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Analysis of the nucleolus in genome organization and function

Periodic Reporting for period 4 - NucleolusChromatin (Analysis of the nucleolus in genome organization and function)

Reporting period: 2023-03-01 to 2024-08-31

In eukaryotic cells, the higher-order organization of genomes is functionally important to ensure correct execution of gene expression programs. For instance, as cells differentiate into specialized cell types, chromosomes undergo diverse structural and organizational changes that affect gene expression and other cellular functions. However, how this process is achieved is still poorly understood. The elucidation of the mechanisms that control the spatial architecture of the genome and its contribution to gene regulation is a key open issue in molecular biology, relevant for physiological and pathological processes.
One important aspect of this regulation is the spatial positioning of genes in the nuclear space. This is exemplified by the location of repressive chromatin domains at the nuclear periphery (i.e. lamina associated domains, LADs) or around nucleoli (nucleolar associated domains, NADs). While LADs have been extensively studied, including at the single cell level, the field has eagerly awaited a deeper understanding of NADs, which have remained underinvestigated mainly due to technical limitations in identifying DNA sequences associated with a membraneless compartment like the nucleolus. However, to fully understand how genome organization regulates chromatin and gene expression states, it is necessary to obtain a comprehensive functional map of genome compartmentalization.
In this project we aimed to develop methods to identify and characterize NADs and analyse the role of the nucleolus in genome organization, moving toward the obtainment of a comprehensive functional map of genome compartmentalization for each cell state. This will also provide novel insights into basic principles of genome organization and its role in gene expression and cell function that yet remain elusive.
Understanding how genome organization affects gene expression and cell state is of high medical relevance and, consequently, has an impact on health and society. This is particularly evident by the fact that alterations in nucleolus size and number have documented in several diseases such as cancer, neurogenerative disorders, and accelerated ageing. Thus, the establishment of technologies to understand the link between nucleolus and the genome has a potential impact to unravel novel pathways linked to disease with nucleolar alterations that might reveal to be useful for diagnosis and therapy.
The overall objectives of this project are to establish precise methods to identify and functionally characterize genomic regions located close to the nucleolus and determine how the nucleolus affects genome organization, chromatin state, and cell fate. This effort will contribute to deciphering how genome structure and position in cells affects gene expression and cell fate.
To obtain a more comprehensive understanding of how the genome is organized within the cell it is necessary to establish methods able to map the genome at the different nuclear compartments. The nucleolus is the largest subnuclear compartment in the cells. Although evidence indicates a role of the nucleolus in genome organization, the genomic regions in contact with the nucleolus (i.e. nucleolar-associated domains, NADs) have remained under-investigated, mainly due to the lack of methods able to map genomic contacts with this compartment.
We established Nucleolar-DamID, a method based on DNA adenine methyltransferase identification method that was successfully used to identify LADs. In Nucleolar-DamID, Dam is fused to an engineered nucleolar histone H2B that has an exclusive nucleolar localization, methylating GATC sequences in contact with nucleoli. We applied this method to identify and functionally characterize NADs in mouse embryonic stem cells (ESCs) and derived neural precursor (NPCs). We have also established and implemented computational methods to identify genomic contacts with rRNA genes using HiC data (HiC-rDNA). Finally, we established nucleolus laser microdissection sequencing (NoLMseq) method that combines laser-capture microdissection (LCM) of nucleoli and DNA sequencing to identify NADs in single nucleoli and under nucleolar stress. Traditional techniques to study genome organization predominantly rely on either microscopy or sequencing. NoLMseq synergistically integrates strengths of both these approaches, enabling precise examination of genome architecture surrounding single nucleoli. The results revealed unprecedented layers of genome compartmentalization by showing distinct, repressive transcriptional and chromatin states based on the interaction with the nucleolus, nuclear lamina, or both. The results also provided unexplored and novel insights into how chromosomes are organized around nucleoli and their reorganization during cell fate commitment and nucleolar structural changes, insights that were undetectable with previous methods. Finally, we dissected the mechanisms by which NADs are anchored to nucleoli and the functional role of these interactions. We found that component of the outer layer of the nucleolus, the granular component, and in particularly NPM1 are required not only for NAD association with nucleoli but also for the establishment of repressive chromatin domains. Thus, we revealed a direct role of the nucleolus in establishing repressive chromatin states, extending beyond its known role to serve as scaffold for positioning repressive chromatin domains in the nuclear space. Altogether, the results obtained in this project, technology advance, and knowledge will feed the study of genome organization and make possible to include the contribution of the nucleolus in future studies investigating the relationship between nuclear space and genome function in health and disease states.
We developed novel methods, of Nucleolar-DamID, HiC-rDNA, and NoLMseq for the identification of genomic contacts with the nucleolus (nucleolar associated domains, NADs) in mouse embryonic stem cells (ESCs) and derived neural precursor (NPCs) and under nucleolar stress. These novel methodologies allowed us to finally distinguish distinct layers of chromatin organization that depend on the interaction with the nucleolus, nuclear lamina, or both. These methodologies can be applied to every cell type, and we predict they will be highly relevant for future works aimed to understand basic principles of genome organization and its role in gene expression and cell function. Considering that structural changes in the nucleolus are often observed in diseases, such as cancer, the results of our work will also have an important biomedical impact for the understanding how changes in nucleolus structure and activity might affect genome function in disease.
Genome organization around the nucleolus