RESUMO
The transcription factor Nanog is essential for mammalian embryogenesis, as well as the pluripotency of embryonic stem (ES) cells. Work with ES cells and embryonal carcinoma (EC) cells previously identified positive and negative cis-regulatory elements that influence the activity of the Nanog promoter, including adjacent cis-regulatory elements that bind Sox2 and Oct-3/4. Given the importance of Nanog during mammalian development, we examined the cis-regulatory elements required for Nanog promoter activity more closely. In this study, we demonstrate that two positive cis-regulatory elements previously shown to be active in F9 EC cells are also active in ES cells. We also identify a novel negative regulatory region that is located in close proximity to two other positive Nanog cis-regulatory elements. Although this negative regulatory region is active in F9 EC cells and ES cells, it is inactive in P19 EC cells. Furthermore, we demonstrate that one of the positive cis-regulatory elements active in F9 EC cells and ES cells is inactive in P19 EC cells. Together, these and other studies suggest that Nanog transcription is regulated by the interplay of positive and negative cis-regulatory elements. Given that P19 appears to be more closely related to a later developmental stage of mammalian development than F9 and ES cells, differential utilization of cis-regulatory elements may reflect mechanisms used during development to achieve the correct level of Nanog expression as embryogenesis unfolds.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Elementos Reguladores de Transcrição/fisiologia , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular Tumoral , Primers do DNA/genética , Camundongos , Dados de Sequência Molecular , Proteína Homeobox Nanog , Regiões Promotoras Genéticas/genética , Elementos Reguladores de Transcrição/genéticaRESUMO
Sox2 and Oct-3/4 function as master regulators during mammalian embryogenesis, where they are believed to regulate a critical gene regulatory network by cooperatively binding to DNA regulatory regions composed of adjacent HMG and POU motifs (HMG/POU cassettes). Previous studies have identified seven genes that contain highly active HMG/POU cassettes (referred to as Sox2:Oct-3/4 target genes). Importantly, nearly all known Sox2:Oct-3/4 target genes appear to be essential for embryogenesis. Recent genome-wide ChIP-chip studies identified over 300 genes that are co-occupied by Sox2 and Oct-3/4, which suggests that most Sox2:Oct-3/4 target genes remain to be identified. The work described here used a 3-step strategy for identifying additional Sox2:Oct-3/4 target genes. First, we employed in silico analysis to search for putative HMG/POU cassettes in 50 genes reported to be co-occupied by Sox2 and Oct-3/4 in embryonic stem cells. We identified 39 genes that contain putative HMG/POU cassettes. Next, we tested the activity of seven of the putative HMG/POU cassettes in a transcription-based assay and determined that nearly all are functional. Finally, as a proof-of-principle, we tested one of the seven cassettes (DPPA4) in the context of its endogenous promoter using a promoter/reporter gene construct. DPPA4 was tested in part because it was shown recently to play an important role in ES cell self-renewal. We determined that the 5' flanking region of the DPPA4 gene contains a functional HMG/POU cassette and behaves as a Sox2:Oct-3/4 target gene. Finally, we used a transcription-based assay to help develop a refined consensus sequence for HMG/POU cassettes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Proteínas HMGB/metabolismo , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Linhagem Celular , Sequência Consenso , Proteínas de Ligação a DNA/genética , Fator 4 de Crescimento de Fibroblastos/genética , Fator 4 de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Proteínas HMGB/genética , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1 , Fatores de Transcrição/genéticaRESUMO
Oct-3/4 is an essential transcription factor that regulates stem cell fate during embryogenesis. Previous reports have shown that the Oct-3/4 gene utilizes different enhancers to regulate its expression as development proceeds. However, the cis-elements contributing to the differential activity of these enhancers require further study. Here, we investigated the function of the HMG/POU cassette and LRH-1 site present in the distal enhancer (DE) and the proximal enhancer, respectively. F9 and P19 EC cells were the focus of this study because their differential utilization of Oct-3/4 enhancers parallels the use of these enhancers during different stages of development. We determined that the LRH-1 site functions as a positive and a negative cis-regulatory element in P19 and F9 EC cells, respectively. Furthermore, we determined that the HMG/POU cassette in the DE strongly activates the Oct-3/4 promoter in F9 cells, but is a much weaker positive regulatory element in P19 cells. Given that HMG/POU cassettes play key roles in the regulation of at least seven essential genes, the Oct-3/4 HMG/POU cassette was examined more closely by focusing on Sox2, which can bind to HMG/POU cassettes. Although chromatin immunoprecipitation demonstrated that Sox2 binds to the Oct-3/4 gene equally well in both EC cell lines, tethering Sox2 to the region of the HMG/POU cassette only activated the Oct-3/4 promoter in F9 EC cells. These and other findings suggest that the differential activity of the HMG/POU cassette of the Oct-3/4 gene in EC cells is due to differential action of Sox2 and its associated co-factors.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fator 3 de Transcrição de Octâmero/metabolismo , Animais , Northern Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Primers do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Componentes do Gene , Proteínas HMGB/metabolismo , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1 , Fatores de Transcrição/metabolismoRESUMO
RNA interference (RNAi) mediated by double stranded small interfering RNA (siRNA) is a novel mechanism of post-transcriptional gene silencing. It is projected as a potential tool to inhibit viral replication. In the present paper, we demonstrate the suppression of replication of an avian herpes virus (Anatid Herpes Virus-1, AHV-1) by siRNA mediated gene silencing in avian cells. The UL-6 gene of AHV-1 that codes for a protein involved in viral packaging was targeted. Both cocktail and unique siRNAs were attempted to evaluate the inhibitory potential of AHV-1 replication in duck embryo fibroblast (DEF) cell line. DEF cells were chemically transfected with different siRNAs in separate experiments followed by viral infection. The observed reduction in virus replication was evaluated by cytopathic effect, viral titration and quantitative real time PCR (QRT-PCR). Among the three siRNA targets used the unique siRNA UL-B sequence was found to be more potent in antiviral activity than the cocktail and UL6-A-siRNA sequences.