Decoding gene regulation in the fly brain.

The Drosophila brain is a frequently used model in neuroscience. Single-cell transcriptome analysis1-6, three-dimensional morphological classification7 and electron microscopy mapping of the connectome8,9 have revealed an immense diversity of neuronal and glial cell types that underlie an array of functional and behavioural traits in the fly. The identities of these cell types are controlled by gene regulatory networks (GRNs), involving combinations of transcription factors that bind to genomic enhancers to regulate their target genes. Here, to characterize GRNs at the cell-type level in the fly brain, we profiled the chromatin accessibility of 240,919 single cells spanning 9 developmental timepoints and integrated these data with single-cell transcriptomes. We identify more than 95,000 regulatory regions that are used in different neuronal cell types, of which 70,000 are linked to developmental trajectories involving neurogenesis, reprogramming and maturation. For 40 cell types, uniquely accessible regions were associated with their expressed transcription factors and downstream target genes through a combination of motif discovery, network inference and deep learning, creating enhancer GRNs. The enhancer architectures revealed by DeepFlyBrain lead to a better understanding of neuronal regulatory diversity and can be used to design genetic driver lines for cell types at specific timepoints, facilitating their characterization and manipulation.

NuRD-independent Mi-2 activity represses ectopic gene expression during neuronal maturation.

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.

Gene expression profiling of epidermal cell types in C. elegans using Targeted DamID.

The epidermis of Caenorhabditis elegans is an essential tissue for survival because it contributes to the formation of the cuticle barrier as well as facilitating 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, which exhibit stem cell-like behaviour during development. How 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. Finally, we 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.

DamID as a versatile tool for understanding gene regulation.

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.

Targeted DamID reveals differential binding of mammalian pluripotency factors.

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.

Regulation of Drosophila intestinal stem cell maintenance and differentiation by the transcription factor Escargot.

Tissue stem cells divide to self-renew and generate differentiated cells to maintain homeostasis. Although influenced by both intrinsic and extrinsic factors, the genetic mechanisms coordinating the decision between self-renewal and initiation of differentiation remain poorly understood. The escargot (esg) gene encodes a transcription factor that is expressed in stem cells in multiple tissues in Drosophila melanogaster, including intestinal stem cells (ISCs). Here, we demonstrate that Esg plays a pivotal role in intestinal homeostasis, maintaining the stem cell pool while influencing fate decisions through modulation of Notch activity. Loss of esg induced ISC differentiation, a decline in Notch activity in daughter enteroblasts (EB), and an increase in differentiated enteroendocrine (EE) cells. Amun, an inhibitor of Notch in other systems, was identified as a target of Esg in the intestine. Decreased expression of esg resulted in upregulation of Amun, while downregulation of Amun rescued the ectopic EE cell phenotype resulting from loss of esg. Thus, our findings provide a framework for further comparative studies addressing the conserved roles of Snail factors in coordinating self-renewal and differentiation of stem cells across tissues and species.

Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells.

Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). We demonstrate here that loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero-endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type-specific transcriptome analysis combined with Dam-ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU-domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation-promoting factors, such as Pdm1.

The transcription factors islet and Lim3 combinatorially regulate ion channel gene expression.

Expression of appropriate ion channels is essential to allow developing neurons to form functional networks. Our previous studies have identified LIM-homeodomain (HD) transcription factors (TFs), expressed by developing neurons, that are specifically able to regulate ion channel gene expression. In this study, we use the technique of DNA adenine methyltransferase identification (DamID) to identify putative gene targets of four such TFs that are differentially expressed in Drosophila motoneurons. Analysis of targets for Islet (Isl), Lim3, Hb9, and Even-skipped (Eve) identifies both ion channel genes and genes predicted to regulate aspects of dendritic and axonal morphology. Significantly, some ion channel genes are bound by more than one TF, consistent with the possibility of combinatorial regulation. One such gene is Shaker (Sh), which encodes a voltage-dependent fast K(+) channel (Kv1.1). DamID reveals that Sh is bound by both Isl and Lim3. We used body wall muscle as a test tissue because in conditions of low Ca(2+), the fast K(+) current is carried solely by Sh channels (unlike neurons in which a second fast K(+) current, Shal, also contributes). Ectopic expression of isl, but not Lim3, is sufficient to reduce both Sh transcript and Sh current level. By contrast, coexpression of both TFs is additive, resulting in a significantly greater reduction in both Sh transcript and current compared with isl expression alone. These observations provide evidence for combinatorial activity of Isl and Lim3 in regulating ion channel gene expression.

Crosstalk between chromatin and Shavenbaby defines transcriptional output along the Drosophila intestinal stem cell lineage.

The transcription factor Shavenbaby (Svb), the only member of the OvoL family in Drosophila, controls the fate of various epithelial embryonic cells and adult stem cells. Post-translational modification of Svb produces two protein isoforms, Svb-ACT and Svb-REP, which promote adult intestinal stem cell renewal or differentiation, respectively. To define Svb mode of action, we used engineered cell lines and develop an unbiased method to identify Svb target genes across different contexts. Within a given cell type, Svb-ACT and Svb-REP antagonistically regulate the expression of a set of target genes, binding specific enhancers whose accessibility is constrained by chromatin landscape. Reciprocally, Svb-REP can influence local chromatin marks of active enhancers to help repressing target genes. Along the intestinal lineage, the set of Svb target genes progressively changes, together with chromatin accessibility. We propose that Svb-ACT-to-REP transition promotes enterocyte differentiation of intestinal stem cells through direct gene regulation and chromatin remodeling.

Neural stem cell transcriptional networks highlight genes essential for nervous system development.

Neural stem cells must strike a balance between self-renewal and multipotency, and differentiation. Identification of the transcriptional networks regulating stem cell division is an essential step in understanding how this balance is achieved. We have shown that the homeodomain transcription factor, Prospero, acts to repress self-renewal and promote differentiation. Among its targets are three neural stem cell transcription factors, Asense, Deadpan and Snail, of which Asense and Deadpan are repressed by Prospero. Here, we identify the targets of these three factors throughout the genome. We find a large overlap in their target genes, and indeed with the targets of Prospero, with 245 genomic loci bound by all factors. Many of the genes have been implicated in vertebrate stem cell self-renewal, suggesting that this core set of genes is crucial in the switch between self-renewal and differentiation. We also show that multiply bound loci are enriched for genes previously linked to nervous system phenotypes, thereby providing a shortcut to identifying genes important for nervous system development.

Transcriptional control of stem cell maintenance in the Drosophila intestine.

Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. Here we find that transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance, and identify genes of the Enhancer of split complex [E(spl)-C] as the major targets of this repression. In addition, we find that the bHLH transcription factor Daughterless is essential to maintain ISC identity and that bHLH binding sites promote ISC-specific enhancer activity. We propose that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation.

Snail-dependent repression of the RhoGEF pebble is required for gastrulation consistency in Drosophila melanogaster.

The Rho GTP exchange factor, Pebble (Pbl), long recognised as an essential activator of Rho during cytokinesis, also regulates mesoderm migration at gastrulation. Like other cell cycle components, pbl expression patterns broadly correlate with proliferative tissue. Surprisingly, in spite of its role in the early mesoderm, pbl is downregulated in the presumptive mesoderm before ventral furrow formation. Here, we show that this mesoderm-specific repression of pbl is dependent on the transcriptional repressor Snail (Sna). pbl repression was lost in sna mutants but was unaffected when Sna was ectopically expressed, showing that Sna is necessary, but not sufficient, for pbl repression. Using DamID, the first intron of pbl was identified as a Sna-binding region. Nine sites with the Sna-binding consensus motif CAGGT[GA] were identified in this intron. Mutating these to TAGGC[GA] abolished the ventral repression of pbl. Surprisingly, Sna-dependent repression of pbl was not essential for viability or fertility. Loss of repression did, however, increase the frequency of low-penetrance gastrulation defects. Consistent with this, expression of a pbl-GFP transgene in the presumptive mesoderm generated similar gastrulation defects. Finally, we show that a cluster of Snail-binding sites in the middle of the first intron of pbl orthologues is a conserved feature in the other 11 sequenced Drosophila species. We conclude that pbl levels are precisely regulated to ensure that there is enough protein available for its role in early mesoderm development but not so much as to inhibit the orderly progression of gastrulation.

Escargot controls somatic stem cell maintenance through the attenuation of the insulin receptor pathway in Drosophila.

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.

Transcriptional memory of dFOXO activation in youth curtails later-life mortality through chromatin remodeling and Xbp1.

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.

An auxin-inducible, GAL4-compatible, gene expression system for Drosophila.

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.

Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells.

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. Here, we show that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. We have identified the in vivo targets of Prospero throughout the entire genome. We show that Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. We further show that in the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation.

FlyORF-TaDa allows rapid generation of new lines for in vivo cell-type-specific profiling of protein-DNA interactions in Drosophila melanogaster.

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 genome.

Dynamic adult tracheal plasticity drives stem cell adaptation to changes in intestinal homeostasis in Drosophila.

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.

Dynamic neurotransmitter specific transcription factor expression profiles during Drosophila development.

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 Caenorhabditis elegans, there are no simple transcription factor codes for neurotransmitter type specification.This article has an associated First Person interview with the first author of the paper.

Maintenance of Cell Fate by the Polycomb Group Gene Sex Combs Extra Enables a Partial Epithelial Mesenchymal Transition in Drosophila.

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 Sexcombs extra (Sce) and Sexcombs midleg (Scm) Depletion of Sce or Scm resulted in internalized 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 toward 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 emphasize 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.