Yushkova A A, Ogienko A A, Andreyeva E N, Pindyurin A V, Letiagina A E, Omelina E S
Institute of Molecular and Cellular Biology of the Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
Vavilovskii Zhurnal Genet Selektsii. 2025 Jun;29(3):401-413. doi: 10.18699/vjgb-25-43.
The nucleolus is a large membraneless subnuclear structure, the main function of which is ribosome biogenesis. However, there is growing evidence that the function of the nucleolus extends beyond this process. While the nucleolus is the most transcriptionally active site in the nucleus, it is also the compartment for the location and regulation of repressive genomic domains and, like the nuclear lamina, is the hub for the organization of inactive heterochromatin. Studies in human and Drosophila cells have shown that a decrease in some nucleolar proteins leads to changes in nucleolar morphology, heterochromatin organization and declustering of centromeres. This work is devoted to the study of the effects of Novel nucleolar protein 3 (Non3) gene mutations in D. melanogaster on the organization of chromatin in the nucleus. Previously, it was shown that partial deletion of the Non3 gene leads to embryonic lethality, and a decrease in NON3 causes an extension of ontogenesis and formation of a Minute-like phenotype in adult flies. In the present work, we have shown that mutations in the Non3 gene suppress the position effect variegation (PEV) and increase the frequency of meiotic recombination. We have analyzed the classical heterochromatin markers in Non3 mutants and shown that the amount of the HP1 protein as well as the modification of the histone H3K9me2 do not change significantly in larval brains and salivary glands compared to the control in Western blot analysis. Immunostaining with antibodies to HP1 and H3K9me2 did not reveal a significant reduction or change in the localization patterns of these proteins in the pericentromeric regions of salivary gland polytene chromosomes either. We analyzed the localization of the HP1 protein in Non3 mutants using DNA adenine methyltransferase identification (DamID) analysis and did not find substantial differences in protein distribution compared to the control. In hemocytes of Non3 mutants, we observed changes in the morphology of the nucleolus and in the size of the region detected by anti-centromere antibodies, but this was not accompanied by declustering of centromeres and their untethering from the nucleolar periphery. Thus, the NON3 protein is important for the formation/function of the nucleolus and is required for the correct chromatin packaging, but the exact mechanism of NON3 involvement in these processes requires further investigations.
核仁是一种大型无膜亚核结构，其主要功能是核糖体生物合成。然而，越来越多的证据表明核仁的功能超出了这一过程。虽然核仁是细胞核中转录活性最高的位点，但它也是抑制性基因组结构域定位和调控的区域，并且与核纤层一样，是无活性异染色质组织的中心。对人类和果蝇细胞的研究表明，某些核仁蛋白的减少会导致核仁形态、异染色质组织和着丝粒去簇集的变化。这项工作致力于研究果蝇中新型核仁蛋白3（Non3）基因突变对细胞核中染色质组织的影响。此前研究表明，Non3基因的部分缺失会导致胚胎致死，而NON3的减少会导致个体发育延长并在成年果蝇中形成类似Minute的表型。在本研究中，我们发现Non3基因突变抑制了位置效应斑驳（PEV）并增加了减数分裂重组的频率。我们分析了Non3突变体中的经典异染色质标记，结果表明，与Western blot分析中的对照相比，幼虫脑和唾液腺中HP1蛋白的含量以及组蛋白H3K9me2的修饰没有显著变化。用抗HP1和H3K9me2抗体进行免疫染色也未发现这些蛋白在唾液腺多线染色体着丝粒周围区域的定位模式有显著减少或变化。我们使用DNA腺嘌呤甲基转移酶鉴定（DamID）分析来分析Non3突变体中HP1蛋白的定位，与对照相比，未发现蛋白质分布有实质性差异。在Non3突变体的血细胞中，我们观察到核仁形态和抗着丝粒抗体检测到的区域大小发生了变化，但这并未伴随着着丝粒的去簇集及其与核仁周边的脱离。因此，NON3蛋白对于核仁的形成/功能很重要，是正确染色质包装所必需的，但NON3参与这些过程的确切机制仍需进一步研究。
