Psoralen Crosslinking-Chromatin Endogenous Cleavage Assay to Examine Histone DNA Interactions of Active and Inactive rRNA Genes

Abstract

In the nucleoli of eukaryotic cells, the multiple copies of ribosomal RNA genes (rRNA genes) coexist in two different forms that have distinct characteristics: transcribed (active) and non-transcribed (inactive) units. "Active" rRNA genes are loaded with RNA polymerase I and are largely depleted of nucleosomes, whereas "inactive" rRNA genes are covered with two copies of the four histone proteins that are folded in nucleosomes. A third form of chromatin is observed in Saccharomyces cerevisiae (here called as yeast) arrested in the G1 phase of the cell cycle. In yeast synchronized before DNA replication, nucleosomes are also absent in the non-transcribed rRNA genes, which are described as "open" units.The presence of two distinct groups of rRNA genes compromises the interpretation of standard biochemical assays that are employed to study the structure of chromatin during DNA transcription, DNA replication, and DNA repair. This chapter describes protocols to investigate the association of histone proteins with rRNA genes in yeast. In addition, it provides a comprehensive list of studies that applied psoralen photo-crosslinking to follow the structure of rRNA gene chromatin in a variety of high eukaryotic cells.

Keywords: Chromatin; Chromatin endogenous cleavage; Histones; Nucleolus; Nucleosomes; Photo-crosslinking; Psoralen; RNA polymerase I; Ribosomal genes; rDNA; rRNA genes.