Cell-type-specific transcriptional profiling often requires the isolation of specific cell types from complex tissues. We have developed “TaDa,” a technique that enables cell-specific profiling ...without cell isolation. TaDa permits genome-wide profiling of DNA- or chromatin-binding proteins without cell sorting, fixation, or affinity purification. The method is simple, sensitive, highly reproducible, and transferable to any model system. We show that TaDa can be used to identify transcribed genes in a cell-type-specific manner with considerable temporal precision, enabling the identification of differential gene expression between neuroblasts and the neuroepithelial cells from which they derive. We profile the genome-wide binding of RNA polymerase II in these adjacent, clonally related stem cells within intact Drosophila brains. Our data reveal expression of specific metabolic genes in neuroepithelial cells, but not in neuroblasts, and highlight gene regulatory networks that may pattern neural stem cell fates.
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•TaDa is a method for cell-type-specific profiling of chromatin binding proteins•TaDa does not require cell sorting, fixation, or affinity purification•This is a highly sensitive and robust technique for transcriptional profiling•We report differential RNA Pol II binding in clonally related stem cells
Southall et al. developed “TaDa,” a technique that enables cell-specific genome-wide profiling of DNA- or chromatin-binding proteins without cell isolation, fixation, or affinity purification. TaDa genome-wide profiles of RNA polymerase II binding reveal spatially and temporally precise distinctions between adjacent, clonally related neural stem cells in intact Drosophila brains.
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
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.
Visible light (400-700 nm) lies outside of the spectral range of what photobiologists define as deleterious radiation and as a result few studies have studied the effects of visible light range of ...wavelengths on skin. This oversight is important considering that during outdoors activities skin is exposed to the full solar spectrum, including visible light, and to multiple exposures at different times and doses. Although the contribution of the UV component of sunlight to skin damage has been established, few studies have examined the effects of non-UV solar radiation on skin physiology in terms of inflammation, and limited information is available regarding the role of visible light on pigmentation. The purpose of this study was to determine the effect of visible light on the pro-pigmentation pathways and melanin formation in skin. Exposure to visible light in ex-vivo and clinical studies demonstrated an induction of pigmentation in skin by visible light. Results showed that a single exposure to visible light induced very little pigmentation whereas multiple exposures with visible light resulted in darker and sustained pigmentation. These findings have potential implications on the management of photo-aggravated pigmentary disorders, the proper use of sunscreens, and the treatment of depigmented lesions.
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.
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.
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.
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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•The BTB-Zn finger factor, Lola, maintains neurons in a differentiated state•Lola represses neural stem cell genes and cell-cycle genes in postmitotic neurons•In lola mutants, neurons dedifferentiate•Dedifferentiated neurons are tumor-initiating cells in lola mutants
Southall et al. show that the BTB-Zn finger transcription factor, Lola, is required to maintain neurons in a differentiated state. 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 divide, forming tumors.
Spatial and temporal cues are required to specify neuronal diversity, but how these cues are integrated in neural progenitors remains unknown.
progenitors (neuroblasts) are a good model: they are ...individually identifiable with relevant spatial and temporal transcription factors known. Here we test whether spatial/temporal factors act independently or sequentially in neuroblasts. We used Targeted DamID to identify genomic binding sites of the Hunchback temporal factor in two neuroblasts (NB5-6 and NB7-4) that make different progeny. Hunchback targets were different in each neuroblast, ruling out the independent specification model. Moreover, each neuroblast had distinct open chromatin domains, which correlated with differential Hb-bound loci in each neuroblast. Importantly, the Gsb/Pax3 spatial factor, expressed in NB5-6 but not NB7-4, had genomic binding sites correlated with open chromatin in NB5-6, but not NB7-4. Our data support a model in which early-acting spatial factors like Gsb establish neuroblast-specific open chromatin domains, leading to neuroblast-specific temporal factor binding and the production of different neurons in each neuroblast lineage.
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.
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.
Synopsis
This paper unravels the functionality of Escargot, so far described as marker for progenitors cells in Drosophila, in intestinal stem cell maintenance and suppressor of differentiation.
Esg is required to maintain intestinal stem cell identity.
Esg acts as a repressor of differentiation genes in progenitor cells.
Pdm1 is a direct target of Esg that is sufficient to trigger EC differentiation.
Esg, Notch and Pdm1 function together in a transcriptional network that controls differentiation in the Drosophila midgut.
This paper unravels the functionality of Escargot, so far described as marker for progenitors cells in Drosophila, in intestinal stem cell maintenance and suppressor of differentiation.
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