RESUMO
Haploinsufficiency of Euchromatin histone methyltransferase 1 (EHMT1), a chromatin modifying enzyme, is the cause of Kleefstra syndrome (KS). KS is an intellectual disability (ID) syndrome, with general developmental delay, hypotonia, and craniofacial dysmorphisms as additional core features. Recent studies have been focused on the role of EHMT1 in learning and memory, linked to the ID phenotype of KS patients. In this study we used the Ehmt1(+/-) mouse model, and investigated whether the core features of KS were mimicked in these mice. When comparing Ehmt1(+/-) mice to wildtype littermates we observed delayed postnatal growth, eye opening, ear opening, and upper incisor eruption, indicating a delayed postnatal development. Furthermore, tests for muscular strength and motor coordination showed features of hypotonia in young Ehmt1(+/-) mice. Lastly, we found that Ehmt1(+/-) mice showed brachycephalic crania, a shorter or bent nose, and hypertelorism, reminiscent of the craniofacial dysmorphisms seen in KS. In addition, gene expression analysis revealed a significant upregulation of the mRNA levels of Runx2 and several other bone tissue related genes in P28 Ehmt1(+/-) mice. Runx2 immunostaining also appeared to be increased. The mRNA upregulation was associated with decreased histone H3 lysine 9 dimethylation (H3K9me2) levels, the epigenetic mark deposited by Ehmt1, in the promoter region of these genes. Together, Ehmt1(+/-) mice indeed recapitulate KS core features and can be used as an animal model for Kleefstra syndrome. The increased expression of bone developmental genes in the Ehmt1(+/-) mice likely contributes to their cranial dysmorphisms and might be explained by diminished Ehmt1-induced H3K9 dimethylation.
Assuntos
Osso e Ossos/metabolismo , Anormalidades Craniofaciais/enzimologia , Anormalidades Craniofaciais/patologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Cardiopatias Congênitas/enzimologia , Cardiopatias Congênitas/patologia , Histona-Lisina N-Metiltransferase/deficiência , Deficiência Intelectual/enzimologia , Deficiência Intelectual/patologia , Crânio/anormalidades , Análise de Variância , Animais , Imunoprecipitação da Cromatina , Deleção Cromossômica , Cromossomos Humanos Par 9/enzimologia , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/patologia , Masculino , Camundongos , Camundongos Knockout , Hipotonia Muscular/genética , Hipotonia Muscular/patologia , Osteopontina , Reação em Cadeia da Polimerase em Tempo RealRESUMO
LRFN5 is a regulator of synaptic development and the only gene in a 5.4 Mb mammalian-specific conserved topologically associating domain (TAD); the LRFN5 locus. An association between locus structural changes and developmental delay (DD) and/or autism was suggested by several cases in DECIPHER and own records. More significantly, we found that maternal inheritance of a specific LRFN5 locus haplotype segregated with an identical type of autism in distantly related males. This autism-susceptibility haplotype had a specific TAD pattern. We also found a male/female quantitative difference in the amount histone-3-lysine-9-associated chromatin around the LRFN5 gene itself (p < 0.01), possibly related to the male-restricted autism susceptibility. To better understand locus behavior, the prevalence of a 60 kb deletion polymorphism was investigated. Surprisingly, in three cohorts of individuals with DD (n = 8757), the number of deletion heterozygotes was 20%-26% lower than expected from Hardy-Weinberg equilibrium. This suggests allelic interaction, also because the conversions from heterozygosity to wild-type or deletion homozygosity were of equal magnitudes. Remarkably, in a control group of medical students (n = 1416), such conversions were three times more common (p = 0.00001), suggesting a regulatory role of this allelic interaction. Taken together, LRFN5 regulation appears unusually complex, and LRFN5 dysregulation could be an epigenetic cause of autism. LAY SUMMARY: LRFN5 is involved with communication between brain cells. The gene sits alone in a huge genomic niche, called the LRFN5 locus, of complex structure and high mammalian conservation. We have found that a specific locus structure increases autism susceptibility in males, but we do not yet know how common this epigenetic cause of autism is. It is, however, a cause that potentially could explain why higher-functioning autism is more common in males than females.
Assuntos
Transtorno do Espectro Autista , Transtorno Autístico , Moléculas de Adesão Celular Neuronais/genética , Animais , Transtorno do Espectro Autista/genética , Transtorno Autístico/genética , Feminino , Haplótipos , Humanos , Masculino , Mamíferos , Polimorfismo GenéticoRESUMO
We describe and evaluate our practice-based learning approach for research in undergraduate students studying Biomedical Sciences at Radboud University Nijmegen, the Netherlands. First-year students who started their study between 2015 and 2018 actively participated in data collection and measurements, including anthropometry, electrocardiogram findings, genetic variants, and lifestyle habits. All data were entered into one anonymous database, which was used by students to analyze their research questions. In 2019, 44 of the 87 students (50%) valued active measurements better than questionnaires. Most students (strongly) agreed that they have learned about data collection and were inspired to learn more about biomedical research.
RESUMO
SSX2 is a member of the family of cancer/testis antigens. The SSX2 derived peptide SSX2(103-111) has been shown to be presented to cytotoxic T-lymphocytes (CTL) by Major-Histocompatibility (MHC) Class-I complexes after endogenous processing, more precisely by the allele HLA-A*0201. The HLA-A*0201- and SSX2-positive melanoma cell line SK-Mel-37 but not Me275 had been shown to elicit reactivity in SSX2(103-111) specific cytotoxic T-lymphocytes. To analyze the correlation between SSX2(103-111) presentation and T-cell stimulation, we intended to visualize presentation of SSX2(103-111) in these melanoma cell lines. Fab-antibodies were established from a human phage library with specificity for SSX2(103-111)/HLA-A*0201 complexes (but non-reactive with HLA-A*0201 or SSX2(103-111) alone) and used to visualize the presentation of SSX2(103-111) in the context of HLA-A*0201 by fluorescence microscopy. Presentation of SSX2(103-111) the context of HLA-A*0201 was demonstrated for the majority of SK-Mel-37, but for only a small fraction (<1%) of Me275 as indicated by a clear membrane-staining pattern in fluorescence microscopy. The presentation of SSX2(103-111) on SK-Mel37 and Me275, but not the expression of the SSX2 protein correlated with the capability of these cells to stimulate cells of an SSX2(103-111)-specific T-cell clone. MHC-peptide specific antibodies are a valuable tool for the analysis of antigenic peptides in the context of MHC-I molecules and for the structural definition of immunodominant epitopes.
Assuntos
Antígenos de Neoplasias/imunologia , Neoplasias da Mama/imunologia , Antígenos HLA-A/imunologia , Melanoma/imunologia , Proteínas de Neoplasias/imunologia , Proteínas Repressoras/imunologia , Células 3T3 , Animais , Especificidade de Anticorpos , Apresentação de Antígeno , Antígenos de Neoplasias/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Antígeno HLA-A2 , Humanos , Fragmentos de Imunoglobulinas/imunologia , Melanoma/genética , Camundongos , Microscopia de Fluorescência , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fragmentos de Peptídeos/imunologia , Reação em Cadeia da Polimerase , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Linfócitos T Citotóxicos/imunologiaRESUMO
Over 95% of all synovial sarcomas (SS) share a unique translocation, t(X;18), however, they show heterogeneous clinical behavior. We analyzed multiple SS to reveal additional genetic alterations besides the translocation. Twenty-six SS from 22 patients were sequenced for 409 cancer-related genes using the Comprehensive Cancer Panel (Life Technologies, USA) on an Ion Torrent platform. The detected variants were verified by Sanger sequencing and compared to matched normal DNAs. Copy number variation was assessed in six tumors using the Oncoscan array (Affymetrix, USA). In total, eight somatic mutations were detected in eight samples. These mutations have not been reported previously in SS. Two of these, in KRAS and CCND1, represent known oncogenic mutations in other malignancies. Additional mutations were detected in RNF213, SEPT9, KDR, CSMD3, MLH1 and ERBB4. DNA alterations occurred more often in adult tumors. A distinctive loss of 6q was found in a metastatic lesion progressing under pazopanib, but not in the responding lesion. Our results emphasize t(X;18) as a single initiating event in SS and as the main oncogenic driver. Our results also show the occurrence of additional genetic events, mutations or chromosomal aberrations, occurring more frequently in SS with an onset in adults.
Assuntos
Análise Mutacional de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Sarcoma Sinovial/genética , Adolescente , Adulto , Idoso , Criança , Variações do Número de Cópias de DNA , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Adulto JovemRESUMO
It was recently shown that duplications of the RevSex element, located 0.5 Mb upstream of SOX9, cause XX-disorder of sex development (DSD), and that deletions cause XY-DSD. To explore how a 148 kb RevSex duplication could have turned on gonadal SOX9 expression in the absence of SRY in an XX-male, we examined the chromatin landscape in primary skin fibroblast cultures from the index, his RevSex duplication-carrier father and six controls. The ENCODE project supports the notion that chromatin state maps show overlap between different cell types, i.e., that our study of fibroblasts could be of biological relevance. We examined the SOX9 regulatory region by high-resolution ChIP-on-chip experiments (a kind of "chromatin-CGH") and DNA methylation investigations. The RevSex duplication was associated with chromatin changes predicting better accessibility of the SRY-responsive TESCO enhancer region 14-15 kb upstream of SOX9. Four kb downstream of the TESCO evolutionary conserved region, a peak of the enhancer/promoter-associated H3K4me3 mark was found together with a major dip of the repressive H3K9me3 chromatin mark. Similar differences were also found when three control males were compared with three control females. A marked male/female difference was a more open chromatin signature in males starting ~400 kb upstream of SOX9 and increasing toward the SOX9 promoter. In the RevSex duplication-carrier father, two positions of DNA hypomethylation were also found, one corresponding to the H3K4me3 peak mentioned above. Our results suggest that the RevSex duplication could operate by inducing long-range epigenetic changes. Furthermore, the differences in chromatin state maps between males and females suggest that the Y chromosome or X chromosome dosage may affect chromatin conformation, i.e., that sex-dependent gene regulation may take place by chromatin modification.
Assuntos
Transtornos 46, XX do Desenvolvimento Sexual/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Duplicação Gênica , Fatores de Transcrição SOX9/genética , Células Cultivadas , Metilação de DNA , Feminino , Disgenesia Gonadal 46 XY/genética , Heterozigoto , Humanos , Masculino , Regiões Promotoras Genéticas , Fatores SexuaisRESUMO
In the last decade, it has become apparent that not only DNA sequence variations but also epigenetic modifications may contribute to disease, including cancer. These epigenetic modifications involve histone modification including acetylation and methylation, DNA methylation, and chromatin remodeling. One of the best-characterized epigenetic changes is aberrant methylation of cytosines that occur in so-called CpG islands. DNA hypomethylation, prevalent as a genome-wide event, usually occurs in more advanced stages of tumor development. In contrast, DNA hypermethylation is often observed as a discrete, targeted event within tumor cells, resulting in specific loss of gene expression. Interestingly, it was found that sporadic and inherited cancers may exhibit similar DNA methylation patterns, and many genes that are mutated in familial cancers have also been found to be hypermethylated, mutated, or deleted in sporadic cancers. In this review, we will focus on DNA methylation events as heritable epimutations predisposing to colorectal cancer development.
Assuntos
Neoplasias Colorretais/genética , Epigênese Genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Polipose Adenomatosa do Colo/genética , Neoplasias Colorretais/etiologia , Metilação de DNA , Genes APC , Mutação em Linhagem Germinativa , Humanos , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS/genética , Proteínas Nucleares/genéticaRESUMO
BACKGROUND: Although the mitotic arrest deficient protein MAD2B (MAD2L2) is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1 (FZR1), its exact role in cell cycle control still remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the human clathrin light chain A (CLTA) as a novel MAD2B binding protein. A direct interaction was established in mammalian cells via GST pull-down and endogenous co-immunoprecipitation during the G2/M phase of the cell cycle. Through subsequent confocal laser scanning microscopy we found that MAD2B and CLTA co-localize at the mitotic spindle. Clathrin forms a trimeric structure, i.e., the clathrin triskelion, consisting of three heavy chains (CLTC), each with an associated light chain. This clathrin structure has previously been shown to be required for the function of the mitotic spindle through stabilization of kinetochore fibers. Upon siRNA-mediated MAD2B depletion, we found that CLTA was no longer concentrated at the mitotic spindle but, instead, diffusely distributed throughout the cell. In addition, we found a marked increase in the percentage of misaligned chromosomes. CONCLUSIONS/SIGNIFICANCE: Previously, we identified MAD2B as an interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and a concomitant failure to shuttle MAD2B to the nucleus. Our current data show that MAD2B interacts with CLTA during the G2/M phase of the cell cycle and that depletion of MAD2B leads to a marked increase in the percentage of misaligned chromosomes and a redistribution of CLTA during mitosis.
Assuntos
Cadeias Leves de Clatrina/metabolismo , Mitose , Proteínas/metabolismo , Fuso Acromático/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cadeias Leves de Clatrina/genética , Fase G2 , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Mad2 , Microscopia Confocal , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Proteínas/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
BACKGROUND: Previously, we identified the mitotic arrest deficient protein MAD2B (MAD2L2) as a bona fide interactor of the renal cell carcinoma (RCC)-associated protein PRCC. In addition, we found that fusion of PRCC with the transcription factor TFE3 in t(X;1)(p11;q21)-positive RCCs results in an impairment of this interaction and, concomitantly, an abrogation of cell cycle progression. Although MAD2B is thought to inhibit the anaphase promoting complex (APC) by binding to CDC20 and/or CDH1(FZR1), its exact role in cell cycle control still remains to be established. METHODOLOGY/PRINCIPAL FINDINGS: Using a yeast two-hybrid interaction trap we identified the small GTPase RAN, a well-known cell cycle regulator, as a novel MAD2B binding protein. Endogenous interaction was established in mammalian cells via co-localization and co-immunoprecipitation of the respective proteins. The interaction domain of RAN could be assigned to a C-terminal moiety of 60 amino acids, whereas MAD2B had to be present in its full-length conformation. The MAD2B-RAN interaction was found to persist throughout the cell cycle. During mitosis, co-localization at the spindle was observed. CONCLUSIONS/SIGNIFICANCE: The small GTPase RAN is a novel MAD2B binding protein. This novel protein-protein interaction may play a role in (i) the control over the spindle checkpoint during mitosis and (ii) the regulation of nucleocytoplasmic trafficking during interphase.
Assuntos
Mitose , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína ran de Ligação ao GTP/química , Animais , Células COS , Proteínas Cdc20 , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Humanos , Proteínas Mad2 , Modelos Biológicos , Mapeamento de Interação de Proteínas , Técnicas do Sistema de Duplo-HíbridoRESUMO
Synovial sarcoma is a soft tissue malignancy characterized by the fusion of SS18 to either SSX1, SSX2, or SSX4 genes. SS18 and SSX are transcriptional cofactors involved in activation and repression of gene transcription, respectively. SS18 interacts with SWI/SNF, whereas SSX associates with the polycomb chromatin remodeling complex. Thus, fusion of these two proteins brings together two opposing effects on gene expression and chromatin structure. Recent studies have shown that a significant number of genes are down-regulated by the SS18-SSX fusion protein and that the clinically applicable histone deacetylase (HDAC) inhibitor romidepsin inhibits synovial sarcoma growth. Therefore, we set out to identify direct targets of SS18-SSX among genes down-regulated in synovial sarcoma and investigated if romidepsin can specifically counteract SS18-SSX-mediated transcriptional dysregulation. Here, we report that the tumor suppressor early growth response 1 (EGR1) is repressed by the SS18-SSX protein through a direct association with the EGR1 promoter. This SS18-SSX binding correlates with trimethylation of Lys(27) of histone H3 (H3K27-M3) and recruitment of polycomb group proteins to this promoter. In addition, we found that romidepsin treatment reverts these modifications and reactivates EGR1 expression in synovial sarcoma cell models. Our data implicate polycomb-mediated epigenetic gene repression as a mechanism of oncogenesis in synovial sarcoma. Furthermore, our work highlights a possible mechanism behind the efficacy of a clinically applicable HDAC inhibitor in synovial sarcoma treatment.
Assuntos
Depsipeptídeos/farmacologia , Proteína 1 de Resposta de Crescimento Precoce/genética , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Proteínas de Fusão Oncogênica/fisiologia , Sequência de Bases , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Primers do DNA , Regulação para Baixo , Humanos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Interferente PequenoRESUMO
Human synovial sarcomas are aggressive soft tissue tumors with relatively high rates of recurrences and metastases. They display a variable response to common treatment protocols such as radiation and chemotherapy. For the development of novel diagnostic, prognostic, and therapeutic approaches, detailed information on the molecular mechanisms underlying the development of these tumors is of imperative importance. Fusion of the SS18 and (one of the) SSX genes is a molecular hallmark of human synovial sarcomas. The SS18 and SSX genes encode nuclear proteins that exhibit opposite transcription regulatory activities, likely through epigenetic mechanisms. The SS18 protein functions as a transcriptional coactivator and interacts directly with members of the epigenetic chromatin remodeling and modification machineries. In contrast, the SSX proteins function as transcriptional corepressors and are associated with several Polycomb group proteins. Since the domains involved in these apparently opposite transcription regulatory activities are retained in the SS18-SSX fusion proteins, we hypothesize that these fusion proteins function as "activator-repressors" of transcription. The implications of this model for human synovial sarcoma development and future treatment are discussed.
Assuntos
Epigênese Genética/fisiologia , Sarcoma Sinovial/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Translocação GenéticaRESUMO
To determine the expression of cancer testis (CT) genes and antibody responses in a nonselected population of patients with primary breast cancer, we investigated the composite expression of 11 CT genes by RT-PCR in fresh biopsies of 100 consecutive cases of primary breast carcinoma and by immunohistology in selected RT-PCR-positive cases. Antibody responses against 7 CT antigens were analyzed using recombinant antigen expression on yeast surface. In 98 evaluable cases, SCP-1 and SSX-4 were expressed most frequently (both 65%), followed by HOM-TES-85/CT-8 (47%), GAGE (26%), SSX-1 (20%), NY-ESO-1 (13%), MAGE-3 (11%), SSX-2 (8%), CT-10 (7%), MAGE-4 (4%) and CT-7 (1%). One CT gene was expressed by 90% of the cases; 79% expressed > or =2, 48% > or =3, 29% > or =4, 12% > or =5, 6% > or =6, 3% > or =7, 2% > or =8 and one case coexpressed 9 antigens. Of 100 serum samples screened for CT antigen-specific antibodies, antibodies against NY-ESO-1 were detected in 4 patients, against SCP-1 in 6 patients and against SSX-2 in 1 patient, while no antibodies were detected against MAGE-3, CT-7 and CT-10. Expression of CT genes or antibody responses was not correlated with clinical parameters (menopausal status, tumor size, nodal involvement, grading, histology and estrogen receptor status) or the demonstration of CT gene expression at the protein level, by immunohistology. Our results show that breast carcinomas are among the tumors with the most frequent expression of CT antigens, rendering many patients potential candidates for vaccine trials.
Assuntos
Anticorpos Antineoplásicos/imunologia , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Neoplasias da Mama/metabolismo , Testículo/metabolismo , Formação de Anticorpos , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/imunologia , Biomarcadores Tumorais/metabolismo , Biópsia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/imunologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/genética , Carcinoma Lobular/imunologia , Carcinoma Lobular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Invasividade Neoplásica/imunologia , Invasividade Neoplásica/patologia , Estudos Prospectivos , RNA Mensageiro , RNA Neoplásico , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Fusion of the SS18 and either one of the SSX genes is a hallmark of human synovial sarcoma. The SS18 and SSX genes encode nuclear proteins that exhibit opposite transcriptional activities. The SS18 protein functions as a transcriptional coactivator and is associated with the SWI/SNF complex, whereas the SSX proteins function as transcriptional corepressors and are associated with the polycomb complex. The domains involved in these opposite transcriptional activities are retained in the SS18-SSX fusion proteins. Here, we set out to determine the direct transcriptional consequences of conditional SS18-SSX2 fusion protein expression using complementary DNA microarray-based profiling. By doing so, we identified several clusters of SS18-SSX2-responsive genes, including a group of genes involved in cholesterol synthesis, which is a general characteristic of malignancy. In addition, we identified a group of SS18-SSX2-responsive genes known to be specifically deregulated in primary synovial sarcomas, including IGF2 and CD44. Furthermore, we observed an uncoupling of EGR1, JUNB, and WNT signaling in response to SS18-SSX2 expression, suggesting that the SWI/SNF-associated coactivation functions of the SS18 moiety are impaired. Finally, we found that SS18-SSX2 expression affects histone modifications in the CD44 and IGF2 promoters and DNA methylation levels in the IGF2 imprinting control region. Together, we conclude that the SS18-SSX2 fusion protein may act as a so-called transcriptional "activator-repressor," which induces downstream target gene deregulation through epigenetic mechanisms. Our results may have implications for both the development and clinical management of synovial sarcomas.
Assuntos
Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica/fisiologia , Sarcoma Sinovial/genética , Transcrição Gênica/genética , Colesterol/metabolismo , Metilação de DNA , Perfilação da Expressão Gênica , Histonas/metabolismo , Humanos , Fator de Crescimento Insulin-Like II/genética , Proteínas de Neoplasias/biossíntese , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/química , Estrutura Terciária de Proteína , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Sarcoma Sinovial/metabolismo , Somatomedinas/fisiologia , Ativação Transcricional/genéticaRESUMO
Cancer testis antigens (CTAs) are tumor-specific antigens that may be useful targets for cancer vaccines. Here, CTA expression was examined in multiple myeloma (MM), a B-cell cancer characterized by malignant plasma cells (PCs) in the bone marrow (BM), and monoclonal gammopathy of undetermined significance (MGUS), a condition that can progress to MM. We screened a panel of patient BMs at different stages of malignancy for CTA expression by reverse transcription polymerase chain reaction RT-PCR. Here, SSX (synovial sarcoma, X chromosome) emerged as a promising candidate for an MM vaccine, having a profile similar to currently studied CTA, NY-ESO-1, and MAGE. SSX1, 2, 4, and 5 expression was studied further in 114 MM (total SSX, 61% of patients; SSX1, 42%; SSX2, 23%; SSX4, 38%; SSX5, 35%), 45 MGUS (total SSX, 24% of patients; SSX1, 9%; SSX4, 20%), and 12 control (0/12, 0%) subjects. Several expression patterns were observed, the most predominant being co-expression of SSX1, 2, 4, and 5 (called group A expression, in 20% of MM), which correlated with reduced survival (P=0.0006). Of the four genes, SSX2 had the strongest association with reduced survival (P=0.0001). SSX protein expression ranged from 13.5% of PCs in an SSX1/SSX4 co-expressor to as high as 88% of PCs in group A expressor, exceeding reported frequencies of NY-ESO-1 and MAGE in MM. In single PCs from group A patients, we detected variable degrees of SSX co-expression, emphasizing the heterogeneity of CTA expression within tumor cell populations. These results demonstrate that SSX is a frequently expressed CTA in MM and highlight its potential as an MM vaccine candidate.
Assuntos
Antígenos de Neoplasias/genética , Mieloma Múltiplo/genética , Proteínas de Neoplasias/genética , Paraproteinemias/genética , Proteínas Repressoras/genética , Antígenos de Neoplasias/biossíntese , Vacinas Anticâncer , Linhagem Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/biossíntese , Paraproteinemias/imunologia , Paraproteinemias/metabolismo , Plasmócitos/imunologia , Plasmócitos/metabolismo , Prognóstico , RNA Neoplásico/biossíntese , Proteínas Repressoras/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sobrevida , Testículo/imunologiaRESUMO
The AF10 gene encodes a putative transcription factor containing an N-terminal LAP/PHD zinc finger motif, a functional nuclear localization signal, an AT-hook domain, and a leucine zipper toward the C-terminus. AF10 is involved in 2 distinct chromosomal translocations associated with hematologic malignancy. The chimeric fusion proteins MLL/AF10 and CALM/AF10, resulting from the t(10;11)(p12;q23) and the t(10;11)(p12;q14), respectively, consistently retain the leucine zipper motif of AF10. This part of the C-terminal region was used as bait in a yeast 2 hybrid screening of a testis complementary DNA library. The leucine zipper interacted with GAS41, a protein previously identified as the product of an amplified gene in a glioblastoma. GAS41 shows significant homology to the Saccharomyces cerevisiae protein ANC1 and to the human MLL fusion partners AF9 and ENL. The interaction was confirmed in vivo. Furthermore, the study showed by coimmunoprecipitation that GAS41 interacts with INI1 (Integrase Interactor 1) and that INI1 was present in the AF10 immunoprecipitate. INI1 is the human homologue of the yeast SNF5 protein, a component of the SWI/SNF complex, which acts to remodel chromatin and to modulate transcription. The retention of the leucine zipper in the MLL and CALM fusions suggests that a key feature of these chimeric proteins may be their ability to interfere in normal gene regulation through interaction with the adenosine triphosphate-dependent chromatinremodeling complexes.
Assuntos
Proteínas de Drosophila , Proteínas de Ligação a RNA , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Núcleo Celular/química , Proteínas Cromossômicas não Histona , Citoplasma/química , DNA Complementar/análise , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/metabolismo , Biblioteca Gênica , Humanos , Técnicas de Imunoadsorção , Zíper de Leucina , Masculino , Dados de Sequência Molecular , Proteína SMARCB1 , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Análise de Sequência de DNA , Homologia de Sequência , Testículo/química , Fatores de Transcrição/análise , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
A global transcriptional co-activator, the SNF/SWI complex, has been characterized as a chromatin remodeling factor that enhances accessibility of the transcriptional machinery to DNA within a repressive chromatin structure. On the other hand, mutations in some human SNF/SWI complex components have been linked to tumor formation. We show here that SYT, a partner protein generating the synovial sarcoma fusion protein SYT-SSX, associates with native human SNF/SWI complexes. The SYT protein has a unique QPGY domain, which is also present in the largest subunits, p250 and the newly identified homolog p250R, of the corresponding SNF/SWI complexes. The C-terminal region (amino acids 310-387) of SSX1, comprising the SSX1 portion of the SYT-SSX1 fusion protein, binds strongly to core histones and oligonucleosomes in vitro and directs nuclear localization of a green fluorescence protein fusion protein. Experiments with serial C-terminal deletion mutants of SSX1 indicate that these properties map to a common region and also correlate with the previously demonstrated anchorage-independent colony formation activity of SYT-SSX in Rat 3Y1 cells. These data suggest that SYT-SSX interferes with the function of either the SNF/SWI complexes or another SYT-interacting co-activator, p300, by changing their targeted localization or by directly inhibiting their chromatin remodeling activities.
Assuntos
Proteínas/química , Proteínas/metabolismo , Fatores de Transcrição/química , Transcrição Gênica , Sequência de Aminoácidos , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/química , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Glutationa Transferase/metabolismo , Células HeLa , Histonas/metabolismo , Humanos , Immunoblotting , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas , Proteínas Recombinantes de Fusão/metabolismo , Proteínas RepressorasRESUMO
The SSX gene family is composed of at least five functional and highly homologous members, SSX1 to SSX5, that are normally expressed in only the testis and thyroid. SSX1, SSX2, or SSX4 may be fused to the SYT gene as a result of the t(X;18) translocation in synovial sarcoma. In addition, the SSX1, SSX2, SSX4, and SSX5 genes were found to be aberrantly expressed in several other malignancies, including melanoma. The SSX proteins are localized in the nucleus and are diffusely distributed. In addition, they may be included in polycomb-group nuclear bodies. Other studies have indicated that the SSX proteins may act as transcriptional repressors. As a first step toward the elucidation of the cellular signaling networks in which the SSX proteins may act, we used the yeast two-hybrid system to identify SSX2-interacting proteins. By doing so, two novel human proteins were detected: RAB3IP, the human homolog of an interactor of the Ras-like GTPase Rab3A; and a novel protein, SSX2IP. RAB3IP did not interact with either SSX1, SSX3, or SSX4 in the yeast two-hybrid system, whereas SSX2IP interacted with SSX3 but not with either SSX1 or SSX4. Further analysis of deletion mutants showed that both RAB3IP and SSX2IP interact with the N-terminal moiety of the SSX2 protein. Immunofluorescence analyses of transfected cells revealed that the RAB3IP protein is normally localized in the cytoplasm. However, coexpression of both RAB3IP and SSX2 led to colocalization of both proteins in the nucleus. Likewise, the SSX2IP protein was found to be colocalizing with SSX2 in the nucleus. By performing glutathione-S-transferase pull-down assays, we found that both RAB3IP and SSX2IP interact directly with SSX2 in vitro. These newly observed protein/protein interactions may have important implications for the mechanisms underlying normal and malignant cellular growth.