RESUMO
Coactivator-associated arginine methyltransferase I (CARM1; PRMT4) regulates gene expression by multiple mechanisms including methylation of histones and coactivation of steroid receptor transcription. Mice lacking CARM1 are small, fail to breathe and die shortly after birth, demonstrating the crucial role of CARM1 in development. In adults, CARM1 is overexpressed in human grade-III breast tumors and prostate adenocarcinomas, and knockdown of CARM1 inhibits proliferation of breast and prostate cancer cell lines. Based on these observations, we hypothesized that loss of CARM1 in mouse embryos would inhibit pulmonary cell proliferation, resulting in respiratory distress. By contrast, we report here that loss of CARM1 results in hyperproliferation of pulmonary epithelial cells during embryonic development. The lungs of newborn mice lacking CARM1 have substantially reduced airspace compared with their wild-type littermates. In the absence of CARM1, alveolar type II cells show increased proliferation. Electron microscopic analyses demonstrate that lungs from mice lacking CARM1 have immature alveolar type II cells and an absence of alveolar type I cells. Gene expression analysis reveals a dysregulation of cell cycle genes and markers of differentiation in the Carm1 knockout lung. Furthermore, there is an overlap in gene expression in the Carm1 knockout and the glucocorticoid receptor knockout lung, suggesting that hyperproliferation and lack of maturation of the alveolar cells are at least in part caused by attenuation of glucocorticoid-mediated signaling. These results demonstrate for the first time that CARM1 inhibits pulmonary cell proliferation and is required for proper differentiation of alveolar cells.
Assuntos
Células Epiteliais/metabolismo , Pulmão/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Proliferação de Células , Células Endoteliais/metabolismo , Glucocorticoides/metabolismo , Camundongos , Alvéolos Pulmonares/metabolismo , Transcrição GênicaRESUMO
Targeted disruption of a highly conserved distal enhancer reduces expression of the PU.1 transcription factor by 80% and leads to acute myeloid leukemia (AML) with frequent cytogenetic aberrations in mice. Here we identify a SNP within this element in humans that is more frequent in AML with a complex karyotype, leads to decreased enhancer activity, and reduces PU.1 expression in myeloid progenitors in a development-dependent manner. This SNP inhibits binding of the chromatin-remodeling transcriptional regulator special AT-rich sequence binding protein 1 (SATB1). Overexpression of SATB1 increased PU.1 expression, and siRNA inhibition of SATB1 downregulated PU.1 expression. Targeted disruption of the distal enhancer led to a loss of regulation of PU.1 by SATB1. Interestingly, disruption of SATB1 in mice led to a selective decrease of PU.1 RNA in specific progenitor types (granulocyte-macrophage and megakaryocyte-erythrocyte progenitors) and a similar effect was observed in AML samples harboring this SNP. Thus we have identified a SNP within a distal enhancer that is associated with a subtype of leukemia and exerts a deleterious effect through remote transcriptional dysregulation in specific progenitor subtypes.
Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas/genética , Transativadores/genética , Animais , Sequência de Bases , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Genoma Humano/genética , Humanos , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Retorno de Linfócitos/genética , Receptores de Retorno de Linfócitos/metabolismo , Células-Tronco/metabolismo , Transativadores/deficiência , Transativadores/metabolismoRESUMO
Respiratory syncytial virus (RSV) is the greatest remaining unmet infant vaccine need in developed countries and an important unmet infant vaccine need worldwide. More than 40 years of effort have yet to result in a licensed RSV vaccine for humans. Key challenges to RSV vaccine development include a peak of severe disease at 2-3 months of age, problematic biochemical behavior of key vaccine antigens, a history of vaccine-mediated disease enhancement, and reliance on animal models that may not accurately reflect human disease processes. Potential paths to overcome these challenges include maternal immunization, structure-based engineering of vaccine antigens, the design of a novel platform for safe infant immunization, and the development of improved animal models for vaccine-enhanced disease.
Assuntos
Infecções por Vírus Respiratório Sincicial/epidemiologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vacinas contra Vírus Sincicial Respiratório/isolamento & purificação , Animais , Pesquisa Biomédica/tendências , Modelos Animais de Doenças , Humanos , Lactente , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinação/métodosRESUMO
The transcription factor PU.1 is an important regulator of hematopoiesis; precise expression levels are critical for normal hematopoietic development and suppression of leukemia. We show here that noncoding antisense RNAs are important modulators of proper dosages of PU.1. Antisense and sense RNAs are regulated by shared evolutionarily conserved cis-regulatory elements, and we can show that antisense RNAs inhibit PU.1 expression by modulating mRNA translation. We propose that such antisense RNAs will likely be important in the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.
Assuntos
Expressão Gênica , Proteínas Proto-Oncogênicas/genética , RNA Antissenso/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Transativadores/genética , Animais , Linhagem Celular , Eletroporação , Granulócitos/citologia , Granulócitos/metabolismo , Células HL-60 , Humanos , Separação Imunomagnética , Células Jurkat , Macrófagos/metabolismo , Camundongos , Modelos Genéticos , Biossíntese de Proteínas , Interferência de RNA , RNA Antissenso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Linfócitos T/metabolismo , Transcrição Gênica , Células U937RESUMO
Activation of JAK tyrosine kinases is an essential step in cell signaling by multiple hormones, cytokines, and growth factors, including growth hormone (GH) and interferon-gamma. Previously, we identified SH2-B beta as a potent activator of JAK2 (Rui, L., and Carter-Su, C. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 7172-7177). Here, we investigated whether the activation of JAK2 by SH2-B beta is specific to JAK2 and SH2-B beta or extends to other JAKs or other members of the SH2-B beta family. When SH2-B beta was overexpressed with JAK1 or JAK3, SH2-B beta failed to increase their activity. However, SH2-B beta bound to both and was tyrosyl-phosphorylated by JAK1. In contrast to SH2-B beta, APS decreased tyrosyl phosphorylation of GH-stimulated JAK2 as well as Stat5B, a substrate of JAK2. APS also decreased tyrosyl phosphorylation of JAK1, but did not affect the activity or tyrosyl phosphorylation of JAK3. Overexpressed APS bound to and was tyrosyl-phosphorylated by all three JAKs. Consistent with these data, in 3T3-F442A adipocytes, endogenous APS was tyrosyl-phosphorylated in response to GH and interferon-gamma. These results suggest that 1) SH2-B beta specifically activates JAK2, 2) APS negatively regulates both JAK2 and JAK1, and 3) both SH2-B beta and APS may serve as adapter proteins for all three JAKs independent of any role they have in JAK activity.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas , Células 3T3 , Animais , Células COS , Linhagem Celular , DNA Complementar/metabolismo , Ativação Enzimática , Humanos , Janus Quinase 1 , Janus Quinase 2 , Janus Quinase 3 , Camundongos , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Transdução de Sinais , Transfecção , Tirosina/metabolismoRESUMO
SH2-Bbeta binds to the activated form of JAK2 and various receptor tyrosine kinases. It is a potent stimulator of JAK2, is required for growth hormone (GH)-induced membrane ruffling, and increases mitogenesis stimulated by platelet-derived growth factor (PDGF) and insulin-like growth factor I. Its domain structure suggests that SH2-Bbeta may act as an adapter protein to recruit downstream signaling proteins to kinase.SH2-Bbeta complexes. SH2-Bbeta is tyrosyl-phosphorylated in response to GH and interferon-gamma, stimulators of JAK2, as well as in response to PDGF and nerve growth factor. To begin to elucidate the role of tyrosyl phosphorylation in the function of SH2-Bbeta, we used phosphopeptide mapping, mutagenesis, and a phosphotyrosine-specific antibody to identify Tyr-439 and Tyr-494 in SH2-Bbeta as targets of JAK2 both in vitro and in intact cells. SH2-Bbeta lacking Tyr-439 and Tyr-494 inhibits GH-induced membrane ruffling but still activates JAK2. We provide evidence that JAK1, like JAK2, phosphorylates Tyr-439 and Tyr-494 in SH2-Bbeta and that PDGF receptor phosphorylates SH2-Bbeta on Tyr-439. Therefore, phosphorylated Tyr-439 and/or Tyr-494 in SH2-Bbeta may provide a binding site for one or more proteins linking cytokine receptor.JAK2 complexes and/or receptor tyrosine kinases to the actin cytoskeleton.