Suppressing the homologous recombination of repetitive DNA sequences is important for maintaining genome stability, and packaging of repeat DNA into silent chromatin was generally thought to protect it from recombination. Here, yeast ribosomal DNA (rDNA) repetitive sequences are shown to associate with the nuclear periphery via inner nuclear membrane proteins, and this tethering is required for rDNA stability. Sir2-dependent silencing is not sufficient to inhibit rDNA recombination. The inner nuclear membrane proteins involved are conserved and have been implicated in chromosome organization in metazoans. These results therefore reveal an ancient mechanism in which interactions between inner nuclear membrane proteins and chromosomal proteins ensure genome stability. Suppressing the homologous recombination of repetitive DNA sequences is important for maintaining genome stability, and packaging of repeat DNA into silent chromatin was generally thought to protect it from recombination. Yeast ribosomal DNA (rDNA) repetitive sequences are shown to associate with the nuclear periphery via inner nuclear membrane proteins, and this tethering is required for rDNA stability. Sir2-dependent silencing is not sufficient to inhibit rDNA recombination. Repetitive DNA sequences, which constitute half the genome in some organisms, often undergo homologous recombination. This can instigate genomic instability resulting from a 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 is the observation, in the budding yeast Saccharomyces cerevisiae, that 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 silencing2,3,4,5. Here we describe a protein network that stabilizes rDNA repeats of budding yeast by means of interactions between rDNA-associated silencing proteins and two proteins of the inner nuclear membrane (INM). 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 that is 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 the perinuclear localization of chromosomes and show that tethering to the nuclear periphery is required for the stability of rDNA repeats. 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 proteins and chromosomal proteins ensure genome stability.

Inactivating mutations in the von Hippel-Lindau (VHL) tumor suppressor gene are associated with clear cell renal cell carcinoma (VHL^−/− RCC), the most frequent malignancy of the human kidney. The VHL protein targets the α subunits of hypoxia-inducible factor (HIF) transcription factor for ubiquitination and degradation. VHL^−/− RCC cells fail to degrade HIF resulting in the constitutive activation of its target genes, a process that is required for tumorigenesis. We recently reported that HIF activates the transforming growth factor-α/epidermal growth factor receptor (TGF-α/EGFR) pathway in VHL-defective RCC cells. Here, we show that short hairpin RNA (shRNA)–mediated inhibition of EGFR is sufficient to abolish HIF-dependent tumorigenesis in multiple VHL^−/− RCC cell lines. The 2α form of HIF (HIF-2α), but not HIF-1α, drives in vitro and in vivo tumorigenesis of VHL^−/− RCC cells by specifically activating the TGF-α/EGFR pathway. Transient incubation of VHL^−/− RCC cell lines with small interfering RNA directed against EGFR prevents autonomous growth in two-dimensional culture as well as the ability of these cells to form dense spheroids in a three-dimensional in vitro tumor assay. Stable expression of shRNA against EGFR does not alter characteristics associated with VHL loss including constitutive production of HIF targets and defects in fibronectin deposition. In spite of this, silencing of EGFR efficiently abolishes in vivo tumor growth of VHL loss RCC cells. These data identify EGFR as a critical determinant of HIF-2α-dependent tumorigenesis and show at the molecular level that EGFR remains a credible target for therapeutic strategies against VHL^−/− renal carcinoma.

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL^-/- RCC). VHL^-/- RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL^-/- RCC cells by activating the transforming growth factor-α (TGF-α)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL^-/- RCC cells. TGF-α is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL^-/- RCC cells is a direct consequence of HIF activation. In contrast to TGF-α, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL^-/- RCC cells. VHL^-/- RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-α and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.