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

Abstract

24Background: Heterochromatin in eukaryotic interphase cells frequently localizes to the nucleolar 25 periphery (nucleolus-associated domains, NADs) and the nuclear lamina (lamina-associated 26 domains, LADs). Gene expression in somatic cell NADs is generally low, but NADs have not 27 been characterized in mammalian stem cells. 28Results: Here, we generated the first genome-wide map of NADs in mouse embryonic stem cells 29 (mESCs) via deep sequencing of chromatin associated with biochemically-purified nucleoli. 30As we had observed in mouse embryonic fibroblasts (MEFs), the large Type I subset of NADs 31 overlaps with constitutive LADs and is enriched for features of constitutive heterochromatin, 32including late replication timing and low gene density and expression levels. Conversely, the 33 Type II NAD subset overlaps with loci that are not lamina-associated, but in mESCs, Type II 34NADs are much less abundant than in MEFs. mESC NADs are also much less enriched in 35H3K27me3 modified regions than are NADs in MEFs. Additionally, comparision of MEF and 36 mESC NADs revealed enrichment of developmentally regulated genes in cell type-specific 37NADs. Together, these data indicate that NADs are a developmentally dynamic component of 38 heterochromatin. 39Conclusions: These studies implicate association with the nucleolar periphery as a mechanism 40 for developmentally-regulated gene silencing, and will facilitate future studies of NADs during 41 mESC differentiation. 42 43 3 are accompanied by spatial separation: heterochromatin is mainly found at the nuclear periphery 47 and nucleolar periphery, where they comprise nucleolus-associated domains (NADs) (Németh et 48 al. 2010; van Koningsbruggen et al. 2010) and lamina-associated domains (LADs) (Pickersgill et 49 al. 2006;Guelen et al. 2008;Peric-Hupkes et al. 2010), respectively. Studies in multiple 50 organisms indicate that sequestration of heterochromatin to the nuclear and nucleolar peripheries 51

Regulation and Roles of the Nucleolus in Embryonic Stem Cells: From Ribosome Biogenesis to Genome Organization

“…Review (Bizhanova et al, 2020;Lu et al, 2020), mouse embryonic fibroblasts (MEFs) (Vertii et al, 2019), a few human somatic cell lines (Dillinger et al, 2017;Nemeth et al, 2010;van Koningsbruggen et al, 2010), and the plant Arabidopsis thaliana (Pontvianne et al, 2016) (Table 1). Although this is the only method that until now can be used for the mapping of NADs at the genome-wide level, some limitations should be taken into consideration.…”

“…This discrepancy could also be due to the different methods applied for the purification of nucleoli of mESCs. Indeed, while in one work nucleoli were purified from cross-linked mESCs (Bizhanova et al, 2020), in other study this step was omitted (Lu et al, 2020). The two initial independent parallel studies that mapped NADs from biochemically purified nucleoli of HeLa, IMR90, and HT1080 human cell lines revealed that NADs contain repressive histone modifications and are enriched in olfactory receptor genes, zincfinger genes, immunoglobulin gene families, and 5S RNA genes (Nemeth et al, 2010;van Koningsbruggen et al, 2010).…”

Computational 3D genome modeling using Chrom3D

“…NADs have been subdivided into type I and II NADs based on their chromatin composition and the type of LAD they correspond to [52,54]. Type-I NADs often associate with the nucleolar periphery and the lamina and contain marks of constitutive heterochromatin.…”

Genome-wide maps of nucleolus interactions reveal distinct layers of repressive chromatin domains

“…Although these methods were based on completely different methodologies, they both revealed similar features of chromosome organization around the nucleolus in ESCs and NPCs. Previous methods for NAD identification used sonication-based biochemical purification of nucleoli, a methodology that is subjected to a certain variation in nucleoli preparation between different cell types and relatively biased toward sonication-resistant heterochromatin (Bizhanova et al, 2020; Dillinger et al, 2017; Lu et al, 2020; Nemeth et al, 2010; van Koningsbruggen et al, 2010; Vertii et al, 2019). Relative to this methodology, the application of Nucleolar-DamID and HiC-rDNA has no bias for any kind of chromatin state and does not vary between cell types, thereby allowing direct comparisons of chromosome architecture around the nucleolus between distinct cell states.…”

“…First, since the heterochromatin is generally resistant to sonication (Becker et al, 2017), the identification of NADs upon sequencing of nucleoli purified through sonication can be biased toward repressive chromatin domains. Secondly, the experimental procedures to isolate nucleoli can be subjected to a certain variation, generating highly divergent NAD maps, even from the same cell types (Bizhanova et al, 2020; Lu et al, 2020). Third, it is difficult to achieve the purification of nucleoli in cells with open genome, such as embryonic stem cells (ESCs), unless protein-DNA crosslinking reagents are used, with a consequent extension of sonication time.…”