Inhibition of RNA polymerase II transcription causes chromatin decondensation, loss of nucleolar structure, and dispersion of chromosomal domains

Abstract

Fluorescence in situ hybridization and immunofluorescence have been used to visualize specific genomic DNA sequences and proteins in interphase nuclei treated with transcriptional inhibitors. The adenosine analog 5,6-dichloro- β-D-ribofuranosylbenzimidazole (DRB) and α-amanitin selectively inhibit transcription by RNA polymerase II at low doses. Upon exposure to DRB or α- amanitin the fibrillar components of the normally compact nucleolus unravel into necklace-like structures which represent highly extended linear arrays of ribosomal (r)RNA genes. Similarly, blocks of tandemly repeated satellite DNAs dissociate into 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 block rRNA synthesis does not cause significant decondensation of nucleolar, heterochromatic, and interphase chromosome domains. Interestingly, both α-amanitin and AMD cause coilin to associate with the nucleolar domain. In AMD-treated cells, coilin is enriched in nucleolar caps abutting upon the residual nucleolus. After α-amanitin treatment, coilin is concentrated in numerous beads closely associated with individual rDNA transcription units within nucleolar necklaces. The changes 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 on a continued transcription of nuclear genes, but not of the rRNA genes.