Posttranslational modification with SUMO1 regulates protein/protein interactions, localization, and stability. SUMOylation requires the E1 enzyme Aos1/Uba2 and the E2 enzyme Ubc9. A family of E3-like factors, PIAS proteins, was discovered recently. Here we show that the nucleoporin RanBP2/Nup358 also has SUMO1 E3-like activity. RanBP2 directly interacts with the E2 enzyme Ubc9 and strongly enhances SUMO1-transfer from Ubc9 to the SUMO1 target Sp100. The E3-like activity is contained within a 33 kDa domain of RanBP2 that lacks RING finger motifs and does not resemble PIAS family proteins. Our findings place SUMOylation at the cytoplasmic filaments of the NPC and suggest that, at least for some substrates, modification and nuclear import are linked events.

Post-translational modification by the ubiquitin-like SUMO protein is emerging as a defining feature of eukaryotic cells. Sumoylation has crucial roles in the regulatory challenges that face nucleate cells, including the control of nucleocytoplasmic signalling and transport and the faithful replication of a large and complex genome, as well as the regulation of gene expression.

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In mammalian cells, as in Schizosaccharomyces pombe and Drosophila, HP1 proteins bind histone H3 tails methylated on lysine 9 (K9). However, whereas K9-methylated H3 histones are distributed throughout the nucleus, HP1 proteins are enriched in pericentromeric heterochromatin. This observation suggests that the methyl-binding property of HP1 may not be sufficient for its heterochromatin targeting. We show that the association of HP1α with pericentromeric heterochromatin depends not only on its methyl-binding chromo domain but also on an RNA-binding activity present in the hinge region of the protein that connects the conserved chromo and chromoshadow domains. Our data suggest the existence of complex heterochromatin binding sites composed of methylated histone H3 tails and RNA, with each being recognized by a separate domain of HP1α.

Post-translational protein modification by small ubiquitin-like modifier (SUMO), termed sumoylation, is an important mechanism in cellular responses to stress and one that appears to be upregulated in many cancers. Here, we examine the role of sumoylation in tumorigenesis as a possibly necessary safeguard that protects the stability and functionality of otherwise easily misregulated gene expression programmes and signalling pathways of cancer cells.

Abstract. The PML protein was first identified as part of a fusion product with the retinoic acid receptor (RARa), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RARa hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4-ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes.T HE eukaryotic nucleus is highly organized into discrete domains which spatially separate different biochemical processes. A variety of metabolic activities such as DNA replication, ribosome assembly, transcription, and pre-mRNA processing localize to distinct subnuclear compartments. The most conspicuous example is the nucleolus in which rRNAs are assembled into ribosomal subunits (reviewed by Scheer and Weisenberger, 1994). DNA replication sites were also shown to concentrate within discrete regions containing the proliferating cell nuclear antigen (PCNA; 1 Bravo and MacdonaldBravo, 1987) as well as DNA methyltransferase (Leonhardt et al., 1992). In addition, the small nuclear ribonucleoprotein particles (snRNPs), which are the major subunits of spliceosomes, were similarly found to localize to T. Carvalho and J.-S. Seeler should be considered as first authors.

Transcriptional repression mediated through histone deacetylation is a critical component of eukaryotic gene regulation. Here we demonstrate that the class II histone deacetylase HDAC4 is covalently modified by the ubiquitin-related SUMO-1 modifier. A sumoylation-deficient point mutant (HDAC4-K559R) shows a slightly impaired ability to repress transcription as well as reduced histone deacetylase activity. The ability of HDAC4 to self-aggregate is a prerequisite for proper sumoylation in vivo. Calcium/calmodulin-dependent protein kinase (CaMK) signalling, which induces nuclear export, abrogates SUMO-1 modification of HDAC4. Moreover, the modification depends on the presence of an intact nuclear localization signal and is catalysed by the nuclear pore complex (NPC) RanBP2 protein, a factor newly identified as a SUMO E3 ligase. These findings suggest that sumoylation of HDAC4 takes place at the NPC and is coupled to its nuclear import. Finally, modification experiments indicate that the MEF2-interacting transcription repressor (MITR) as well as HDAC1 and -6 are similarly SUMO modified, indicating that sumoylation may be an important regulatory mechanism for the control of transcriptional repression mediated by both class I and II HDACs.

The PML͞SP100 nuclear bodies (NBs) were first described as discrete subnuclear structures containing the SP100 protein. Subsequently, they were shown to contain the PML protein which is part of the oncogenic PML-RAR␣ hybrid produced by the t(15;17) chromosomal translocation characteristic of acute promyelocytic leukemia. Yet, the physiological role of these nuclear bodies remains unknown. Here, we show that SP100 binds to members of the heterochromatin protein 1 (HP1) families of non-histone chromosomal proteins. Further, we demonstrate that a naturally occurring splice variant of SP100, here called SP100-HMG, is a member of the high mobility group-1 (HMG-1) protein family and may thus possess DNA-binding potential. Both HP1 and SP100-HMG concentrate in the PML͞SP100 NBs, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When bound to a promoter, SP100, SP100-HMG and HP1 behave as transcriptional repressors in transfected mammalian cells. These observations present molecular evidence for an association between the PML͞ SP100 NBs and the chromatin nuclear compartment. They support a model in which the NBs may play a role in certain aspects of chromatin dynamics.