Date Published: December 19, 2018
Publisher: John Wiley and Sons Inc.
Author(s): Natalia Dworak, Dawid Makosa, Mandovi Chatterjee, Kasey Jividen, Chun‐Song Yang, Chelsi Snow, William C. Simke, Isaac G. Johnson, Joshua B. Kelley, Bryce M. Paschal.
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.
The Ran GTPase plays a central role in regulating nuclear import and export in eukaryotic cells. By analogy with other GTPases, distinct conformations of Ran associated with its GTP‐ and GDP‐bound states are the basis for selective binding to the nuclear transport machinery (Pemberton & Paschal, 2005). Ran regulation of key steps in nuclear transport has been defined using biological, biochemical, and structural approaches (Chook et al., 1999; Pemberton & Paschal, 2005). Proteins that contain a nuclear localization signal (NLS) bind an NLS receptor (termed importin‐α or KPNA) and assemble into a cytoplasmic NLS‐KPNA‐importin‐β complex that translocates through the nuclear pore complex (NPC). Upon reaching the nucleoplasm, RanGTP binding to a single, high‐affinity site on importin‐β triggers disassembly of the NLS‐KPNA‐importin‐β complex (Görlich, Panté, Kutay, Aebi, & Bischoff, 1996), thus releasing the NLS‐containing proteins for nuclear function. RanGTP, therefore, regulates nuclear import by controlling complex disassembly, the terminal step in this pathway. By contrast, RanGTP regulates the initial step of nuclear export by promoting export complex assembly. The major pathway for transporting nuclear export signal (NES)‐containing proteins from the nucleus to the cytoplasm is mediated by the NES receptor Crm1. The NES‐Crm1‐RanGTP complex forms in the nucleoplasm, translocates through the NPC, and is disassembled in the cytoplasm because the complex encounters the GTPase‐activating protein (GAP) for Ran (Askjaer et al., 1999; Bischoff, Klebe, Kretschmer, Wittinghofer, & Ponstingl, 1994). GAP stimulation of GTP hydrolysis promotes disassembly of NES‐Crm1‐RanGTP complex, which releases the NES‐containing protein for function in the cytoplasm.
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). Our group showed that the reduction in heterochromatin in Progeria was correlated with changes in the nuclear:cytoplasmic (N:C) levels of the Ran GTPase, the master regulator of nuclear transport (Datta, Snow, & Paschal, 2014; Kelley et al., 2011). This led us to suggest that heterochromatin might regulate the Ran GTPase gradient through RCC1, a chromatin‐binding protein that mediates nucleotide exchange on Ran (Ohtsubo et al., 1989). Consistent with this possibility, genomewide localization of the RCC1 homologue in yeast, Prp20, showed preferential binding to inactive genes (Casolari et al., 2004). From biochemical analysis, it is known that RCC1 binds histones and DNA, and the nucleotide exchange reaction that converts RanGDP to RanGTP involves transient formation of a Ran:RCC1:chromatin complex (Hao & Macara, 2008; Nemergut, 2001). Histones can stimulate RCC1‐mediated nucleotide exchange on Ran, and the crystal structure of RCC1 bound to the nucleosome revealed the specific contacts between RCC1, histones, and DNA (Makde, England, Yennawar, & Tan, 2010; Nemergut, 2001). These data lend strong support for the model that RCC1 uses chromatin as a scaffold for the nucleotide exchange reaction. If this reaction requires or is biased toward heterochromatin, then conditions that reduce heterochromatin levels in the nucleus could affect Ran:RCC1:chromatin formation, generation of RanGTP, and Ran regulation of nuclear transport pathways. These relationships can be viewed as an axis that links the structure of the nuclear lamina and chromatin state to the activity of the nuclear transport machinery (Figure 1a).
A large body of literature indicates that the nuclear lamina helps organize and regulate nuclear pathways including transcription, DNA replication, nuclear size control, apoptosis, and mechanical stability (Burke & Stewart, 2013). Determining exactly how lamina proteins function in these pathways has been difficult, owing to the structural complexity and dynamic nature of the lamina (Turgay et al., 2017). One of the best‐defined functions of the nuclear lamina is its contribution to heterochromatin organization in the interphase nucleus. This involves heterochromatin contact with lamina proteins, including LAP2β, the lamin B receptor, and emerin (Gruenbaum, Margalit, Goldman, Shumaker, & Wilson, 2005). Genomewide mapping of DNA sequences that are in close proximity to the lamina has identified lamina‐associated domains (LADs), which are gene‐poor or transcriptionally silent regions constituting ~30% of nuclear DNA (Guelen et al., 2008). Thus, LADs are part of the peripheral heterochromatin that is anchored to the nuclear lamina. Physical interactions between the nuclear lamina and chromatin probably contribute to heterochromatin maintenance and provide part of the explanation for why disruption of lamina structure in HGPS causes the striking reduction in Histone H3 marks H3K9me3 and H3K27me3 (McCord et al., 2013). Loss of heterochromatin has also been shown to occur in Werner’s syndrome, which is caused by mutation of a DNA helicase that is important for heterochromatin maintenance (Zhang et al., 2015). These observations suggest that heterochromatin levels could play a cellular role in premature aging (Kubben & Misteli, 2017).