Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin.
Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin.
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DOI：
10.1371/journal.pone.0155558
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Takihara Y
中科院分区：
文献类型：
--
作者：
Ohno Y;Suzuki-Takedachi K;Yasunaga S;Kurogi T;Santo M;Masuhiro Y;Hanazawa S;Ohtsubo M;Naka K;Takihara Y
Geminin调节染色质重塑和DNA复制许可，在调节细胞增殖和分化中起重要作用。Geminin基因的转录通过E2 F反应区调节，而蛋白质则受到泛素-蛋白酶体系统的密切调节。我们的目的是将Geminin蛋白直接导入细胞。通过将Geminin与来自FGF 4的膜转位基序融合产生重组细胞穿透Geminin（CP-Geminin），并有效地掺入NIH 3 T3细胞和小鼠胚胎成纤维细胞。撤药研究表明，从培养基中取出后，掺入的CP-Geminin迅速减少。我们证实CP-双子蛋白掺入后被输入细胞核，并且掺入的CP-双子蛋白以与双子蛋白相同的方式直接与Cdt 1或Brahma/Brg 1相互作用。我们进一步证明，纳入CP-Geminin抑制细胞周期的S期进展，并减少染色质中的核酸酶可及性，可能通过抑制染色质重塑，表明CP-Geminin构成了一种新的工具，用于控制染色质构型和细胞周期。由于已显示Geminin参与干细胞和癌细胞的调节，因此预期CP-Geminin可用于阐明Geminin在干细胞和癌细胞中的作用，并用于操纵它们的活性。
Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin) was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.