Maintenance of highly compact heterochromatin at ribosomal DNA (rDNA) segments is essential to prevent homologous recombination between rDNA repeats and for preserving genomic stability and nucleolar architecture. Here, we investigated the role of Sirtuin 7 (Sirt7) in the regulation of rDNA chromatin structure, rDNA repeat stability and nucleolar organization. We found that Sirt7 mediates heterochromatin formation at rRNA genes through recruitment of DNA methyltransferase 1 and another member of the sirtuin family, Sirt1. Lack of Sirt7 leads to nucleolar fragmentation associated with hypomethylation of rDNA and hyperacetylation of histones at rDNA loci resulting in rDNA and genomic instability. Our findings suggest a novel role of Sirt7 in preventing cellular transformation by mediating maintenance of rDNA repeats and nucleolar integrity. (C) 2017 Elsevier Inc. All rights reserved.