Project description:The three-dimensional architecture of the eukaryotic genome is a critical determinant in the regulation of gene expression programs. Repressive chromatin regions are localized either at the nuclear lamina or the nucleolus. While the role of the nuclear lamina in genome organization has been widely studied, the role of the nucleolus has only recently come under investigation. Here, we present a new method called nucleolus architecture mapping (NAM) for identifying nucleolus-associated genomic domains (NADs) at a single nucleolus resolution. NAM combines laser capture microdissection and DNA sequencing. We applied NAM to embryonic stem cells (ESCs) and neural progenitor cells and observed a distinct pattern of nucleolus-to-nucleolus heterogeneity for NADs. NAM not only identified characteristic features of NADs, such as high levels of H3K9me2, depletion of H3K27me3, low active histone marks, and repressed gene expression but also revealed that the genomic domains seen in cell population studies to contact both nucleoli and nuclear lamina (NAD/LAD regions) can contact the nucleolus in one cell and the NL in another cell, but rarely both nucleolus and NL in the same cell. Additionally, we found that ribosomal protein (RP) genes are frequently associated with the nucleolus, suggesting a potential direct crosstalk between the nucleolus and the regulation of RP genes, and hence ribosome biogenesis. We also performed NAM upon perturbation of the nucleolus structure by inhibiting PolI transcription with Actinomycin D. This showed a loss of mostly NADs from the nucleolus other than the chromosomes containing rRNA genes, demonstrating that nucleolar integrity is required for genomic contacts with the nucleoli. Additionally, NAM applied to hybrid ESCs demonstrates a predominantly monoallelic distribution of NADs in single cells. We identify parental-specific NADs exhibiting a non-uniform distribution across different chromosomes. Our findings underscore NAM as an invaluable tool for investigating nucleolar organization in individual cells and, potentially, genome organization in other large phase-separated organelles in the future.

			Action	DRS
	SRR29531268.fastq.gz	Fastqsanger.gz
	SRR29531269.fastq.gz	Fastqsanger.gz
	SRR29531270.fastq.gz	Fastqsanger.gz
	SRR29531271.fastq.gz	Fastqsanger.gz
	SRR29531272.fastq.gz	Fastqsanger.gz

Dataset Information

The nucleolar granular component is required for NAD association with nucleoli and the establishment of repressive chromatin architecture [RNAseq_NPM1KD]

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