36 results found
Mundorf J, Donohoe CD, McClure CD, et al., 2019, Ets21c governs tissue renewal, stress tolerance, and aging in the drosophila intestine, Cell Reports, Vol: 27, Pages: 3019-3033.e5, ISSN: 2211-1247
Homeostatic renewal and stress-related tissue regeneration rely on stem cell activity, which drives the replacement of damaged cells to maintain tissue integrity and function. The Jun N-terminal kinase (JNK) signaling pathway has been established as a critical regulator of tissue homeostasis both in intestinal stem cells (ISCs) and mature enterocytes (ECs), while its chronic activation has been linked to tissue degeneration and aging. Here, we show that JNK signaling requires the stress-inducible transcription factor Ets21c to promote tissue renewal in Drosophila. We demonstrate that Ets21c controls ISC proliferation as well as EC apoptosis through distinct sets of target genes that orchestrate cellular behaviors via intrinsic and non-autonomous signaling mechanisms. While its loss appears dispensable for development and prevents epithelial aging, ISCs and ECs demand Ets21c function to mount cellular responses to oxidative stress. Ets21c thus emerges as a vital regulator of proliferative homeostasis in the midgut and a determinant of the adult healthspan.
Sharrock J, Estacio Gomez A, Jacobson J, et al., 2019, fs(1)h controls metabolic and immune function and enhances survival via AKT and FOXO in Drosophila, Disease Models & Mechanisms, Vol: 12, ISSN: 1754-8403
The Drosophila fat body is the primary organ of energy storage as well as being responsible for the humoral response to infection. Its physiological function is of critical importance to the survival of the organism; however, many molecular regulators of its function remain ill-defined. Here, we show that the Drosophila melanogaster bromodomain-containing protein FS(1)H is required in the fat body for normal lifespan as well as metabolic and immune homeostasis. Flies lacking fat body fs(1)h exhibit short lifespan, increased expression of immune target genes, an inability to metabolize triglyceride, and low basal AKT activity, mostly resulting from systemic defects in insulin signalling. Removal of a single copy of the AKT-responsive transcription factor foxo normalises lifespan, metabolic function, uninduced immune gene expression and AKT activity. We suggest that the promotion of systemic insulin signalling activity is a key in vivo function of fat body fs(1)h.
Southall T, Aughey G, Cheetham S, 2019, DamID as a versatile tool for understanding gene regulation, Development, Vol: 146, ISSN: 0950-1991
The interaction of proteins and RNA with chromatin underlies the regulation of gene expression. The ability to profile easily these interactions is fundamental for understanding chromatin biology in vivo. DNA adenine methyltransferase identification (DamID) profiles genome-wide protein-DNA interactions without antibodies, fixation or protein pull-downs. Recently, DamID has been adapted for applications beyond simple assaying of protein-DNA interactions, such as for studying RNA-chromatin interactions, chromatin accessibility and long-range chromosome interactions. Here, we provide an overview of DamID and introduce improvements to the technology, discuss their applications and compare alternative methodologies.
Sen SQ, Chanchani S, Southall TD, et al., 2019, Neuroblast-specific open chromatin allows the temporal transcription factor, Hunchback, to bind neuroblast-specific loci, ELIFE, Vol: 8, ISSN: 2050-084X
Cheetham SW, Gruhn WH, van den Ameele J, et al., 2018, Targeted DamID reveals differential binding of mammalian pluripotency factors, Development, Vol: 145, ISSN: 0950-1991
The precise control of gene expression by transcription factor networks is crucial to organismal development. The predominant approach for mapping transcription factor-chromatin interactions has been chromatin immunoprecipitation (ChIP). However, ChIP requires a large number of homogeneous cells and antisera with high specificity. A second approach, DamID, has the drawback that high levels of Dam methylase are toxic. Here, we modify our targeted DamID approach (TaDa) to enable cell type-specific expression in mammalian systems, generating an inducible system (mammalian TaDa or MaTaDa) to identify genome-wide protein/DNA interactions in 100 to 1000 times fewer cells than ChIP-based approaches. We mapped the binding sites of two key pluripotency factors, OCT4 and PRDM14, in mouse embryonic stem cells, epiblast-like cells and primordial germ cell-like cells (PGCLCs). PGCLCs are an important system for elucidating primordial germ cell development in mice. We monitored PRDM14 binding during the specification of PGCLCs, identifying direct targets of PRDM14 that are key to understanding its crucial role in PGCLC development. We show that MaTaDa is a sensitive and accurate method for assessing cell type-specific transcription factor binding in limited numbers of cells.
Southall TD, Aughey GN, Estacio Gomez A, et al., 2018, CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo, eLife, Vol: 7, ISSN: 2050-084X
During development eukaryotic gene expression is coordinated by dynamic changes in chromatin structure. Measurements of accessible chromatin are used extensively to identify genomic regulatory elements. Whilst chromatin landscapes of pluripotent stem cells are well characterised, chromatin accessibility changes in the development of somatic lineages are not well defined. Here we show that cell-specific chromatin accessibility data can be produced via ectopic expression of E. coli Dam methylase in vivo, without the requirement for cell-sorting (CATaDa). We have profiled chromatin accessibility in individual cell-types of Drosophila neural and midgut lineages. Functional cell-type specific enhancers were identified, as well as novel motifs enriched at different stages of development. Finally, we show global changes in the accessibility of chromatin between stem-cells and their differentiated progeny. Our results demonstrate the dynamic nature of chromatin accessibility in somatic tissues during stem cell differentiation and provide a novel approach to understanding gene regulatory mechanisms underlying development.
Dinges N, Morin V, Kreim N, et al., 2017, Comprehensive Characterization of the Complex lola Locus Reveals a Novel Role in the Octopaminergic Pathway via Tyramine Beta-Hydroxylase Regulation, Cell Reports, Vol: 21, Pages: 2911-2925, ISSN: 2211-1247
Longitudinals lacking (lola) is one of the most complex genes in Drosophila melanogaster, encoding up to 20 protein isoforms that include key transcription factors involved in axonal pathfinding and neural reprogramming. Most previous studies have employed loss-of-function alleles that disrupt lola common exons, making it difficult to delineate isoform-specific functions. To overcome this issue, we have generated isoform-specific mutants for all isoforms using CRISPR/Cas9. This enabled us to study specific isoforms with respect to previously characterized roles for Lola and to demonstrate a specific function for one variant in axon guidance via activation of the microtubule-associated factor Futsch. Importantly, we also reveal a role for a second variant in preventing neurodegeneration via the positive regulation of a key enzyme of the octopaminergic pathway. Thus, our comprehensive study expands the functional repertoire of Lola functions, and it adds insights into the regulatory control of neurotransmitter expression in vivo.
Aughey GN, Estacio Gomez A, Thomson J, et al., 2017, CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo, Publisher: Cold Spring Harbor Laboratory
<jats:title>Abstract</jats:title><jats:p>Regulation of eukaryotic gene expression is coordinated by dynamic changes to chromatin states throughout development. Measurements of accessible chromatin are used extensively to identify genomic regulatory elements. Whilst the chromatin landscapes of pluripotent stem cells are well characterised, chromatin accessibility changes in the development of somatic stem cell lineages are not well defined. Here we show that tissue specific chromatin accessibility data can be produced via ectopic expression of <jats:italic>E. coli</jats:italic> Dam methylase <jats:italic>in vivo</jats:italic>, without the requirement for cell-sorting. We have profiled chromatin accessibility in individual cell types of the <jats:italic>Drosophila</jats:italic> neural and midgut stem cell lineages. Functional cell-type specific enhancers were identified, as well as novel motifs enriched at diferent stages of development. Finally, we show global changes in the accessibility of chromatin between stem-cells and their diferentiated progeny. Our results demonstrate the dynamic nature of chromatin accessibility in somatic tissues during stem cell diferentiation and provide a novel approach to understanding the gene regulatory mechanisms underlying development.</jats:p>
Southall TD, Marshall OJ, Brand AH, 2016, Cell type-specific profiling of protein-DNA interactions without cellisolation using Targeted DamID with next-generation sequencing, Nature Protocols, Vol: 11, Pages: 1586-1598, ISSN: 1750-2799
The ability to profile transcription and chromatin binding in a cell type-specific manner is a powerfulapproach for understanding cell fate specification and cellular function in multicellular organisms.We recently developed Targeted DamID (TaDa) to enable genome-wide, cell-type-specific profiling ofDNA- and chromatin-binding proteins in vivo without cell isolation. As a Protocol Extension, thisarticle describes substantial modifications to an existing Protocol and offers additional applications.TaDa builds upon DamID, a technique for detecting genome-wide DNA binding profiles of proteins,by coupling it with the GAL4 system in Drosophila to enable both temporal and spatial resolution.TaDa ensures that Dam-fusion proteins are expressed at very low levels, avoiding toxicity andpotential artefacts from over-expression. The modifications to the core DamID technique presentedhere also increase the speed of sample processing and throughput, and adapt the method to NextgenerationSequencing technology. TaDa is robust, reproducible, and highly sensitive. Compared toother methods for cell-type specific profiling, the technique requires no cell-sorting, crosslinking orantisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profilingthe genome-wide binding of RNA polymerase II, TaDa can also identify transcribed genes in a celltype-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments andpreparing the material for next generation sequencing. Although we developed TaDa in Drosophila, itshould be easily adapted to other organisms with an inducible expression system. Once transgenicanimals are obtained, the entire experimental procedure – from collecting tissue samples togenerating sequencing libraries – can be accomplished within 5 days.
Cattenoz PB, Popkova A, Southall TD, et al., 2016, Functional conservation of the Glide/Gcm regulatory network controlling glia, hemocyte, and tendon cell differentiation in drosophila, Genetics, Vol: 202, Pages: 191-219, ISSN: 1943-2631
High-throughput screens allow us to understand how transcription factors trigger developmental processes, including cell specification. A major challenge is identification of their binding sites because feedback loops and homeostatic interactions may mask the direct impact of those factors in transcriptome analyses. Moreover, this approach dissects the downstream signaling cascades and facilitates identification of conserved transcriptional programs. Here we show the results and the validation of a DNA adenine methyltransferase identification (DamID) genome-wide screen that identifies the direct targets of Glide/Gcm, a potent transcription factor that controls glia, hemocyte, and tendon cell differentiation in Drosophila. The screen identifies many genes that had not been previously associated with Glide/Gcm and highlights three major signaling pathways interacting with Glide/Gcm: Notch, Hedgehog, and JAK/STAT, which all involve feedback loops. Furthermore, the screen identifies effector molecules that are necessary for cell-cell interactions during late developmental processes and/or in ontogeny. Typically, immunoglobulin (Ig) domain–containing proteins control cell adhesion and axonal navigation. This shows that early and transiently expressed fate determinants not only control other transcription factors that, in turn, implement a specific developmental program but also directly affect late developmental events and cell function. Finally, while the mammalian genome contains two orthologous Gcm genes, their function has been demonstrated in vertebrate-specific tissues, placenta, and parathyroid glands, begging questions on the evolutionary conservation of the Gcm cascade in higher organisms. Here we provide the first evidence for the conservation of Gcm direct targets in humans. In sum, this work uncovers novel aspects of cell specification and sets the basis for further understanding of the role of conserved Gcm gene regulatory cascades.
Southall TD, Aughey GN, 2015, Dam it's good! DamID profiling of protein-DNA interactions, Wiley Interdisciplinary Reviews: Developmental Biology, Vol: 5, Pages: 25-37, ISSN: 1759-7692
The interaction of proteins with chromatin is fundamental for several essential cellular processes. During the development of an organism, genes must to be tightly regulated both temporally and spatially. This is achieved through the action of chromatin-binding proteins such as transcription factors, histone modifiers, nucleosome remodelers, and lamins. Furthermore, protein–DNA interactions are important in the adult, where their perturbation can lead to disruption of homeostasis, metabolic dysregulation, and diseases such as cancer. Understanding the nature of these interactions is of paramount importance in almost all areas of molecular biological research. In recent years, DNA adenine methyltransferase identification (DamID) has emerged as one of the most comprehensive and versatile methods available for profiling protein–DNA interactions on a genomic scale. DamID has been used to map a variety of chromatin-binding proteins in several model organisms and has the potential for continued adaptation and application in the field of genomic biology
McClure CD, Southall TD, 2015, Getting Down to Specifics: Profiling Gene Expression and Protein-DNA Interactions in a Cell Type-Specific Manner., Advances in Genetics, Vol: 91, Pages: 103-151, ISSN: 0065-2660
The majority of multicellular organisms are comprised of an extraordinary range of cell types, with different properties and gene expression profiles. Understanding what makes each cell type unique and how their individual characteristics are attributed are key questions for both developmental and neurobiologists alike. The brain is an excellent example of the cellular diversity expressed in the majority of eukaryotes. The mouse brain comprises of approximately 75million neurons varying in morphology, electrophysiology, and preferences for synaptic partners. A powerful process in beginning to pick apart the mechanisms that specify individual characteristics of the cell, as well as their fate, is to profile gene expression patterns, chromatin states, and transcriptional networks in a cell type-specific manner, i.e., only profiling the cells of interest in a particular tissue. Depending on the organism, the questions being investigated, and the material available, certain cell type-specific profiling methods are more suitable than others. This chapter reviews the approaches presently available for selecting and isolating specific cell types and evaluates their key features.
Loza-Coll MA, Southall TD, Sandall SL, et al., 2014, Regulation of Drosophila intestinal stem cell maintenance and differentiation by the transcription factor Escargot, EMBO JOURNAL, Vol: 33, Pages: 2983-2996, ISSN: 0261-4189
Korzelius J, Naumann SK, Loza-Coll MA, et al., 2014, Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells, EMBO JOURNAL, Vol: 33, Pages: 2967-2982, ISSN: 0261-4189
Southall TD, Davidson CM, Miller C, et al., 2014, Dedifferentiation of Neurons Precedes Tumor Formation in lola Mutants, DEVELOPMENTAL CELL, Vol: 28, Pages: 685-696, ISSN: 1534-5807
Wolfram V, Southall TD, Guenay C, et al., 2014, The Transcription Factors Islet and Lim3 Combinatorially Regulate Ion Channel Gene Expression, JOURNAL OF NEUROSCIENCE, Vol: 34, Pages: 2538-2543, ISSN: 0270-6474
Neville MC, Nojima T, Ashley E, et al., 2014, Male-Specific Fruitless Isoforms Target Neurodevelopmental Genes to Specify a Sexually Dimorphic Nervous System, Current Biology, Vol: 24, Pages: 229-241, ISSN: 0960-9822
Southall TD, Gold KS, Egger B, et al., 2013, Cell-Type-Specific Profiling of Gene Expression and Chromatin Binding without Cell Isolation: Assaying RNA Pol II Occupancy in Neural Stem Cells, DEVELOPMENTAL CELL, Vol: 26, Pages: 101-112, ISSN: 1534-5807
Wolfram V, Southall TD, Brand AH, et al., 2012, The homeodomain transcription factors islet and lim3 synergize to regulate sh expression in motoneurons, JOURNAL OF NEUROGENETICS, Vol: 26, Pages: 36-36, ISSN: 0167-7063
Murray MJ, Southall TD, Liu W, et al., 2012, Snail-dependent repression of the RhoGEF pebble is required for gastrulation consistency in Drosophila melanogaster, DEVELOPMENT GENES AND EVOLUTION, Vol: 222, Pages: 361-368, ISSN: 0949-944X
Wolfram V, Southall TD, Brand AH, et al., 2012, The LIM-Homeodomain Protein Islet Dictates Motor Neuron Electrical Properties by Regulating K+ Channel Expression, NEURON, Vol: 75, Pages: 663-674, ISSN: 0896-6273
Bardin AJ, Perdigoto CN, Southall TD, et al., 2010, Transcriptional control of stem cell maintenance in the Drosophila intestine, DEVELOPMENT, Vol: 137, Pages: 705-714, ISSN: 0950-1991
Southall TD, Brand AH, 2009, Neural stem cell transcriptional networks highlight genes essential for nervous system development, EMBO JOURNAL, Vol: 28, Pages: 3799-3807, ISSN: 0261-4189
Southall TD, Elliott DA, Brand AH, 2008, The GAL4 System: A Versatile Toolkit for Gene Expression in Drosophila., CSH Protoc, Vol: 2008
INTRODUCTIONThe generation of gain-of-function phenotypes by ectopic expression of known genes provides a powerful complement to the genetic approach, in which genes are studied or identified through mutations that generally reduce or eliminate gene function. The GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. A key advantage of the system is the separation of the GAL4 protein from its target gene in distinct transgenic lines, which ensures that the target gene is silent until the introduction of GAL4. Recent modifications and adaptations of the GAL4 system to make the system inducible have further expanded its scope, enabling greater temporal control over the activity of GAL4. There are now large resources for the community, including thousands of GAL4 lines and a wide selection of reporter lines. Here we present an overview of the GAL4 system, highlighting recent developments and discussing methods for generating and analyzing transgenic flies for GAL4-mediated ectopic expression.
Southall TD, Brand AH, 2008, Generation of Driver and Reporter Constructs for the GAL4 Expression System in Drosophila., CSH Protoc, Vol: 2008
INTRODUCTIONThe GAL4 system is a method for ectopic gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. This protocol describes the generation of driver and reporter lines for use with the GAL4 system in Drosophila. A promoter-GAL4 fusion is constructed using a P-element transformable vector, and a GAL4-responsive target gene is created via generation of an upstream activation sequence (UAS)-reporter construct. An alternative strategy for integration using the phiC31 system is also provided. Transformant lines are generated using standard procedures for microinjection.
Southall TD, Egger B, Gold KS, et al., 2008, Regulation of Self-renewal and Differentiation in the Drosophila Nervous System, 73rd Cold Spring Harbor Symposium on Quantitative Biology, Publisher: COLD SPRING HARBOR LABORATORY PRESS, Pages: 523-+, ISSN: 0091-7451
Southall TD, Brand AH, 2007, Chromatin profiling in model organisms., Brief Funct Genomic Proteomic, Vol: 6, Pages: 133-140, ISSN: 1473-9550
The correct control of gene expression is essential for the proper development of organisms. Abnormal expression of genes can lead to cancerous growth and certain diseases. To understand how gene expression is controlled on a genome-wide scale, methods for assaying transcription factor binding sites are required. There are two prevailing techniques for mapping protein-chromatin interactions, ChIP (chromatin immunoprecipitation) and DamID (DNA adenine methyltransferase identification). Both of these methods, when combined with microarray technology, can provide powerful insights into transcription factor function, higher order chromatin structure and gene regulatory networks. In vivo chromatin profiling studies are now being performed on model organisms, targeting specific tissues to help generate more accurate maps of protein-DNA interactions.
Choksi SP, Southall TD, Bossing T, et al., 2006, Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells, DEVELOPMENTAL CELL, Vol: 11, Pages: 775-789, ISSN: 1534-5807
Pym ECG, Southall TD, Mee CJ, et al., 2006, The homeobox transcription factor Even-skipped regulates acquisition of electrical properties in Drosophila neurons, NEURAL DEVELOPMENT, Vol: 1, ISSN: 1749-8104
Terhzaz S, Southall TD, Lilley KS, et al., 2006, Differential gel electrophoresis and transgenic mitochondrial calcium reporters demonstrate spatiotemporal filtering in calcium control of mitochondria, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 281, Pages: 18849-18858
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