The nucleolar granular component is required for NAD association with nucleoli and the establishment of repressive chromatin architecture [CHIPseq_NPM1]

Repressive chromatin domains often localize to the nuclear lamina or nucleolus. Although nucleolar-associated domains (NADs) have recently been mapped, their mechanisms of nucleolar association and functional significance remain unclear. Here, we show that nucleophosmin (NPM1), a factor located in the granular component of the nucleolus, mediates NAD association in mouse embryonic stem cells. NPM1 binds NADs, interacts with the histone methyltransferase G9a (EHMT2), and is required for establishing H3K9me2 at NADs. Loss of NPM1 or expression of a DNA-binding-deficient mutant disrupts NAD-nucleolus association and reduces H3K9me2 specifically at NADs. G9a is dispensable for NAD-nucleolus contacts, indicating that H3K9me2 is acquired after NADs associate with NPM1 at nucleoli. These findings reveal mechanistic insights into how genomic domains associate with nucleoli and form repressive chromatin and indicate that the nucleolus not only serves as a scaffold for positioning repressive domains but also plays a direct role in establishing their repressive chromatin states. Overall design: RNAseq in mouse ESCs.

抑制性染色质结构域通常定位于核纤层或核仁。尽管近年来已对核仁相关结构域（nucleolar-associated domains, NADs）完成图谱绘制，但其与核仁结合的机制及功能意义仍不明确。本研究证实，定位于核仁颗粒组分的核仁素（nucleophosmin, NPM1）可介导小鼠胚胎干细胞中NADs与核仁的结合。NPM1能够结合NADs，并与组蛋白甲基转移酶G9a（EHMT2）相互作用，同时是在NADs处建立组蛋白H3赖氨酸9二甲基化（H3K9me2）修饰的必需因子。敲除NPM1或表达DNA结合缺陷型突变体，均会破坏NAD-核仁结合，并特异性降低NADs处的H3K9me2水平。G9a对于NAD-核仁接触并非必需，这表明H3K9me2修饰是在NADs与核仁处的NPM1结合后才获得的。上述发现揭示了基因组结构域与核仁结合并形成抑制性染色质的机制性见解，同时表明核仁不仅可作为定位抑制性结构域的支架，还可直接参与建立其抑制性染色质状态。整体实验设计：小鼠胚胎干细胞中的RNA测序（RNA-seq）。