Two contrasting classes of nucleolus-associated domains in mouse fibroblast heterochromatin

Abstract

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining nucleolus-associated domains (NADs) and lamina-associated domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, owing to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery and the nuclear lamina, and generally display characteristics of constitutive heterochromatin, including late DNA replication, enrichment of H3K9me3, and little gene expression. In contrast, Type II NADs associate with nucleoli but do not overlap with LADs. Type II NADs tend to replicate earlier, display greater gene expression, and are more often enriched in H3K27me3 than Type I NADs. The nucleolar associations of both classes of NADs were confirmed via DNA-FISH, which also detected Type I but not Type II probes enriched at the nuclear lamina. Type II NADs are enriched in distinct gene classes, including factors important for differentiation and development. In keeping with this, we observed that a Type II NAD is developmentally regulated, and present in MEFs but not in undifferentiated embryonic stem (ES) cells.

Distinct features of nucleolus-associated domains in mouse embryonic stem cells

“…We sequenced approximately 50 million reads from each nucleolar and genomic DNA sample. We note that subsampling analyses of larger MEF datasets previously showed that the number of peaks detected had reached a plateau at this sequencing depth (Vertii et al 2019). Genomic reads were mostly uniformly distributed across the genome, whereas nucleolar reads contained well-defined peaks and valleys, with peaks overlapping known heterochromatic regions, such as constitutive LADs (cLADs) (Peric-Hupkes et al 2010) and late replicating regions (Hiratani et al 2010) (Fig 2A, B).…”

“…cLADs were previously defined as LADs that are lamina-associated in mESCs, and also in neural precursor cells (NPCs) and astrocytes differentiated from these mESCs (Peric-Hupkes et al 2010). Previous studies of NADs have identified frequent overlap of NADs with LADs (van Koningsbruggen et al 2010;Németh et al 2010;Dillinger et al 2017;Vertii et al 2019) and with late-replicating regions (Dillinger et al 2017;Vertii et al 2019), thus we concluded that the nucleolar reads are enriched with bona fide nucleolar heterochromatic regions in F121-9 mESCs.…”

“…We isolated nucleoli from formaldehydecrosslinked hybrid F121-9 mES cells using methods previously shown to yield reproducible data using MEF cells (Vertii et al 2019). In those studies, crosslinked and non-crosslinked MEFs were directly compared, and shown to yield highly overlapping results, with crosslinked samples detecting a greater proportion of the genome associated with nucleoli (Vertii et al 2019). This suggests crosslinking could assist detection of weak or transient nucleolar interactions.…”

“…NADfinder (Vertii et al 2019) to call NAD peaks in F121-9 mESCs. This software uses local background correction, which was important for detection of validated NAD peaks distal from centromeres in MEFs (Vertii et al 2019).…”

“…LADs have been mapped and studied in multiple species and cell types (Pickersgill et al 2006;Guelen et al 2008;Peric-Hupkes et al 2010; Kind et al 2013;Borsos et al 2019). In contrast, NADs have been characterized in a few human somatic cell lines (Németh et al 2010;van Koningsbruggen et al 2010;Dillinger et al 2017), in the plant Arabidopsis thaliana (Pontvianne et al 2016), and recently, in mouse embryonic fibroblasts (MEFs) (Vertii et al 2019). Several experiments indicate that LADs can be redistributed to the nucleolar periphery after passage through mitosis, and vice versa (van Koningsbruggen et al 2010;Kind et al 2013).…”