However, 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. Indeed it has recently been suggested that both of these organizational principals could be in operation at sites of RNA polymerase II (pol II) transcription (Hnisz et al. The other deals with the physical and functional organization of the interchromatin space, a prominent feature of which is the presence of a variety of nuclear bodies that are now thought to reflect the formation of liquid-liquid phase-separated compartments (Mao et al. One is that chromosome looping and loop structures at various levels underpin both the spatial organization of genomes in interphase nuclei and the establishment and regulation of gene transcription (Denker and de Laat 2016 Fudenberg et al. Two fundamental aspects of nuclear organization have recently been substantiated using novel experimental approaches. Overall, it appears that on transcription loops, nascent transcripts contribute to a dynamic self-organizing structure that exemplifies a phase-separated nuclear compartment. 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. In stable loops and loop-derived structures, the molecular dynamics of the visible nascent RNP component were addressed using photokinetic approaches. 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. Some individual loops remained extended and essentially static structures over time courses of up to an hour. 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. The approach described here allows for extended imaging of individual transcription loops and transcription units under conditions in which loop RNA synthesis continues. 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. 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).
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