Repression of SOX6 transcriptional activity by SUMO modification.

SOX6 plays key functions in several developmental processes, including neurogenesis and skeleton formation. In this report, we show that SOX6 is modified in vitro and in vivo by small ubiquitin-related modifier (SUMO) on two distinct sites. Mutation of both sites abolished SOX6 sumoylation and increased SOX6 transcriptional activity. SUMO dependent repression of SOX6 transcription was promoted by UBC9 whereas siRNA to UBC9, cotransfection of inactive UBC9 or a SUMO protease increased SOX6 transcriptional activity. Furthermore, co-expression of SOX6 with SUMO2 results in the appearance of SOX6 in a punctate nuclear pattern that colocalized with promyelocytic leukemia protein, which was partially abolished by mutations in SOX6 sumoylation sites.

Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9.

Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence psiKXE, which represents the consensus SUMO modification site. As a consequence SAE1/SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detected in vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences.

Lamins in disease: why do ubiquitously expressed nuclear envelope proteins give rise to tissue-specific disease phenotypes?

The nuclear lamina is a filamentous structure composed of lamins that supports the inner nuclear membrane. Several integral membrane proteins including emerin, LBR, LAP1 and LAP2 bind to nuclear lamins in vitro and can influence lamin function and dynamics in vivo. Results from various studies suggest that lamins function in DNA replication and nuclear envelope assembly and determine the size and shape of the nuclear envelope. In addition, lamins also bind chromatin and certain DNA sequences, and might influence chromosome position. Recent evidence has revealed that mutations in A-type lamins give rise to a range of rare, but dominant, genetic disorders, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy with conduction-system disease and Dunnigan-type familial partial lipodystrophy. An examination of how lamins A/C, emerin and other integral membrane proteins interact at the INM provides the basis for a novel model for how mutations that promote disease phenotypes are likely to influence these interactions and therefore cause cellular pathology through a combination of weakness of the lamina or altered gene expression.

Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells.

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.

Lamina-associated polypeptide 2alpha binds intranuclear A-type lamins.

The nucleoskeletal protein lamina-associated polypeptide 2(&agr;) (LAP2*) contains a large, unique C terminus and differs significantly from its alternatively spliced, mostly membrane-integrated isoforms, such as LAP2beta. Unlike lamin B-binding LAP2beta, LAP2alpha was found by confocal immunofluorescence microscopy to colocalize preferentially with A-type lamins in the newly formed nuclei assembled after mitosis. While only a subfraction of lamins A and C (lamin A/C) was associated with the predominantly nuclear LAP2alpha in telophase, the majority of lamin A/C colocalized with LAP2alpha in G(1)-phase nuclei. Furthermore, selective disruption of A-type lamin structures by overexpression of lamin mutants in HeLa cells caused a redistribution of LAP2alpha. Coimmunoprecipitation experiments revealed that a fraction of lamin A/C formed a stable, SDS-resistant complex with LAP2alpha in interphase cells and in postmetaphase cell extracts. Blot overlay binding studies revealed a direct binding of LAP2alpha to exclusively A-type lamins and located the interaction domains to the C-terminal 78 amino acids of LAP2alpha and to residues 319-566 in lamin A/C, which include the C terminus of the rod and the entire tail common to lamin A/C. These findings suggest that LAP2alpha and A-type lamins cooperate in the organization of internal nuclear structures.