Periodic Reporting for period 3 - NucleolusChromatin (Analysis of the nucleolus in genome organization and function)
Okres sprawozdawczy: 2021-09-01 do 2023-02-28
Increasing evidence indicated that large-scale folding of chromatin affects gene expression by locating genes to specific sub-nuclear compartments that are either stimulatory or inhibitory to transcription. Nuclear periphery (NP) and nucleolus are two important nuclear landmarks where repressive chromatin domains are often located. The interaction of chromosomes with NP and nucleolus is thought to contribute to a basal chromosome architecture and genome function. However, while the role of NP in genome organization has been well documented, the function of the nucleolus remains yet elusive.
To fully understand how genome organization regulates chromatin and gene expression states, it is necessary to obtain a comprehensive functional map of genome compartmentalization. However, so far, only domains associating with NP (LADs) have been identified and characterized while nucleolar-associated domains (NADs) remained under-investigated. The aim of this project is to fill this gap by developing 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 historically been used by pathologists as a prognostic indicator of cancerous lesions, suggesting that changes in nucleolus structure can affect genome organization and have an impact in disease. Furthermore, nucleolus structure undergoes changes during the early developmental phases, a time where the genome undergoes drastic remodeling in order to establish totipotency and then pluripotency after the fusion of the parental genomes. Thus, the understanding of the link between nucleolus and the genome has the potential to provide information relevant to regenerative biomedicine.
The overall objectives of this project are to establish robust and 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 in deciphering how genome structure and position in cells affects gene expression and cell fate.
We have successfully established methods for the identification of NADs. Nucleolar-DamID is based on in vivo chemical modification of genomic regions in contact with the nucleolus. This modification can be then measured with next generation sequencing methods, allowing the identification of NADs. 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). 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. Remarkably, contacts with the nucleolus demarcate repressed regions of the genome that are enriched in H3K9me2 and depleted in H3K27me3 relative to sequences only contacting the NL, highlighting a specific repressive chromatin landscape that depends on the interactions with the nucleolus. Furthermore, we showed that genes moving away from the nucleolus upon ESC differentiation into NPCs are implicated in neuron development, indicating that the detachment from the repressive nucleolar compartment marks a first step toward activation in later stages of differentiation. The results revealed the role of the nucleolus as repressive compartment that is implicated in the control of gene expression program during lineage commitment. The establishment of these method and generation of high-quality maps of NADs that can be finally used by the community and will feed the study of genome organization will finally make possible to include the contribution of the nucleolus in future studies investigating the relationship between nuclear space and genome function.