Anastasia McKinlay

Understanding how the three-dimensional (3D) organization of the genome relates to chromatin and epigenetic landscapes requires methods capable of measuring chromosomal DNA interactions and chromatin modifications in an integrated manner. Current approaches typically use separate assays to assess chromatin conformation and epigenetic state, thus these features are not assessed on the same DNA molecules. Here, we describe Epi-PoreC, an Oxford Nanopore long-read sequencing-based method that combines the chromosome conformation capture method, PoreC with Fiber-seq, a method for detecting accessible chromatin regions based on their ability to be labeled using an exogenous adenosine methyltransferase. The positions of chromosome contacts, 6-methyladenosines, and endogenous 5-methylcytosines are then detected by nanopore sequencing, all on the same DNA molecules. Using Arabidopsis thaliana nuclei, we demonstrate that Epi-PoreC yields genome-wide chromatin contact maps comparable to those obtained by standard PoreC and DNA methylation and accessibility profiles comparable to standard Fiber-seq experiments. Epi-PoreC is thus an efficient means for conducting epigenetic and 3D genome profiling on individual DNA sequences in a single assay. Multidisciplinary Understanding genome regulation is aided by integrating information about chromosome architecture, DNA methylation, and chromatin accessibility. However, most experimental methods measure these features separately, precluding their detection on the same DNA molecules. Here we describe Epi-PoreC, an nanopore sequencing–based method that simultaneously measures chromatin contacts, DNA methylation, and chromatin accessibility in a single assay. The approach integrates chromosome conformation capture (PoreC) with Fiber-seq–based adenine methyltransferase labeling of accessible DNA. Using Arabidopsis thaliana nuclei, we demonstrate that Epi-PoreC produces chromatin contact maps highly concordant with those obtained by standard PoreC assays and DNA methylation and accessibility profiles comparable to those obtained by Fiber-seq. Because long nanopore sequencing reads can traverse multi-way chromatin ligation products, Epi-PoreC enables detection of chromatin interactions and modifications on the same DNA molecules, yielding genome folding and epigenetic landscape information relevant to studies of gene and genome function. Epi-PoreC therefore expands the toolkit for multi-omic singlemolecule genome analysis, particularly for complex or repetitive genomic loci where long-read sequencing is needed to identify sub-repeats or sequence elements that map to specific chromosomal locations. Method summary: Epi-PoreC integrates Fiber-seq–based adenine methyltransferase labeling of accessible DNA with the PoreC chromosome conformation capture protocol. Long-read Oxford Nanopore sequencing then allows the simultaneously detection of chromatin contacts, cytosine methylation and chromatin accessibility on individual DNA molecules. The method thus provides an efficient workflow for integrated single-molecule analyses of genome architecture and epigenetic state.

Arabidopsis thaliana has two ribosomal RNA (rRNA) gene loci, nucleolus organizer regions NOR2 and NOR4 , whose complete sequences are missing in current genome assemblies. Ultralong DNA sequences assembled using an unconventional approach yielded ~5.5- and 3.9-Mbp sequences for NOR2 and NOR4 in the reference strain, Col-0. The distinct rRNA gene subtype compositions of the NORs enabled the positional mapping of their active and inactive regions, using RNA sequencing to identify subtype-specific transcripts and DNA sequencing to identify subtypes associated with flow-sorted nucleoli. Comparisons of wild-type and silencing-defective plants revealed that most rRNA gene activity occurs in the central region of NOR4 , whereas most, but not all, genes of NOR2 are epigenetically silenced. Intervals of low CG and CHG methylation overlap regions where gene activity and gene subtype homogenization are high. Collectively, the data reveal the genetic and epigenetic landscapes underlying nucleolar dominance (differential NOR activity) and implicate transcription as a driver of rRNA gene concerted evolution.