Poster Presentation 43rd Lorne Genome Conference 2022

A cellular model for the study of rDNA class switching (#158)

Anran Lin 1 , Tess Morrison 1 , Nadine Hein 1 , Amee J George 1 2 , Dominik Spensberger 3 , Kate M Hannan 1 4 , Ross D Hannan 1 5 , Rita Ferreira 5
  1. Division of Genome Sciences and Cancer, John Curtin School of Medical Research, Australian National University, Canberra, ACT, Australia
  2. ANU Centre for Therapeutic Discovery, John Curtin School of Medical Research, Australian National University, Australia , Canberra, ACT, Australia
  3. ANU Phenogenomics Targeting Facility, Australian National University, Canberra, ACT, Australia
  4. Dept. Biochemistry & Molecular Biology, Monash University, Monash University, Melbourne, Victoria, Australia
  5. Division of Cancer Research, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia

The ribosomal DNA gene (rDNA) encodes the central RNA component of ribosomes, ribosomal RNA (rRNA). rDNA is a multicopy gene (200-400 copies per haploid genome) organized in large arrays of tandem repeats. rRNA transcription, in the nucleolus, is mediated by RNA Polymerase I (PolI) and inhibition of PolI transcription affects nucleolar morphology. rDNA repeats exist in three distinct chromatin configurations: transcriptionally active (euchromatic), pseudo-silent (heterochromatic, hypo-CpG methylated) and silent (heterochromatic and hyper-CpG methylated). In a normal cell, only ~50% of the total rDNA repeats are actively transcribed. We recently demonstrated that malignant transformation is associated with the conversion of pseudo-silent to transcriptionally active rDNA repeats mediated by the binding of the cytoarchitectural transcription factor Upstream Binding Factor (UBF). This process is called rDNA class switching.

 

In order to study rDNA class switching, we established a cellular model where UBF binding to rDNA can be visualized. Using CRISPR/Cas9 gene-editing technology we knocked in EGFP in the endogenous UBF locus in U2OShuman osteosarcoma  cells. The UBF-EGFP fusion protein retains the expression and function of the wild-type UBF protein.

 

To validate that UBF-EGFP can serve as a biomarker for transcriptionally active rDNA repeats, we’ve mimicked the inactivation of transcriptionally active rDNA repeats by knocking down (KD) genes involved in PolI-mediated transcription. Using small interference RNA (siRNA) we’ve KD 5 PolI-associated genes (e.g. POLRIA, RRN3, TAF1A) and 7 genes known to affect nucleolar morphology. The effect of individual gene KD on UBF-EGFP intensity and nucleolar morphology was visualized by high content imaging microscopy. This data set is being used to develop an analysis algorithm to quantify changes in UBF-EGFP intensity that correlate with changes in the number of transcriptionally active rDNA repeats.

 

Therefore, the U2OS UBF-EGFP cell line can be used as a model system to study the mechanism of rDNA class switching.