Project description:Eukaryotic genome is compartmentalized into structural and functional domains. One of the concepts of higher order organization of chromatin posits that the DNA is organized in constrained loops that behave as independent functional domains. A predominantly ribo-proteinaceous nucleoskeleton, termed as Nuclear Matrix (NuMat) is proposed to provide the structural platform for attachment of these loops. The DNA sequence located at the base of the loops are known as the Matrix Attachment Regions (MARs). NuMat relates to all nuclear processes and has been shown to be cell type specific in composition. It is a biochemically defined structure and several protocols have been used to isolate the NuMat where some of the steps have been critically evaluated. In the present study we have looked into the dynamics of MARs when the isolation process is varied and also during embryonic development of D. melanogaster. Our results show that a subset of MARs termed here as “Core-MARs” are fixed and unalterable anchor points in the Drosophila genome as they remain associated with NuMat at all developmental stages and do not depend on the isolation procedure used. Core-MARs are abundant in the pericentromeric heterochromatin. On the other hand, MARs in the euchromatin are dynamic and reflect the transcriptomic profile of the developmental stage of the host cell. New MARs are generated by nuclear stabilization (a critical step in the isolation procedure), and during development, mostly at the paused RNA polymerase II (Pol II) promoters. Paused Pol II MARs depend on RNA transcription for NuMat association. RNase A treatment leads to collapse of the NuMat and loss of paused Pol II promoter MARs. Our data reveals the role of MARs in functional compartmentalization of D. melanogaster genome and adds to the current understanding of nuclear architecture and 3D organization of a functionally dynamic nucleus.
Project description:This project’s aim was to compare the transcriptional profiles of olfactory sensory neurons in Drosophila melanogaster in order to identify novel genes that specify neuron-specific functions/phenotypes or may otherwise be involved in the development of the olfactory system. The isolation of sufficient numbers of intact olfactory sensory neurons (OSN) from the antenna of Drosophila melanogaster has so far limited single-cell transcriptomic approaches being applied to the adult fly antenna. Targeted DamID (TaDa) provides an alternative approach for profiling transcriptional activity in a cell-specific manor that bypasses the need for isolating OSN. Using the Gal4/UAS system, we applied TaDa to seven OSN populations and compared differences in Pol II occupancy for genes across these datasets.
Project description:Targeted DamID (TaDa) measures gene expression using a DNA adenine methyltransferase (Dam) fused to RNA polymerase II (Dam Pol II) to methylate DNA as the genome is transcribed (Southall, Gold et al. 2013, PMID: 23792147). Here we use traditional Illumina RNA-Seq to determine if a cell’s expression profile is affected by Dam Pol II expression. Twenty-four hours prior to collection, we ubiquitously drove UAS Dam or UAS Dam Pol II using a tubulin (tub) GAL4 regulated by a temperature sensitive tub GAL80[ts] (McGuire et al. 2003, PMID: 14657498) in adult Drosophila melanogaster (taxon: 7227) whole males, whole females, testes, and ovaries.
Project description:Argonaute2 and LaminB repress transcription by controlling RNA Pol II recruitment and chromatin topology in Drosophila melanogaster (mRNA)
Project description:ChIP-seq study analysing adult Drosophila melanogaster head, glial, neuronal and fat body, as well as embryonic RNA pol II and H2A.v binding by employing the GAL4-UAS system to generate GFP-fusion proteins and ChIP-seq
