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

Abstract

Eukaryotic chromosomes are folded into hierarchical domains, enabling the organization of the genome into functional compartments. Nuclear periphery and nucleolus are two nuclear landmarks thought to contribute to repressive chromosome architecture. However, while the role of nuclear lamina (NL) in genome organization has been well documented, the function of the nucleolus remains under-investigated due to the lack of methods for genome-wide maps of nucleolar associated domains (NADs). Here we established a method based on a Dam-fused engineered nucleolar histone H2B that marks DNA contacting the nucleolus. NAD-maps of ESCs and neural progenitors revealed layers of genome compartmentalization with distinct, repressive chromatin states based on the interaction with the nucleolus, NL, or both. NADs showed higher H3K9me2 and lower H3K27me3 content than regions exclusively interacting with NL. Upon ESC differentiation, chromosomes around the nucleolus acquire a more compact, rigid architecture whereas NADs specific for ESCs decrease their interaction strength within the repressive B-compartment strength, unlocking neural genes from repressive nuclear environment. The methodologies here developed will make possible to include the contribution of the nucleolus in future studies investigating the relationship between nuclear space and genome function.

Nucleolar-based Dux repression is essential for 2-cell stage exit

“…Future work is needed to understand if these distinct NAD classes show differences in their association strength with the nucleolus, or if their activation depends on further mechanisms or factors upon dissociation. For example, neuronal NAD genes detach from the nucleolus upon neural progenitor cell differentiation yet do not yet become activated, supporting the model that their release might poise them for later expression (Bersaglieri et al 2020).…”

Stress as a Chromatin Landscape Architect

“…Additionally, a number of studies have detected HS- and HSF1-dependent activation of typically silent constitutive heterochromatin regions, the satellite repeats on human chromosome 9 ( Jolly et al, 2002 , 2004 ; Rizzi et al, 2003 ; Valgardsdottir et al, 2008 ; Sengupta et al, 2009 ; Eymery et al, 2010 ; Pezer and Ugarkovic, 2012 ; Feliciello et al, 2015 ; Col et al, 2017 ; Feliciello et al, 2020 ). These repeats are often closely associated with the nucleoli periphery, one of the major locations of heterochromatin domains within the nucleus ( Politz et al, 2016 ; Vertii et al, 2019 ; Bersaglieri et al, 2020 ; Bury et al, 2020 ; Wang et al, 2021 ). Satellite III repeats are located in the pericentromeric regions of acrocentric chromosomes and in response to HS produce long noncoding RNA transcripts that accumulate at the site of transcription, primarily at chromosome 9, and help to mediate HS response.…”

Visualization of chromatin higher-order structures and dynamics in live cells

“…These limitations led to the establishment of another novel method, called 'Nucleolar-DamID'. By applying the existing DamID, Dam is fused with the histone, H2B, where in this adaptation, a nucleolar localization signal (NoLS, RKK RKKK) is inserted at the C-terminus (60). Through these sequencing approaches, the genome-wide maps and heterochromatic characteristics of LADs and NADs have been defined.…”

“…This implicates that there seems to be no possible candidate molecules as scaffold to bind in and around the nucleoli, while there are the stable lamins under the nuclear envelope for mapping LADs. However, Nucleolar-DamID using Dam-fused and NoLS inserted histone, which can bind DNA sequences near nucleoli without motif specificity, has recently been proposed (60), thereby applying it may allow insight to be gained into the dynamics of NADs, as has been done for LADs. In addition to Nucleolar-DamID, other creative technologies for visualizing NADs need to be established.…”