RESUMEN
Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.
Asunto(s)
Elementos sin Sentido (Genética)/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Transcripción Genética , Elementos sin Sentido (Genética)/biosíntesis , Cromatina/genética , Islas de CpG/genética , Regulación Fúngica de la Expresión Génica , Genómica , Código de Histonas/genética , Histonas/genética , Humanos , Proteínas Nucleares/biosíntesis , Nucleosomas/genética , Unión Proteica/genética , Alineación de SecuenciaRESUMEN
Macroautophagy is an intracellular degradation system for the majority of proteins and some organelles that is conserved in all eukaryotic species. The precise role of autophagy in mammalian development and potential involvement in disease remain to be discerned. Yeast Atg9p is the first integral membrane protein shown to be essential for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas its mammalian functional orthologue has yet to be identified. We have identified two human genes homologous to yeast Atg9p and designated these as APG9L1 and APG9L2. We have previously identified APG9L2 as NOS3AS, which participates in the post-transcriptional regulation of the endothelial nitric-oxide synthase (NOS3) gene on chromosome 7 through its antisense overlap. In human adult tissues, APG9L1 was ubiquitously expressed, whereas APG9L2 was highly expressed in placenta (trophoblast cells) and pituitary gland. In transient transfection assays we found that both proteins were primarily localized to the perinuclear region and also scattered throughout the cytosol as dots, a subset of which colocalized with an autophagosome-specific marker LC3 under starvation conditions. Finally, by the small interfering RNA-mediated knockdown of APG9L1 in HeLa cells, we demonstrated that APG9L1 is essential for starvation-induced autophagosome formation. In addition, APG9L2 can functionally complement APG9L1 in this process. These results, taken together with those of phylogenetic and sequence analyses, suggest that both APG9L1 and APG9L2 are functionally orthologous to the yATG9 in autophagosome formation. Moreover, APG9L2 is a vertebrate-specific gene that may have gained critical roles in mammalian-specific developmental events, such as placentation, through rapid evolution.
Asunto(s)
Elementos sin Sentido (Genética)/genética , Autofagia/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Óxido Nítrico Sintasa/genética , Trofoblastos/enzimología , Animales , Elementos sin Sentido (Genética)/biosíntesis , Autofagia/fisiología , Proteínas Relacionadas con la Autofagia , Pollos , Perros , Drosophila melanogaster , Células HeLa , Humanos , Proteínas de la Membrana/biosíntesis , Ratones , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/biosíntesis , Óxido Nítrico Sintasa de Tipo II , Óxido Nítrico Sintasa de Tipo III , Pan troglodytes , Ratas , Saccharomyces cerevisiae , Takifugu , Pez CebraRESUMEN
OBJECTIVE: To evaluate the effects of transforming growth factor beta1 (TGFbeta1) autocrine blockage on proliferation activity and drug sensitivity of osteosarcoma. METHODS; Northern blot, MTT determination, and 3H thymidine incorporation were used to investigate the effects of antisense TGF beta1 gene on osteosarcoma. RESULTS: The proliferation of osteosarcoma cells transfected by antisense TGF beta1 gene was suppressed markedly, and adriamycin sensitivity was significantly increased. CONCLUSION: Blockage of osteosarcoma cells TGF beta1 autocrine loop inhibits cell proliferation and enhances chemotherapy sensitivity.
Asunto(s)
Comunicación Autocrina/efectos de los fármacos , Neoplasias Óseas/patología , Osteosarcoma/patología , Factor de Crecimiento Transformador beta/genética , Antineoplásicos/farmacología , Elementos sin Sentido (Genética)/biosíntesis , Elementos sin Sentido (Genética)/genética , Neoplasias Óseas/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Doxorrubicina/farmacología , Humanos , Osteosarcoma/metabolismo , ARN Mensajero/genética , Transfección , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta1RESUMEN
To construct the antisense transforming growth factor beta 1 (TGF beta 1) gene and investigate the effect of TGF beta 1 autocrine loop blockage on the proliferation of osteosarcoma cells. TGF beta 1 cDNA was cloned by RT-PCR from human osteosarcoma cells (MG-63) and inserted into pcDNA3 to construct an antisense expression vector, which was dubbed pcDNA3-TGF beta 1(-). MTT was used to detect the proliferation of osteosarcoma cells transfected by antisense TGF beta 1 gene. Our results showed that the proliferation of the transfected osteosarcoma cells was suppressed markedly. It is concluded that TGF beta 1 autocrine loop blockage in osteosarcoma cells could inhibit cell proliferation, which might be helpful for gene therapy of osteosarcoma.
Asunto(s)
Neoplasias Óseas/genética , Osteosarcoma/genética , Factor de Crecimiento Transformador beta/genética , Elementos sin Sentido (Genética)/biosíntesis , Elementos sin Sentido (Genética)/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , División Celular , Humanos , Osteosarcoma/patología , Transcripción Genética , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta1 , Células Tumorales CultivadasRESUMEN
Wolfram syndrome is a rare condition in which the pancreatic beta-cells of patients are selectively deleted during the early years of life by a non-autoimmune-mediated mechanism. The condition is associated with mutations in the gene encoding wolframin, suggesting that this protein exerts a critical, but currently unknown, function in beta-cells. We have used an antisense strategy to modulate the expression of wolframin in insulin-secreting BRIN-BD11 cells to study its function. Stably transfected clones were established expressing full-length human wolframin antisense transcripts. These cells exhibited a dramatic reduction in cell proliferation rate and changes in morphology, although insulin secretion was not modified. The results imply that wolframin expression is required to sustain normal rates of beta-cell proliferation.
