A nuclear lamina‐chromatin‐Ran GTPase axis modulates nuclear import and DNA damage signaling

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.

Comprehensive histochemical profiles of histone modification in male germline cells during meiosis and spermiogenesis: Comparison of young and aged testes in mice

“…anti-H3K4me2 rabbit polyclonal 1/500 Millipore (07-030) [13][14][15] anti-H3K4me3 rabbit polyclonal 1/500 Abcam (ab8580) [16][17][18] anti-H3K9me3 rabbit polyclonal 1/500 Abcam (ab8898) [19][20][21] anti-H3K27ac rabbit polyclonal 1/500 Abcam (ab4729) [22][23][24] anti-H3K27me2 rabbit polyclonal 1/500 Abcam (ab24684) [25,26] anti-H3K27me3 rabbit polyclonal 1/500 Millipore (07-449) [24,[27][28][29] anti-H3K79me2 rabbit polyclonal 1/500 Abcam (ab3594) [30][31][32] anti-H3K79me3 rabbit polyclonal 1/500 Abcam (ab2621) [33][34][35] anti-SCP3 mouse monoclonal 1/500 Abcam (ab97672) [36][37][38] anti-α-Tubulin (S2 Fig) mouse monoclonal 1/500 Sigma-Aldrich (T6199) [39,40] All antibodies are commercially available, and their specificity has been reported in previous studies.…”

Enhanced nuclear protein export in premature aging and rescue of the progeria phenotype by modulation of CRM1 activity

“…We therefore investigated whether enhanced nuclear export activity via CRM1 overexpression can occur in cells with a deficient Ran gradient. Since depletion of the Ran import factor NTF2 (nuclear transport factor 2) is sufficient to disrupt the Ran gradient (Datta et al, ; Dworak et al, ), HeLa cells were double‐transfected to express CRM1 and a shRNA against NTF2. Interestingly, stably transfected HeLa cells having both overexpressed levels of CRM1 and depleted levels of NTF2 (Figure a) still showed elevated nuclear export activity, as shown by cytoplasmic mislocalization of the NES‐containing protein STAT3 (right panel).…”

The Multifaceted Roles of RCC1 in Tumorigenesis

“…The biological function of RCC1 nuclear transport is to generate a RanGTP gradient through the nuclear pore, which is then used to drive various cargo molecules to overcome their concentration gradients for transportation ( Kahana and Cleveland, 1999 ; Dworak et al, 2019 ). During the early stages of apoptosis, histone modification regulates RCC1 to inhibit nuclear transport.…”