Project description:Chromatin remodeling complexes control the availability of DNA binding sites to transcriptional regulators. Two distinct forms of the major SWI/SNF-related complex that have different activities in vitro can be distinguished by the presence of specific accessory subunits. In Drosophila, the core Brahma complex associates either with Osa to form the BAP complex, or with Bap170 and Bap180 to form the PBAP complex. Mutations affecting the core subunits have stronger developmental phenotypes than osa mutations; these differences could be due to PBAP complex activity . We have generated mutations in the genes encoding the PBAP-specific subunits Bap170 and Bap180 in order to study their functions in vivo. Bap180 is not essential for viability, but is required in ovarian follicle cells for normal eggshell development. Bap170 is necessary to stabilize the Bap180 protein; however, a mutant form that retains this function is sufficient for survival and fertility. The two subunits act redundantly to allow metamorphosis; using gene expression profiling of double mutants, we have found that the PBAP complex regulates genes involved in tissue remodeling and immune system function. Finally, we have generated mutants that lack Bap170, Bap180 and Osa in the germline to demonstrate that the function of the core Brahma complex in oogenesis does not require any of these accessory subunits. To study PBAP specific subunits Bap170 and Bap180 role in Drosophila melanogaster development Experiment Overall Design: Two males and two females for each genotype (wild type and bap170;bap180 double mutant) were processed at white prepupae stage (0 hour APF)
Project description:Chromatin remodeling complexes control the availability of DNA binding sites to transcriptional regulators. Two distinct forms of the major SWI/SNF-related complex that have different activities in vitro can be distinguished by the presence of specific accessory subunits. In Drosophila, the core Brahma complex associates either with Osa to form the BAP complex, or with Bap170 and Bap180 to form the PBAP complex. Mutations affecting the core subunits have stronger developmental phenotypes than osa mutations; these differences could be due to PBAP complex activity . We have generated mutations in the genes encoding the PBAP-specific subunits Bap170 and Bap180 in order to study their functions in vivo. Bap180 is not essential for viability, but is required in ovarian follicle cells for normal eggshell development. Bap170 is necessary to stabilize the Bap180 protein; however, a mutant form that retains this function is sufficient for survival and fertility. The two subunits act redundantly to allow metamorphosis; using gene expression profiling of double mutants, we have found that the PBAP complex regulates genes involved in tissue remodeling and immune system function. Finally, we have generated mutants that lack Bap170, Bap180 and Osa in the germline to demonstrate that the function of the core Brahma complex in oogenesis does not require any of these accessory subunits. To study PBAP specific subunits Bap170 and Bap180 role in Drosophila melanogaster development Keywords: genetic modification, chromatin remodelling, gene regulation, microarray
Project description:The Drosophila pioneer factor GAF is known to be essential for RNA Pol II promoter-proximal pausing and the removal of nucleosomes from a set of target promoters with GAGAG motifs. We and others have speculated that GAF recruits the ISWI family ATP-dependent chromatin remodeling complex NURF, on the basis that NURF and GAF are both required to remodel nucleosomes on an hsp70 promoter in vitro and that GAF interacts physically with NURF. However, GAF was also recently shown to interact with PBAP, a SWI/SNF family remodeler. To test which of these remodeling complexes GAF works with, we depleted GAF, NURF301, BAP170, and NURF301+BAP170 in Drosophila S2 cells using RNAi. We used a combination of PRO-seq, ATAC-seq, 3'RNA-seq, and CUT&RUN to demonstrate that while GAF and PBAP synergistically open chromatin at target promtoers which allows Pol II recruitment and pausing to proceed, GAF and NURF also synergistically position the +1 nucleosome to ensure efficient pause release and transition to productive elongation.
Project description:The Drosophila pioneer factor GAF is known to be essential for RNA Pol II promoter-proximal pausing and the removal of nucleosomes from a set of target promoters with GAGAG motifs. We and others have speculated that GAF recruits the ISWI family ATP-dependent chromatin remodeling complex NURF, on the basis that NURF and GAF are both required to remodel nucleosomes on an hsp70 promoter in vitro and that GAF interacts physically with NURF. However, GAF was also recently shown to interact with PBAP, a SWI/SNF family remodeler. To test which of these remodeling complexes GAF works with, we depleted GAF, NURF301, BAP170, and NURF301+BAP170 in Drosophila S2 cells using RNAi. We used a combination of PRO-seq, ATAC-seq, 3'RNA-seq, and CUT&RUN to demonstrate that while GAF and PBAP synergistically open chromatin at target promoters which allows Pol II recruitment and pausing to proceed, GAF and NURF also synergistically position the +1 nucleosome to ensure efficient pause release and transition to productive elongation.
Project description:The Drosophila pioneer factor GAF is known to be essential for RNA Pol II promoter-proximal pausing and the removal of nucleosomes from a set of target promoters with GAGAG motifs. We and others have speculated that GAF recruits the ISWI family ATP-dependent chromatin remodeling complex NURF, on the basis that NURF and GAF are both required to remodel nucleosomes on an hsp70 promoter in vitro and that GAF interacts physically with NURF. However, GAF was also recently shown to interact with PBAP, a SWI/SNF family remodeler. To test which of these remodeling complexes GAF works with, we depleted GAF, NURF301, BAP170, and NURF301+BAP170 in Drosophila S2 cells using RNAi. We used a combination of PRO-seq, ATAC-seq, 3'RNA-seq, and CUT&RUN to demonstrate that while GAF and PBAP synergistically open chromatin at target promoters which allows Pol II recruitment and pausing to proceed, GAF and NURF also synergistically position the +1 nucleosome to ensure efficient pause release and transition to productive elongation.
Project description:The Drosophila pioneer factor GAF is known to be essential for RNA Pol II promoter-proximal pausing and the removal of nucleosomes from a set of target promoters with GAGAG motifs. We and others have speculated that GAF recruits the ISWI family ATP-dependent chromatin remodeling complex NURF, on the basis that NURF and GAF are both required to remodel nucleosomes on an hsp70 promoter in vitro and that GAF interacts physically with NURF. However, GAF was also recently shown to interact with PBAP, a SWI/SNF family remodeler. To test which of these remodeling complexes GAF works with, we depleted GAF, NURF301, BAP170, and NURF301+BAP170 in Drosophila S2 cells using RNAi. We used a combination of PRO-seq, ATAC-seq, 3'RNA-seq, and CUT&RUN to demonstrate that while GAF and PBAP synergistically open chromatin at target promoters which allows Pol II recruitment and pausing to proceed, GAF and NURF also synergistically position the +1 nucleosome to ensure efficient pause release and transition to productive elongation.
Project description:The Drosophila pioneer factor GAF is known to be essential for RNA Pol II promoter-proximal pausing and the removal of nucleosomes from a set of target promoters with GAGAG motifs. We and others have speculated that GAF recruits the ISWI family ATP-dependent chromatin remodeling complex NURF, on the basis that NURF and GAF are both required to remodel nucleosomes on an hsp70 promoter in vitro and that GAF interacts physically with NURF. However, GAF was also recently shown to interact with PBAP, a SWI/SNF family remodeler. To test which of these remodeling complexes GAF works with, we depleted GAF, NURF301, BAP170, and NURF301+BAP170 in Drosophila S2 cells using RNAi. We used a combination of PRO-seq, ATAC-seq, 3'RNA-seq, and CUT&RUN to demonstrate that while GAF and PBAP synergistically open chromatin at target promoters which allows Pol II recruitment and pausing to proceed, GAF and NURF also synergistically position the +1 nucleosome to ensure efficient pause release and transition to productive elongation.
Project description:Drosophila PBAP complex, a form of SWI/SNF class of complexes, played a important role in metamorphosis. We conducted next-generation sequencing (NGS) to analyse the expression profile in both control and Brm knockdown fly larvae.
Project description:Drosophila PBAP complex, a form of SWI/SNF class of complexes, played a important role in metamorphosis. We conducted MNase digestion followed by next-generation sequencing (NGS) to analyse the nucleosome profile in both control and Brm knockdown fly larvae.
Project description:The Spt-Ada-Gcn5-acetyltransferase (SAGA) chromatin-modifying complex is a transcriptional coactivator that contains four different modules of subunits. The intact SAGA complex has been well characterized for its function in transcription regulation and development. However, little is known about the roles of individual modules within SAGA and if they have any SAGA independent functions. Here we demonstrate that the two enzymatic modules of Drosophila SAGA are differently required in oogenesis. Loss of the HAT activity blocks oogenesis, while loss of the DUB activity does not. However, the DUB module regulates a subset of genes in early embryogenesis and loss of the DUB subunits causes defects in embryogenesis. ChIP-seq analysis of ada2b, spt3, nonstop, and sgf11revealed that both the DUB and HAT modules bind most SAGA target genes even though many of these targets do not require the DUB module for expression. Furthermore, we found that the DUB module can bind to chromatin and regulate transcription independently of the rest of SAGA. Our results suggest that the DUB module has functions within SAGA as well as independent functions.