Because the formation of the Ran protein gradient depends on its nucleotide exchange factor RCC1 (72
), which is enriched in heterochromatin (chromatin immunoprecipitation [12
]), we considered whether the distribution of Ran might be linked to the epigenetic state of chromatin. Primary fibroblasts from progeria patients provide a setting for the testing of this model, as the heterochromatin marks H3K9me3 and H3K27me3 are reduced in HGPS (64
). The distribution of Ran was examined in primary fibroblasts from three progeria patients (HGPS 1972, HGPS 1498, and HGPS 3199) and, as a control, in primary fibroblasts of a similar passage number from an unaffected father of a child with progeria (Normal 8469). By IF microscopy, Ran is predominantly nuclear in Normal 8469 fibroblasts, which is the characteristic distribution of Ran, but in HGPS fibroblasts there was a striking reduction in the level of nuclear Ran and an increase in the level of cytoplasmic Ran ( A). In HGPS cells where the Ran distribution was defective, the pattern of Ran localization ranged from cells where the Ran appeared to equilibrate between the nucleus and cytoplasm to cells that displayed a reversal in the N/C Ran gradient.
Fig. 1. The Ran protein gradient in interphase cells depends on the nuclear lamina and is correlated with markers of heterochromatin. (A) Ran distribution in primary fibroblasts from a control individual (Normal 8469) and three progeria patients (HGPS 1972, HGPS (more ...)
To better assess the Ran distribution changes in HGPS cells, we combined IF microscopy with digital imaging to measure the nuclear and cytoplasmic levels in patient and normal fibroblasts (n > 50 cells per sample). Ran N/C levels were reduced in 3/3 HGPS patient cell lines (P < 0.0005), an effect that we refer to as a disruption of the Ran gradient (B). To our knowledge, this is the first example of a human disease that displays a disruption of the Ran gradient. By double-label IF microscopy, Ran N/C ratios were correlated with nuclear H3K9me3 levels in both patient and normal fibroblasts (Spearman's rank correlation coefficient [ρ] values of 0.81 for Normal 8469, 0.74 for HGPS 1972, 0.81 for HGPS 1498, and 0.29 for HGPS 3199), suggesting that the Ran distribution in these cells is potentially linked to the epigenetic state of chromatin (C). Nuclear Ran levels were also correlated with nuclear levels of heterochromatin protein 1γ (HP1γ), which binds the chromatin mark H3K9me3 (ρ values of 0.98 for Normal 8469, 0.98 for HGPS 1972, 0.96 for HGPS 1498, and 0.97 for HGPS 3199) (D). These correlations could reflect a chromatin regulation of the Ran N/C distribution, the Ran-dependent transport of enzymes that modify chromatin, or a combination of these processes.
By immunoblotting, Ran protein levels were similar in the HGPS (HGPS 1498, HGPS 3199, and HGPS 1972) and normal (Normal 8469) fibroblasts (E). An antibody that recognizes progerin (but not wild-type [WT] lamin A) was generated and used to show that comparable levels of progerin are expressed in the three HGPS fibroblast lines used in this study (E). All three HGPS fibroblast lines displayed a reduction in H3K9me3 levels by immunoblotting, which is consistent with data from previous reports (64
) and our IF microscopy.
We tested whether the ectopic expression of progerin is sufficient to disrupt the distribution of endogenous Ran in HeLa cells. By double-label IF microscopy, HA-progerin expression disrupted the Ran gradient, while at comparable expression levels, HA-lamin A did not have this effect (F). Cell-to-cell variation in the extent to which progerin disrupted the Ran distribution was apparent in HeLa cells, consistent with what was observed for HGPS primary fibroblasts. Cell-to-cell variation in the penetrance of progerin was noted previously by others (68
), including Scaffidi and Misteli (64
), who reported that 60% of HGPS 1972 cells (passages 25 to 30) had reduced levels of HP1α by IF microscopy.
Given the central role played by Ran in nucleocytoplasmic transport (36
), we examined whether the disruption of the Ran gradient in HGPS fibroblasts affects nuclear transport. We measured importin β-mediated import into the nucleus by using a microinjection assay with a fluorescent cargo that contains an importin β binding (IBB) domain (from an importin α) fused to β-galactosidase (IBB-β-Gal) labeled with Alexa Fluor 555. Because the molecular mass of the IBB-β-Gal tetramer (~400 kDa) (44
) is well above the diffusion limit of the NPC, the nuclear localization of the IBB-β-Gal reporter is strictly dependent on binding to importin β and the translocation of the importin β/IBB-β-Gal complex through the NPC. Following microinjection into the cytoplasm, IBB-β-Gal underwent rapid nuclear import, both in the Normal 8469 fibroblasts and in the HGPS 1498 fibroblasts (). Plotting of the initial import rate versus the initial concentration (measured in the cytoplasm of the injected cell) revealed a slight reduction in the nuclear import rate in HGPS 1498 fibroblasts (n
= 30 cells). The reduced level of import in HGPS cells was observed only at the higher concentrations of IBB-β-Gal used (). Thus, while there is a measureable defect in importin β-dependent transport into the nucleus, the fact that the disruption of the Ran protein gradient in HGPS does not cause a large reduction in transport is unexpected.
Fig. 2. The importin β-dependent import pathway is functional in HGPS fibroblasts. (A) Rate of importin β-dependent transport measured in living cells. A fluorescent reporter protein (Alexa 544-labeled IBB-β-galactosidase) that undergoes (more ...) Import of the nucleoporin TPR is defective in HGPS.
We first considered whether the disruption of the Ran gradient in HGPS cells and in HeLa cells expressing progerin () might be explained by progerin-induced defects in the NPC. Nucleoporins within the central channel of the NPC form a meshwork that acts as a permeability barrier that restricts the free exchange of proteins between the cytoplasm and the nucleus, and age-related changes in nucleoporin composition that disrupt the permeability barrier were described previously (21
). It seemed plausible that Ran would leak out of the nucleus if the permeability barrier was defective in HGPS cells. We tested this possibility by cotransfecting progerin with a reporter protein (Myc-tagged pyruvate kinase [PK]) that is normally excluded from the nucleus because it lacks transport signals and is too large to diffuse through the NPC. Myc-pyruvate kinase was completely excluded from the nuclei of cells expressing progerin, which supports the conclusion that the NPC diffusion barrier is functional in this setting (data available for inspection from the authors). Additionally, a disruption of the diffusion barrier would be predicted to result in Ran equilibration between the nucleus and the cytoplasm, whereas in a subset of progerin-expressing cells the cytoplasmic level of Ran exceeded that of the nucleus (F).
We then considered whether the Ran distribution defects might arise because structural components (i.e., nucleoporins) necessary for Ran import are missing from the NPC in HGPS cells. IF microscopy with antibodies to nucleoporins that comprise the nuclear (Nup153 and TPR), cytoplasmic (Nup358/RanBP2), and central (p62) domains of the NPC was performed. The nucleoporins Nup153, RanBP2, and p62 each localized to the NPC (data not shown). The nucleoporin TPR, however, accumulated in the cytoplasm of HGPS cells and displayed a reduced level of NPC localization ( A). To determine if progerin expression is sufficient to cause TPR localization to the cytoplasm, we transfected HeLa cells with HA-progerin and HA-lamin A and examined the samples by double-label IF microscopy. HA-progerin expression resulted in a clear loss of TPR “rim staining” (B and C). Thus, progerin expression is sufficient to disrupt the subcellular distribution of TPR.
Fig. 3. Progerin inhibits TPR import. (A) Distribution of endogenous TPR in control (Normal 8469) and progeria (HGPS 1972, HGPS 1498, and HGPS 3199) fibroblasts. (B) HA-lamin A and HA-progerin transfection in HeLa cells costained for endogenous TPR (red) and (more ...)
Although TPR is a component of the NPC, it contains an NLS and undergoes Ran-dependent nuclear import following the postmitotic assembly of the NPC (5
). While progerin could induce the cytoplasmic localization of TPR (A and B) by any one of several mechanisms, arguably the simplest explanation was that progerin inhibited the nuclear import of TPR. We tested this by using a PK fusion protein engineered with the 56-amino-acid NLS from TPR (PK-NLSTPR
was localized to the nucleus in cells cotransfected with HA-lamin A (C). In contrast, PK-NLSTPR
showed a clear cytoplasmic localization when coexpressed with HA-progerin, and in the same cells there was a nuclear import defect in endogenous TPR (C). Because the progerin-induced TPR transport defect can be recapitulated by using the NLS from TPR, we conclude that cytoplasmic localization of TPR occurs because progerin inhibits the TPR import pathway.
The fact that the Ran N/C gradient is disrupted in HGPS cells () led us to test whether the TPR import defect is a downstream consequence of the Ran defect. To test this prediction, we disrupted the Ran protein gradient by depleting the Ran import factor NTF2 in HeLa cells and analyzing the distribution of endogenous Ran and TPR by double-label IF microscopy. HeLa cells treated with siRNA to NTF2 showed a marked defect in the Ran N/C distribution, similar to the Ran localization that we observed for HGPS fibroblasts and HeLa cells expressing progerin (). HeLa cells that had a disrupted Ran gradient displayed a predominantly cytoplasmic distribution of TPR (D), again similar to that observed for HGPS cells.
Given that TPR is a key architectural component of the NPC, we tested whether the loss of TPR (independent of progerin expression) affects the Ran N/C distribution. In this experiment we used siRNA to deplete TPR and performed double-label IF microscopy for TPR and Ran. We found that the nuclear distribution of Ran was unaffected by the loss of TPR from the NPC (E). Thus, TPR undergoes Ran-dependent import, TPR cytoplasmic localization in HGPS cells is a consequence of the disruption of the Ran gradient, and nuclear TPR is not required for the Ran gradient.
Progerin reduces nuclear SUMO2/3 levels.
The NPC and the nuclear lamina provide anchoring sites for enzymes that regulate SUMOylation (31
). For example, TPR orthologues in S. cerevisiae
and Arabidopsis thaliana
direct the subnuclear localization and/or activity of the deSUMOylating SENP enzymes (78
). Moreover, the SUMO-conjugating enzyme Ubc9 binds to nucleoporins on the cytoplasmic and nuclear sides of the NPC (82
). Given these considerations, we postulated that nuclear SUMOylation might be altered by the progerin perturbation of the nuclear lamina structure or by the progerin inhibition of TPR import. To test this idea, we transfected HeLa cells with HA-tagged WT lamin A and HA-tagged progerin and examined the levels of endogenous SUMO1- and SUMO2/3-modified proteins by quantitative IF microscopy. Levels of SUMO1-conjugated proteins in the nucleus detected by IF were unchanged by the expression of HA-WT lamin A and HA-progerin ( A, top). In contrast, HA-progerin expression reduced the levels of SUMO2/3-conjugated proteins detected by IF microscopy (A, bottom). The effect of progerin on SUMO2/3 levels in the nucleus was validated by using HGPS patient cells, which showed a statistically significant reduction in SUMO2/3 levels compared with those of Normal 8469 fibroblasts (B and C). The nuclear signal for SUMO2/3 was correlated with the Ran protein gradient in HGPS cells (ρ values of 0.19 for Normal 8469, 0.82 for HGPS 1972, 0.70 for HGPS 1498, and 0.53 for HGPS 3199) (C and D). As was the case with Ran, there was cell-to-cell variation in HGPS cells, which ranged from normal nuclear levels of SUMO2/3 to significantly reduced levels.
Fig. 4. SUMO2/3 levels in the nucleus are reduced in response to progerin expression. (A) Overlay of SUMO1 and SUMO2/3 distributions (red) with HA-progerin and HA-WT lamin A (green) in HeLa cells. (B) SUMO2/3 (red) and Ran (green) levels in control (Normal 8469) (more ...)
These data suggest that progerin reduces the nuclear levels of SUMO2/3-modified proteins. Immunoblotting with HGPS cell extracts failed to reveal an obvious reduction in levels of SUMO2/3-modified proteins (n = 6 experiments) (data are available upon request). This finding might be explained by a cell population that is heterogeneous with regard to SUMO2/3 levels. Differences between the cell lines could also be obscured by the loss of the SUMO2/3 modification during handling, despite the inclusion of N-ethylmaleimide to inhibit postlysis deSUMOylation by SENPs.
Two approaches were used to corroborate our immunofluorescence data showing that progerin reduces nuclear levels of SUMO2/3. First, we employed a fluorescent protein fusion (mCherry-SUMO2) as a direct readout of progerin effects on the SUMO2 distribution in the cell. mCherry-SUMO2 was cotransfected with HA-progerin and HA-lamin A, and the distributions of mCherry-SUMO2, HA-tagged protein, and endogenous Ran were visualized by triple labeling. We measured both nuclear fluorescence and total cellular fluorescence generated by mCherry-SUMO2 in HA-positive cells and plotted the data as a ratio ( A and B). The mCherry-SUMO2 fluorescence (ratio of nuclear to total fluorescence) was reduced in cells cotransfected with HA-progerin compared with cells cotransfected with HA-lamin A. This result suggests that progerin expression reduces nuclear SUMOylation or reduces the nuclear import of SUMOylated proteins or that nuclear SUMOylation is reduced by a combination of mechanisms.
Fig. 5. Membrane attachment underlies progerin effects on SUMOylation. (A) Images from HeLa cells triple labeled for HA-progerin and HA-WT lamin A (purple), endogenous Ran (green), and mCherry-SUMO2 (red). (B) Histograms (bin size = 0.1) of Cherry-SUMO2 levels (more ...)
Second, we examined SUMOylation by an approach that does not rely on transfection but instead takes advantage of an aspartyl protease inhibitor shown previously by other groups to inhibit lamin A processing (15
). The HIV protease inhibitor lopinavir blocks the Zmpste24 cleavage of prelamin A, and as a consequence, prelamin A remains membrane anchored. Since the lopinavir-induced accumulation of WT prelamin A induces nuclear morphology defects similar to those reported for progerin, we reasoned that lopinavir should mimic the effects of progerin expression with regard to Ran system and SUMOylation defects. Lopinavir treatment induced the appearance of a slower-migrating form of lamin A that was recognized by a prelamin A-specific antibody (E). By double-label IF we found that lopinavir treatment disrupted the Ran gradient and reduced nuclear levels of endogenous SUMO2/3 (C and D). Thus, membrane-tethered WT prelamin A (endogenous) has progerin-like effects on Ran and SUMOylation.
Inhibition of nuclear SUMOylation disrupts the Ran protein gradient.
The correlation between SUMO2/3 levels and the Ran protein gradient in HGPS cells led us to explore whether there is a link between nuclear SUMOylation and the Ran protein gradient. We transfected HeLa cells with a catalytic mutant of Ubc9 (C93S mutant) that reduces SUMOylation by acting as a dominant negative protein (13
), and we transfected the catalytic domain (CD) of the SUMO protease SENP2, which cleaves SUMO from target proteins (50
). FLAG-tagged WT and C93S forms of Ubc9 and the FLAG-tagged SENP CD were introduced into HeLa cells and examined by double-label IF microscopy for FLAG and Ran or SUMO2/3 and Ran. The expression of FLAG-Ubc9 C93S and the FLAG-SENP CD reduced nuclear levels of Ran ( A). Cells with reduced levels of nuclear SUMO2/3 signal showed a loss of the Ran protein gradient (A). Thus, a reduction of the level of nuclear SUMOylation is sufficient to reduce the level of Ran in the nucleus.
RanGAP is one of the key regulators of the Ran system, and the SUMOylation of RanGAP regulates its anchorage to the NPC (45
). We examined whether RanGAP targeting to the NPC is disrupted by progerin and SENP CD expression, settings where SUMOylation is markedly reduced. In cells where the SENP CD and progerin reduced nuclear SUMOylation and disrupted the Ran protein gradient, RanGAP was still targeted to the NPC (B and C). This result together with the fact that RanGAP is localized to the NPC in HGPS patient cells (Kelley and Paschal, unpublished) indicate that the Ran protein gradient disruption in HGPS is not caused by a failure of RanGAP targeting to the NPC.
Defective nuclear localization of Ubc9 in progeria.
The reduced nuclear levels of SUMO2/3 caused by progerin expression () led us to hypothesize that progerin inhibits the activity or localization of a component that is critical for SUMOylation. The sole E2 for SUMO conjugation is Ubc9, an enzyme found in both the nucleus and the cytoplasm (40
). In normal human fibroblasts, Ubc9 was mostly nuclear, with a small pool detected in the cytoplasm ( A). In progeria fibroblasts, however, Ubc9 was localized predominantly to the cytoplasm. Moreover, in cells where the distribution of Ubc9 was predominantly cytoplasmic, there was a strong disruption of the Ran gradient (HGPS 3199) (A and data not shown). The reduced nuclear localization of Ubc9 was linked to progerin expression by showing that HA-progerin transfection is sufficient to cause a quantitative reduction in the N/C distribution of endogenous Ubc9 in HeLa cells (B and data not shown).
Fig. 7. Reduced nuclear localization of Ubc9 in HGPS. (A) Endogenous Ran (green) and Ubc9 (red) in control (Normal 8469) and progeria (HGPS 3199) fibroblasts. (B) Localization of endogenous Ubc9 (green) and SUMO2/3 (red) in HeLa cells cotransfected with HA-WT (more ...) Constitutive nuclear localization of Ubc9 restores the Ran gradient in cells expressing progerin.
The overexpression of factors that reduce nuclear SUMOylation (SENP CD and dominant negative Ubc9) disrupted the Ran gradient (), suggesting a potential link between SUMOylation and the Ran system. Since progerin induced the cytoplasmic localization of Ubc9, we considered whether the loss of Ubc9 from the nucleus is responsible for the disruption of the Ran gradient in HGPS. We engineered Ubc9 with transport signal fusions (SV40 NLS and PKI NES) to force its subcellular localization to the nucleus and cytoplasm ( A); we then tested whether compartment-specific forms of Ubc9 could rescue the Ran gradient. We also introduced the C93S mutation into the NLS form of Ubc9 to address whether the catalytic function of Ubc9 is necessary for effects on the Ran gradient. Based on the transfection efficiency (~25%), expression levels of FLAG-tagged forms of Ubc9 were approximately 2-fold that of endogenous Ubc9 (B). As expected, progerin inhibited the nuclear localization of transfected FLAG-tagged WT Ubc9; however, the SV40 NLS fusion rendered both the WT and the catalytically inactive forms of Ubc9 resistant to this effect of progerin (A). The PKI NES fusion with WT Ubc9 was exclusively cytoplasmic in the absence and presence of progerin (A).
Fig. 8. Transport signal fusions that direct nuclear and cytoplasmic targeting of Ubc9. (A) FLAG-tagged SV40 NLS and PKI NES fusions with Ubc9 (green) were expressed in HeLa cells in the absence and presence of HA-progerin (red). Scale bar, 20 μm. (B) (more ...)
The four engineered forms of Ubc9 () were each cotransfected with progerin into HeLa cells, and IF microscopy was used to measure Ran N/C levels and the nuclear levels of SUMO2/3. The controls for the experiment were WT Ubc9 cotransfected with WT lamin A (, black histograms) and WT Ubc9 cotransfected with progerin (red histograms). Progerin induced a quantitative reduction in the Ran N/C and SUMO2/3 levels (, red histograms), both of which were rescued by NLS-Ubc9 expression (green histograms). The catalytically inactive Ubc9 (C93S mutant) (blue histograms) targeted to the nucleus and cytoplasmic WT Ubc9 (NES fusion) (plum histograms) targeted to the cytoplasm both failed to restore the Ran N/C and nuclear SUMO2/3 levels in the presence of progerin (). These data show that the SUMO2/3 reduction and Ran gradient disruption by progerin (measured by IF microscopy) can be rescued by forcing Ubc9 into the nucleus and that the restoration of the Ran gradient requires the catalytic activity of Ubc9.
Fig. 9. Forcing nuclear localization of Ubc9 rescues the Ran gradient in progerin-expressing cells. HeLa cells were cotransfected with HA-WT lamin A and HA-progerin, together with the transport signal fusions of Ubc9. Histograms show the nuclear levels of SUMO2 (more ...) Constitutive nuclear localization of Ubc9 restores Ran-dependent transport of TPR.
To determine whether the Ubc9-mediated rescue of the Ran gradient in progerin-expressing cells also rescued Ran-dependent nuclear import, we analyzed the distribution of endogenous TPR in HeLa cells cotransfected with progerin, WT Ubc9, and NLS-Ubc9. The reduction in the nuclear import of TPR caused by progerin expression ( A, middle) was restored by the cotransfection of NLS-Ubc9 (A, bottom). By measuring the N/C ratios of TPR in cells expressing WT lamin A and WT Ubc9 (B, black histograms), progerin and WT Ubc9 (red histograms), and progerin and NLS-Ubc9 (green histograms), we determined that NLS-Ubc9 expression results in a quantitative increase in TPR import (B).
Fig. 10. Nuclear localization of Ubc9 restores TPR import in cells expressing progerin. (A) IF localization of HA-progerin and endogenous TPR in cells cotransfected with Ubc9. Scale bar, 20 μm. (B) Histograms showing TPR N/C levels (bin size = 0.5) in (more ...)
The correlation between the Ran gradient and nuclear levels of the chromatin mark H3K9me3 () suggested that these pathways might be linked. We tested whether the restoration of the Ran gradient via NLS-Ubc9 could restore H3K9me3 levels. By IF microscopy, HA-progerin transfected into HeLa cells with WT Ubc9 caused a statistically significant reduction of H3K9me3 levels ( A, middle). Significantly, the cotransfection of HA-progerin with NLS-Ubc9 restored nuclear levels of H3K9me3 (A, bottom). Our data suggest that the reduced level of H3K9me3 induced by progerin expression is a consequence of either a Ran gradient disruption, a reduced level of Ubc9 function in the nucleus, or the combined effect of these changes.
Fig. 11. Nuclear localization of Ubc9 restores H3K9me3 levels in cells expressing progerin. (A) IF localization of HA-progerin and nuclear H3K9me3 in cells cotransfected with Ubc9. Scale bar, 20 μm. (B) Histograms showing H3K9me3 levels (bin size = 1) (more ...) Ran system defects require progerin attachment to the nuclear membrane.
Farnesyl transferase inhibitors (FTIs) block the prenylation of progerin, which prevents its attachment to the inner nuclear membrane (81
). Nuclear morphology defects in cultured cells and body weight gain in a mouse model of progeria are both improved by FTIs (11
). We tested whether the HGPS phenotypes described in this study are dependent on the constitutive attachment of progerin to the nuclear membrane. We treated HGPS 3199 cells with FTI-277 or vehicle for 3 days and subsequently examined Ran, TPR, SUMO2/3, and H3K9me3 by IF microscopy. The nuclear levels of these proteins and modifications were increased by FTI-277 treatment, indicating that these nuclear phenotypes are induced by the constitutive attachment of progerin to the inner nuclear membrane (). These observations are consistent with the data shown in .
Fig. 12. Farnesylation is required for the inhibitory effects of progerin on Ran, TPR, SUMO2/3, and H3K9me3. Progeria fibroblasts (HGPS 3199) were treated with FTI-277 (3 μM) or DMSO (0.1%) for 72 h and examined by IF microscopy for the indicated proteins. (more ...) Progerin expression and SUMOylation alter RCC1-chromatin interactions in living cells.
Several properties of RCC1 led us to examine whether progerin transduces its effects to the Ran gradient by modulating RCC1 activity: (i) RCC1 activity is required for the Ran protein gradient (60
), (ii) RCC1 binds and dissociates from chromatin during nucleotide exchange on Ran (43
), (iii) RCC1 binds preferentially to heterochromatin (12
), and (iv) progerin reduces the levels of the heterochromatin marks H3K9me3 and H3K27me3. Additionally, RCC1 remains nuclear in progerin-expressing cells. We used fluorescence recovery after photobleaching (FRAP) to test whether progerin affects RCC1, as the nuclear mobility of RCC1 reflects its binding and dissociation from chromatin (41
). We also used FRAP to test whether RCC1 mobility is sensitive to nuclear levels of SUMOylation by transfecting the FLAG-SENP2 CD. HeLa cells expressing HA-progerin and the FLAG-SENP CD were selected for FRAP analysis based on the distribution of cotransfected mCherry-SUMO2, which we found was nuclear in HA-lamin A-transfected cells but additionally localizes to the cytoplasm when cotransfected with HA-progerin and the FLAG-SENP CD ( A). In cells transfected with HA-progerin and the FLAG-SENP CD, the t1/2
values for the recovery of RCC1-GFP were increased by 38% and 50%, respectively (B). Because progerin is mostly restricted to the nuclear membrane but the FRAP measurements reflect RCC1 mobility throughout the nucleus, our data suggest that progerin-induced changes in the nuclear lamina are transduced to RCC1 throughout the nucleoplasm. Finally, the SENP-induced reduction in RCC1 mobility suggests that nuclear SUMOylation can regulate RCC1-chromatin dynamics.
Fig. 13. SUMOylation regulates RCC1-chromatin interactions. (A) Localizations of mCherry-SUMO2 (red) and RCC1-GFP (green) in HeLa cells cotransfected HA-WT lamin A, HA-progerin, or the FLAG-SENP CD. (B) Nuclear mobility of RCC1-GFP measured by FRAP of cells expressing (more ...) SUMOylation promotes RCC1 release from chromatin in vitro.
The current model for the nucleotide exchange cycle includes the formation of a Ran-RCC1-chromatin complex that is dissociated upon GTP binding to Ran (32
). The fact that SENP2 CD expression reduced RCC1 mobility (B) suggested that SUMOylation levels in the nucleus influence RCC1-chromatin interactions, which would be predicted to affect nucleotide exchange on Ran. We used a biochemical assay to examine whether SUMOylation affects the interaction between RCC1 and chromatin. Chromatin from HeLa cells was incubated with recombinant Ran (His tagged), which binds to the endogenous RCC1. Following two wash steps, the Ran-RCC1-chromatin complexes were incubated with recombinant factors that mediate SUMOylation (C). A maximal release of RCC1 from chromatin was observed in the presence of SUMO2, E1, E2 (Ubc9), ATP, and GTP (C, lane 8). The substitution of a nonconjugable form of SUMO2 (denoted SUMO2-G) (C, lane 9) or the omission of GTP from the reaction mixture (lane 7) reduced the amount of RCC1 released from chromatin, consistent with the interpretation that, in this assay, RCC1 release is regulated by SUMOylation and GTP binding to Ran. As an additional means of testing whether Ran release from RCC1 is necessary for the SUMOylation-dependent dissociation of RCC1 from chromatin, we performed the experiments using chromatin from HeLa cells prebound with recombinant WT and T24N forms of Ran (both GST tagged). The T24N mutant of Ran was used because it “locks” RCC1 into chromatin (41
). The T24N mutant of Ran rendered endogenous RCC1 resistant to dissociation by in vitro
SUMOylation (D, compare lanes 6 and 12). This finding is consistent with the possibility that nucleotide binding to Ran and dissociation from RCC1 could be linked to the SUMOylation-stimulated release of these proteins from chromatin. RCC1 itself does not appear to be SUMOylated under the conditions of this assay, leading us to posit that RCC1 dissociation is promoted by a chromatin-associated factor that is SUMOylated.