RESUMO
Meningioma-1 (MN1) overexpression in AML is associated with poor prognosis, and forced expression of MN1 induces leukemia in mice. We sought to determine how MN1 causes AML. We found that overexpression of MN1 can be induced by translocations that result in hijacking of a downstream enhancer. Structure predictions revealed that the entire MN1 coding frame is disordered. We identified the myeloid progenitor-specific BAF complex as the key interaction partner of MN1. MN1 over-stabilizes BAF on enhancer chromatin, a function directly linked to the presence of a long polyQ-stretch within MN1. BAF over-stabilization at binding sites of transcription factors regulating a hematopoietic stem/progenitor program prevents the developmentally appropriate decommissioning of these enhancers and results in impaired myeloid differentiation and leukemia. Beyond AML, our data detail how the overexpression of a polyQ protein, in the absence of any coding sequence mutation, can be sufficient to cause malignant transformation.
Assuntos
Carcinogênese/genética , DNA Helicases/genética , Proteínas Intrinsicamente Desordenadas/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Sequência de Bases , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Cromatina/patologia , DNA Helicases/metabolismo , Elementos Facilitadores Genéticos , Feminino , Regulação Leucêmica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Nucleares/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Transporte Proteico , Transdução de Sinais , Análise de Sobrevida , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Recent studies indicate that chromatin regulatory complexes produce biological specificity in the way that letters produce meanings by combinations into words. Combinatorial assembly of chromatin regulatory complexes may be critical for maximizing the information content provided by arrays of histone modifications.
Assuntos
Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Animais , Proteínas Cromossômicas não Histona/metabolismo , Código das Histonas , Humanos , Vertebrados/genéticaRESUMO
Stem cells of all types are characterized by a stable, heritable state permissive of multiple developmental pathways. The past five years have seen remarkable advances in understanding these heritable states and the ways that they are initiated or terminated. Transcription factors that bind directly to DNA and have sufficiency roles have been most easy to investigate and, perhaps for this reason, are most solidly implicated in pluripotency. In addition, large complexes of ATP-dependent chromatin-remodeling and histone-modification enzymes that have specialized functions have also been implicated by genetic studies in initiating and/or maintaining pluripotency or multipotency. Several of these ATP-dependent remodeling complexes play non-redundant roles, and the esBAF complex facilitates reprogramming of induced pluripotent stem cells. The recent finding that virtually all histone modifications can be rapidly reversed and are often highly dynamic has raised new questions about how histone modifications come to play a role in the steady state of pluripotency. Another surprise from genetic studies has been the frequency with which the global effects of mutations in chromatin regulators can be largely reversed by a single target gene. These genetic studies help define the arena for future mechanistic studies that might be helpful to harness pluripotency for therapeutic goals.
Assuntos
Montagem e Desmontagem da Cromatina , Células-Tronco Pluripotentes/citologia , Animais , Células-Tronco Embrionárias/citologia , HumanosRESUMO
In mammals, combinatorial assembly of alternative families of subunits confers functional specificity to adenosine triphosphate (ATP)-dependent SWI/SNF-like Brg/Brm-associated factor (BAF) chromatin remodeling complexes by creating distinct polymorphic surfaces for interaction with regulatory elements and DNA-binding factors. Although redundant in terms of biochemical activity, the core ATPase subunits, BRG/SMARCA4 and BRM/SMARCA2, are functionally distinct and may contribute to complex specificity. Here we show using quantitative proteomics that BAF complexes expressed in leukemia are specifically assembled around the BRG ATPase. Moreover, using a mouse model of acute myeloid leukemia, we demonstrate that BRG is essential for leukemia maintenance, as leukemic cells lacking BRG rapidly undergo cell-cycle arrest and apoptosis. Most importantly, we show that BRG is dispensable for the maintenance of immunophenotypic long-term repopulating hematopoietic stem cells, suggesting that adroit targeting of BRG in leukemia may have potent and specific therapeutic effects.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , DNA Helicases/fisiologia , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Animais , Southern Blotting , Cromatografia Líquida , DNA Helicases/genética , Modelos Animais de Doenças , Citometria de Fluxo , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas em Tandem , Fatores de Transcrição/genéticaRESUMO
ATP-dependent SWI/SNF-like BAF chromatin remodeling complexes are emerging as key regulators of embryonic and adult stem cell function. Particularly intriguing are the findings that specialized assemblies of BAF complexes are required for establishing and maintaining pluripotent and multipotent states in cells. However, little is known on the importance of these complexes in normal and leukemic hemopoiesis. Here we provide the first evidence that the actin-related protein BAF53a, a subunit of BAF complexes preferentially expressed in long-term repopulating stem cells, is essential for adult hemopoiesis. Conditional deletion of BAF53a resulted in multilineage BM failure, aplastic anemia, and rapid lethality. These severe hemopoietic defects originate from a proliferative impairment of BM HSCs and progenitors and decreased progenitor survival. Using hemopoietic chimeras, we show that the impaired function of BAF53a-deficient HSCs is cell-autonomous and independent of the BM microenvironment. Altogether, our studies highlight an unsuspected role for BAF chromatin remodeling complexes in the maintenance of HSC and progenitor cell properties.
Assuntos
Actinas/genética , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/metabolismo , Sistema Hematopoético/metabolismo , Transcriptoma , Actinas/metabolismo , Anemia Aplástica/genética , Anemia Aplástica/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/fisiologia , Proteínas de Ciclo Celular/genética , Proliferação de Células , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Feto , Citometria de Fluxo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/fisiologia , Sistema Hematopoético/crescimento & desenvolvimento , Fígado , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Lymphocyte development from murine hematopoietic stem cells (HSCs) entails a loss of self-renewal capacity and a progressive restriction of developmental potential. Previous research from our laboratory suggests that specialized assemblies of ATP-dependent SWI/SNF chromatin-remodeling complexes play lineage-specific roles during murine hematopoiesis. Here, we demonstrate that the Smarcd1 subunit is essential for specification of lymphoid cell fate from multipotent progenitors. Acute deletion of Smarcd1 in murine adult hematopoiesis leads to lymphopenia, characterized by a near-complete absence of early lymphoid progenitors and mature B and T cells, while the myeloid and erythroid lineages remain unaffected. Mechanistically, we demonstrate that Smarcd1 is essential for the coordinated activation of a lymphoid gene signature in murine multipotent progenitors. This is achieved by interacting with the E2a transcription factor at proximal promoters and by regulating the activity of distal enhancers. Globally, these findings identify Smarcd1 as an essential chromatin remodeler that governs lymphoid cell fate.
RESUMO
It is believed that hemopoietic stem cells (HSC), which colonize the fetal liver (FL) rapidly, expand to establish a supply of HSCs adequate for maintenance of hemopoiesis throughout life. Accordingly, FL HSCs are actively cycling as opposed to their predominantly quiescent bone marrow counterparts, suggesting that the FL microenvironment provides unique signals that support HSC proliferation and self-renewal. We now report the generation and characterization of mice with a mutant allele of Baf250a lacking exons 2 and 3. Baf250a(E2E3/E2E3) mice are viable until E19.5, but do not survive beyond birth. Most interestingly, FL HSC numbers are markedly higher in these mice than in control littermates, thus raising the possibility that Baf250a determines the HSC pool size in vivo. Limit dilution experiments indicate that the activity of Baf250a(E2E3/E2E3) HSC is equivalent to that of the wild-type counterparts. The Baf250a(E2E3/E2E3) FL-derived stroma, in contrast, exhibits a hemopoiesis-supporting potential superior to the developmentally matched controls. To our knowledge, this demonstration is the first that a mechanism operating in a cell nonautonomous manner canexpand the pool size of the fetal HSC populations.
Assuntos
Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/metabolismo , Fígado/metabolismo , Mutação , Proteínas Nucleares/genética , Alelos , Animais , Western Blotting , Contagem de Células , Proliferação de Células , Ensaio de Unidades Formadoras de Colônias , Proteínas de Ligação a DNA/metabolismo , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Fígado/embriologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Tempo , Fatores de TranscriçãoRESUMO
Mammalian SWI/SNF [also called BAF (Brg/Brahma-associated factors)] ATP-dependent chromatin remodeling complexes are essential for formation of the totipotent and pluripotent cells of the early embryo. In addition, subunits of this complex have been recovered in screens for genes required for nuclear reprogramming in Xenopus and mouse embryonic stem cell (ES) morphology. However, the mechanism underlying the roles of these complexes is unclear. Here, we show that BAF complexes are required for the self-renewal and pluripotency of mouse ES cells but not for the proliferation of fibroblasts or other cells. Proteomic studies reveal that ES cells express distinctive complexes (esBAF) defined by the presence of Brg (Brahma-related gene), BAF155, and BAF60A, and the absence of Brm (Brahma), BAF170, and BAF60C. We show that this specialized subunit composition is required for ES cell maintenance and pluripotency. Our proteomic analysis also reveals that esBAF complexes interact directly with key regulators of pluripotency, suggesting that esBAF complexes are specialized to interact with ES cell-specific regulators, providing a potential explanation for the requirement of BAF complexes in pluripotency.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/fisiologia , Animais , Proliferação de Células , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/análise , Fibroblastos/citologia , Camundongos , Proteínas Musculares/análise , Proteômica , Fatores de Transcrição/análiseRESUMO
The diversity of dendritic patterns is one of the fundamental characteristics of neurons and is in part regulated by transcriptional programs initiated by electrical activity. We show that dendritic outgrowth requires a family of combinatorially assembled, neuron-specific chromatin remodeling complexes (nBAF complexes) distinguished by the actin-related protein BAF53b and based on the Brg/Brm ATPases. nBAF complexes bind tightly to the Ca(2+)-responsive dendritic regulator CREST and directly regulate genes essential for dendritic outgrowth. BAF53b is not required for nBAF complex assembly or the interaction with CREST, yet is required for their recruitment to the promoters of specific target genes. The highly homologous BAF53a protein, which is a component of neural progenitor and nonneural BAF complexes, cannot replace BAF53b's role in dendritic development. Remarkably, we find that this functional specificity is conferred by the actin fold subdomain 2 of BAF53b. These studies suggest that the genes encoding the individual subunits of BAF complexes function like letters in a ten-letter word to produce biologically specific meanings (in this case dendritic outgrowth) by combinatorial assembly of their products.
Assuntos
Actinas/genética , Montagem e Desmontagem da Cromatina/fisiologia , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Dendritos/fisiologia , Neurônios/citologia , Actinas/deficiência , Animais , Cálcio/metabolismo , Células Cultivadas , Montagem e Desmontagem da Cromatina/genética , Imunoprecipitação da Cromatina/métodos , Proteínas Cromossômicas não Histona/deficiência , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Dendritos/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/citologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismoRESUMO
Mammalian neural stem cells (NSCs) have the capacity to both self-renew and to generate all the neuronal and glial cell-types of the adult nervous system. Global chromatin changes accompany the transition from proliferating NSCs to committed neuronal lineages, but the mechanisms involved have been unclear. Using a proteomics approach, we show that a switch in subunit composition of neural, ATP-dependent SWI/SNF-like chromatin remodeling complexes accompanies this developmental transition. Proliferating neural stem and progenitor cells express complexes in which BAF45a, a Krüppel/PHD domain protein and the actin-related protein BAF53a are quantitatively associated with the SWI2/SNF2-like ATPases, Brg and Brm. As neural progenitors exit the cell cycle, these subunits are replaced by the homologous BAF45b, BAF45c, and BAF53b. BAF45a/53a subunits are necessary and sufficient for neural progenitor proliferation. Preventing the subunit switch impairs neuronal differentiation, indicating that this molecular event is essential for the transition from neural stem/progenitors to postmitotic neurons. More broadly, these studies suggest that SWI/SNF-like complexes in vertebrates achieve biological specificity by combinatorial assembly of their subunits.
Assuntos
Diferenciação Celular/fisiologia , Montagem e Desmontagem da Cromatina/fisiologia , Complexos Multienzimáticos/metabolismo , Células-Tronco Multipotentes/metabolismo , Neurônios/citologia , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina/genética , Epigênese Genética/genética , Epigênese Genética/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Camundongos , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Células-Tronco Multipotentes/citologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Fatores de Transcrição/genéticaRESUMO
We set out to examine the pathophysiological mechanisms of fibrosis in diffuse systemic sclerosis (SSc) using a tissue engineering approach. Skin fibroblasts were isolated from lesional skin of SSc patients with a disease duration of less than 1 year (early-stage SSc) or more than 10 years (late-stage SSc). Fibroblasts were also isolated from non-lesional skin and compared with normal fibroblasts isolated from healthy adults. Cells were cultured using a tissue engineering method to reconstruct a human dermis, and histologically observed. Dermal thickness was measured, as it reflects the global and intrinsic capacity of cells to reconstitute matrix. Collagen I, MMP-1, and MMP activity were evaluated. Cells were treated with TGFbeta1 or CTGF during dermis formation to study their fibrogenic role. Clinical severity of skin involvement was measured by a modified Rodnan score. Thickness of the dermis generated with non-lesional early-stage SSc fibroblasts was similar to normal cells. In contrast, reconstructed dermis from lesional early-stage SSc fibroblasts and non-lesional late-stage SSc cells was thinner, while lesional late-stage SSc fibroblasts made a thicker dermis. Dermis was always thicker when produced with TGFbeta1-treated cells, except when lesional late-stage SSc fibroblasts from patients with high Rodnan skin scores were used. CTGF did not affect dermal thickness. Measurements of collagen I and collagenases in the culture medium of the various reconstructed dermis could explain some of the changes observed. We conclude that the fibrotic phenotype of SSc fibroblasts varies with disease duration and with severity of skin involvement, and this is clearly visualized during in vitro dermis reconstruction.
Assuntos
Escleroderma Sistêmico/patologia , Pele/patologia , Adulto , Idoso , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/análise , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Progressão da Doença , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Masculino , Metaloproteinase 1 da Matriz/análise , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinases da Matriz/análise , Metaloproteinases da Matriz/metabolismo , Pessoa de Meia-Idade , Fenótipo , Escleroderma Sistêmico/metabolismo , Pele/metabolismo , Engenharia Tecidual/métodos , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Androgen-induced bZIP (AIbZIP/CREB3L4) is a transcription factor of the bZIP family that associates with the membrane of the endoplasmic reticulum (ER). In humans, AIbZIP RNA is most abundant in the prostate gland where the protein is produced in luminal cells of the glandular epithelium. AIbZIP could play an important role in prostate cancer because its expression is up-regulated by androgens in LNCaP prostate cancer cells and the protein is more abundant in cancerous than in non-cancerous prostate cells. We recently added 74 adenocarcinomas and 43 specimens of prostatic intraepithelial neoplasia (PIN) to our survey of AIbZIP expression in prostate tumours. This study showed that AIbZIP is expressed in all grades of adenocarcinoma and that it is more abundant in high-grade PIN and in adenocarcinoma than in normal prostate. The physiological function of AIbZIP remains unknown but its association with the ER and its structural homology to transcription factors such as ATF6 suggest that AIbZIP could be activated by regulated intramembrane proteolysis during the cellular response to ER stress. This review will describe the characteristics of human and mammalian AIbZIP, its relationship to prostate cancer, and our recent efforts to characterize the transcriptional properties and targets of AIbZIP.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Proteínas Nucleares/fisiologia , Neoplasias da Próstata/fisiopatologia , Sequência de Aminoácidos , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Análise Mutacional de DNA , Retículo Endoplasmático/fisiologia , Humanos , Masculino , Dados de Sequência Molecular , Alinhamento de Sequência , Transcrição Gênica/fisiologiaRESUMO
The androgen-regulated protein androgen-induced bZIP (AIbZIP) is a bZIP transcription factor that localizes to the membrane of the endoplasmic reticulum (ER). The physiological role of AIbZIP is unknown, but other ER-bound transcription factors such as ATF6 and SREBPs play a crucial role in the regulation of protein processing and lipid synthesis, respectively. In response to alterations in the intracellular milieu, ATF6 and SREBPs are processed to their transcriptionally active forms by regulated intramembrane proteolysis. In humans, AIbZIP mRNA is expressed in several organs including the pancreas, liver, and gonads, but it is especially abundant in prostate epithelial cells. We therefore used LNCaP human prostate cancer cells as a model to identify stimuli that lead to AIbZIP activation and define the transcriptional targets of AIbZIP. In LNCaP cells, AIbZIP was processed to its transcriptionally active form by drugs that deplete ER calcium stores (i.e., A23187 and caffeine), but it was unaffected by an inhibitor of protein glycosylation (tunicamycin). To identify AIbZIP-regulated genes, we generated LNCaP cell lines that conditionally express the processed form of AIbZIP and used Affymetrix microarrays to screen for AIbZIP-regulated transcripts. Selected genes (n = 48) were validated by Northern blot hybridization. The results reveal that the downstream targets of AIbZIP include genes that are implicated in protein processing (e.g., BAG3, DNAJC12, KDELR3). Strikingly, a large number of AIbZIP-regulated transcripts encode proteins that are involved in transcriptional regulation, small molecule transport, signal transduction, and metabolism. These results suggest that AIbZIP plays a novel role in cell homeostasis.
Assuntos
Adenocarcinoma/patologia , Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/fisiologia , Neoplasias da Próstata/patologia , Transcrição Gênica , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica/genética , Brefeldina A/farmacologia , Cafeína/farmacologia , Calcimicina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Complexo de Golgi/enzimologia , Homeostase/genética , Humanos , Masculino , Dados de Sequência Molecular , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Tapsigargina/farmacologia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Tunicamicina/farmacologiaRESUMO
BACKGROUND: Trauma care of thoracic and abdominal injuries is currently in turmoil because of both a decrease in the number of these injuries and a concomitant increase in their nonsurgical management. The goal of this study was to evaluate the incidence of thoracic and abdominal injuries in the province of Quebec and the number of associated surgical procedures. METHODS: Patients with blunt thoracic or abdominal injuries taken to a tertiary trauma center in the province of Quebec from April 1, 1998 to March 31, 2002 were identified. Patients who were dead on arrival were excluded. Only patients with an Abbreviated Injury Scale score > or =2 for the thoracic or abdominal regions were included. RESULTS: During the study period, a total of 16,430 blunt trauma patients were admitted to one of the four trauma centers. A total of 2,660 (16.2%) patients sustained thoracic and/or abdominal injuries with an Abbreviated Injury Scale score >1. Among these, the median Injury Severity Score was 24 (range: 4-75) and the in-hospital mortality rate was 11.0%. There were 2,196 patients (82.5%) with thoracic injuries, 977 patients (36.7%) with abdominal injuries, and 520 patients (19.5%) with injuries to both regions. A surgical intervention was undertaken in 76 patients with thoracic injuries (3.5%) and in 414 patients with abdominal injuries (42.3%). On average, 4.7 thoracic and 28.8 abdominal trauma procedures were performed per center, yearly. Each trauma surgeon performed, on average, less than one thoracic and less than five abdominal trauma procedures yearly. CONCLUSIONS: The incidence of blunt thoracic and abdominal injuries needing surgical intervention is low in Quebec tertiary trauma centers. The competence of general surgeons in trauma-related procedures might be compromised by such low patient volume unless they frequently perform non-trauma surgical procedures. We think that in Quebec, trauma care must be provided by surgeons who practice both acute emergency and elective surgical care in addition to trauma care. These findings should have an important impact on the development of on-going education and resident training programs.
Assuntos
Traumatismos Abdominais/epidemiologia , Traumatismos Torácicos/epidemiologia , Ferimentos não Penetrantes/epidemiologia , Traumatismos Abdominais/cirurgia , Acidentes por Quedas/estatística & dados numéricos , Acidentes de Trânsito/estatística & dados numéricos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Quebeque/epidemiologia , Procedimentos Cirúrgicos Operatórios/estatística & dados numéricos , Traumatismos Torácicos/cirurgia , Centros de Traumatologia/estatística & dados numéricos , Ferimentos não Penetrantes/cirurgiaRESUMO
The ability of hemopoietic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that establish and maintain stage-specific patterns of gene expression. However, the epigenetic factors responsible for mediating these regulatory events remain poorly defined. Here we provide evidence that BAF45a/PHF10, a subunit of SWI/SNF-like chromatin remodeling complexes, is essential for adult hemopoietic stem cell maintenance and myeloid lineage development. Deletion of BAF45a in the mouse is embryonic lethal. Acute deletion of BAF45a in the adult hemopoietic system causes a dose-dependent decrease in the frequency of long-term repopulating hemopoietic stem cells and committed myeloid progenitors without affecting their rate of proliferation. BAF45a-deficient hemopoietic stem cells and myeloid progenitors are selectively lost from mixed bone marrow chimeras, indicating their impaired function even in an intact microenvironment. Together, these studies suggest that the BAF45a subunit of SWI/SNF-like chromatin remodeling complexes plays nonredundant and specialized roles within the developing hemopoietic tissue.
Assuntos
Autorrenovação Celular , Proteínas Cromossômicas não Histona/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/metabolismo , Animais , Biomarcadores , Diferenciação Celular/genética , Autorrenovação Celular/genética , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ordem dos Genes , Genes Letais , Loci Gênicos , Transplante de Células-Tronco Hematopoéticas , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Complexos Multiproteicos/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Fenótipo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas , Ativação Transcricional , Dedos de ZincoRESUMO
Dedifferentiation of adipocytes contributes to the generation of a proliferative cell population that could be useful in cellular therapy or tissue engineering. Adipocytes can dedifferentiate into precursor cells to acquire a fibroblast-like phenotype using ceiling culture, in which the buoyancy of fat cells is exploited to allow them to adhere to the inner surface of a container. Ceiling culture is usually performed in flasks, which limits the ability to test various culture conditions. Using a new six-well plate ceiling culture approach, we examined the relevance of TGF-ß signaling during dedifferentiation. Adipose tissue samples from patients undergoing bariatric surgery were digested with collagenase, and cell suspensions were used for ceiling cultures. Using the six-well plate approach, cells were treated with SB431542 (an inhibitor of TGF-ß receptor ALK5) or human TGF-ß1 during dedifferentiation. Gene expression was measured in these cultures and in whole adipose tissue, the stromal-vascular fraction (SVF), mature adipocytes, and dedifferentiated fat (DFAT) cells. TGF-ß1 and collagen type I alpha 1 (COL1A1) gene expression was significantly higher in DFAT cells compared to whole adipose tissue samples and SVF cells. TGF-ß1, COL1A1, and COL6A3 gene expression was significantly higher at day 12 of dedifferentiation compared to day 0. In the six-well plate model, treatment with TGF-ß1 or SB431542, respectively, stimulated and inhibited the TGF-ß pathway as shown by increased TGF-ß1, TGF-ß2, COL1A1, and COL6A3 gene expression and decreased expression of TGF-ß1, COL1A1, COL1A2, and COL6A3, respectively. Treatment of DFAT cells with TGF-ß1 increased the phosphorylation level of SMAD 2 and SMAD 3. Thus, a new six-well plate model for ceiling culture allowed us to demonstrate a role for TGF-ß in modulating collagen gene expression during dedifferentiation of mature adipocytes.
RESUMO
Recent studies suggest that individual subunits of chromatin-remodeling complexes produce biologically specific meaning in different cell types through combinatorial assembly. Here we show that granulocyte development requires SMARCD2, a subunit of ATP-dependent SWI/SNF (BAF) chromatin-remodeling complexes. Smarcd2-deficient mice fail to generate functionally mature neutrophils and eosinophils, a phenotype reminiscent of neutrophil-specific granule deficiency (SGD) in humans, for which loss-of-function mutations in CEBPE (encoding CEBPÉ) have been reported. SMARCD2-containing SWI/SNF complexes are necessary for CEBPÉ transcription factor recruitment to the promoter of neutrophilic secondary granule genes and for granulocyte differentiation. The homologous SMARCD1 protein (63% identical at the amino acid level) cannot replace the role of SMARCD2 in granulocyte development. We find that SMARCD2 functional specificity is conferred by its divergent coiled-coil 1 and SWIB domains. Strikingly, both CEBPE and SMARCD2 loss-of-function mutations identified in patients with SGD abolish the interaction with SWI/SNF and thereby secondary granule gene expression, thus providing a molecular basis for this disease.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/genética , Cromatina/genética , Granulócitos/metabolismo , Hematopoese/genética , Mutação , Fatores de Transcrição/genética , Animais , Animais Recém-Nascidos , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica , Granulócitos/citologia , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismoRESUMO
Objective. To characterize changes in gene expression profile during human mature adipocyte dedifferentiation in ceiling culture. Methods. Subcutaneous (SC) and omental (OM) adipose tissue samples were obtained from 4 participants paired for age and BMI. Isolated adipocytes were dedifferentiated in ceiling culture. Gene expression analysis at days 0, 4, 7, and 12 of the cultures was performed using Affymetrix Human Gene 2.0 STvi arrays. Hierarchical clustering according to similarity of expression changes was used to identify overrepresented functions. Results. Four clusters gathered genes with similar expression between day 4 to day 7 but decreasing expression from day 7 to day 12. Most of these genes coded for proteins involved in adipocyte functions (LIPE, PLIN1, DGAT2, PNPLA2, ADIPOQ, CEBPA, LPL, FABP4, SCD, INSR, and LEP). Expression of several genes coding for proteins implicated in cellular proliferation and growth or cell cycle increased significantly from day 7 to day 12 (WNT5A, KITLG, and FGF5). Genes coding for extracellular matrix proteins were differentially expressed between days 0, 4, 7, and 12 (COL1A1, COL1A2, and COL6A3, MMP1, and TGFB1). Conclusion. Dedifferentiation is associated with downregulation of transcripts encoding proteins involved in mature adipocyte functions and upregulation of genes involved in matrix remodeling, cellular development, and cell cycle.
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The maturation of haploid spermatids into spermatozoa relies on the timely production of proteins required for spermatid differentiation. The mammalian CREB3L4 (cAMP responsive element binding protein 3-like 4) gene encodes a bZIP transcription factor that associates with the membrane of the endoplasmic reticulum. CREB3L4 is presumed to play an important role in protein maturation via its involvement in the cellular response to endoplasmic reticulum stress. In mice, the Creb3l4 gene gives rise to 2 distinct classes of mRNAs through the use of alternate promoters. Transcripts that initiate upstream of the first coding exon encode a 370-amino acid (aa) protein designated Tisp40beta, whereas transcripts that initiate downstream of the first coding exon encode Atce1/Tisp40alpha, a truncated (315-aa) form of Tisp40beta. In the mouse testis, Creb3l4 transcripts are known to be expressed exclusively in postmeiotic spermatids but the presence of CREB3L4 protein in spermatids has not been formally demonstrated. We produced an antibody directed against the carboxy terminus of mouse CREB3L4 and used it in immunostaining experiments to document that CREB3L4 protein accumulates in post-meiotic spermatids in a stage-specific manner. Moreover, we show that Atce1/Tisp40alpha is the major form of CREB3L4 in mouse testis. These findings suggest that testis-specific isoforms of Creb3l4 could play an important role in spermatid differentiation.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Espermátides/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Zíper de Leucina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/biossíntese , CoelhosRESUMO
All mature blood cells originate from a small population of self-renewing pluripotent hematopoietic stem cells (HSCs). The capacity to self-renew characterizes all stem cells, whether normal or neoplastic. Interestingly, recent studies suggest that self-renewal is essential for tumor cell maintenance, implicating that this process has therapeutic relevance. Unfortunately, the molecular bases for self-renewal of vertebrate cells remain poorly defined. This article will focus on the developmental mechanisms underlying fetal and adult HSC homeostasis. Specifically, distinctions between genetic programs regulating HSC specification (identity), self-renewal (in both fetal and adult) and differentiation/commitment will be discussed with a special emphasis on transcriptional and chromatin regulators.