INHIBITION OF RNA-POLYMERASE-II TRANSCRIPTION CAUSES CHROMATIN DECONDENSATION, LOSS OF NUCLEOLAR STRUCTURE, AND DISPERSION OF CHROMOSOMAL DOMAINS

Fluorescence in situ hybridization and immunofluorescence have been us ed to visualize specific genomic DNA sequences and proteins in interph ase nuclei treated with transcriptional inhibitors. The adenosine anal og 5,6-dichloro-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-aman itin selectively inhibit transcription by RNA polymerase II at low dos es, Upon exposure to DRB or alpha-amanitin the fibrillar components of the normally compact nucleolus unravel into necklace-like structures which represent highly extended linear arrays of ribosomal (r)RNA gene s. Similarly, blocks of tandemly repeated satellite DNAs dissociate in to extended beaded strands. Localized (euchromatic) chromosome domains and even whole chromosome territories disperse throughout the nuclear interior. Treatment of cells with actinomycin D (AMD) at doses that b lock rRNA synthesis does not cause significant decondensation of nucle olar, heterochromatic, and interphase chromosome domains. Interestingl y, both alpha-amanitin and ARID cause coilin to associate with the nuc leolar domain, In AMD-treated cells, coilin is enriched in nucleolar c aps abutting upon the residual nucleolus, After alpha-amanitin treatme nt, coilin is concentrated in numerous beads closely associated with i ndividual rDNA transcription units within nucleolar necklaces, The cha nges in higher-order nuclear structure are reversible in cell cultures exposed to nontoxic doses of transcriptional inhibitors. It therefore may be concluded that nuclear topographic organization is dependent o n a continued transcription of nuclear genes, but not of the rRNA gene s. (C) 1996 Academic Press, Inc.