Project description:To determine the Cdk9 targets of VSV-induced genes in Drosophila cells at 4 hours post-infection Drosophila cells were treated with Bgal or Cdk9 and infected with VSV (MOI 10) for 4 hours, in two independent biological replicates. Total RNA was isolated using Trizol and microarray experiments were performed at the University of Pennsylvania Microarray Facility (U. Penn MF) following directions according to the manufacturerM-bM-^@M-^Ys protocol (Affymetrix). In brief, 100 ng RNA were amplified with the Ovation RNA Amplification System v2 (NuGen), the Encore Biotin Module (NuGen) was used for fragmentation and labeling, Protocol FS450 002 was used for hybridization, washing, and staining. Images were scanned using the GeneChipM-BM-. Scanner 3000 and image analysis was performed using the AffymetrixM-BM-. GeneChipM-BM-. Command ConsoleM-BM-. Software (AGCC).
Project description:Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. In this study, we show that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, we performed differential ChIP-seq analysis for RNA Polymerase II (Pol II) upon depletion of AGO2. Interestingly, we find specific accumulation of the elongating but not initiating form of Pol II after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (CDK9) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.
Project description:Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. In this study, we show that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, we performed differential ChIP-seq analysis for RNA Polymerase II (Pol II) upon depletion of AGO2. Interestingly, we find specific accumulation of the elongating but not initiating form of Pol II after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (CDK9) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.
Project description:Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. In this study, we show that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, we performed differential ChIP-seq analysis for RNA Polymerase II (Pol II) upon depletion of AGO2. Interestingly, we find specific accumulation of the elongating but not initiating form of Pol II after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (CDK9) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.
Project description:Increasing lines of evidence support that Argonaute2 (AGO2) harbors several nuclear functions in metazoa. In particular, Drosophila AGO2 modulates transcription of developmentally regulated genes; however, the molecular mechanisms behind AGO2 recruitment into chromatin and its function in transcription have not been deeply explored. In this study, we show that Drosophila AGO2 chromatin association depends on active transcription. In order to gain insight into how AGO2 controls transcription, we performed differential ChIP-seq analysis for RNA Polymerase II (Pol II) upon depletion of AGO2. Interestingly, we find specific accumulation of the elongating but not initiating form of Pol II after AGO2 knockdown, suggesting that AGO2 impairs transcription elongation. Finally, AGO2 also affects Negative Elongation Factor (CDK9) chromatin association but not the Cyclin Dependent Kinase 9 (CDK9). Altogether, these results provide key insights into the molecular role of AGO2 in attenuating elongation of certain actively transcribed genes.
