RESUMO
The activation of naïve B lymphocyte involves rapid and major changes in chromatin organization and gene expression; however, the complete repertoire of nuclear factors affecting these genomic changes is not known. We report that HMGN proteins, which bind to nucleosomes and affect chromatin structure and function, co-localize with, and maintain the intensity of DNase I hypersensitive sites genome wide, in resting but not in activated B cells. Transcription analyses of resting and activated B cells from wild-type and Hmgn(-/-) mice, show that loss of HMGNs dampens the magnitude of the transcriptional response and alters the pattern of gene expression during the course of B-cell activation; defense response genes are most affected at the onset of activation. Our study provides insights into the biological function of the ubiquitous HMGN chromatin binding proteins and into epigenetic processes that affect the fidelity of the transcriptional response during the activation of B cell lymphocytes.
Assuntos
Linfócitos B/metabolismo , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Proteínas HMGN/metabolismo , Ativação Linfocitária/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Desoxirribonuclease I/metabolismo , Epigênese Genética , Proteínas HMGN/deficiência , Proteínas HMGN/genética , Proteína HMGN1/metabolismo , Proteína HMGN2/metabolismo , Masculino , Camundongos , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Baço/citologia , Baço/imunologiaRESUMO
HMGN5 is a typical member of the HMGN (high mobility group nucleosome-binding protein) family which may function as a nucleosomal binding and transcriptional activating protein. Overexpression of HMGN5 has been observed in several human tumors but its role in tumorigenesis has not been fully clarified. To investigate its significance for human lung cancer progression, we successfully constructed a shRNA expression lentiviral vector in which sense and antisense sequences targeting the human HMGN5 were linked with a 9-nucleotide loop. Inhibitory effects of siRNA on endogenous HMGN5 gene expression and protein synthesis were demonstrated via real-time RT-PCR and western blotting. We found HMGN5 silencing to significantly inhibit A549 and H1299 cell proliferation assessed by MTT, BrdU incorporation and colony formation assays. Furthermore, flow cytometry analysis showed that specific knockdown of HMGN5 slowed down the cell cycle at the G0/G1 phase and decreased the populations of A549 and H1299 cells at the S and G2/M phases. Taken together, these results suggest that HMGN5 is directly involved in regulation cell proliferation in A549 and H1299 cells by influencing signaling pathways involved in cell cycle progression. Thus, our finding suggests that targeting HMGN5 may be an effective strategy for human lung cancer treatment.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Ciclo Celular/genética , Proteínas HMGN/deficiência , Proteínas HMGN/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Transativadores/deficiência , Transativadores/genética , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Proteínas HMGN/biossíntese , Humanos , Interferência de RNA , RNA Interferente Pequeno/genética , Transativadores/biossínteseRESUMO
Improper glucose-stimulated insulin secretion from pancreatic beta cells is a major factor in the onset of type 2 diabetes. We now report that HMGN3, a nuclear protein that binds to nucleosomes and affects chromatin function, is highly expressed in beta cells and that in mice, loss of HMGN3 impairs glucose-stimulated insulin secretion and leads to a diabetic phenotype. In pancreatic beta cells, loss of HMGN3 affects the transcription of several genes involved in glucose-stimulated insulin secretion, including that of the Glut2 glucose transporter. Chromatin immunoprecipitation reveals that HMGN3 and the transcription factor PDX1 mutually reinforce their specific binding to the chromatin in the promoter of the Glut2 gene, thereby regulating GLUT2 protein levels in pancreatic islets and in beta cells. Our results identify a new regulator of glucose homeostasis and demonstrate a link between the activity of a nucleosome binding structural protein and the regulation of insulin secretion.
Assuntos
Perfilação da Expressão Gênica , Proteínas HMGN/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Nucleossomos/metabolismo , Animais , Glicemia/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Transportador de Glucose Tipo 2/genética , Transportador de Glucose Tipo 2/metabolismo , Proteínas HMGN/deficiência , Proteínas HMGN/genética , Humanos , Secreção de Insulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição GênicaRESUMO
HMGN (high-mobility-group N) family members are vertebrate proteins that unfold chromatin and promote transcription and replication of chromatin templates in vitro. However, their precise roles in vivo have been elusive until recently. This paper summarizes recent advances from studies of Hmgn1 knockout mice and genetically engineered cell lines that are beginning to reveal the diverse roles that HMGN proteins play in DNA repair and transcription within mammalian cells.