In eukaryotes, the genes encoding ribosomal RNAs (rDNA) exist in two distinct epigenetic states that can be distinguished by a specific chromatin structure that is maintained throughout the cell cycle and is inherited from one cell to another. The fact that even in proliferating cells with a high demand of protein synthesis a fraction of rDNA is silenced provides a unique possibility to decipher the mechanism underlying epigenetic regulation of rDNA. This chapter summarizes our knowledge of the molecular mechanisms that establish and propagate the epigenetic state of rRNA genes, unraveling a complex interplay of DNA methyltransferases and histone-modifying enzymes that act in concert with chromatin remodeling complexes and RNA-guided mechanisms to define the transcriptional state of rDNA. We also review the critical role of the RNA polymerase I transcription factor UBF in the formation of active nucleolar organizer regions (NORs) and maintenance of the euchromatic state of rRNA genes.

DNA double-strand breaks (DSBs) are repaired by two main pathways: nonhomologous end-joining and homologous recombination (HR). Repair pathway choice is thought to be determined by cell cycle timing and chromatin context. Nucleoli, prominent nuclear subdomains and sites of ribosome biogenesis, form around nucleolar organizer regions (NORs) that contain rDNA arrays located on human acrocentric chromosome p-arms. Actively transcribed rDNA repeats are positioned within the interior of the nucleolus, whereas sequences proximal and distal to NORs are packaged as heterochromatin located at the nucleolar periphery. NORs provide an opportunity to investigate the DSB response at highly transcribed, repetitive, and essential loci. Targeted introduction of DSBs into rDNA, but not abutting sequences, results in ATM-dependent inhibition of their transcription by RNA polymerase I. This is coupled with movement of rDNA from the nucleolar interior to anchoring points at the periphery. Reorganization renders rDNA accessible to repair factors normally excluded from nucleoli. Importantly, DSBs within rDNA recruit the HR machinery throughout the cell cycle. Additionally, unscheduled DNA synthesis, consistent with HR at damaged NORs, can be observed in G1 cells. These results suggest that HR can be templated in cis and suggest a role for chromosomal context in the maintenance of NOR genomic stability.

The HMG box containing protein UBF binds to the promoter of vertebrate ribosomal repeats and is required for their transcription by RNA polymerase I in vitro. UBF can also bind in vitro to a variety of sequences found across the intergenic spacer in Xenopus and mammalian ribosomal DNA (rDNA) repeats. The high abundance of UBF, its colocalization with rDNA in vivo, and its DNA binding characteristics, suggest that it plays a more generalized structural role over the rDNA repeat. Until now this view has not been supported by any in vivo data. Here, we utilize chromatin immunoprecipitation from a highly enriched nucleolar chromatin fraction to show for the first time that UBF binding in vivo is not restricted to known regulatory sequences but extends across the entire intergenic spacer and transcribed region of Xenopus, human, and mouse rDNA repeats. These results are consistent with a structural role for UBF at active nucleolar organizer regions in addition to its recognized role in stable transcription complex formation at the promoter.