Super-resolution in situ analysis of active ribosomal DNA chromatin organization in the nucleolus

Abstract

Ribosomal RNA (rRNA) transcription by RNA polymerase I (Pol I) is the first key step of ribosome biogenesis. While the molecular mechanisms of rRNA transcription regulation have been elucidated in great detail, the functional organization of the multicopy rRNA gene clusters (rDNA) in the nucleolus is less well understood. Here we apply super-resolution 3D structured illumination microscopy (3D-SIM) to investigate the spatial organization of transcriptionally competent active rDNA chromatin at size scales well below the diffraction limit by optical microscopy. We identify active rDNA chromatin units exhibiting uniformly ring-shaped conformations with diameters of ~240 nm in mouse and ~170 nm in human fibroblasts, consistent with rDNA looping. The active rDNA chromatin units are clearly separated from each other and from the surrounding areas of rRNA processing. Simultaneous imaging of all active genes bound by Pol I and the architectural chromatin protein Upstream Binding Transcription Factor (UBF) reveals a random spatial orientation of regular repeats of rDNA coding sequences within the nucleoli. These observations imply rDNA looping and exclude potential formation of systematic spatial assemblies of the well-ordered repetitive arrays of transcription units. Collectively, this study uncovers key features of the 3D organization of active rDNA chromatin units and their nucleolar clusters providing a spatial framework of nucleolar chromatin organization at unprecedented detail.

Figure 1

Super-resolution 3D-SIM imaging of nucleolar organization. Immunofluorescence labeling of MEF and IMR90 cells. Single nucleoli are shown in the zoom-in images. (a) Transcriptionally competent rRNA genes localized by UBF and the GC marker protein nucleophosmin (NPM1). (b) Simultaneous immunostaining of the DFC marker protein FBL with UBF and NPM1. (c) Immuno-FISH localization of transcriptionally active enhancer/coding rDNA (UBF) and rDNA intergenic spacer sequences (rDNA IGS). (d) Simultaneous immunostaining of UBF and RPA194, the largest subunit of Pol I. DNA was stained with DAPI in panels a, c, d. Scale bars: 5 µm on the large images and 1 µm on zoom-in images. (e) Schematic linear map of a single mammalian rDNA repeat unit. P_sp: spacer promoter; P: 47S promoter; coding region: 47S rRNA gene (black brackets label 18S, 5.8S and 28S rRNA coding regions); IGS: intergenic spacer. The positions of the UBF-bound region (in active genes) and the FISH hybridization probe are shown as grey and green lines below the rDNA scheme, respectively.

Figure 2

Colocalization analysis of nucleolar and extra-nucleolar UBF and Pol I. Colocalization analysis of UBF and Pol I (RPA194) signals in whole cells, nuclei and nucleoli in IMR90 (n = 23) and MEF (n = 11) cells summarized in box plots. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. Notches are defined as +/−1.58*IQR/sqrt(n).

Figure 3

Visualization of irregularly oriented active rRNA gene loops in the nucleolus. (a) GFP-UBF expressing MEF cells immunofluorescent labeled with antibodies against UBF. DNA was stained with DAPI. The upper row shows a single section, the lower a maximum intensity projection (MIP) of the same nucleolus. Scale bars: 5 µm on the large image and 1 µm on the zoom-in image. (b) 3D volume renderings of selected UBF rings indicated in panel a were rotated to comparable, planar orientation with their relative XYZ orientations in the nucleolus shown on the left. Scale bar: 0.2 µm.

Figure 4

Size determination of active rRNA gene loops. (a) Enlarged views of individual UBF foci from MEF cells. The loops were rotated to a planar position and the diameter of each loop was determined at three positions by 60° rotation as indicated. Distances between the peaks of the fluorescence profiles are shown below the images. Scale bar: 0.2 µm. (b) Box plots of ring diameter measurements. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots; data points are plotted as open circles. n = 36 (MEF) and n = 30 (IMR90) sample points. (c) Simplified hypothetic models of active rDNA chromatin loops with one or two rRNA genes per loop. Arrows label promoter and spacer promoter sites that demarcate the enhancer region, T labels the transcription termination site, red drops mark RNA polymerase I, green squares indicate UBF, and the discontinued blue line illustrates part of the IGS. The factors are not drawn to scale. The transcribed rRNA genes in the grey areas that can be correlated with the FC/DFC border represent the SIM view.