Periodic Reporting for period 1 - NucleolusChromatin (Analysis of the nucleolus in genome organization and function)
Reporting period: 2018-09-01 to 2020-02-29
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.
In this 1st period (18 months), we successfully established a method for the identification of NAD that is based on in vivo chemical modification of genomic regions in contact with the nucleolus. This modification can be then measure with next generation sequencing methods, allowing the identification of NADs. We applied this method to identify NADs in mouse embryonic stem cells (ESCs). We are currently performing functional analyses to define their features by combining the analysis of gene expression and chromatin states. The analysis of NADs in neural progenitors is currently on going and will serve to determine how genome architecture changes during differentiation.
We have also established other additional methods for the identification of NADs that allow to measure genomic contacts with the ribosomal rRNA genes, which localize within nucleoli. We used this technology to determine how genomic contacts with rRNA genes change during ESC differentiation and under conditions of nucleolus structure alteration. In this first period of this ERC project, we have also established and implemented computational methods to identify genomic contacts with rRNA genes using HiC data. This pipeline will be very helpful to interrogate HiC data from cancer studies to determine the role of the nucleolus in pathological genomic alterations.