Center for Cell Signaling, University of Virginia, Charlottesville, Virginia.
Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia.
Aging Cell. 2019 Feb;18(1):e12851. doi: 10.1111/acel.12851. Epub 2018 Dec 19.
The Ran GTPase regulates nuclear import and export by controlling the assembly state of transport complexes. This involves the direct action of RanGTP, which is generated in the nucleus by the chromatin-associated nucleotide exchange factor, RCC1. Ran interactions with RCC1 contribute to formation of a nuclear:cytoplasmic (N:C) Ran protein gradient in interphase cells. In previous work, we showed that the Ran protein gradient is disrupted in fibroblasts from Hutchinson-Gilford progeria syndrome (HGPS) patients. The Ran gradient disruption in these cells is caused by nuclear membrane association of a mutant form of Lamin A, which induces a global reduction in heterochromatin marked with Histone H3K9me3 and Histone H3K27me3. Here, we have tested the hypothesis that heterochromatin controls the Ran gradient. Chemical inhibition and depletion of the histone methyltransferases (HMTs) G9a and GLP in normal human fibroblasts reduced heterochromatin levels and caused disruption of the Ran gradient, comparable to that observed previously in HGPS fibroblasts. HMT inhibition caused a defect in nuclear localization of TPR, a high molecular weight protein that, owing to its large size, displays a Ran-dependent import defect in HGPS. We reasoned that pathways dependent on nuclear import of large proteins might be compromised in HGPS. We found that nuclear import of ATM requires the Ran gradient, and disruption of the Ran gradient in HGPS causes a defect in generating nuclear γ-H2AX in response to ionizing radiation. Our data suggest a lamina-chromatin-Ran axis is important for nuclear transport regulation and contributes to the DNA damage response.
Ran GTPase 通过控制运输复合物的组装状态来调节核输入和输出。这涉及到 RanGTP 的直接作用，RanGTP 由染色质相关的核苷酸交换因子 RCC1 在核内产生。Ran 与 RCC1 的相互作用有助于在有丝分裂细胞中形成核质（N:C）Ran 蛋白梯度。在之前的工作中，我们表明，Hutchinson-Gilford 早衰综合征（HGPS）患者的成纤维细胞中的 Ran 蛋白梯度被破坏。这些细胞中的 Ran 梯度破坏是由核膜结合的突变形式的 lamin A 引起的，这会导致异染色质的整体减少，异染色质标记有组蛋白 H3K9me3 和组蛋白 H3K27me3。在这里，我们测试了这样一个假设，即异染色质控制着 Ran 梯度。在正常的人类成纤维细胞中，化学抑制和耗尽组蛋白甲基转移酶（HMTs）G9a 和 GLP 会降低异染色质水平，并导致 Ran 梯度破坏，与之前在 HGPS 成纤维细胞中观察到的破坏相当。HMT 抑制导致 TPR 的核定位缺陷，TPR 是一种高分子量蛋白，由于其体积较大，在 HGPS 中表现出依赖 Ran 的输入缺陷。我们推断，依赖于大分子蛋白核输入的途径可能在 HGPS 中受到损害。我们发现，ATM 的核输入需要 Ran 梯度，而 HGPS 中 Ran 梯度的破坏会导致核内 γ-H2AX 的产生缺陷，以响应电离辐射。我们的数据表明，核膜-染色质-Ran 轴对于核转运调节很重要，并有助于 DNA 损伤反应。
