Imaging the dynamics of transcription loops in living chromosomes

Garry T Morgan

doi:10.1007/s00412-018-0667-8

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.

Highlights

Two fundamental aspects of nuclear organization have recently been substantiated using novel experimental approaches
Both these fundamental features of nuclear organization can be directly addressed by investigating the unusual lampbrush configuration that chromosomes adopt in the oocytes of animals such as amphibians that form large, yolky eggs. (Even mammalian chromosomes, which as in other organisms that produce small eggs, do not exhibit a lampbrush configuration naturally, can be reprogrammed to adopt it by their being injected into amphibian oocytes (Liu and Gall 2012))
Fluorescent protein fusions are correctly targeted to nascent transcripts of lampbrush chromosome loops

Summary

Introduction

Two fundamental aspects of nuclear organization have recently been substantiated using novel experimental approaches. Many questions still remain about the formation and dynamics of individual loop structures and the detailed structure and organization of transcription sites in living cells. Both these fundamental features of nuclear organization can be directly addressed by investigating the unusual lampbrush configuration that chromosomes adopt in the oocytes of animals such as amphibians that form large, yolky eggs (reviewed in Callan 1986; Gaginskaya et al 2009; Gall 2014; Morgan 2002). (Even mammalian chromosomes, which as in other organisms that produce small eggs, do not exhibit a lampbrush configuration naturally, can be reprogrammed to adopt it by their being injected into amphibian oocytes (Liu and Gall 2012)) Both these fundamental features of nuclear organization can be directly addressed by investigating the unusual lampbrush configuration that chromosomes adopt in the oocytes of animals such as amphibians that form large, yolky eggs (reviewed in Callan 1986; Gaginskaya et al 2009; Gall 2014; Morgan 2002). (Even mammalian chromosomes, which as in other organisms that produce small eggs, do not exhibit a lampbrush configuration naturally, can be reprogrammed to adopt it by their being injected into amphibian oocytes (Liu and Gall 2012))

Methods

Results

Conclusion