Cell type-specific profiling of protein-DNA interactions without cell isolation using Targeted DamID with next-generation sequencing

The ability to profile transcription and chromatin binding in a cell type-specific manner is a powerful approach 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 of DNA- and chromatin-binding proteins in vivo without cell isolation. As a Protocol Extension, this article 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 and potential artefacts from over-expression. The modifications to the core DamID technique presented here also increase the speed of sample processing and throughput, and adapt the method to Nextgeneration Sequencing technology. TaDa is robust, reproducible, and highly sensitive. Compared to other methods for cell-type specific profiling, the technique requires no cell-sorting, crosslinking or antisera, and binding profiles can be generated from as few as 10,000 total induced cells. By profiling the genome-wide binding of RNA polymerase II, TaDa can also identify transcribed genes in a cell type-specific manner. Here we describe a detailed protocol for carrying out TaDa experiments and preparing the material for next generation sequencing. Although we developed TaDa in Drosophila, it should be easily adapted to other organisms with an inducible expression system. Once transgenic animals are obtained, the entire experimental procedure – from collecting tissue samples to generating sequencing libraries – can be accomplished within 5 days

The LIM-Homeodomain Protein Islet Dictates Motor Neuron Electrical Properties by Regulating K+ Channel Expression

Neuron electrical properties are critical to function and generally subtype specific, as are patterns of axonal and dendritic projections. Specification of motoneuron morphology and axon pathfinding has been studied extensively, implicating the combinatorial action of Lim-homeodomain transcription factors. However, the specification of electrical properties is not understood. Here, we address the key issues of whether the same transcription factors that specify morphology also determine subtype specific electrical properties. We show that Drosophila motoneuron subtypes express different K(+) currents and that these are regulated by the conserved Lim-homeodomain transcription factor Islet. Specifically, Islet is sufficient to repress a Shaker-mediated A-type K(+) current, most likely due to a direct transcriptional effect. A reduction in Shaker increases the frequency of action potential firing. Our results demonstrate the deterministic role of Islet on the excitability patterns characteristic of motoneuron subtypes

Dedifferentiation of neurons precedes tumor gormation in lola mutants

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

miR-7 Buffers Differentiation in the Developing Drosophila Visual System

The 40,000 neurons of the medulla, the largest visual processing center of the $\textit{Drosophila}$ brain, derive from a sheet of neuroepithelial cells. During larval development, a wave of differentiation sweeps across the neuroepithelium, converting neuroepithelial cells into neuroblasts that sequentially express transcription factors specifying different neuronal cell fates. The switch from neuroepithelial cells to neuroblasts is controlled by a complex gene regulatory network and is marked by the expression of the proneural gene $\textit{l’sc}$. We discovered that microRNA $\textit{miR-7}$ is expressed at the transition between neuroepithelial cells and neuroblasts. We showed that $\textit{miR-7}$ promotes neuroepithelial cell-to-neuroblast transition by targeting downstream Notch effectors to limit Notch signaling. $\textit{miR-7}$ acts as a buffer to ensure that a precise and stereotypical pattern of transition is maintained, even under conditions of environmental stress, echoing the role that $\textit{miR-7}$ plays in the eye imaginal disc. This common mechanism reflects the importance of robust visual system development.This work was funded by a Wellcome Trust Programme grant (092545), a Wellcome Trust Senior Investigator Award (103792), and a BBSRC Project Grant (BB/L007800/1) to A.H.B. A.H.B. acknowledges core funding to the Gurdon Institute from the Wellcome Trust (092096) and CRUK (C6946/A14492)

Cell cycle heterogeneity directs the timing of neural stem cell activation from quiescence.

Quiescent stem cells in adult tissues can be activated for homeostasis or repair. Neural stem cells (NSCs) in Drosophila are reactivated from quiescence in response to nutrition by the insulin signaling pathway. It is widely accepted that quiescent stem cells are arrested in G0 In this study, however, we demonstrate that quiescent NSCs (qNSCs) are arrested in either G2 or G0 G2-G0 heterogeneity directs NSC behavior: G2 qNSCs reactivate before G0 qNSCs. In addition, we show that the evolutionarily conserved pseudokinase Tribbles (Trbl) induces G2 NSCs to enter quiescence by promoting degradation of Cdc25String and that it subsequently maintains quiescence by inhibiting Akt activation. Insulin signaling overrides repression of Akt and silences trbl transcription, allowing NSCs to exit quiescence. Our results have implications for identifying and manipulating quiescent stem cells for regenerative purposes.This work was funded by the Royal Society Darwin Trust Research Professorship, Wellcome Trust Senior Investigator Award 103792 and Wellcome Trust Programme grant 092545 to A.H.B., and Wellcome Trust PhD Studentship 097423 to L.O. A.H.B acknowledges core funding to the Gurdon Institute from the Wellcome Trust (092096) and CRUK (C6946/A14492)

Identification and characterization of novel factors that act in the nonsense-mediated mRNA decay pathway in nematodes, flies and mammals

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNAs harboring premature termination codons (PTCs). We have conducted a genome-wide RNAi screen in Caenorhabditis elegans that resulted in the identification of five novel NMD genes that are conserved throughout evolution. Two of their human homologs, GNL2 (ngp-1) and SEC13 (npp-20), are also required for NMD in human cells. We also show that the C. elegans gene noah-2, which is present in Drosophila melanogaster but absent in humans, is an NMD factor in fruit flies. Altogether, these data identify novel NMD factors that are conserved throughout evolution, highlighting the complexity of the NMD pathway and suggesting that yet uncovered novel factors may act to regulate this process

Complex roles of myoglianin in regulating adult performance and lifespan

Myoglianin, the Drosophila homolog of the secreted vertebrate proteins Myostatin and GDF-11, is an important regulator of neuronal modelling, and synapse function and morphology. While Myoglianin suppression during development elicits positive effects on the neuromuscular system, genetic manipulations of myoglianin expression levels have a varied effect on the outcome of performance tests in aging flies. Specifically, Myoglianin preserves jumping ability, has no effect on negative geotaxis, and negatively regulates flight performance in aging flies. In addition, Myoglianin exhibits a tissue-specific effect on longevity, with myoglianin upregulation in glial cells increasing the median lifespan. These findings indicate complex role for this TGF-β-like protein in governing neuromuscular signalling and consequent behavioural outputs and lifespan in adult flies

Rate, characteristics, and factors associated with high emergency department utilization

Background: Patients with high emergency department (ED) utilization account for a disproportionate number of ED visits. The existing research on high ED utilization has raised doubts about the homogeneity of the frequent ED user. Attention to differences among the subgroups of frequent visitors (FV) and highly frequent visitors (HFV) is necessary in order to plan more effective interventions. In the Netherlands, the incidence of high ED utilization is unknown. The purpose of this study was to investigate if the well-documented international high ED utilization also exists in the Netherlands and if so, to characterize these patients. Therefore, we assessed the proportion of FV and HFV; compared age, sex, and visit outcomes between patients with high ED utilization and patients with single ED visits; and explored the factors associated with high ED utilization. Methods: A 1-year retrospective descriptive correlational study was performed in two Dutch EDs, using thresholds of 7 to 17 visits for frequent ED use, and greater than or equal to 18 visits for highly frequent ED use. Results: FV and HFV (together accounting for 0.5% of total ED patients) attended the ED 2,338 times (3.3% of the total number of ED visits). FV and HFV were equally likely to be male or female, were less likely to be self-referred, and they suffered from urgent complaints more often compared to patients with single visits. FV were significantly older than patients with single visits and more often admitted than patients with single visits. Several chief complaints were indicative for frequent and highly frequent ED use, such as shortness of breath and a psychiatric disorder. Conclusions: Based on this study, high ED utilization in the Netherlands seems to be less a problem than outlined in international literature. No major differences were found between FV and HFV, they presented with the same, often serious, problems. Our study supports the notion that most patients with high ED utilization visit the ED for significant medical problems. © 2014 van der Linden et al.; licensee Springer

Antiprion drugs 6-aminophenanthridine and guanabenz reduce PABPN1 toxicity and aggregation in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset syndrome characterized by progressive degeneration of specific muscles. OPMD is caused by extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). Insoluble nuclear inclusions form in diseased muscles. We have generated a Drosophila model of OPMD that recapitulates the features of the disorder. Here, we show that the antiprion drugs 6-aminophenanthridine (6AP) and guanabenz acetate (GA), which prevent formation of amyloid fibers by prion proteins in cell models, alleviate OPMD phenotypes in Drosophila, including muscle degeneration and nuclear inclusion formation. The large ribosomal RNA and its activity in protein folding were recently identified as a specific cellular target of 6AP and GA. We show that deletions of the ribosomal DNA locus reduce OPMD phenotypes and act synergistically with sub-effective doses of 6AP. In a complementary approach, we demonstrate that ribosomal RNA accelerates in vitro fibril formation of PABPN1 N-terminal domain. These results reveal the conserved role of ribosomal RNA in different protein aggregation disorders and identify 6AP and GA as general anti-aggregation molecules