RESUMO
STUDY QUESTION: Are sequence variants in the stromal antigen 3 (STAG3) gene a cause for non-obstructive azoospermia (NOA) in infertile human males? SUMMARY ANSWER: Sequence variants affecting protein function of STAG3 cause male infertility due to meiotic arrest. WHAT IS KNOWN ALREADY: In both women and men, STAG3 encodes for a meiosis-specific protein that is crucial for the functionality of meiotic cohesin complexes. Sequence variants in STAG3 have been reported to cause meiotic arrest in male and female mice and premature ovarian failure in human females, but not in infertile human males so far. STUDY DESIGN, SIZE, DURATION: The full coding region of STAG3 was sequenced directly in a cohort of 28 men with NOA due to meiotic arrest. In addition, a larger group of 275 infertile men that underwent whole-exome sequencing (WES) was screened for potential STAG3 sequence variants. Furthermore, meiotic spreads, immunohistochemistry, WES and population sampling probability (PSAP) have been conducted in the index case. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 28 infertile but otherwise healthy human males who underwent Sanger sequencing of the full coding region of STAG3. Additionally, WES data of 275 infertile human males with different infertility phenotypes have been screened for relevant STAG3 variants. All participants underwent karyotype analysis and azoospermia factor (AZF) screening in advance. In the index patient, segregation analysis, WES data, PSAP, lab parameters, testis histology and nuclear spreads have been added to suplort the findings. MAIN RESULTS AND THE ROLE OF CHANCE: Two compound-heterozygous variants in STAG3 (c.[1262T>G];[1312C>T], p.[(Leu421Arg)];[(Arg438Ter)]) have been found to cause male infertility due to complete bilateral meiotic arrest in an otherwise healthy human male. Compound heterozygosity was confirmed by Sanger sequencing of the parents and the patient's brother. Other variants which may affect spermatogenesis have been ruled out through analysis of the patient's WES data and application of the PSAP pipeline. As expected from Stag3 knockout-mice meiotic spreads, germ cells did not develop further than zygotene and showed drastic chromosome aberrations. No rare variants in STAG3 were found in the 275 infertile males with other phenotypes. Our results indicate that STAG3 variants that negatively affect its protein function are a rare cause of NOA (<1% of cases). LIMITATIONS, REASONS FOR CAUTION: We identified only one patient with compound-heterozygous variants in STAG3 causing NOA due to meiotic arrest. Future studies should evaluate STAG3 variants in larger cohorts to support this finding. WIDER IMPLICATIONS OF THE FINDINGS: Identification of STAG3 sequence variants in infertile human males should improve genetic counselling as well as diagnostics and treatment. Especially before testicular sperm extraction (TESE) for ICSI, STAG3 variants should be ruled out to prevent unnecessary interventions with frustrating outcomes for both patients and clinicians. STUDY FUNDING/COMPETING INTEREST(S): This work was carried out within the frame of the German Research Foundation (DFG) Clinical Research Unit 'Male Germ Cells: from Genes to Function' (CRU326). Work in the laboratory of R.J. is supported by a grant of the European Union H2020 program GermAge. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.
Assuntos
Azoospermia/genética , Proteínas de Ciclo Celular/genética , Infertilidade Masculina/genética , Meiose , Cromossomos/ultraestrutura , Exoma , Heterozigoto , Humanos , Cariotipagem , Masculino , Linhagem , Fenótipo , Probabilidade , Análise de Sequência de DNA , Espermatócitos/metabolismo , Testículo/patologiaRESUMO
Recent years have witnessed a number of new findings with significant implications for our understanding of the development of germ cell tumors. This communication reviews some of these recent insights with an emphasis on mechanisms that may convert a germ cell into a tumor cell. Three aspects are discussed in this review: (1) the early origin of germ cell tumors from primordial germ cells through an aberrant mitosis-to-meiosis switch; (2) errors during meiosis, which promote tumorigenic transformation of germ cells; and (3) the role of small RNAs such as oncomirs (miRNAs) and oncopirs (piRNAs) in germ cell tumor formation. Since much has been learned using a variety of organismal models, data obtained in experiments with mice, nematodes, fruit flies, and human data will be considered. Only exemplary references are included.
Assuntos
Transformação Celular Neoplásica/metabolismo , Células Germinativas/metabolismo , Meiose , Mitose , Neoplasias Embrionárias de Células Germinativas/metabolismo , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Modelos Animais de Doenças , Células Germinativas/patologia , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/patologia , RNA Neoplásico/genética , RNA Neoplásico/metabolismoRESUMO
Meiosis poses unique challenges to chromosome dynamics. Before entry into meiosis, each chromosome is duplicated and gives rise to two sister chromatids linked to each other by cohesion. Production of haploid gametes requires segregation of homologous chromosomes in the first meiotic division and of sister chromatids in the second. To ensure precise distribution of chromosomes to the daughter cells, sister chromatid cohesion (SCC) has to be dissolved in two steps. Maintenance and regulation of SCC is performed by the cohesin protein complex. This short review will primarily focus on the core cohesin proteins before venturing into adjacent territories with an emphasis on interacting proteins and complexes. It will also concentrate on mammalian meiosis and only occasionally discuss cohesion in other organisms.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas Fúngicas/fisiologia , Mamíferos/fisiologia , Meiose/fisiologia , Proteínas Nucleares/fisiologia , Oócitos/citologia , Troca de Cromátide Irmã/fisiologia , Animais , Proteínas Cromossômicas não Histona , Segregação de Cromossomos , Feminino , CoesinasRESUMO
Structural maintenance of chromosomes (SMC) proteins fulfill pivotal roles in chromosome dynamics. In yeast, the SMC1-SMC3 heterodimer is required for meiotic sister chromatid cohesion and DNA recombination. Little is known, however, about mammalian SMC proteins in meiotic cells. We have identified a novel SMC protein (SMC1beta), which-except for a unique, basic, DNA binding C-terminal motif-is highly homologous to SMC1 (which may now be called SMC1alpha) and is not present in the yeast genome. SMC1beta is specifically expressed in testes and coimmunoprecipitates with SMC3 from testis nuclear extracts, but not from a variety of somatic cells. This establishes for mammalian cells the concept of cell-type- and tissue-specific SMC protein isoforms. Analysis of testis sections and chromosome spreads of various stages of meiosis revealed localization of SMC1beta along the axial elements of synaptonemal complexes in prophase I. Most SMC1beta dissociates from the chromosome arms in late-pachytene-diplotene cells. However, SMC1beta, but not SMC1alpha, remains chromatin associated at the centromeres up to metaphase II. Thus, SMC1beta and not SMC1alpha is likely involved in maintaining cohesion between sister centromeres until anaphase II.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , Proteoglicanas de Sulfatos de Condroitina , Proteínas Fúngicas/química , Meiose , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Northern Blotting , Bovinos , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/biossíntese , Mapeamento Cromossômico , Cromossomos/metabolismo , Clonagem Molecular , Relação Dose-Resposta a Droga , Feminino , Proteínas Fúngicas/fisiologia , Masculino , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Filogenia , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas , Homologia de Sequência de Aminoácidos , Testículo/metabolismo , Distribuição TecidualRESUMO
Protein SWAP-70 was initially isolated from nuclei of activated B cells and was implicated in the immunoglobulin class switch process. After B cell activation the protein translocates from the cytoplasm to the nucleus, and may serve to signal nuclear processes. We have generated mice deficient in SWAP-70 and found three main differences when compared to wild-type mice: (i) their B lymphocytes are two- to threefold more sensitive to gamma-irradiation than B cells of wild type; (ii) SWAP-70-deficient mice developed autoantibodies at a much higher frequency; and (iii) the CD40 signaling pathway is compromised in the mutant mice. CD40-dependent switching to the IgE isotype is reduced five- to eightfold in vitro. In SWAP-70-deficient mice, IgE levels prior to immunization were six- to sevenfold lower than in wild-type mice, and after immunization three- to fourfold lower. CD40-induced proliferation was transiently increased in the mutant. LPS-induced switching to other isotypes, however, and LPS-induced proliferation were normal. We propose that SWAP-70 serves a specific role in the CD40 signaling pathway, in particular in the IgE response.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Antígenos CD40/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/fisiologia , Fatores de Troca do Nucleotídeo Guanina , Imunoglobulina E/imunologia , Proteínas Nucleares/deficiência , Proteínas Nucleares/fisiologia , Animais , Apoptose , Autoanticorpos/biossíntese , Linfócitos B/citologia , Linfócitos B/metabolismo , Linfócitos B/efeitos da radiação , Divisão Celular , Proteínas de Ligação a DNA/genética , Raios gama , Marcação de Genes , Switching de Imunoglobulina/genética , Switching de Imunoglobulina/imunologia , Imunoglobulina E/biossíntese , Imunoglobulina E/genética , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Lipopolissacarídeos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor , Mutação/genética , Proteínas Nucleares/genética , Tolerância a Radiação , Transdução de SinaisRESUMO
The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.
Assuntos
Meiose/fisiologia , Proteínas Nucleares/fisiologia , Sinapses/fisiologia , Animais , Células COS , Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona , Cromossomos/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas , Proteínas Recombinantes/genética , CoesinasRESUMO
DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), has a phosphoinositol 3-kinase (PI 3-K) domain close to its C-terminus. Cell lines derived from the SCID mouse have been utilised as a model DNA-PKcs-defective system. The SCID mutation results in truncation of DNA-Pkcs at the extreme C-terminus leaving the PI 3-K domain intact. The mutated protein is expressed at low levels in most SCID cell lines, leaving open the question of whether the mutation abolishes kinase activity. Here, we show that a SCID cell line that expresses the mutant protein normally has dramatically impaired kinase activity. We estimate that the residual kinase activity typically present in SCID fibroblast cell lines is at least two orders of magnitude less than that found in control cells. Our results substantiate evidence that DNA-PKcs kinase activity is required for DSB rejoining and V(D)J recombination and show that the extreme C-terminal region of DNA-PKcs, present in PI 3-K-related protein kinases but absent in bona fide PI 3 lipid kinases, is required for DNA-PKcs to function as a protein kinase. We also show that expression of mutant DNA-PKcs protein confers a growth disadvantage, providing an explanation for the lack of DNA-PKcs expression in most SCID cell lines.
Assuntos
Proteínas de Ligação a DNA , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linfócitos B/enzimologia , Sequência de Bases , Células CHO , Linhagem Celular , Cromossomos Artificiais de Levedura/genética , Sequência Conservada , Cricetinae , Primers do DNA/genética , Reparo do DNA/genética , Reparo do DNA/fisiologia , Proteína Quinase Ativada por DNA , Células-Tronco Hematopoéticas/enzimologia , Camundongos , Camundongos SCID , Mutagênese Sítio-Dirigida , Fosfatidilinositol 3-Quinases/química , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de ProteínaRESUMO
In somatic cells, the heterodimeric Structural Maintenance of Chromosomes (SMC) proteins are involved in chromosome condensation and gene dosage compensation (SMC2 and 4), and sister chromatid cohesion and DNA recombination (SMC1 and 3). We report here evidence for an involvement of mammalian SMC1 and SMC3 proteins in meiosis. Immunofluorescence analysis of testis sections showed intense chromatin association in meiotic prophase cells, weaker staining in round spermatids and absence of the SMC proteins in elongated spermatids. In spermatocyte nuclei spreads, the SMC1 and SMC3 proteins localize in a beaded structure along the axial elements of synaptonemal complexes of pachytene and diplotene chromosomes. Both SMC proteins are present in rat spermatocytes and enriched in preparations of synaptonemal complexes. Several independent experimental approaches revealed interactions of the SMC proteins with synaptonemal complex-specific proteins SCP2 and SCP3. These results suggest a model for the arrangement of SMC proteins in mammalian meiotic chromatin.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteoglicanas de Sulfatos de Condroitina , Proteínas Cromossômicas não Histona/metabolismo , Espermatócitos/citologia , Espermatócitos/metabolismo , Complexo Sinaptonêmico/fisiologia , Sequência de Aminoácidos , Animais , Bovinos , Proteínas de Ciclo Celular/análise , Cromatografia de Afinidade , Proteínas Cromossômicas não Histona/análise , Cromossomos/metabolismo , Proteínas de Ligação a DNA/análise , Imunofluorescência , Masculino , Mamíferos , Dados de Sequência Molecular , Proteínas Nucleares/análise , Ligação Proteica/fisiologia , Ratos , Espermatócitos/químicaRESUMO
SWAP-70 is a component of an enzyme complex that recombines Ig switch regions in vitro. We report here the cloning of the human cDNA and its B lymphocyte-specific expression. Although its sequence contains three nuclear localization signals, in small resting B cells, SWAP-70 is mainly found in the cytoplasm. On stimulation, SWAP-70 translocates to the nucleus. In activated, class-switching B cell cultures, it is associated with membrane IgG, but not IgM. The membrane Ig association requires a functional pleckstrin homology domain and is controlled by the C terminus. We suggest that SWAP-70 is involved not only in nuclear events but also in signaling in B cell activation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Proteínas Nucleares/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico , Antígenos CD40/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Mutagênese , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Coelhos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
The protein SWAP-70 was isolated as part of a DNA recombination complex in B lymphocytes, where it is predominantly expressed. In resting B cells, SWAP-70 is found in the cytoplasm; upon B-cell activation, it is transported both into the nucleus and to the cell membrane, where it is associated with the B-cell receptor complex and may play a role in signal transduction. In the nucleus, its involvement in heavy-chain class switch recombination has been suggested. In this report, using restriction fragment length polymorphism, simple sequence length polymorphism, and fluorescence in situ hybridization, we map the chromosomal localization of the mouse and the human genes to syntenic regions of mouse mid Chromosome (Chr) 7 and human Chr 11p15.
Assuntos
Cromossomos Humanos Par 11/genética , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina , Proteínas Nucleares/genética , Mapeamento Físico do Cromossomo , Animais , Haplótipos , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos BALB C , Repetições de Microssatélites , Antígenos de Histocompatibilidade Menor , Polimorfismo de Fragmento de RestriçãoRESUMO
The C-terminal domains of yeast structural maintenance of chromosomes (SMC) proteins were previously shown to bind double-stranded DNA, which generated the idea of the antiparallel SMC heterodimer, such as the SMC1/3 dimer, bridging two DNA molecules. Analysis of bovine SMC1 and SMC3 protein domains now reveals that not only the C-terminal domains, but also the coiled-coil region, binds DNA, while the N terminus is inactive. Duplex DNA and DNA molecules with secondary structures are highly preferred substrates for both the C-terminal and coiled-coil domains. Contrasting other cruciform DNA-binding proteins like HMG1, the SMC3 C-terminal and coiled-coil domains do not bend DNA, but rather prevent bending in ring closure assays. Phosphatase, exonuclease, and ligase assays showed that neither domain renders DNA ends inaccessible for other enzymes. These observations allow modifications of models for SMC-DNA interactions.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Conformação de Ácido Nucleico , Animais , Sequência de Bases , Bovinos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , DNA/química , Primers do DNA , Proteínas de Ligação a DNA/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The evolutionarily-conserved eukaryotic SMC (structural maintenance of chromosomes) proteins are ubiquitous chromosomal components in prokaryotes and eukaryotes. The eukaryotic SMC proteins form two kind of heterodimers: the SMC1/SMC3 and the SMC2/SMC4 types. These heterodimers constitute an essential part of higher order complexes, which are involved in chromatin and DNA dynamics. The two most prominent and best-characterized complexes are cohesin and condensin, necessary for sister chromatid cohesion and chromosome condensation. Here we discuss these functions together with additional roles in gene dosage compensation and DNA recombination.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Ciclo Celular/genética , Cromátides/fisiologia , Proteínas Cromossômicas não Histona/genética , DNA/genética , DNA/metabolismo , Dimerização , Mecanismo Genético de Compensação de Dose , Humanos , Substâncias Macromoleculares , Conformação Proteica , Recombinação GenéticaRESUMO
SWAP-70 is part of a protein complex that catalyzes cell-free DNA recombination between immunoglobulin heavy chain gene switch region substrates. This report studies the expression pattern of SWAP-70 in mouse tissues, sorted cells, and cultured primary cells. SWAP-70 RNA is strongly increased upon switch-induction of spleen cells, and very weakly expressed in thymus and bone marrow. SWAP-70 protein is specifically expressed in B cells, and levels increase rapidly after stimulation. Tissue staining shows strong expression in germinal center B cells, while macrophages and T lymphocytes do not stain. SWAP-70 is not detected in early B cells in the bone marrow. Its expression during mouse ontogeny after birth correlates with the appearance of non-IgM isotypes. While SWAP-70 localizes to the cell nucleus in activated B cells, it is not tightly associated with the chromatin and is found in the cytoplasm as well. SWAP-70 expression is not increased by gamma or UV irradiation of spleen cells, nor does it depend on p53. These characteristics are consistent with the putative role of SWAP-70 in immunoglobulin class switching.
Assuntos
Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina , Proteínas Nucleares/genética , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Diferenciação Celular , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Proteínas Nucleares/biossíntese , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Recombinação Genética , Frações Subcelulares/metabolismo , Distribuição TecidualRESUMO
Members of the evolutionary conserved Structural Maintenance of Chromosomes (SMC) protein family are involved in chromosome condensation and gene dosage compensation with the SMC2 and SMC4 subtypes, and sister chromatid cohesion with the SMC1 and SMC3 subtypes. The bovine recombination protein complex RC-1, which catalyzes DNA transfer reactions, contains two heterodimeric SMC polypeptides, the genes of which have now been cloned, sequenced, and classified as bovine (b)SMC1 and bSMC3. Both proteins display all the characteristic features of the SMC family. FISH analysis localized the mouse SMC3 gene to chromosome 19D2-D3. Mono- and polyclonal antibodies specific for either subtype detected high levels of protein expression in lymphoid tissues, lung, testis and ovary. No change in levels of bSMC1 and bSMC3 proteins occurred after X-ray or UV-light irradiation of various cell lines or primary cells, and the amounts of individual proteins and the heterodimer are roughly constant throughout the cell cycle. Immunofluorescence of mouse cells detected the SMC1 protein in foci associated with the chromatin. These foci dissolve and the SMC protein dissociates from the chromatin during M phase.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona , DNA Ligases/genética , DNA Polimerase Dirigida por DNA/genética , Endodesoxirribonucleases/genética , Proteínas Fúngicas/genética , Genes/genética , Mamíferos/genética , Complexos Multienzimáticos/genética , Sequência de Aminoácidos , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Linfócitos B/efeitos da radiação , Bovinos , Ciclo Celular , Núcleo Celular/química , Células Cultivadas , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Imunofluorescência , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Germinal centers (GC) are the sites of antigen-driven B cell switch recombination, V(D)J gene hypermutation, and selection to generate high-afinity CD38+ memory B cells. A marked expansion of GC associated with hypergammaglobulinemia followed by complete disruption of normal splenic architecture and a striking drop in immunoglobulin levels are prominent features of the murine retrovirus-induced immunodeficiency syndrome, MAIDS. B cell lymphomas are frequent in long-term infected mice. Normal GC formation is critically dependent on a number of genes including the transcription factor, Bcl6. Deregulated expression of BCL6 protein has been implicated in the development of human and mouse B cell lymphomas. Another nuclear protein, SWAP-70, has been identified as a subunit of the protein complex, SWAP, that recombines switch regions in vitro. To develop a fuller understanding of B cell biology in MAIDS, we examined the characteristics of BCL6, SWAP-70, CD38, and peanut agglutinin (PNA)-staining cells during the course of the disease. The levels of both nuclear proteins increased rapidly until 6-8 weeks after infection. During this time frame, BCL6 was expressed at highest levels in the usually rare CD4+ Thyl- T cell subset as well as in B cells. At later times. BCL6 levels dropped to undetectable levels while SWAP-70 levels continued to increase. Changes in the levels of either protein could not be ascribed to transcriptional regulation. PNA-reactive cells decreased in concert with BCL6 while CD38 staining increased with SWAP-70. These results demonstrate that progression of MAIDS results in the massive accumulation of B cells with the morphology of secretory cells that behave like post-GC cells for expression of BCL6 and CD38, and for PNA-staining but with abnormally high-level expression of SWAP-70.
Assuntos
Antígenos CD , Proteínas de Ligação a DNA/genética , Centro Germinativo/imunologia , Fatores de Troca do Nucleotídeo Guanina , Síndrome de Imunodeficiência Adquirida Murina/genética , Síndrome de Imunodeficiência Adquirida Murina/imunologia , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Fatores de Transcrição/genética , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Antígenos de Diferenciação/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/patologia , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Linhagem Celular , Primers do DNA/genética , Feminino , Regulação da Expressão Gênica , Genes de Troca , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Glicoproteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Síndrome de Imunodeficiência Adquirida Murina/patologia , NAD+ Nucleosidase/genética , Proteínas Proto-Oncogênicas c-bcl-6 , Recombinação GenéticaRESUMO
Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the C-terminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for double-stranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffold-associated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona , DNA/química , DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Nucleares/metabolismo , Conformação de Ácido Nucleico , Proteínas de Saccharomyces cerevisiae , Composição de Bases , Sequência de Bases , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Clonagem Molecular , DNA/genética , Primers do DNA , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Dosagem de Genes , Cinética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
We have purified and biochemically characterized a multiprotein complex designated SWAP. In a DNA transfer assay, SWAP preferentially recombines ("swaps") sequences derived from Ig heavy chain switch regions. We identified four of the proteins in the SWAP complex: B23 (nucleophosmin), C23 (nucleolin), poly(ADP-ribose) polymerase (PARP), and SWAP-70. The first three are proteins known to be present in most cells. B23 promotes single-strand DNA reannealing and the formation of joint molecules in a D-loop assay between homologous, but also between Smu and Sgamma sequences. SWAP-70 is a novel protein of 70 kDa. Its cDNA was cloned and sequenced, and the protein was overexpressed in Escherichia coli. SWAP-70 protein expression was found only in B lymphocytes that had been induced to switch to various Ig isotypes and in switching B-cell lines. SWAP-70 is a nuclear protein, has a weak affinity for DNA, binds ATP, and forms specific, high affinity complexes with B23, C23, and poly(ADP-ribose) polymerase. These findings are consistent with SWAP being the long elusive "switch recombinase" and with SWAP-70 being the specific recruiting element that assembles the switch recombinase from universal components.
Assuntos
Linfócitos B/metabolismo , DNA Recombinante/genética , Proteínas de Ligação a DNA/genética , Fatores de Troca do Nucleotídeo Guanina , Proteínas Nucleares/genética , Recombinação Genética , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Temperatura Alta , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Nucleofosmina , Fosfoproteínas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação ProteicaRESUMO
Recent evidence suggests that members of the structural maintenance of chromosomes (SMC) protein family are involved in a much broader spectrum of chromosome and DNA metabolic reactions than was originally thought. Other than their role in chromosome condensation, SMC proteins are essential for sister chromatid cohesion and gene dosage compensation, and are involved in DNA recombination. This diversity of function is achieved both through the formation of different heterodimers and through their participation in higher-order protein complexes adapted to achieve specific ends.
Assuntos
Proteínas Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Cromatina , Reparo do DNA , Mitose , Proteínas Nucleares/metabolismo , Recombinação GenéticaRESUMO
Withdrawal of interleukin-7 from cultured murine preB lymphocytes induces cell differentiation including V(D)J immunoglobulin gene rearrangements and cell cycle arrest. Advanced steps of the V(D)J recombination reaction involve processing of coding ends by several largely unidentified DNA metabolic enzymes. We have analyzed expression and activity of DNA polymerases alpha, beta, delta and epsilon, proliferating cell nuclear antigen (PCNA), topoisomerases I and II, terminal deoxynucleotidyl transferase (TdT) and DNA ligases I, III and IV upon induction of preB cell differentiation. Despite the immediate arrest of cell proliferation, DNA polymerase delta protein levels remained unchanged for approximately 2 days and its activity was up-regulated several-fold, while PCNA was continuously present. Activity of DNA polymerases alpha,beta and epsilon decreased. Expression and activity of DNA ligase I were drastically reduced, while those of DNA ligases III and IV remained virtually constant. No changes in DNA topoisomerases I or II expression and activity occurred and TdT expression was moderately increased early after induction. Our results render DNA polymerase delta a likely candidate acting in DNA synthesis related to V(D)J recombination in lymphocytes.