51 results found
Aughey G, Grimes K, Forsberg E, et al., 2023, NuRD independent Mi-2 activity represses ectopic gene expression during neuronal maturation, EMBO Reports, Vol: 24, Pages: 1-15, ISSN: 1469-221X
During neuronal development, extensive changes to chromatin states occur to regulate lineage-specific gene expression. The molecular factors underlying the repression of non-neuronal genes in differentiated neurons are poorly characterised. The Mi2/NuRD complex is a multiprotein complex with nucleosome remodelling and histone deacetylase activity. Whilst NuRD has previously been implicated in the development of nervous system tissues, the precise nature of the gene expression programmes that it coordinates is ill-defined. Furthermore, evidence from several species suggests that Mi-2 may be incorporated into multiple complexes that may not possess histone deacetylase activity. We show that Mi-2 activity is required for suppressing ectopic expression of germline genes in neurons independently of HDAC1/NuRD, whilst components of NuRD, including Mi-2, regulate neural gene expression to ensure proper development of the larval nervous system. We find that Mi-2 binding in the genome is dynamic during neuronal maturation, and Mi-2-mediated repression of ectopic gene expression is restricted to the early stages of neuronal development, indicating that Mi-2/NuRD is required for establishing stable neuronal transcriptomes during the early stages of neuronal differentiation.
Martinez Corrales G, Li M, Svermova T, et al., 2022, Transcriptional memory of dFOXO activation in youth curtails later-life mortality through chromatin remodeling and Xbp1, Nature Aging, Vol: 2, Pages: 1176-1190, ISSN: 2662-8465
A transient, homeostatic transcriptional response can result in transcriptional memory, programming subsequent transcriptional outputs. Transcriptional memory has great but unappreciated potential to alter animal aging as animals encounter a multitude of diverse stimuli throughout their lifespan. Here we show that activating an evolutionarily conserved, longevity-promoting transcription factor, dFOXO, solely in early adulthood of female fruit flies is sufficient to improve their subsequent health and survival in midlife and late life. This youth-restricted dFOXO activation causes persistent changes to chromatin landscape in the fat body and requires chromatin remodelers such as the SWI/SNF and ISWI complexes to program health and longevity. Chromatin remodeling is accompanied by a long-lasting transcriptional program that is distinct from that observed during acute dFOXO activation and includes induction of Xbp1. We show that this later-life induction of Xbp1 is sufficient to curtail later-life mortality. Our study demonstrates that transcriptional memory can profoundly alter how animals age.
Grimes K, Beckwith EJ, Pearson WH, et al., 2022, A serine-folate metabolic unit controls resistance and tolerance of infection
<jats:title>Abstract</jats:title><jats:p>Immune activation drives metabolic change in most animals. Immune-induced metabolic change is most conspicuous as a driver of pathology in serious or prolonged infection, but it is normally expected to be important to support immune function and recovery. Many of the signalling mechanisms linking immune detection with metabolic regulation, and their specific consequences, are unknown. Here, we show that<jats:italic>Drosophila melanogaster</jats:italic>respond to many bacterial infections by altering expression of genes of the folate cycle and associated enzymes of amino acid metabolism. The net result of these changes is increased flow of carbon from glycolysis into serine and glycine synthesis and a shift of folate cycle activity from the cytosol into the mitochondrion. Immune-induced transcriptional induction of<jats:italic>astray</jats:italic>and<jats:italic>Nmdmc</jats:italic>, the two most-induced of these enzymes, depends on<jats:italic>Dif</jats:italic>and<jats:italic>foxo</jats:italic>. Loss of<jats:italic>astray</jats:italic>or<jats:italic>Nmdmc</jats:italic>results in infection-specific immune defects. Our work thus shows a key mechanism that connects immune-induced changes in metabolic signalling with the serine-folate metabolic unit to result in changed immune function.</jats:p>
Demarco RS, Stack BJ, Tang AM, et al., 2022, Escargot controls somatic stem cell maintenance through the attenuation of the insulin receptor pathway in Drosophila, Cell Reports, Vol: 39, Pages: 1-20, ISSN: 2211-1247
Adult stem cells coordinate intrinsic and extrinsic, local and systemic, cues to maintain the proper balance between self-renewal and differentiation. However, the precise mechanisms stem cells use to integrate these signals remain elusive. Here, we show that Escargot (Esg), a member of the Snail family of transcription factors, regulates the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis by attenuating the activity of the pro-differentiation insulin receptor (InR) pathway. Esg positively regulates the expression of an antagonist of insulin signaling, ImpL2, while also attenuating the expression of InR. Furthermore, Esg-mediated repression of the InR pathway is required to suppress CySC loss in response to starvation. Given the conservation of Snail-family transcription factors, characterizing the mechanisms by which Esg regulates cell-fate decisions during homeostasis and a decline in nutrient availability is likely to provide insight into the metabolic regulation of stem cell behavior in other tissues and organisms.
McClure C, Aughey G, Hassan A, et al., 2022, An auxin-inducible, GAL4-compatible, gene expression system for Drosophila, eLife, Vol: 11, Pages: 1-18, ISSN: 2050-084X
The ability to control transgene expression, both spatially and temporally, is essential for studying model organisms. In Drosophila, spatial control is primarily provided by the GAL4/UAS system, whilst temporal control relies on a temperature-sensitive GAL80 (which inhibits GAL4) and drug-inducible systems. However, these are not ideal. Shifting temperature can impact on many physiological and behavioural traits, and the current drug-inducible systems are either leaky, toxic, incompatible with existing GAL4-driver lines, or do not generate effective levels of expression. Here, we describe the auxin-inducible gene expression system (AGES). AGES relies on the auxin-dependent degradation of a ubiquitously expressed GAL80, and therefore, is compatible with existing GAL4-driver lines. Water-soluble auxin is added to fly food at a low, non-lethal, concentration, which induces expression comparable to uninhibited GAL4 expression. The system works in both larvae and adults, providing a stringent, non-lethal, cost-effective, and convenient method for temporally controlling GAL4 activity in Drosophila.
Katsanos D, Ferrando-Marco M, Razzaq I, et al., 2021, Gene expression profiling of epidermal cell types in C. elegans using Targeted-DamID., Development, Vol: 148, Pages: 1-16
The epidermis of Caenorhabditis elegans is an essential tissue for survival as it contributes to the formation of the cuticle barrier, as well as facilitates developmental progression and animal growth. Most of the epidermis consists of the hyp7 hypodermal syncytium, the nuclei of which are largely generated by the seam cells that exhibit stem cell-like behaviour during development. How the seam cell progenitors differ transcriptionally from the differentiated hypodermis is poorly understood. Here, we introduce Targeted DamID (TaDa) in C. elegans as a method for identifying genes expressed within a tissue of interest without cell isolation. We show that TaDa signal enrichment profiles can be used to identify genes transcribed in the epidermis and use this method to resolve differences in gene expression between the seam cells and the hypodermis. We finally predict and functionally validate new transcription and chromatin factors acting in seam cell development. These findings provide insights into cell-type-specific gene expression profiles likely associated with epidermal cell fate patterning.
Perochon J, Yu Y, Aughey GN, et al., 2021, Dynamic adult tracheal plasticity drives stem cell adaptation to changes in intestinal homeostasis in Drosophila, NATURE CELL BIOLOGY, Vol: 23, Pages: 485-496, ISSN: 1465-7392
Coordination of stem cell function by local and niche-derived signals is essential to preserve adult tissue homeostasis and organismal health. The vasculature is a prominent component of multiple stem cell niches. However, its role in adult intestinal homeostasis remains largely understudied. Here we uncover a previously unrecognised crosstalk between adult intestinal stem cells in Drosophila and the vasculature-like tracheal system, which is essential for intestinal regeneration. Following damage to the intestinal epithelium, gut-derived reactive oxygen species activate tracheal HIF-1α and bidirectional FGF/FGFR signalling, leading to reversible remodelling of gut-associated terminal tracheal cells and intestinal stem cell proliferation following damage. Unexpectedly, reactive oxygen species-induced adult tracheal plasticity involves downregulation of the tracheal specification factor trachealess (trh) and upregulation of IGF2 messenger RNA-binding protein (IGF2BP2/Imp). Our results reveal an intestine–vasculature inter-organ communication programme that is essential to adapt the stem cell response to the proliferative demands of the intestinal epithelium.
Southall T, Marshall OJ, Aughey GN, et al., 2021, FlyORF-TaDa allows rapid generation of new lines for in vivo cell-type specific profiling of protein-DNA interactions in Drosophila melanogaster, G3: Genes, Genomes, Genetics, Vol: 11, Pages: 1-6, ISSN: 2160-1836
Targeted DamID (TaDa) is an increasingly popular method of generating cell-type specific DNA binding profiles in vivo. Although sensitive and versatile, TaDa requires the generation of new transgenic fly lines for every protein that is profiled, which is both time-consuming and costly. Here, we describe the FlyORF-TaDa system for converting an existing FlyORF library of inducible open reading frames (ORFs) to TaDa lines via a genetic cross, with recombinant progeny easily identifiable by eye color. Profiling the binding of the H3K36me3-associated chromatin protein MRG15 in larval neural stem cells using both FlyORF-TaDa and conventional TaDa demonstrates that new lines generated using this system provide accurate and highly-reproducible DamID binding profiles. Our data further show that MRG15 binds to a subset of active chromatin domains in vivo. Courtesy of the large coverage of the FlyORF library, the FlyORF-TaDa system enables the easy creation of TaDa lines for 74% of all transcription factors and chromatin modifying proteins within the Drosophila 26 genome.
Jefferies G, Somers J, Lohrey I, et al., 2020, Maintenance of cell fate by the polycomb group gene sex combs extra enables a partial epithelial mesenchymal transition in drosophila., G3: Genes, Genomes, Genetics, Vol: 10, Pages: 1-18, ISSN: 2160-1836
Epigenetic silencing by Polycomb group (PcG) complexes can promote epithelial-mesenchymal transition (EMT) and stemness and is associated with malignancy of solid cancers. Here we report a role for Drosophila PcG repression in a partial EMT event that occurs during wing disc eversion, an early event during metamorphosis. In a screen for genes required for eversion we identified the PcG genes Sex combs extra (Sce) and Sex combs midleg (Scm) Depletion of Sce or Scm resulted in internalised wings and thoracic clefts, and loss of Sce inhibited the EMT of the peripodial epithelium and basement membrane breakdown, ex vivo. Targeted DamID (TaDa) using Dam-Pol II showed that Sce knockdown caused a genomic transcriptional response consistent with a shift towards a more stable epithelial fate. Surprisingly only 17 genes were significantly upregulated in Sce-depleted cells, including Abd-B, abd-A, caudal, and nubbin Each of these loci were enriched for Dam-Pc binding. Of the four genes, only Abd-B was robustly upregulated in cells lacking Sce expression. RNAi knockdown of all four genes could partly suppress the Sce RNAi eversion phenotype, though Abd-B had the strongest effect. Our results suggest that in the absence of continued PcG repression peripodial cells express genes such as Abd-B, which promote epithelial state and thereby disrupt eversion. Our results emphasise the important role that PcG suppression can play in maintaining cell states required for morphogenetic events throughout development and suggest that PcG repression of Hox genes may affect epithelial traits that could contribute to metastasis.
Aughey GN, Delandre C, Southall TD, et al., 2020, FlyORF-TaDa allows rapid generation of new lines for in vivo cell-type specific profiling of protein-DNA interactions in Drosophila melanogaster, Publisher: bioRxiv
Targeted DamID (TaDa) is an increasingly popular method of generating cell-type specific DNA binding profiles in vivo. Although sensitive and versatile, TaDa requires the generation of new transgenic fly lines for every protein that is profiled, which is both time-consuming and costly. Here, we describe the FlyORF-TaDa system for converting an existing FlyORF library of inducible open reading frames (ORFs) to TaDa lines via a genetic cross, with recombinant progeny easily identifiable by eye colour. Profiling the binding of the H3K36me3-associated chromatin protein MRG15 in larval neural stem cells using both FlyORF-TaDa and conventional TaDa demonstrates that new lines generated using this system provide accurate and highly-reproducible DamID binding profiles. Our data further show that MRG15 binds to a subset of active chromatin domains in vivo. Courtesy of the large coverage of the FlyORF library, the FlyORF-TaDa system enables the easy creation of TaDa lines for 74% of all transcription factors and chromatin modifying proteins within the Drosophila genome.
Southall T, Estacio Gomez A, Hassan A, et al., 2020, Dynamic neurotransmitter specific transcription factor expression profiles during Drosophila development, Biology Open, Vol: 9, Pages: 1-14, ISSN: 2046-6390
The remarkable diversity of neurons in the nervous system is generated during development, when properties such as cell morphology, receptor profiles and neurotransmitter identities are specified. In order to gain a greater understanding of neurotransmitter specification we profiled the transcription state of cholinergic, GABAergic and glutamatergic neurons in vivo at three developmental time points. We identified 86 differentially expressed transcription factors that are uniquely enriched, or uniquely depleted, in a specific neurotransmitter type. Some transcription factors show a similar profile across development, others only show enrichment or depletion at specific developmental stages. Profiling of Acj6 (cholinergic enriched) and Ets65A (cholinergic depleted) binding sites in vivo reveals that they both directly bind the ChAT locus, in addition to a wide spectrum of other key neuronal differentiation genes. We also show that cholinergic enriched transcription factors are expressed in mostly non-overlapping populations in the adult brain, implying the absence of combinatorial regulation of neurotransmitter fate in this context. Furthermore, our data underlines that, similar to C. elegans, there are no simple transcription factor codes for neurotransmitter type specification.
Hassan A, Walmsley E, Araguas Rodriguez P, et al., 2020, Condensin I subunit Cap-G is essential for proper gene expression during the maturation of post-mitotic neurons, eLife, Vol: 9, ISSN: 2050-084X
Condensin complexes are essential for mitotic chromosome assembly and segregation during cell divisions, however, little is known about their functions in post-mitotic cells. Here we report a role for the condensin I subunit Cap-G in Drosophila neurons. We show that, despite not requiring condensin for mitotic chromosome compaction, post-mitotic neurons express Cap-G. Knockdown of Cap-G specifically in neurons (from their birth onwards) results in developmental arrest, behavioural defects, and dramatic gene expression changes, including reduced expression of a subset of neuronal genes and aberrant expression of genes that are not normally expressed in the developing brain. Knockdown of Cap-G in mature neurons results in similar phenotypes but to a lesser degree. Furthermore, we see dynamic binding of Cap-G at distinct loci in progenitor cells and differentiated neurons. Therefore, Cap-G is essential for proper gene expression in neurons and plays an important role during the early stages of neuronal development.
Hassan A, Rodriguez PA, Heidmann SK, et al., 2020, Condensin I subunit Cap-G is essential for proper gene expression during the maturation of post-mitotic neurons
<jats:title>Abstract</jats:title><jats:p>The condensin complex is essential for mitotic chromosome assembly and segregation during cell divisions, however, little is known about its function in post-mitotic, differentiated cells. Here we report a novel role for the condensin I subunit Cap-G in<jats:italic>Drosophila</jats:italic>neurons. We show that, despite not requiring condensin for mitotic chromosome compaction, post-mitotic neurons express Cap-G and that knockdown of Cap-G specifically in neurons (from their birth onwards) results in developmental arrest, behavioural defects, and dramatic gene expression changes. These include reduced expression of a subset of neuronal genes and aberrant expression of genes that are not normally expressed in the developing brain. Knockdown of Cap-G in more mature neurons also results in similar phenotypes but to a lesser degree. Furthermore, we see dynamic binding of Cap-G to chromatin at distinct loci in neural stem cells and differentiated neurons. Therefore, Cap-G is essential for proper gene expression in neurons and plays an important role during the early stages of neuronal development.</jats:p><jats:p><jats:fig id="ufig1" position="float" fig-type="figure" orientation="portrait"><jats:graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="904409v1_ufig1" position="float" orientation="portrait" /></jats:fig></jats:p>
Brown P, Tan A-C, El-Esawi MA, et al., 2019, Large expert-curated database for benchmarking document similarity detection in biomedical literature search, Database: the journal of biological databases and curation, Vol: 2019, Pages: 1-66, ISSN: 1758-0463
Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency–Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.
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
<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.
Korzelius J, Naumann SK, Loza-Coll MA, et al., 2014, <i>Escargot</i> maintains stemness and suppresses differentiation in <i>Drosophila</i> intestinal stem cells, EMBO JOURNAL, Vol: 33, Pages: 2967-2982, ISSN: 0261-4189
Loza-Coll MA, Southall TD, Sandall SL, et al., 2014, Regulation of <i>Drosophila</i> intestinal stem cell maintenance and differentiation by the transcription factor Escargot, EMBO JOURNAL, Vol: 33, Pages: 2983-2996, ISSN: 0261-4189
Southall TD, Davidson CM, Miller C, et al., 2014, Dedifferentiation of neurons precedes tumor gormation in lola mutants, Developmental Cell, Vol: 28, Pages: 685-696, ISSN: 1534-5807
The ability to reprogram differentiated cells into a pluripotent state has revealed that the differentiated state is plastic and reversible. It is evident, therefore, that mechanisms must be in place to maintain cells in a differentiated state. Transcription factors that specify neuronal characteristics have been well studied, but less is known about the mechanisms that prevent neurons from dedifferentiating to a multipotent, stem cell-like state. Here, we identify Lola as a transcription factor that is required to maintain neurons in a differentiated state. We show that Lola represses neural stem cell genes and cell-cycle genes in postmitotic neurons. In lola mutants, neurons dedifferentiate, turn on neural stem cell genes, and begin to divide, forming tumors. Thus, neurons rather than stem cells or intermediate progenitors are the tumor-initiating cells in lola mutants.
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