A model for the topology of active ribosomal RNA genes

Abstract

The Christmas tree view of active ribosomal RNA (rRNA) genes suggests a gene topology in which a large number of nascent rRNA transcripts are prevented from intertwining. The way in which this is achieved has remained unclear. By using a combination of chromatin immunoprecipitation and chromosome conformation capture techniques, we show that the promoter, upstream region and terminator R3 of active rRNA genes are held together spatially throughout the cell cycle, forming a stable core around which the transcribed region is organized. We suggest a new core-helix model for the topology of rRNA genes, that provides a structural basis for the productive synthesis or rRNA.

Figure 1

Binding of RNA polymerase I transcription factors to ribosomal DNA. (A) DNA fragments precipitated by TBP-ChIP were cloned (Denissov et al, 2007), sequenced and aligned to the rRNA unit. The number of reads is counted per 500 bp. Organization of rDNA: the upstream region (U), the promoter (P), the terminators R1–R3 and the rDNA coding regions are shown by grey boxes. Arrowheads point to genomic locations (A–E) that were validated by ChIP-qPCR analysis. The arrow indicates the transcription start site at +1 position. (B) Enrichment of TBP by ChIP-qPCR (grey bars, right scale) was plotted against the numbers of cloned DNA fragments (black bars, left scale) at the positions shown in (A). (C) Graph showing the enrichment of RNAP I factors as in (B). (D) Binding of TBP to rDNA in different phases of the cell cycle. ChIP, chromatin immunoprecipitation; qPCR, quantitative PCR; rDNA, ribosomal DNA; RNAP I, RNA polymerase I; rRNA, ribosomal RNA; TAF_I, TBP-associated factor; TBP, TATA binding protein; UBF, upstream binding factor.

Figure 2

Spatial proximity of the ribosomal DNA regions. (A) Schematic presentation of the 3C assay. The thickness of the arcs linking the promoter to other regions of rDNA represents the ligation efficiency. Arrows indicate the primers used to amplify two regions flanking the promoter (P_Left and P_Right), the coding region, the terminator R3 (T) and a region in the intergenic spacer (IS). The location of the primers (in kb) is indicated next to the arrows. The organization of rRNA gene is shown with grey boxes and named below. Arrowheads point to the HpyCH4IV restriction sites. (B) Ligation frequencies between promoter (P_Left and P_Right) and other rDNA regions are plotted as fold over background (intergenic spacer). 3C, chromosome conformation capture; CR, coding region, IS, intergenic spacer; rDNA, ribosomal DNA; rRNA, ribosomal RNA.

Figure 3

Genotoxic stress leads to enhanced association of SL1 with the coding region. (A–C) Binding of RNAP I (RPA116), SL1 (TBP) and UBF to rDNA in untreated cells and cells treated with different transcription inhibitors. The error bars represent a standard deviation of ChIP-enrichment values. (D) Metabolic labelling of pre-rRNA after treatment with H₂O₂. (E) Immunofluorescent imaging of RNAP I (green) and fibrillarin (red) at different times after H₂O₂ treatment. ChIP, chromatin immunoprecipitation; DAPI, 4′-6-diamidino-2-phenylindole; EtBr, ethidium bromide; rDNA, ribosomal DNA; rRNA, ribosomal RNA; RNAP I, RNA polymerase I; TBP, TATA binding protein; UBF, upstream binding factor.

Figure 4

The core–helix model describing the topology of active ribosomal RNA genes. (A) The core–helix rDNA structure. The anchoring core is formed by SL1 bound to the promoter (P), the upstream region (U) and the terminator R3 (T). Non-intersecting DNA rings comprising the transcribed region are cylindrically wrapped around the core; the 18S genomic region locates close to SL1. DNA fibrils (green arrows) enter into the core of the helix from its lateral sides. (B) Core–helix organization of transcribed rRNA genes. RNA polymerases initiate at the promoter in the core and elongate along the cylindrical helix (long arrow). Zig-zag black lines represent growing rRNA chains attached to RNA polymerases that are cotranscriptionally assembled into processing ribonucleoprotein particles. After termination, RNA polymerases dissociate from rDNA. Topoisomerase I removes torsional stress in the intergenic spacer resulting from the rotation of the rDNA solenoid. In addition, Topoisomerase I is required throughout the transcribed region (not indicated). (C) Scheme of the nucleolar compartments in frontal projection of the core–helix structure. The central area corresponds to the FCs with SL1 and DNA fibrils of the anchoring core. The transcribed region and elongating RNA polymerases are at the periphery of the FC. Nascent rRNA chains of increasing length are assembled into processing RNPs that form the DFC compartment. DFC, dense fibrillar centre; FC, fibrillar centre; GC, granular component; IS, intergenic spacer; rDNA, ribosomal DNA; RNAP, RNA polymerase; RNP, ribonucleoprotein complex; rRNA, ribosomal RNA; UBF, upstream binding factor.