Periodic Reporting for period 2 - NucleolusChromatin (Analysis of the nucleolus in genome organization and function)
Reporting period: 2020-03-01 to 2021-08-31
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.
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.
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.
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 catch genomic contact with this compartment. To date, the only method employed to identify NADs is based on the sonication of cells followed by purification of nucleoli. However, this method is biased toward heterochromatin, which is resistance to sonication-mediate DNA fragmentation, thereby affecting the complete identification of NADs. Furthermore, the purity of isolated nucleoli is subjected to great variations that depend on cell types. In this proposal, we aimed to establish a robust method for the identification of NADs, overcoming the technical limitation of previous methods. This will allow us 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 and providing novel insights into basic principles of genome organization and its role in gene expression and cell function that yet remain elusive.
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.
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.
This ERC project started in September 2018. Within 1.5 years, we were able to establish and implement methods for the identification of NADs. The functional analysis of these results is currently going on and we expected to obtain novel and important results that will instruct us how the nucleolus acts as a regulator of genome architecture. This will serve to obtain a comprehensive functional map of genome compartmentalization for each cell state and provide novel insights into basic principles of genome organization and its role in gene expression and cell function that yet remain elusive. Considering that alterations of nucleolus structure are a hallmark of cancer, the results of our work will also have a biomedical impact for the understanding how changes in nucleolus structure and activity might affect genome function in disease.