Nucleolar DNA Double-Strand Break Responses Underpinning rDNA Genomic Stability

Sluis, Marjolein van; McStay, Brian

doi:10.1016/j.tig.2019.07.001

Cited by 40 publications

(31 citation statements)

References 73 publications

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“…This conclusion was further supported by appearance of a subset of DSB foci associated with nucleolar markers (colocalization of 53BP1 foci with fibrillarin and UBF in Figure 6A and Supplementary Figure S6A–C ), suggesting that a subset of these breaks occurred within the rDNA itself. It is well established that persistent DSBs correlate with nucleolar disruption ( 32 , 67–69 ).…”

Section: Resultsmentioning

confidence: 99%

The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability

et al. 2020

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In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex. CX-5461 does not achieve this by preventing formation of the pre-initiation complex nor does it affect the promoter recruitment of the SL1 TBP complex or the HMGB-box upstream binding factor (UBF/UBTF). CX-5461 also does not prevent the subsequent recruitment of the initiation-competent RPI–Rrn3 complex. Rather, CX-5461 blocks promoter release of RPI–Rrn3, which remains irreversibly locked in the pre-initiation complex even after extensive drug removal. Unexpectedly, this results in an unproductive mode of RPI recruitment that correlates with the onset of nucleolar stress, inhibition of DNA replication, genome-wide DNA damage and cellular senescence. Our data demonstrate that the cytotoxicity of CX-5461 is at least in part the result of an irreversible inhibition of RPI transcription initiation and hence are of direct relevance to the design of improved strategies of chemotherapy.

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Section: Resultsmentioning

confidence: 99%

The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability

et al. 2020

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“…Released pre-rRNA species are processed and ribosome subunits assembled in the granular component (GC). Nucleoli are dynamic; induction of nucleolar stress by inhibition of RNA Pol I with actinomycin D (AMD), or by introduction of rDNA double strand breaks, results in rapid large-scale reorganization, including the formation of nucleolar caps (19). During cap formation, FC and DFC components move together with the rDNA to the nucleolar periphery (20).…”

Section: Significancementioning

confidence: 99%

NORs on human acrocentric chromosome p-arms are active by default and can associate with nucleoli independently of rDNA

Sluis

Vuuren

Mangan

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Nucleoli, the sites of ribosome biogenesis and the largest structures in human nuclei, form around nucleolar organizer regions (NORs) comprising ribosomal DNA (rDNA) arrays. NORs are located on the p-arms of the five human acrocentric chromosomes. Defining the rules of engagement between these p-arms and nucleoli takes on added significance as describing the three-dimensional organization of the human genome represents a major research goal. Here we used fluorescent in situ hybridization (FISH) and immuno-FISH on metaphase chromosomes from karyotypically normal primary and hTERT-immortalized human cell lines to catalog NORs in terms of their relative rDNA content and activity status. We demonstrate that a proportion of acrocentric p-arms in cell lines and from normal human donors have no detectable rDNA. Surprisingly, we found that all NORs with detectable rDNA are active, as defined by upstream binding factor loading. We determined the nucleolar association status of all NORs during interphase, and found that nucleolar association of acrocentric p-arms can occur independently of rDNA content, suggesting that sequences elsewhere on these chromosome arms drive nucleolar association. In established cancer lines, we characterize a variety of chromosomal rearrangements involving acrocentric p-arms and observe silent, rDNA-containing NORs that are dissociated from nucleoli. In conclusion, our findings indicate that within human nuclei, positioning of all 10 acrocentric chromosomes is dictated by nucleolar association. Furthermore, these nucleolar associations are buffered against interindividual variation in the distribution of rDNA.

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“…However, recent efforts to map the nucleolar proteome have revealed a plethora of proteins with roles beyond canonical nucleolar processes, including DNA repair and apoptosis (30)(31)(32). Further work has also shown the nucleolus as a dynamic hub capable of regulating protein function in response to specific stimuli, including DNA double-strand breaks (DSBs) (33)(34)(35).…”

Section: Significancementioning

confidence: 99%

Nucleolar localization of RAG1 modulates V(D)J recombination activity

Brecht

Liu

Beilinson

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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V(D)J recombination assembles and diversifies Ig and T cell receptor genes in developing B and T lymphocytes. The reaction is initiated by the RAG1-RAG2 protein complex which binds and cleaves at discrete gene segments in the antigen receptor loci. To identify mechanisms that regulate V(D)J recombination, we used proximity-dependent biotin identification to analyze the interactomes of full-length and truncated forms of RAG1 in pre-B cells. This revealed an association of RAG1 with numerous nucleolar proteins in a manner dependent on amino acids 216 to 383 and allowed identification of a motif required for nucleolar localization. Experiments in transformed pre-B cell lines and cultured primary pre-B cells reveal a strong correlation between disruption of nucleoli, reduced association of RAG1 with a nucleolar marker, and increased V(D)J recombination activity. Mutation of the RAG1 nucleolar localization motif boosts recombination while removal of the first 215 amino acids of RAG1, required for efficient egress from nucleoli, reduces recombination activity. Our findings indicate that nucleolar sequestration of RAG1 is a negative regulatory mechanism in V(D)J recombination and identify regions of the RAG1 N-terminal region that control nucleolar association and egress.

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Nucleolar DNA Double-Strand Break Responses Underpinning rDNA Genomic Stability

Cited by 40 publications

References 73 publications

The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability

The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability

NORs on human acrocentric chromosome p-arms are active by default and can associate with nucleoli independently of rDNA

Nucleolar localization of RAG1 modulates V(D)J recombination activity

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