RESUMO
OBJECTIVE: Patients with bicuspid aortic valve (BAV) have an increased risk of developing ascending aortic aneurysms. Transforming growth factor-ß (TGFß) is a crucial factor of vascular remodeling, the impaired signaling of which can alter the structure and composition of the extracellular matrix. In this study, we analyzed the activity of TGFß in aneurysmal and nonaneurysmal ascending aorta from BAV patients, using tricuspid aortic valve (TAV) patients as a reference group. APPROACH AND RESULTS: The response to exogenous TGFß was analyzed with regard to gene expression in primary aortic smooth muscle cells that were isolated from 7 BAV and 5 TAV patients and in valve fibroblasts from 7 BAV and 8 TAV patients. The set of genes that were significantly changed by TGFß (217 genes) was compared with gene expression profiles of the ascending aorta from BAV and TAV patients (139 arrays). By principle component analysis, based on the 217 genes, gene expression differed significantly in the intima/media region between aneurysmal BAV and TAV aortas, driven by the response in TAV patients. During aneurysm development the levels of phosphorylated SMADs and the availability of free TGFß were lower in BAV patients compared with TAV. Confocal microscopy analysis showed a higher colocalization of latency associated peptide and latent TGFß binding protein 3 in BAV aortas. CONCLUSIONS: Our findings suggest that TGFß activation during aneurysm formation is muted in patients with BAV, possibly as a result of an increased TGFß sequestration in the extracellular space.
Assuntos
Aneurisma Aórtico/etiologia , Valva Aórtica/anormalidades , Valva Aórtica/metabolismo , Doenças das Valvas Cardíacas/complicações , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Aneurisma Aórtico/genética , Aneurisma Aórtico/metabolismo , Aneurisma Aórtico/patologia , Valva Aórtica/patologia , Doença da Válvula Aórtica Bicúspide , Células Cultivadas , Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Doenças das Valvas Cardíacas/genética , Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/patologia , Humanos , Proteínas de Ligação a TGF-beta Latente/genética , Proteínas de Ligação a TGF-beta Latente/metabolismo , Masculino , Pessoa de Meia-Idade , Fosforilação , Análise de Componente Principal , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismoRESUMO
Thoracic aortic aneurysm is a pathological local dilatation of the aorta, potentially leading to aortic rupture or dissection. The disease is a common complication of patients with bicuspid aortic valve, a congenital disorder present in 1-2% of the population. Using two dimensional fluorescence difference gel electrophoresis proteomics followed by mRNA expression, and alternative splicing analysis of the identified proteins, differences in dilated and nondilated aorta tissues between 44 patients with bicuspid and tricuspid valves was examined. The pattern of protein expression was successfully validated with LC-MS/MS. A multivariate analysis of protein expression data revealed diverging protein expression fingerprints in patients with tricuspid compared with the patients with bicuspid aortic valves. From 302 protein spots included in the analysis, 69 and 38 spots were differentially expressed between dilated and nondilated aorta specifically in patients with tricuspid and bicuspid aortic valve, respectively. 92 protein spots were differentially expressed between dilated and nondilated aorta in both phenotypes. Similarly, mRNA expression together with alternative splicing analysis of the identified proteins also showed diverging fingerprints in the two patient groups. Differential splicing was abundant but the expression levels of differentially spliced mRNA transcripts were low compared with the wild type transcript and there was no correlation between splicing and the number of spots. Therefore, the different spots are likely to represent post-translational modifications. The identification of differentially expressed proteins suggests that dilatation in patients with a tricuspid aortic valve involves inflammatory processes whereas aortic aneurysm in patients with BAV may be the consequence of impaired repair capacity. The results imply that aortic aneurysm formation in patients with bicuspid and tricuspid aortic valves involve different biological pathways leading to the same phenotype.
Assuntos
Aneurisma da Aorta Torácica/genética , Regulação da Expressão Gênica , Doenças das Valvas Cardíacas/metabolismo , Proteoma/metabolismo , Transcriptoma , Valva Tricúspide/metabolismo , Processamento Alternativo , Aneurisma da Aorta Torácica/congênito , Aneurisma da Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/patologia , Valva Aórtica/anormalidades , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Doença da Válvula Aórtica Bicúspide , Biópsia , Estudos de Casos e Controles , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Feminino , Perfilação da Expressão Gênica , Doenças das Valvas Cardíacas/patologia , Humanos , Masculino , Análise de Componente Principal , Proteoma/química , Espectrometria de Massas em Tandem , Valva Tricúspide/patologiaRESUMO
Environmental exposures filtered through the genetic make-up of each individual alter the transcriptional repertoire in organs central to metabolic homeostasis, thereby affecting arterial lipid accumulation, inflammation, and the development of coronary artery disease (CAD). The primary aim of the Stockholm Atherosclerosis Gene Expression (STAGE) study was to determine whether there are functionally associated genes (rather than individual genes) important for CAD development. To this end, two-way clustering was used on 278 transcriptional profiles of liver, skeletal muscle, and visceral fat (n = 66/tissue) and atherosclerotic and unaffected arterial wall (n = 40/tissue) isolated from CAD patients during coronary artery bypass surgery. The first step, across all mRNA signals (n = 15,042/12,621 RefSeqs/genes) in each tissue, resulted in a total of 60 tissue clusters (n = 3958 genes). In the second step (performed within tissue clusters), one atherosclerotic lesion (n = 49/48) and one visceral fat (n = 59) cluster segregated the patients into two groups that differed in the extent of coronary stenosis (P = 0.008 and P = 0.00015). The associations of these clusters with coronary atherosclerosis were validated by analyzing carotid atherosclerosis expression profiles. Remarkably, in one cluster (n = 55/54) relating to carotid stenosis (P = 0.04), 27 genes in the two clusters relating to coronary stenosis were confirmed (n = 16/17, P<10(-27 and-30)). Genes in the transendothelial migration of leukocytes (TEML) pathway were overrepresented in all three clusters, referred to as the atherosclerosis module (A-module). In a second validation step, using three independent cohorts, the A-module was found to be genetically enriched with CAD risk by 1.8-fold (P<0.004). The transcription co-factor LIM domain binding 2 (LDB2) was identified as a potential high-hierarchy regulator of the A-module, a notion supported by subnetwork analysis, by cellular and lesion expression of LDB2, and by the expression of 13 TEML genes in Ldb2-deficient arterial wall. Thus, the A-module appears to be important for atherosclerosis development and, together with LDB2, merits further attention in CAD research.
Assuntos
Movimento Celular/genética , Doença da Artéria Coronariana/genética , Células Endoteliais/patologia , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/genética , Leucócitos/patologia , Fatores de Transcrição/metabolismo , Idoso , Animais , Aterosclerose/genética , Artérias Carótidas/patologia , Análise por Conglomerados , Estudos de Coortes , Biologia Computacional , Células Endoteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Proteínas com Domínio LIM , Leucócitos/metabolismo , Masculino , Camundongos , Especificidade de Órgãos/genética , Reprodutibilidade dos Testes , Suécia , Fatores de Transcrição/genéticaRESUMO
Thoracic aortic aneurysm (TAA) is a common complication in patients with a bicuspid aortic valve (BAV), the most frequent congenital heart disorder. For unknown reasons TAA occurs at a younger age, with a higher frequency in BAV patients than in patients with a tricuspid aortic valve (TAV), resulting in an increased risk for aortic dissection and rupture. To investigate the increased TAA incidence in BAV patients, we obtained tissue biopsy samples from nondilated and dilated aortas of 131 BAV and TAV patients. Global gene expression profiles were analyzed from controls and from aortic intima-media and adventitia of patients (in total 345 samples). Of the genes found to be differentially expressed with dilation, only a few (<4%) were differentially expressed in both BAV and TAV patients. With the use of gene set enrichment analysis, the cell adhesion and extracellular region gene ontology sets were identified as common features of TAA in both BAV and TAV patients. Immune response genes were observed to be particularly overexpressed in the aortic media of dilated TAV samples. The divergent gene expression profiles indicate that there are fundamental differences in TAA etiology in BAV and TAV patients. Immune response activation solely in the aortic media of TAV patients suggests that inflammation is involved in TAA formation in TAV but not in BAV patients. Conversely, genes were identified that were only differentially expressed with dilation in BAV patients. The result has bearing on future clinical studies in which separate analysis of BAV and TAV patients is recommended.
Assuntos
Aorta Torácica/patologia , Aneurisma da Aorta Torácica/genética , Perfilação da Expressão Gênica , Doenças das Valvas Cardíacas/genética , Valva Mitral/patologia , Valva Tricúspide/patologia , Idoso , Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/complicações , Aneurisma da Aorta Torácica/imunologia , Biomarcadores/metabolismo , Antígenos CD4/metabolismo , Bases de Dados Genéticas , Dilatação Patológica , Feminino , Regulação da Expressão Gênica , Doenças das Valvas Cardíacas/complicações , Doenças das Valvas Cardíacas/imunologia , Humanos , Imunidade/genética , Imuno-Histoquímica , Inflamação/complicações , Inflamação/genética , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Valva Mitral/metabolismo , Análise de Componente Principal , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Valva Tricúspide/metabolismo , Túnica Íntima/metabolismo , Túnica Íntima/patologia , Túnica Média/metabolismo , Túnica Média/patologiaRESUMO
Despite the well-documented effects of plasma lipid lowering regimes halting atherosclerosis lesion development and reducing morbidity and mortality of coronary artery disease and stroke, the transcriptional response in the atherosclerotic lesion mediating these beneficial effects has not yet been carefully investigated. We performed transcriptional profiling at 10-week intervals in atherosclerosis-prone mice with human-like hypercholesterolemia and a genetic switch to lower plasma lipoproteins (Ldlr(-/-)Apo(100/100)Mttp(flox/flox) Mx1-Cre). Atherosclerotic lesions progressed slowly at first, then expanded rapidly, and plateaued after advanced lesions formed. Analysis of lesion expression profiles indicated that accumulation of lipid-poor macrophages reached a point that led to the rapid expansion phase with accelerated foam-cell formation and inflammation, an interpretation supported by lesion histology. Genetic lowering of plasma cholesterol (e.g., lipoproteins) at this point all together prevented the formation of advanced plaques and parallel transcriptional profiling of the atherosclerotic arterial wall identified 37 cholesterol-responsive genes mediating this effect. Validation by siRNA-inhibition in macrophages incubated with acetylated-LDL revealed a network of eight cholesterol-responsive atherosclerosis genes regulating cholesterol-ester accumulation. Taken together, we have identified a network of atherosclerosis genes that in response to plasma cholesterol-lowering prevents the formation of advanced plaques. This network should be of interest for the development of novel atherosclerosis therapies.
Assuntos
Aterosclerose/genética , Aterosclerose/metabolismo , Colesterol/metabolismo , Animais , Apolipoproteína B-100/genética , Aterosclerose/etiologia , Aterosclerose/patologia , Proteínas de Transporte/genética , Células Espumosas/metabolismo , Perfilação da Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Receptores de LDL/deficiência , Receptores de LDL/genéticaRESUMO
Thoracic aortic aneurysm (TAA) is the progressive enlargement of the aorta due to destructive changes in the connective tissue of the aortic wall. Aneurysm development is silent and often first manifested by the drastic events of aortic dissection or rupture. As yet, therapeutic agents that halt or reverse the process of aortic wall deterioration are absent, and the only available therapeutic recommendation is elective prophylactic surgical intervention. Being born with a bicuspid instead of the normal tricuspid aortic valve (TAV) is a major risk factor for developing aneurysm in the ascending aorta later in life. Although the pathophysiology of the increased aneurysm susceptibility is not known, recent studies are suggestive of a transformation of aortic endothelium into a more mesenchymal state i.e., an endothelial-to-mesenchymal transition in these individuals. This process involves the loss of endothelial cell features, resulting in junction instability and enhanced vascular permeability of the ascending aorta that may lay the ground for increased aneurysm susceptibility. This finding differentiates and further emphasizes the specific characteristics of aneurysm development in individuals with a bicuspid aortic valve (BAV). This review discusses the possibility of a developmental fate shared between the aortic endothelium and aortic valves. It further speculates about the impact of aortic endothelium phenotypic shift on aneurysm development in individuals with a BAV and revisits previous studies in the light of the new findings.
RESUMO
Cellular and molecular mechanisms of thoracic aortic aneurysm are still not clear and therapeutic approaches are mostly absent. The role of endothelial cells in aortic wall integrity is emerging from recent studies. Although Notch pathway ensures endothelial development and integrity, and NOTCH1 mutations have been associated with thoracic aortic aneurysms, the role of this pathway in aneurysm remains elusive. The purpose of the present work was to study functions of Notch genes in endothelial cells of patients with sporadic thoracic aortic aneurysm. Aortic endothelial cells were isolated from aortic tissue of patients with thoracic aortic aneurysm and healthy donors. Gene expression of Notch and related BMP and WNT/ß-catenin pathways was estimated by qPCR; WNT/ß-catenin signaling was studied by TCF-luciferase reporter. To study the stress-response the cells were subjected to laminar shear stress and the expression of corresponding genes was estimated by qPCR. Analyses of mRNA expression of Notch genes, Notch target genes and Notch related pathways showed that endothelial cells of aneurysm patients have dysregulated Notch/BMP/WNT pathways compared to donor cells. Activity of Wnt pathway was significantly elevated in endothelial cells of the patients. Cells from patients had attenuated activation of DLL4, SNAIL1, DKK1 and BMP2 in response to shear stress. In conclusion endothelial cells of the patients with thoracic aortic aneurysm have dysregulated Notch, BMP and WNT/ß-catenin related signaling. Shear stress-response and cross-talk between Notch and Wnt pathways that normally ensures aortic integrity and resistance of endothelial cells to stress is impaired in aneurysmal patients.
Assuntos
Aorta Torácica/metabolismo , Aneurisma da Aorta Torácica/metabolismo , Proteína Morfogenética Óssea 2/metabolismo , Células Endoteliais/metabolismo , Receptores Notch/metabolismo , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal , Idoso , Idoso de 80 Anos ou mais , Aorta Torácica/patologia , Aorta Torácica/fisiopatologia , Aneurisma da Aorta Torácica/genética , Aneurisma da Aorta Torácica/patologia , Aneurisma da Aorta Torácica/fisiopatologia , Proteína Morfogenética Óssea 2/genética , Proteínas de Ligação ao Cálcio , Células Cultivadas , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Mecanotransdução Celular , Pessoa de Meia-Idade , Receptores Notch/genética , Fluxo Sanguíneo Regional , Fatores de Transcrição da Família Snail/metabolismo , Estresse Mecânico , Via de Sinalização Wnt/genéticaRESUMO
Disturbed flow has been suggested to contribute to aneurysm susceptibility in bicuspid aortic valve (BAV) patients. Lately, flow has emerged as an important modulator of DNA methylation. Hear we combined global methylation analysis with in vitro studies of flow-sensitive methylation to identify biological processes associated with BAV-aortopathy and the potential contribution of flow. Biopsies from non-dilated and dilated ascending aortas were collected from BAV (n = 21) and tricuspid aortic valve (TAV) patients (n = 23). DNA methylation and gene expression was measured in aortic intima-media tissue samples, and in EA.hy926 and primary aortic endothelial cells (ECs) isolated from BAV and TAV exposed to oscillatory (±12 dynes/cm2) or laminar (12 dynes/cm2) flow. We show methylation changes related to epithelial-mesenchymal-transition (EMT) in the non-dilated BAV aorta, associated with oscillatory flow related to endocytosis. The results indicate that the flow-response in BAV ECs involves hypomethylation and increased expression of WNT/ß-catenin genes, as opposed to an angiogenic profile in TAV ECs. The EMT-signature was exasperated in dilated BAV aortas. Aberrant EMT in BAV aortic walls could contribute to increased aneurysm susceptibility, and may be due to disturbed flow-exposure. Perturbations during the spatiotemporally related embryonic development of ascending aorta and semilunar valves can however not be excluded.
Assuntos
Aorta , Valva Aórtica/anormalidades , Circulação Sanguínea , Metilação de DNA , Células Endoteliais/metabolismo , Transição Epitelial-Mesenquimal , Doenças das Valvas Cardíacas/metabolismo , Valva Tricúspide/metabolismo , Aorta/citologia , Aorta/metabolismo , Valva Aórtica/metabolismo , Doença da Válvula Aórtica Bicúspide , Dilatação Patológica , Células Endoteliais/citologia , Humanos , TranscriptomaRESUMO
Individuals with a bicuspid aortic valve (BAV) are at significantly higher risk of developing aortic complications than individuals with tricuspid aortic valves (TAV) and defective signaling during the embryonic development and/or life time exposure to abnormal hemodynamic have been proposed as underlying factors. However, an explanation for the molecular mechanisms of aortopathy in BAV has not yet been provided. We combined proteomics, RNA analyses, immunohistochemistry, and electron microscopy to identify molecular differences in samples of non-dilated ascending aortas from BAV (N = 62) and TAV (N = 54) patients. Proteomic analysis was also performed for dilated aortas (N = 6 BAV and N = 5 TAV) to gain further insight into the aortopathy of BAV. Our results collectively showed the molecular signature of an endothelial/epithelial-mesenchymal (EndMT/EMT) transition-like process, associated with instability of intimal cell junctions and activation of RHOA pathway in the intima and media layers of ascending aorta in BAV patients. We propose that an improper regulation of EndMT/EMT during the spatiotemporally related embryogenesis of semilunar valves and ascending aorta in BAV individuals may result in aortic immaturity and instability prior to dilation. Exasperation of EndMT/EMT state in post embryonic life and/or exposure to non-physiological hemodynamic could lead to the aneurysm of ascending aorta in BAV individuals.
Assuntos
Aneurisma Aórtico/etiologia , Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/patologia , Túnica Íntima/patologia , Valva Aórtica/anormalidades , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Doença da Válvula Aórtica Bicúspide , Endocitose , Transição Epitelial-Mesenquimal , Doenças das Valvas Cardíacas/complicações , Humanos , Artéria Torácica Interna/metabolismo , Artéria Torácica Interna/patologia , Células-Tronco Mesenquimais/patologia , Proteoma , Receptores Notch/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Individuals with bicuspid aortic valve (BAV) are at significantly higher risk of developing serious aortic complications than individuals with tricuspid aortic valves (TAV). Studies have indicated an altered aortic blood flow in patients with BAV; however, the extent to which altered flow influences the pathological state of BAV aorta is unclear. In the present study, we dissected flow-mediated aortic gene expression in patients undergoing elective open heart surgery. A large collection of public microarray data sets were firstly screened for consistent co-expression with five well-characterized flow-regulated genes (query genes). Genes with co-expression probability of >0.5 were selected and further analysed in expression profiles (127 arrays) from ascending aorta of BAV and TAV patients. Forty-four genes satisfied two filtering criteria: a significant correlation with one or more of the query genes (R > 0.40) and differential expression between patients with BAV and TAV. No gene fulfilled the criteria in mammary artery (88 arrays), an artery not in direct contact with the valve. Fifty-five percent of the genes significantly altered between BAV and TAV patients showed differential expression between two identified flow regions in the rat aorta. A large proportion of the identified genes were related to angiogenesis and/or wound healing, with pro-angiogenesis genes downregulated and inhibitory genes upregulated in patients with BAV. Moreover, differential expression of ZFP36, GRP116 and PKD2 was confirmed using immunohistochemistry. Implementing a new strategy, we have demonstrated an angiostatic gene expression signature in patients with BAV, indicating impaired wound healing in these patients, potentially involved in BAV-associated aortopathy.
Assuntos
Estenose da Valva Aórtica/genética , Doenças das Valvas Cardíacas/metabolismo , Canais de Cátion TRPP/genética , Transcriptoma , Tristetraprolina/genética , Animais , Aorta/metabolismo , Valva Aórtica/anormalidades , Valva Aórtica/metabolismo , Valva Aórtica/patologia , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Estenose da Valva Aórtica/cirurgia , Doença da Válvula Aórtica Bicúspide , Velocidade do Fluxo Sanguíneo , Perfilação da Expressão Gênica , Doenças das Valvas Cardíacas/patologia , Humanos , Masculino , Artéria Torácica Interna/metabolismo , Ratos , Ratos Wistar , Canais de Cátion TRPP/metabolismo , Valva Tricúspide/metabolismo , Valva Tricúspide/patologia , Tristetraprolina/metabolismoRESUMO
OBJECTIVE: Shear forces play a key role in the maintenance of vessel wall integrity. Current understanding regarding shear-dependent gene expression is mainly based on in vitro or in vivo observations with experimentally deranged shear, hence reflecting acute molecular events in relation to flow. Our objective was to combine computational fluid dynamic (CFD) simulations with global microarray analysis to study flow-dependent vessel wall biology in the aortic wall under physiological conditions. METHODS AND RESULTS: Male Wistar rats were used. Animal-specific wall shear stress (WSS) magnitude and vector direction were estimated using CFD based on aortic geometry and flow information acquired by magnetic resonance imaging. Two distinct flow pattern regions were identified in the normal rat aortic arch; the distal part of the lesser curvature being exposed to low WSS and a non-uniform vector direction, and a region along the greater curvature being subjected to markedly higher levels of WSS and a uniform vector direction. Microarray analysis identified numerous novel mechanosensitive genes, including Trpc4 and Fgf12, and confirmed well-known ones, e.g. Klf2 and Nrf2. Gene ontology analysis revealed an over-representation of genes involved in transcriptional regulation. The most differentially expressed gene, Hand2, is a transcription factor previously shown to be involved in extracellular matrix remodeling. HAND2 protein was endothelial specific and showed higher expression in the regions exposed to low WSS with disturbed flow. CONCLUSIONS: Microarray analysis validated the CFD-defined WSS regions in the rat aortic arch, and identified numerous novel shear-sensitive genes. Defining the functional importance of these genes in relation to atherosusceptibility may provide important insight into the understanding of vascular pathology.
Assuntos
Aorta/metabolismo , Aorta/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Resistência ao Cisalhamento/fisiologia , Estresse Mecânico , Animais , Aorta Torácica/metabolismo , Aorta Torácica/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos WistarRESUMO
In most sexually reproducing organisms, meiotic recombination is initiated by DNA double-strand breaks (DSBs) formed by the Spo11 protein. In budding yeast, nine other proteins are also required for DSB formation, but the roles of these proteins and the interactions among them are poorly understood. We report further studies of the behaviors of these proteins. Consistent with other studies, we find that Mei4 and Rec114 bind to chromosomes from leptonema through early pachynema. Both proteins showed only limited colocalization with the meiotic cohesin subunit Rec8, suggesting that Mei4 and Rec114 associated preferentially with chromatin loops. Rec114 localization was independent of other DSB factors, but Mei4 localization was strongly dependent on Rec114 and Mer2. Systematic deletion analysis identified protein regions important for a previously described two-hybrid interaction between Mei4 and Rec114. We also report functional characterization of a previously misannotated 5' coding exon of REC102. Sequences encoded in this exon are essential for DSB formation and for Rec102 interaction with Rec104, Spo11, Rec114, and Mei4. Finally, we also examined genetic requirements for a set of previously described two-hybrid interactions that can be detected only when the reporter strain is induced to enter meiosis. This analysis reveals new functional dependencies for interactions among the DSB proteins. Taken together, these studies support the view that Mei4, Rec114, and Mer2 make up a functional subgroup that is distinct from other subgroups of the DSB proteins: Spo11-Ski8, Rec102-Rec104, and Mre11-Rad50-Xrs2. These studies also suggest that an essential function of Rec102 and Rec104 is to connect Mei4 and Rec114 to Spo11.
Assuntos
Quebras de DNA de Cadeia Dupla , Meiose/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Complexo Sinaptonêmico/genética , Quebra Cromossômica , DNA Fúngico , Endodesoxirribonucleases , Esterases , Meiose/fisiologia , Proteínas Nucleares , Estágio Paquíteno , Recombinases , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexo Sinaptonêmico/metabolismoRESUMO
Meiosis is a specialized cell division that halves the genome complement, producing haploid gametes/spores from diploid cells. Proper separation of homologous chromosomes at the first meiotic division requires the production of physical connections (chiasmata) between homologs through recombinational exchange of chromosome arms after sister-chromatid cohesion is established but before chromosome segregation takes place. The events of meiotic prophase must thus occur in a strictly temporal order, but the molecular controls coordinating these events have not been well elucidated. Here, we demonstrate that the budding yeast cyclin-dependent kinase Cdc28 directly regulates the formation of the DNA double-strand breaks that initiate recombination by phosphorylating the Mer2/Rec107 protein and thereby modulating interactions of Mer2 with other proteins required for break formation. We propose that this function of Cdc28 helps to coordinate the events of meiotic prophase with each other and with progression through prophase.
Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Meiose/genética , Meiose/fisiologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Cromatina/metabolismo , Cromossomos Fúngicos/metabolismo , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Prófase , Recombinação Genética , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
It is well documented that the formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination is influenced by chromatin and larger scale chromosome organization, but the molecular nature of this influence has remained elusive. Several recent studies, including (this issue of Cell), shed light on this issue by revealing roles for posttranslational histone modifications in promoting DSB formation.
Assuntos
Histonas/química , Meiose , Recombinação Genética , Animais , Caenorhabditis elegans , Cromatina/metabolismo , Cromatina/fisiologia , DNA/química , Dano ao DNA , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/metabolismoRESUMO
The yeast Rad27 protein is homologous to mammalian Fen1 and is involved in the processing of replication intermediates. Enhanced instability of various artificial repetitive DNA sequences in RAD27-deficient yeast strains has been observed previously and shown to involve preferentially expansion mutations. In the present investigation, we characterised the mitotic instability of alleles of the naturally occurring human minisatellites MS1, MS32, MS205 and CEB1 and the modified MS1 alleles containing more highly homogeneous repeat regions than the original alleles. These minisatellites demonstrated more pronounced instability in rad27 Delta strains, with increases in the frequencies of both expansion and contraction mutants. In RAD27 strains, MS32 and MS205 were relatively stable, while MS1 and CEB1 were unstable, indicating that the effect of RAD27 on stability is influenced by intrinsic properties of the repeat array. This conclusion received further support from the remarkably high frequency of length-mutants observed for the modified allele of MS1. Thus, our findings emphasise the importance of: (1) comparing results obtained with various naturally occurring minisatellites and (2) manipulating their sequences in attempts to understand the molecular basis for mitotic stability/instability of minisatellite DNA.
Assuntos
Reparo do DNA/fisiologia , Endodesoxirribonucleases/fisiologia , Repetições Minissatélites , Saccharomyces cerevisiae/genética , Sequência de Bases , Endonucleases Flap , Genoma Fúngico , Humanos , Dados de Sequência Molecular , Organismos Geneticamente Modificados , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Meiotic recombination initiates with double-strand breaks (DSBs) catalyzed by Spo11 in conjunction with accessory proteins whose roles are not understood. Two-hybrid analysis reveals a network of interactions connecting the yeast DSB proteins to one another. Of these proteins, Ski8 was known to function in cytoplasmic RNA metabolism, suggesting that its role in recombination might be indirect. However, obligate partners of Ski8 in RNA metabolism are dispensable for recombination and Ski8 relocalizes to the nucleus and associates with chromosomes specifically during meiosis. Interaction of Ski8 with Spo11 is essential for DSB formation and Ski8 relocalization. Thus, Ski8 plays distinct roles in RNA metabolism and, as a direct partner of Spo11, in DSB formation. Ski8 works with Spo11 to recruit other DSB proteins to meiotic chromosomes, implicating Ski8 as a scaffold protein mediating assembly of a multiprotein complex essential for DSB formation.