Abstract
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.
Highlights
The Ran GTPase plays a central role in regulating nuclear import and export in eukaryotic cells
Based on chromatin immunoprecipitation (ChIP) data indicating that regulator of chromatin condensation 1 (RCC1) can be enriched on chromatin marked with H3K9me3 and H3K27me3, we propose that RCC1 senses heterochromatin levels, which, in turn, is transduced to Ran
In primary fibroblasts from Progeria patients, alterations in the structure of the nuclear lamina are associated with reduced levels of the heterochromatin marks Histone H3K9me3 and Histone H3K27me3, as well as lower nuclear levels of HP1 (Kelley et al, 2011; Scaffidi & Misteli, 2006)
Summary
The Ran GTPase plays a central role in regulating nuclear import and export in eukaryotic cells. Based on chromatin immunoprecipitation (ChIP) data indicating that RCC1 can be enriched on chromatin marked with H3K9me and H3K27me, we propose that RCC1 senses heterochromatin levels, which, in turn, is transduced to Ran. Decrease in heterochromatin levels and disruption of the Ran gradient with the HMT inhibitor Bix01294 were sufficient to induce a defect in nuclear import of the “large cargo” protein TPR, an effect elicited by progerin (Snow, Dar, Dutta, Kehlenbach, & Paschal, 2013). Decrease in heterochromatin levels and disruption of the Ran gradient with the HMT inhibitor Bix01294 were sufficient to induce a defect in nuclear import of the “large cargo” protein TPR, an effect elicited by progerin (Snow, Dar, Dutta, Kehlenbach, & Paschal, 2013) This led us to explore whether DNA damage signaling, a pathway that relies on nuclear import of large proteins cargoes, is affected by the state of the nuclear lamina, heterochromatin, and Ran. In cells where the Ran gradient is disrupted, we observed a striking reduction in nuclear localization of ATM and H2AX protein and reduced generation of nuclear γ‐H2AX in response to ionizing radiation. Our data suggest that chromatin regulation of Ran is a conserved mechanism and that the lamina–chromatin–Ran axis in mammalian cells promotes the DNA damage response through nuclear transport‐based mechanisms
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