Project description:Genome-wide maps of nucleolar-associated domains (NADs) of mouse embryonic stem cells (ESCs) have recently been identified using several methodologies, such as Nucleolar-DamID and HiC-rDNA. These data revealed that the association with nucleoli is not only limited to the heterochromatic centromeric repeats and that these NAD sequences are generally in a repressive chromatin state. However, it remains yet unclear how NADs are recruited to the nucleolus and whether and how their repressive chromatin state is established. By using a combination of DNA-FISH and Nucleolar-DamID analyses in ESCs, we show that NAD association with nucleoli is mediated by components of the GC, NPM1, and Nucleolin, whereas the DFC component Fibrillarin is not involved in this process. ChIPseq analyses showed that NPM1 specifically binds to NADs, suggesting a direct involvement of NPM1 in NAD association with nucleoli. Moreover, ChIPseq analyses showed that NPM1 is also required to establish H3K9me2 specifically at NADs. Accordingly, IF analyses showed that nucleoli of ESCs are surrounded by H3K9me2-marked chromatin, forming a ring-like shape around nucleoli that could not be detected for any other repressive histone modification. However, loss of NPM1 did not alter either the association of chromocenters with nucleoli and their characteristic H3K9me3 mark, indicating that NPM1 only regulates the class of NADs not composed of centromeric repeats. We show that H3K9me2 at NADs is deposited by the histone lysine methyltransferase G9a (EHMT2). Importantly, mass spectrometry analysis of NPM1-interactome revealed that NPM1 interacts with G9a in mouse embryonic stem cells. Finally, loss of H3K9me2 via G9a depletion did not affect the association of NADs with nucleoli. This signifies that H3K9me2 at NADs is established only when they contact nucleoli through the binding to NPM1 and the subsequent recruitment of G9a. These results provide mechanistic insights into how genomic domains associate with nucleoli and the establishment of repressive chromatin structures on them.

			Action	DRS
	SRR29536149.fastq.gz	Fastqsanger.gz
	SRR29536150.fastq.gz	Fastqsanger.gz
	SRR29536151.fastq.gz	Fastqsanger.gz
	SRR29536152.fastq.gz	Fastqsanger.gz

Dataset Information

The nucleolar granular component is required for NAD association with nucleoli and the establishment of repressive chromatin architecture [CHIPSeq_H3K9me2uponNPM1KD]

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