Positional mapping of active versus silenced rRNA gene clusters within A. thaliana nucleolus organizer regions

Abstract Nucleolus organizer regions, or NORs, are chromosomal loci at which ribosomal RNA (rRNA) genes are repeated in long tandem arrays, spanning millions of basepairs. In Arabidopsis thaliana, NORs are located at the distal ends of Chr2 and Chr4 and consist of hundreds of rRNA genes. Individual rRNA genes are too similar to discriminate from one another using short-read sequencing approaches, which has hindered the ability to understand how individual rRNA genes within a NOR are regulated. However, using Oxford Nanopore sequencing technology to obtain ultra-long reads, we succeeded in assembling the two Arabidopsis thaliana NORs, NOR2, and NOR4. Knowing the positions of more than 70 rRNA gene subtypes, we then used RNA sequencing, and fluorescence-activated nucleolus sorting, to determine which regions of the NORs are transcriptionally active. Complementary assays, including cytosine methylation detection, DNaseI accessibility, and single-molecule long-read sequencing of chromatin fibers (Fiber-seq), confirm that active rRNA genes, primarily located in the center of NOR4, exist in a hypomethylated and open chromatin state. To understand how NOR regions interact with other genomic loci, potentially playing a role in regional NOR activation or silencing, we have used Oxford Nanopore Pore-C, which couples chromatin conformation capture with long-read sequencing. This analysis revealed that NORs do not exhibit strong interactions with other loci but form separate compartments that encompass each NOR on the short arms of Chromosomes 2 and 4. Biography Anastasia McKinlay is a Research Associate in Prof. Craig Pikaard’s lab, in the Department of Biology, Indiana University. With a background in molecular biology and genomics, she adapts ultra-long sequencing technologies and develops tools to study the highly repetitive ribosomal RNA gene-containing genomic loci, known as nucleolus organizer regions, or NORs.