SSX genes show extensive nucleotide sequence conservation but little is known of their function. Disruption of SSX1 or SSX2, by chromosome translocation and`inframe' fusion to SYT, is a consistent feature of synovial sarcomas. The resulting SYT-SSX1/SSX2 proteins are activators of transcription; transactivation function is located in SYT. Unrearranged SSX1 can repress transcription, and this has been attributed to a putative KruÈ ppel associated box (KRAB) repression domain at the N-terminus. Here we isolated SSX-KRAB domains to speci®cally measure repression activity, using a previously characterized KOX1-KRAB domain as a control. In our repressor assay SSX1-and SSX2-KRAB domains down-modulated the transactivation of a reporter gene by threefold, compared with 83-fold repression achieved by KOX1-KRAB in the assay. Yeast two-hybrid analysis showed that SSX1-KRAB, unlike KOX1-KRAB, fails to interact with the KRAB corepressor TIF1b. These results raise questions about the evolutionary and functional relationship of SSX-KRAB and typical KRAB domains of KruÈ ppel zinc ®nger genes. We found that full-length SSX1 showed potent (74-fold) repression in our repressor assay, indicating the existence of a repression domain distinct from SSX-KRAB. By assaying deletion constructs of SSX1 we localized repression activity to 33 amino acids at the C-terminus. This novel domain is conserved between SSX family members, and, unlike the KRAB-related domain, is retained on fusion with SYT. This has important implications in understanding the mechanism by which the SYT-SSX fusion protein could contribute to neoplasia.Keywords: sarcoma; SSX; KRAB; transcription Chromosome translocation t(X;18)(p11.2;q11.2) (Clark et al., 1994;Crew et al., 1995) is a diagnostic feature of human synovial sarcomas, in some cases representing the sole cytogenetic abnormality (Sandberg and Bridge, 1994). All the available evidence indicates that this translocation is a key event in tumorigenesis. In all tumours characterized, the SYT gene on chromosome 18 is juxtaposed`in-frame' with either the SSX1 gene or SSX2 gene on chromosome X (Figure 1). SSX1 and SSX2 are now known to be members of a highly conserved multigene family Gure et al., 1997), and the SSX loci that have been mapped are all located in chromosome band Xp11.2 (Crew et al., 1995;. In contrast to SYT, which is a widely expressed gene (de Bruijn et al., 1996; J Knight., unpublished data), SSX transcripts show a very restricted distribution in adult human tissues. So far, SSX1 and SSX2 expression has only been detected in testis and thyroid (Crew et al., 1995;Tureci et al., 1996;Gure et al., 1997).As yet, little is known about the normal biological functions of the SYT and SSX gene products. No DNA binding sequences are recognizable in the SYT or SSX proteins. However, when coupled to a GAL4 DNA binding domain in in vitro reporter assays, SYT can activate transcription (70-fold activation) and SSX1 can repress transcription (50-fold repression) from a minimal promoter in NIH3T3 ®broblasts (Brett ...
In order to study to what extent and at which stage serum response factor (SRF) is indispensable for myogenesis, we stably transfected C2 myogenic cells with, successively, a glucocorticoid receptor expression vector and a construct allowing for the expression of an SRF antisense RNA under the direction of the mouse mammary tumor virus long terminal repeat. In the clones obtained, SRF synthesis is reversibly down-regulated by induction of SRF antisense RNA expression by dexamethasone, whose effect is antagonized by the antihormone RU486. Two kinds of proliferation and differentiation patterns have been obtained in the resulting clones. Some clones with a high level of constitutive SRF antisense RNA expression are unable to differentiate into myotubes; their growth can be blocked by further induction of SRF antisense RNA expression by dexamethasone. Other clones are able to differentiate and are able to synthesize SRF, MyoD, myogenin, and myosin heavy chain at confluency. When SRF antisense RNA expression is induced in proliferating myoblasts by dexamethasone treatment, cell growth is blocked and cyclin A concentration drops. When SRF antisense RNA synthesis is induced in arrested confluent myoblasts cultured in a differentiation medium, cell fusion is blocked and synthesis of not only SRF but also MyoD, myogenin, and myosin heavy chain is inhibited. Our results show, therefore, that SRF synthesis is indispensable for both myoblast proliferation and myogenic differentiation.
Chromosome translocation t(X;18)(p11.2;q11.2) is unique to synovial sarcomas and results in an`in frame' fusion of the SYT gene with the SSX1 or closely-related SSX2 gene. Wild-type SYT and SSX proteins, and the SYT-SSX chimaeric proteins, can modulate transcription in gene reporter assays. To help elucidate the role of these proteins in cell function and neoplasia we have performed immunolabelling experiments to determine their subcellular localization in three cell types. Transient expression of epitope-tagged proteins produced distinctive nuclear staining patterns. The punctate staining of SYT and SYT-SSX proteins showed some similarities. We immunolabelled a series of endogenous nuclear antigens and excluded the SYT and SYT-SSX focal staining from association with these domains (e.g. sites of active transcription, snRNPs). In further experiments we immunolabelled the Polycomb group (PcG) proteins RING1 or BMI-1 and showed that SSX and SYT-SSX proteins, but not SYT, co-localized with these markers. Consistent with this we show that SSX and SYT-SSX associate with chromatin, and also associate with condensed chromatin at metaphase. Noteably, SSX produced a dense signal over the surface of metaphase chromosomes whereas SYT-SSX produced discrete focal staining. Our data indicate that SSX and SYT-SSX proteins are recruited to nuclear domains occupied by PcG complexes, and this provides us with a new insight into the possible function of wild-type SSX and the mechanism by which the aberrant SYT-SSX protein might disrupt fundamental mechanisms controlling cell division and cell fate.
Oligonucleotide microarrays were used to analyse gene expression profiles in human ZR75-1 breast cancer cells in the presence of 17 -oestradiol and oestrogen antagonists. Differential gene expression of a number of genes was confirmed by quantitative RNA analysis. In addition to known oestrogenresponsive genes, an appreciable number of novel targets were identified, including growth factors and components of the cell cycle, adhesion molecules, enzymes, signalling molecules and transcription factors. The most pronounced oestrogen-sensitive gene was that for the cytochrome P450-IIB enzyme, involved in metabolising steroids and xenobiotics, which was increased 100-fold over a 24 h period. It is a direct target gene for oestrogens, because its expression was increased in the presence of cyclohexamide. In contrast, expression of cytochrome P450-IIB was not detected in human MCF7 breast cancer cells. Expressions of the cationic amino acid transporter E16, gap junction protein and insulin-like growth factor binding protein 4 were also markedly increased by oestrogens, but the kinetics of induction varied according to the target gene. With the exception of the cationic amino acid transporter E16 and the insulin-like growth factor binding protein 4, the expression of the majority of the genes was unaffected by antioestrogen treatment. Further analysis of this set of markers will provide alternative approaches to the investigation of the mitogenicity of oestrogens in breast cancer cells.
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