SUMMARY ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases conjugate either a single ADP-ribose to a target, or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+- dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly-ADP-ribose binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly- ADP-ribose regulation of E3 activity.

The mutant form of lamin A responsible for the premature aging disease Hutchinson-Gilford progeria syndrome (termed progerin) acts as a dominant negative protein that changes the structure of the nuclear lamina. How the perturbation of the nuclear lamina in progeria is transduced into cellular changes is undefined. Using patient fibroblasts and a variety of cell-based assays, we determined that progerin expression in Hutchinson-Gilford progeria syndrome inhibits the nucleocytoplasmic transport of several factors with key roles in nuclear function. We found that progerin reduces the nuclear/cytoplasmic concentration of the Ran GTPase and inhibits the nuclear localization of Ubc9, the sole E2 for SUMOylation, and of TPR, the nucleoporin that forms the basket on the nuclear side of the nuclear pore complex. Forcing the nuclear localization of Ubc9 in progerin-expressing cells rescues the Ran gradient and TPR import, indicating that these pathways are linked. Reducing nuclear SUMOylation decreases the nuclear mobility of the Ran nucleotide exchange factor RCC1 in vivo, and the addition of SUMO E1 and E2 promotes the dissociation of RCC1 and Ran from chromatin in vitro. Our data suggest that the cellular effects of progerin are transduced, at least in part, through reduced function of the Ran GTPase and SUMOylation pathways.The nuclear lamina provides an architectural framework that defines the size, shape, and physical properties of the nucleus (29). A critical function of the nuclear lamina is to provide a scaffold for chromatin attachment, and there is a growing body of evidence linking the nuclear lamina to the regulation of gene expression and chromosome positioning within interphase cells (49). The nuclear periphery, including the region proximal to the lamina, is rich in heterochromatin and provides a nuclear subcompartment that promotes transcriptional silencing (19). The mechanisms responsible for transcriptional silencing associated with the lamina appear to involve epigenetic regulation and modulation of the higherorder chromatin structure (2). Other functions of the lamina include roles in DNA replication and apoptosis (22,29). The principal components of the lamina are lamin A/C and lamin B, which are encoded by the LMNA and LMNB genes, respectively (22, 29). More than 300 mutations in LMNA have been described (http://www.umd.be/LMNA/) and have been linked to 12 diseases collectively known as laminopathies. These diseases include dilated cardiomyopathy with conduction defects (DCM-CD), familial partial lipodystrophy (FPLD), atypical Werner's syndrome, Emery-Dreifuss muscular dystrophy (EDMD), and Hutchinson-Gilford progeria syndrome (HGPS) (9, 70, 77).The nuclear lamina also provides a scaffold for organizing nuclear pore complexes (NPCs) within the nuclear membrane (1). NPCs span the nuclear lamina and both the inner and outer nuclear membranes and serve as conduits for nuclear import and export (73). Nucleoporins that comprise the NPC are organized into subcomplexes that disassemble and reassemble duri...

The N-terminal tail of Ndc80 is essential for kinetochore-microtubule binding in human cells but is not required for viability in yeast. We show that the yeast Ndc80 tail is required for timely mitotic progression and accurate chromosome segregation. The tail is essential when cells are limited for Dam1, demonstrating a redundant function for the Ndc80 and Dam1 complexes in vivo.

Transport regulation by the Ran GTPase requires its nuclear localization and GTP loading by the chromatin-associated exchange factor RCC1. These reactions generate Ran protein and Ran nucleotide gradients between the nucleus and the cytoplasm. Cellular stress disrupts the Ran gradients, but the specific mechanisms underlying this disruption have not been elucidated. We used biochemical approaches to determine how oxidative stress disrupts the Ran system. RCC1 exchange activity was reduced by diamide-induced oxidative stress and restored with dithiothreitol. Using mass spectrometry, we found that multiple solventexposed cysteines in RCC1 are oxidized in cells treated with diamide. The cysteines oxidized in RCC1 included Cys93, which is solvent exposed and unique because it becomes buried upon contact with Ran. A Cys93Ser substitution dramatically reduced exchange activity through an effect on RCC1 binding to RanGDP. Diamide treatment reduced the size of the mobile fraction of RCC1-green fluorescent protein in cells and inhibited nuclear import in digitonin-permeabilized cell assays. The Ran protein gradient was also disrupted by UV-induced stress but without affecting RCC1 exchange activity. Our data suggest that stress can disrupt the Ran gradients through RCC1-dependent and RCC1-independent mechanisms, possibly dependent on the particular stress condition. R egulation of nuclear transport by the Ran GTPase involves two integrated cycles, a nucleotide cycle and a nucleocytoplasmic shuttling cycle. The nucleotide cycle, which is a general feature of GTPases, depends on GTP loading onto Ran and a subsequent step of GTP hydrolysis (1, 2). GTP loading onto Ran occurs in the nucleus through the action of the nucleotide exchange factor RCC1, which, upon binding Ran, promotes GDP release and GTP binding (3,4). GTP binding to Ran is favored over GDP rebinding because of the GTP/GDP ratio (ϳ10:1) in cells (4, 5). The nuclear export phase of the nucleocytoplasmic shuttling cycle occurs as a result of high-affinity binding of RanGTP to nuclear transport receptors that translocate from the nucleus to the cytoplasm. In the cytoplasm, GTP hydrolysis by Ran, which promotes release from the transport receptors, occurs through association with the Ran GTPase-activating protein (GAP) (6). RanGDP can then engage with a dedicated import receptor, NTF2, and undergo reimport (7-10). Thus, Ran undergoes import, nucleotide exchange, export, and nucleotide hydrolysis. These reactions generate two nuclear/cytoplasmic (N/C) Ran gradients, a Ran protein gradient (ϳ3:1) and a RanGTP gradient (ϳ100:1) (11, 12). Disruption of the Ran protein gradient by depletion of NTF2 reduces Ran GTPdependent import (13,14), and inhibition of GTP loading with a temperature-sensitive allele of RCC1 disrupts the Ran protein gradient (15). Because disruption of the Ran protein gradient perturbs the nucleotide cycle and vice versa, the two cycles appear to be linked in the cell.RCC1 is the only known nucleotide exchange factor for Ran, and the exclusive nuclea...

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.

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