Project description:In Drosophila, defects in asymmetric cell division can result in the formation of stem cell derived tumors. Here, we reveal a different mechanism that can result in the formation of very similar terminal brain tumor phenotypes. We demonstrate that brain tumors in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by de-repression of target genes in the Salvador-Warts-Hippo (SWH) pathway. We use ChIP-seq to identify L(3)mbt-binding sites and show that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting the insulator protein mod(mdg4) results in upregulation of SWH pathway reporters. As l(3)mbt tumors are rescued by mutations in bantam or yorkie or by overexpression of expanded the deregulation of SWH pathway target genes is an essential step in brain tumor formation. Our data reveal that very different primary defects can result in the formation of brain tumors, which behave quite similarly in their advanced stages. Examination of l(3)mbt binding in third instar larval brain and wing/haltere/third leg imaginal discs tissue.
Project description:In Drosophila, defects in asymmetric cell division can result in the formation of stem cell derived tumors. Here, we reveal a different mechanism that can result in the formation of very similar terminal brain tumor phenotypes. We demonstrate that brain tumors in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by de-repression of target genes in the Salvador-Warts-Hippo (SWH) pathway. We use ChIP-seq to identify L(3)mbt-binding sites and show that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting the insulator protein mod(mdg4) results in upregulation of SWH pathway reporters. As l(3)mbt tumors are rescued by mutations in bantam or yorkie or by overexpression of expanded the deregulation of SWH pathway target genes is an essential step in brain tumor formation. Our data reveal that very different primary defects can result in the formation of brain tumors, which behave quite similarly in their advanced stages.
Project description:Early zebrafish embryo development proceeds first from a maternally transcribed and stored mRNAs, and zygotic gene activation (ZGA) is initiated at the mid-blastula transition (MBT; 1000-cell stage), 3.3 h post-fertilization. Very little is known on how the zygotic genome is programmed for transcriptional activation at the MBT. To start addressing this issue, we have mapped by ChIP-chip genome-wide promoter histone methylation (H3K4me3, H3K9me3, H3K27me3, H3K36me3) and RNA Pol II profiles before ZGA (256-cell stage; 2.5 hpf), during ZGA (MBT; 3.5 hpf)) and after ZGA (Post-MBT; 5.3 hpf) . We used a custom 2.1M probe HD promoter array (Nimblegen) for ChIP and input DNA hybridization. Peak detection was done using MA2C with P=10e-4 as cutoff.
Project description:During animal development, a fertilized egg is initially under the control of maternal products and only starts zygotic transcription after several cell divisions. In animals such as Xenopus, zebrafish and Drosophila, a massive increase in zygotic transcription occurs during the mid-blastula transition (MBT), when cells shift from rapid, synchronous cell divisions without gap phases to prolonged asynchronous divisions. Before MBT, only a few so-called pre-MBT genes are expressed. How transcription is set up during these early stages is poorly understood. For example, paused RNA Polymerase (Pol II) is frequently found at developmental control genes in mammalian embryonic stem cells and Drosophila embryos but when Pol II pausing is first established in the embryo is unknown. We have analyzed the genome-wide Pol II occupancy during the maternal-to-zygotic transition in hand-staged Drosophila embryos. The results show that massive Pol II recruitment and pausing is established during MBT. The ~100 genes that are transcribed before MBT are particularly short, consistent with a need for rapid transcription during these early cell divisions. Remarkably, most of these genes are transcribed without Pol II pausing and this correlates with a TATA-enriched promoter type. This suggests that distinct strategies are used for activation in the early Drosophila embryo and this may reflect general dynamic properties of promoters used throughout development. Mnase-seq in staged Drosophila embryos
Project description:During animal development, a fertilized egg is initially under the control of maternal products and only starts zygotic transcription after several cell divisions. In animals such as Xenopus, zebrafish and Drosophila, a massive increase in zygotic transcription occurs during the mid-blastula transition (MBT), when cells shift from rapid, synchronous cell divisions without gap phases to prolonged asynchronous divisions. Before MBT, only a few so-called pre-MBT genes are expressed. How transcription is set up during these early stages is poorly understood. For example, paused RNA Polymerase (Pol II) is frequently found at developmental control genes in mammalian embryonic stem cells and Drosophila embryos but when Pol II pausing is first established in the embryo is unknown. We have analyzed the genome-wide Pol II occupancy during the maternal-to-zygotic transition in hand-staged Drosophila embryos. The results show that massive Pol II recruitment and pausing is established during MBT. The ~100 genes that are transcribed before MBT are particularly short, consistent with a need for rapid transcription during these early cell divisions. Remarkably, most of these genes are transcribed without Pol II pausing and this correlates with a TATA-enriched promoter type. This suggests that distinct strategies are used for activation in the early Drosophila embryo and this may reflect general dynamic properties of promoters used throughout development. ChIP-seq for Pol II, TBP, H3K4me3, H3K27me3 and H3Ac in Drosophila embryos
Project description:L(3)mbt regulate the transcription of germ-specific genes in somatic cells but the detailed mechanism remains unclear. We performed shotgun LC/MS/MS of L(3)mbt-IP samples and identified a new interactor in OSCs.