Periodic Reporting for period 4 - NucleolusChromatin (Analysis of the nucleolus in genome organization and function)
Reporting period: 2023-03-01 to 2024-08-31
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.
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.