Repetitive DNA sequences, which constitute half the genome in some organisms, often undergo homologous recombination. This can instigate genomic instability due to gain or loss of DNA1. Assembly of DNA into silent chromatin is generally thought to serve as a mechanism ensuring repeat stability by limiting access to the recombination machinery2. Consistent with this notion, in the budding yeast Saccharomyces cerevisiae, stability of the highly repetitive ribosomal DNA (rDNA) sequences requires a Sir2-containing chromatin silencing complex that also inhibits transcription from foreign promoters and transposons inserted within the repeats by a process called rDNA silencing2-5. Here, we describe a protein network that stabilizes rDNA repeats of budding yeast via interactions between rDNA-associated silencing proteins and two inner nuclear membrane (INM) proteins. Deletion of either the INM or silencing proteins reduces perinuclear rDNA positioning, disrupts the nucleolus-nucleoplasm boundary, induces the formation of recombination foci, and destabilizes the repeats. In addition, artificial targeting of rDNA repeats to the INM suppresses the instability observed in cells lacking an rDNA-associated silencing protein typically required for peripheral tethering of the repeats. Moreover, in contrast to Sir2 and its associated nucleolar factors, the INM proteins are not required for rDNA silencing, indicating that Sir2-dependent silencing is not sufficient to inhibit recombination within the rDNA locus. These findings demonstrate a role for INM proteins in perinuclear chromosome localization and show that tethering to the nuclear periphery is required for rDNA repeat stability. The INM proteins studied here are conserved and have been implicated in chromosome organization in metazoans6,7. Our results therefore reveal an ancient mechanism in which interactions between INM and chromosomal proteins ensure genome stability.

Non-random positioning of chromosomal domains relative to each other and to nuclear landmarks is a common feature of eukaryotic genomes. In particular, the distribution of DNA loci relative to the nuclear periphery has been linked to both transcriptional activation and repression. Nuclear pores and other integral membrane protein complexes are key players in the dynamic organization of the genome in the nucleus, and recent advances in our understanding of the molecular networks that organize genomes at the nuclear periphery point to a further role for non-random locus positioning in DNA repair, recombination and stability.

Hypoxia and acidosis occur in a wide variety of physiological and pathological settings that include muscle stress, tumour development and ischaemic disorders. A central element in the adaptive response to cellular hypoxia is HIF (hypoxia-inducible factor), a transcription factor that activates an array of genes implicated in oxygen homeostasis, tumour vascularization and ischaemic preconditioning. HIF is activated by hypoxia, but undergoes degradation by the VHL (von Hippel-Lindau) tumour suppressor protein in the presence of oxygen. Here, we demonstrate that hypoxia induction or normoxic acidosis can neutralize the function of VHL by triggering its nucleolar sequestration, a regulatory mechanism of protein function that is observed rarely. VHL is confined to nucleoli until neutral pH conditions are reinstated. Nucleolar sequestration of VHL enables HIF to evade destruction in the presence of oxygen and activate its target genes. Our findings suggest that an increase in hydrogen ions elicits a transient and reversible loss of VHL function by promoting its nucleolar sequestration.

Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli 1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease. Various proteins self-organize via liquid-liquid phase separation (LLPS) into nucleolar subdomains, which are needed for highly stereotyped ribosome assembly 1,2. At fibrillar centres in the heart of mammalian nucleoli, the major rRNA molecules needed to assemble ribosomes are generated by Pol-I-dependent transcription of rRNA genes within ribosomal DNA (rDNA) repeats 1,3. Within rDNA, rRNA genes are separated by large intergenic spacers (IGSs) (Extended Data Fig. 1a). At nucleolar rRNA genes, Pol I synthesizes precursor rRNAs (pre-rRNAs) that are processed into mature 28S, 18S and 5.8S rRNA molecules as they migrate to the granular component at the nucleolar periphery. Outside nucleoli, Pol III synthesizes 5S rRNA molecules that are targeted to nucleoli for processing. Mature rRNAs are packaged into 40S and 60S ribosomal subunits for export to the cytoplasm. Traditionally, the nucleolar Pol I and nucleoplasmic Pol III are viewed as the sole mammalian RNA polymerases that directly mediate housekeeping ribosome biogenesis. Interestingly, in the budding yeast Saccharomyces cerevisiae, Pol II is physically enriched at rDNA IGSs, but this phenomenon is deleterious because it drives ageing without affecting rRNA expression 3-5. It is unclear whether nucleolar Pol II exists in higher organisms or directly promotes ribosome biogenesis in any species. Active Pol II at rDNA IGSs To investigate whether Pol II exists within human nucleoli, we first used immunofluorescence coupled to super-resolution microscopy. Within nucleoli, which we...