Nucleolar Stress and Disease

Recent studies have shown that specific parts of the genome are more susceptible to DNA damage and therefore of particular interest in the context of genome instability and tumorigenesis.

In our research group, Nucleolar stress and disease, we investigate how a specialized, repetitive and highly unstable genomic region, namely the ribosomal RNA genes, is maintained and how its instability promotes tumorigenesis. These genes cluster in the largest substructure of the nucleus called the nucleolus and are essential for the production of ribosomes that synthesise proteins. In spite of the instability of ribosomal DNA (rDNA), its contribution to tumorigenesis is poorly understood.

To address this question, we study the specialised nucleolar DNA damage response (n-DDR) that functions to maintain the integrity of rDNA. We analyse the consequences of nDDR defects and aim to clarify its clinical value.

The study of DNA damage in specific genomic regions have been technically challenging in the past, but the discovery of CRISPR/Cas9 provided new opportunities.

To study DNA repair specifically in rDNA, we have engineered the osteosarcoma cell line (U2OS) to stably express the Cas9 endonuclease and a DNA repair protein, NBS1-GFP, that marks rDNA DSBs (Korsholm et al., 2019). By introducing guide RNAs targeting rDNA, we can create site specific DNA damage recognized by NBS1-GFP and we can quantify rDNA repair by measuring GFP-intensity in the nucleolus (See Figure 1A and B) 

Ongoing research projects:

The nucleolar DNA damage response 
The nucleolar DNA damage response is unique and in addition to break detection, signalling and repair it also involves transcriptional silencing and a prominent restructuring of the nucleolus that leads to accumulation of rDNA at the nucleolar periphery in so-called nucleolar caps (See Figure 1B).

We use siRNA screening and proximity-based labelling techniques to obtain a deeper understanding of the nucleolar DDR and to identify yet uncharacterised nDDR factors. We aim to identify the biological function of candidate proteins in the nDDR and to determine their anti-cancer potential.

Mutations in a number of well-characterized DDR proteins lead to hyper-instability of rDNA. By depletion of such DDR proteins, we also study mechanisms maintaining rDNA stability and the faulty mechanisms that become activated in their absence and lead to rDNA destabilisation.

The nucleolus and cancer 
Nucleolar activity is upregulated in almost all cancers and altered nucleolar morphology is a characteristic of cancer. In spite of this its clinical use is very limited.

We aim to establish methods that will enable evaluation of nucleolar status in cancer patients and thereby allow new investigation of its predictive and/or prognostic value.  

Selected publications:

Korsholm L, Gal Z, Nieto B, Quevedo Oliver, Boukoura S, Lund C, Larsen DH: Recent advances in the nucleolar responses to DNA double-strand breaks. Nucleic Acids Res 2020;48(17):9449-9461

Korsholm LM, Gál Z, Lin L, Quevedo O, Ahmad DA, Dulina E, Luo Y, Bartek J, Larsen DH: Double-strand breaks in ribosomal RNA genes activate a distinct signaling and chromatin response to facilitate nucleolar restructuring and repair. Nucleic Acids Res 2019;47(15):8019-8035

Larsen DH, Stucki M: Nucleolar responses to DNA double-strand breaks. Nucleic Acids Res 2016;44(2):538-544

Larsen DH, Hari F, Clapperton JA, Gwerder M, Gutsche K, Altmeyer M, Jungmichel S, Toledo LI, Fink D, Rask MB, Grøfte M, Lukas C, Nielsen ML, Smerdon SJ, Lukas J, Stucki M: The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage. Nat Cell Biol 2014;16(8):792-803

Toledo LI, Altmeyer M, Rask MB, Lukas C, Larsen DH, Povlsen LK, Bekker-Jensen S, Mailand N, Bartek J, Lukas J: ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 2013;155(5):1088-1103