RESUMO
We have previously isolated and characterized a mouse cDNA orthologous to the human synovial sarcoma associated SS18 (formerly named SSXT and SYT) cDNA. Here, we report the characterization of the genomic structure of the mouse Ss18 gene. Through in silico methods with sequence information contained in the public databases, we did the same for the human SS18 gene and two human SS18 homologous genes, SS18L1 and SS18L2. In addition, we identified a mouse Ss18 processed pseudogene and mapped it to chromosome 1, band A2-3. The mouse Ss18 gene, which is subject to extensive alternative splicing, is made up of 11 exons, spread out over approximately 45 kb of genomic sequence. The human SS18 gene is also composed of 11 exons with similar intron-exon boundaries, spreading out over about 70 kb of genomic sequence. One alternatively spliced exon, which is not included in the published SS18 cDNA, corresponds to a stretch of sequence which we previously identified in the mouse Ss18 cDNA. The human SS18L1 gene, which is also made up of 11 exons with similar intron-exon boundaries, was mapped to chromosome 20 band q13.3. The smaller SS18L2 gene, which is composed of three exons with similar boundaries as the first three exons of the other three genes, was mapped to chromosome 3 band p21. Through sequence and mutation analyses this gene could be excluded as a candidate gene for 3p21-associated renal cell cancer. In addition, we created a detailed BAC map around the human SS18 gene, placing it unequivocally between the CA-repeat marker AFMc014wf9 and the dihydrofolate reductase pseudogene DHFRP1. The next gene in this map, located distal to SS18, was found to be the TBP associated factor TAFII-105 (TAF2C2). Further analogies between the mouse Ss18 gene, the human SS18 gene and its two homologous genes were found in the putative promoter fragments. All four promoters resemble the promoters of housekeeping genes in that they are TATA-less and embedded in canonical CpG islands, thus explaining the high and widespread expression of the SS18 genes.
Assuntos
Cromossomos Humanos Par 20/genética , Cromossomos Humanos Par 3/genética , Proteínas/genética , Pseudogenes/genética , Sarcoma Sinovial/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Mapeamento de Sequências Contíguas , Ilhas de CpG/genética , Éxons/genética , Humanos , Hibridização in Situ Fluorescente , Íntrons/genética , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Proteínas/química , Proteínas Proto-Oncogênicas , Sítios de Splice de RNA/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Proteínas Repressoras , Elementos de Resposta/genética , Homologia de Sequência , Fatores de Transcrição/metabolismoRESUMO
The SSX genes, located on the X chromosome, encode a family of highly homologous nuclear proteins. The SSX1 and SSX2 genes were initially identified as fusion partners of the SYT gene in t(X;18)-positive synovial sarcomas. Recently, however, it was found that these two genes, as well as the highly homologous SSX4 and SSX5 genes, are aberrantly expressed in different types of cancers, including melanomas. Because normal SSX expression has been detected only in the testis and, at very low levels, the thyroid, these proteins are considered as new members of the still growing family of cancer/testis antigens. These antigens are presently considered as targets for the development of cancer immunotherapy protocols. In the present study, we developed a monoclonal antibody found to recognize SSX2, SSX3, and SSX4 proteins expressed in formaldehyde-fixed and paraffin-embedded tissues. This antibody was used to investigate SSX expression in normal testis and thyroid, benign melanocytic lesions, melanoma lesions, and melanoma cell lines. SSX nuclear expression in the testis was found to be restricted to spermatogenic cells, mainly spermatogonia. Of 18 melanoma cell lines analyzed, 9 showed SSX RNA and protein expression, although heterogeneously and at variable levels. Treatment of an SSX-negative cell line with 5-aza-2'-deoxycytidine, a demethylating agent, led to SSX RNA and protein expression, indicating a role for methylation in transcription regulation. Thirty-four of 101 primary and metastatic melanoma cases and 2 of 24 common nevocellular and atypical nevus cases showed SSX nuclear staining. Again, SSX expression was heterogeneous, ranging from widespread to scarce. Our findings stress the importance of assessing the a priori SSX expression status of melanoma cases that may be selected for immunotherapeutic trials.
Assuntos
Melanoma/genética , Proteínas de Neoplasias/genética , Proteínas Repressoras/genética , Testículo/metabolismo , Adulto , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Células CACO-2 , Núcleo Celular/química , Criança , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Imuno-Histoquímica , Células K562 , Masculino , Melanoma/metabolismo , Melanoma/patologia , Microscopia de Fluorescência , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismo , RNA Neoplásico/efeitos dos fármacos , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Repressoras/imunologia , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Deleção de Sequência , Testículo/química , Células Tumorais CultivadasRESUMO
In the vast majority of synovial sarcomas the N-terminal part of the SYT protein is fused to the C-terminal part of an SSX protein, either SSX1 or SSX2. The wild-type proteins, as well as the resultant SYT-SSX1 and SYT-SSX2 fusion proteins, are localized in the nucleus. Recent studies in experimental systems indicated that the SYT protein may function as a transcriptional activator whereas the SSX proteins may act as transcriptional repressors. In the present work we created a series of deletion mutants and found that SYT and SSX depend on N-terminal and highly conserved C-terminal domains for nuclear localization, respectively. Our results also show that the SYT-SSX proteins colocalize with SSX2, a feature that depends on the presence of the C-terminal SSX sequences in the chimeric proteins. Absence of these sequences led to an altered subcellular localization, coinciding with that of SYT. Besides, we found that endogenously expressed SSX proteins colocalize with polycomb-group proteins and condensed chromosomes during mitosis, features that are also conferred by the C-terminus of SSX. Taken together, these results led us to conclude that the SSX moiety, especially the most C-terminal 34 amino acids, of the SYT-SSX fusion proteins is crucial for aberrant spatial targeting and transcriptional control within the nucleus.