Double-strand breaks in ribosomal RNA genes activate a distinct signaling and chromatin response to facilitate nucleolar restructuring and repair

Nucleic Acids Res. 2019 Sep 5;47(15):8019-8035. doi: 10.1093/nar/gkz518.

Abstract

The nucleolus is a nuclear sub-domain containing the most highly transcribed genes in the genome. Hundreds of human ribosomal RNA (rRNA) genes, located in the nucleolus, rely on constant maintenance. DNA double-strand breaks (DSBs) in rRNA genes activate the ATM kinase, repress rRNA transcription and induce nucleolar cap formation. Yet how ribosomal-DNA (rDNA) lesions are detected and processed remains elusive. Here, we use CRISPR/Cas9-mediated induction of DSBs and report a chromatin response unique to rDNA depending on ATM-phosphorylation of the nucleolar protein TCOF1 and recruitment of the MRE11-RAD50-NBS1 (MRN) complex via the NBS1-subunit. NBS1- and MRE11-depleted cells fail to suppress rRNA transcription and to translocate rDNA into nucleolar caps. Furthermore, the DNA damage response (DDR) kinase ATR operates downstream of the ATM-TCOF1-MRN interplay and is required to fully suppress rRNA transcription and complete DSB-induced nucleolar restructuring. Unexpectedly, we find that DSBs in rDNA neither activate checkpoint kinases CHK1/CHK2 nor halt cell-cycle progression, yet the nucleolar-DDR protects against genomic aberrations and cell death. Our data highlight the concept of a specialized nucleolar DNA damage response (n-DDR) with a distinct protein composition, spatial organization and checkpoint communication. The n-DDR maintains integrity of ribosomal RNA genes, with implications for cell physiology and disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acid Anhydride Hydrolases
  • Ataxia Telangiectasia Mutated Proteins / genetics
  • Ataxia Telangiectasia Mutated Proteins / metabolism
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Cell Line, Tumor
  • Cell Nucleolus / metabolism*
  • Checkpoint Kinase 1 / genetics
  • Checkpoint Kinase 1 / metabolism
  • Chromatin / genetics*
  • DNA Breaks, Double-Stranded*
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism
  • DNA Repair*
  • DNA, Ribosomal / genetics
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Genes, rRNA / genetics*
  • HEK293 Cells
  • Humans
  • MRE11 Homologue Protein / genetics
  • MRE11 Homologue Protein / metabolism
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phosphorylation
  • RNA Interference
  • Signal Transduction / genetics
  • Transcription, Genetic

Substances

  • Cell Cycle Proteins
  • Chromatin
  • DNA, Ribosomal
  • DNA-Binding Proteins
  • MRE11 protein, human
  • NBN protein, human
  • Nuclear Proteins
  • Phosphoproteins
  • TCOF1 protein, human
  • Ataxia Telangiectasia Mutated Proteins
  • Checkpoint Kinase 1
  • MRE11 Homologue Protein
  • Acid Anhydride Hydrolases
  • Rad50 protein, human
  • DNA Repair Enzymes