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.

Mutual dependency between lncRNA LETN and protein NPM1 in controlling the nucleolar structure and functions sustaining cell proliferation

“…2f ), apparently the GC. This strong colocalization was nicely supported by the Pearson’s correlation coefficients (PCC) 46 between the distributions of the two factors in multiple cells (Supplementary information, Fig. S 13c ).…”

Bridging scales: From cell biology to physiology using in situ single-cell technologies

“…New avenues of spatial multi-omics are just now being explored and could have a great impact on the construction of atlases with many maps. Furthermore, these technologies open up new avenues to study gene perturbations (Wang et al, 2019), cell lineage tracing (Chen et al, 2018;Frieda et al, 2017), and other aspects of functional DNA and RNA biology (Cai et al, 2020;Maiser et al, 2020).…”

The Ribosomal Gene Loci—The Power behind the Throne

“…A number of factors, including TTF-1 and c-Myc, have also been identified as being involved in this loop formation. Direct evidence of active rDNA looping was recently presented by Maiser and colleagues [ 81 ]. Ray et al showed that, in human cells, Top IIα altered the rDNA topology at the rRNA core promoter and this was required for the assembly of functional PICs [ 58 ].…”