41] , and looping of mammalian rDNA repeat units has also been hypothesized based on biochemical analyses 42 , detailed 3D structure analyses of individual rDNA units were lacking. Optical microscopy approaches have been successfully applied to visualize DNA loops that are very actively transcribed by RNA polymerase II on lampbrush chromosomes 43,44 . However, the looping of active rRNA or other genes on normal interphase chromosomes has not yet been observed by optical microscopy.…”

Spatial organization of transcribed eukaryotic genes

“…Importantly, in lateral loops of lampbrushes, Y-loops and puffs, such as Balbiani rings, the level of transcription is so high that, due to dense decoration by thousands of RNAPIIs with nRNAs, transcribed regions gain a noticeable width. This in combination with substantial length, allows their visualization even under a phase contrast microscope (Bjork and Wieslander, 2015;Grond et al, 1983;Morgan, 2018). Therefore, for studying expressed genes by light microscopy, both sufficient length and sufficiently high expression are essential prerequisites.…”

“…The discrepancy between these two views on the mechanisms of transcription, can be explained to a great extent by the limitations of light microscopy resolution. Lateral lampbrush loops and polytene puffs are visible even under a phase contrast microscope 7, 8, 14 , owing to the significant length of transcription units (up to hundreds of kilobases) combined with their high transcriptional level. Therefore, for the visualization of expressed genes within interphase nuclei both a sufficient length and a sufficiently high expression are essential.…”

Identification of Genomic Loci Responsible for the Formation of Nuclear Domains Using Lampbrush Chromosomes

“…Thus, using Fluorescence in situ hybridization (FISH) on lampbrush chromosomes makes it possible to map genomic sequences with high cytogenetic resolution and to supplement and/or to correct genome sequence assembly [21,22]. The high level of transcriptional activity on lateral loops of lampbrush chromosomes allows the visualization of nascent transcripts and components of RNA processing machinery at the level of individual transcription units [23,24]. Another benefit of the lampbrush stage of oogenesis is the larger sized nuclear bodies and domains which form on lampbrush chromosomes.…”