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The extent of transposable element (TE) mobilization in different somatic tissues and throughout diverse species is not well understood. Somatic transposition is often challenging to study as it generates de novo TE insertions that represent rare genetic variants present in heterogenous tissues. Here, we describe experimental approaches that can be applied to address TE mobility in somatic tissues with the use of short- and long-read whole-genome DNA sequencing. Focusing on the analysis of the Drosophila melanogaster intestinal and head tissues, we provide instructions on how to design, perform, and validate experiments that aim at detecting somatic transposition. In addition to providing examples of protocols, this chapter intends to deliver general experimental guidelines that may be adapted to other fly tissues or to other species.

Spontaneous mutations can alter tissue dynamics and lead to cancer initiation. While large-scale sequencing projects have illustrated processes that influence somatic mutation and subsequent tumour evolution, the mutational dynamics operating in the very early stages of cancer development are currently not well understood. In order to explore mutational dynamics in the early stages of cancer evolution we exploited neoplasia arising spontaneously in the Drosophila intestine. We analysed whole-genome sequencing data through the development of a dedicated bioinformatic pipeline to detect structural variants, single nucleotide variants, and indels. We found neoplasia formation to be driven largely through the inactivation of Notch by structural variants, many of which involve highly complex genomic rearrangements. Strikingly, the genome-wide mutational burden of neoplasia - at six weeks of age - was found to be similar to that of several human cancers. Finally, we identified genomic features associated with spontaneous mutation and defined the evolutionary dynamics and mutational landscape operating within intestinal neoplasia over the short lifespan of the adult fly. Our findings provide unique insight into mutational dynamics operating over a short time scale in the genetic model system, Drosophila melanogaster.

Transposable elements (TEs) play a significant role in evolution by contributing to genetic variation through germline insertional activity. However, how TEs act in somatic cells and tissues is not well understood. Here, we address the prevalence of transposition in a somatic tissue, exploiting theDrosophilamidgut as a model system. Using whole-genome sequencing ofin vivoclonally expanded gut tissue, we map hundreds of high-confidence somatic TE integration sites genome-wide. We show that somatic retrotransposon insertions are associated with inactivation of the tumor suppressorNotch, likely contributing to neoplasia formation. Moreover, by applying Oxford Nanopore long-read sequencing technology, as well as by mapping germline TE activity, we provide evidence suggesting tissue-specific differences in retrotransposition. By comparing somatic TE insertional activity with transcriptomic and small RNA sequencing data, we demonstrate that transposon mobility cannot be simply predicted by whole tissue TE expression levels or by small RNA pathway activity. Finally, we reveal that somatic TE insertions in the adult fly intestine are found preferentially in genic regions and open, transcriptionally active chromatin. Together, our findings provide clear evidence of ongoing somatic transposition inDrosophilaand delineate previously unknown underlying features of somatic TE mobilityin vivo.

International audience; Transposable elements (TEs) play a significant role in evolution, contributing to genetic variation. However, TE mobilization in somatic cells is not well understood. Here, we address the prevalence of transposition in a somatic tissue, exploiting the Drosophila midgut as a model. Using whole-genome sequencing of in vivo clonally expanded gut tissue, we have mapped hundreds of highconfidence somatic TE integration sites genome-wide. We show that somatic retrotransposon insertions are associated with inactivation of the tumor suppressor Notch, likely contributing to neoplasia formation. Moreover, applying Oxford Nanopore longread sequencing technology we provide evidence for tissue-specific differences in retrotransposition. Comparing somatic TE insertional activity with transcriptomic and small RNA sequencing data, we demonstrate that transposon mobility cannot be simply predicted by whole tissue TE expression levels or by small RNA pathway activity. Finally, we reveal that somatic TE insertions in the adult fly intestine are enriched in genic regions and in transcriptionally active chromatin. Together, our findings provide clear evidence of ongoing somatic transposition in Drosophila and delineate previously unknown features underlying somatic TE mobility in vivo.

How terminal cell fates are specified in dynamically renewing adult tissues is not well understood. Here we explore terminal cell fate establishment during homeostasis using the enteroendocrine cells (EEs) of the adult Drosophila midgut as a paradigm. Our data argue against the existence of local feedback signals, and we identify Numb as an intrinsic regulator of EE fate. Our data further indicate that Numb, with alpha‐adaptin, acts upstream or in parallel of known regulators of EE fate to limit Notch signaling, thereby facilitating EE fate acquisition. We find that Numb is regulated in part through its asymmetric and symmetric distribution during stem cell divisions; however, its de novo synthesis is also required during the differentiation of the EE cell. Thus, this work identifies Numb as a crucial factor for cell fate choice in the adult Drosophila intestine. Furthermore, our findings demonstrate that cell‐intrinsic control mechanisms of terminal cell fate acquisition can result in a balanced tissue‐wide production of terminally differentiated cell types.

Pantothenate kinase-associated neurodegeneration (PKAN is a neurodegenerative disease with unresolved pathophysiology. Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN. Coenzyme A is required for acetyl-Coenzyme A synthesis and acyl groups from the latter are transferred to lysine residues of proteins, in a reaction regulated by acetyltransferases. The tight balance between acetyltransferases and their antagonistic counterparts histone deacetylases is a well-known determining factor for the acetylation status of proteins. However, the influence of Coenzyme A levels on protein acetylation is unknown. Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models. We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival. Decreased protein acetylation and the concurrent phenotypes are partly rescued by pantethine and HDAC inhibitors, suggesting possible directions for future PKAN therapy development.

Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall (stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 (two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress.

Aneuploidy is associated with a variety of diseases such as cancer and microcephaly. Although many studies have addressed the consequences of a non-euploid genome in cells, little is known about their overall consequences in tissue and organism development. Here we use two different mutant conditions to address the consequences of aneuploidy during tissue development and homeostasis in Drosophila. We show that aneuploidy causes brain size reduction due to a decrease in the number of proliferative neural stem cells (NSCs), but not through apoptosis. Instead, aneuploid NSCs present an extended G1 phase, which leads to cell cycle exit and premature differentiation. Moreover, we show that this response to aneuploidy is also present in adult intestinal stem cells but not in the wing disc. Our work highlights a neural and intestine stem cell-specific response to aneuploidy, which prevents their proliferation and expansion., It is unclear why certain tissues are more susceptible to the consequences of aneuploidy. Here, in Drosophila, Gogendeau et al. identify aneuploidy as the cause of lengthened G1 and premature differentiation in both neural and adult intestinal stem cells, which prevents cells with abnormal genomes from cycling.

Porphyromonas gingivalis acquires iron and heme from the host environment using gingipains, lipoproteins, and outer-membrane receptors. Recently, we identified and characterized a heme receptor HmuR. The hmuR gene is localized in an operon together with a hmuY gene encoding a putative heme-binding protein. The aim of this study was to overexpress and perform a preliminary analysis of the recombinant HmuY protein. We constructed and examined several recombinant HmuY variants which were overexpressed and purified from Escherichia coli and insect cells. Recombinant HmuY protein was expressed in insect cells at levels similar to those in E. coli cells. This protein is predominantly present in a monomeric form but also dimerizes and several other oligomerization forms were found. Hemin and ATP binding to the purified HmuY showed that this protein may play a regulatory function in hemin utilization in P. gingivalis.

SummaryAdult stem cells may acquire mutations that modify cellular behavior, leading to functional declines in homeostasis or providing a competitive advantage resulting in premalignancy. However, the frequency, phenotypic impact, and mechanisms underlying spontaneous mutagenesis during aging are unclear. Here, we report two mechanisms of genome instability in adult Drosophila intestinal stem cells (ISCs) that cause phenotypic alterations in the aging intestine. First, we found frequent loss of heterozygosity arising from mitotic homologous recombination in ISCs that results in genetic mosaicism. Second, somatic deletion of DNA sequences and large structural rearrangements, resembling those described in cancers and congenital diseases, frequently result in gene inactivation. Such modifications induced somatic inactivation of the X-linked tumor suppressor Notch in ISCs, leading to spontaneous neoplasias in wild-type males. Together, our findings reveal frequent genomic modification in adult stem cells and show that somatic genetic mosaicism has important functional consequences on aging tissues.

Coenzyme A (CoA) is a pantothenic acid-derived metabolite essential for many fundamental cellular processes including energy, lipid and amino acid metabolism. Pantothenate kinase (PANK), which catalyses the first step in the conversion of pantothenic acid to CoA, has been associated with a rare neurodegenerative disorder PKAN. However, the consequences of impaired PANK activity are poorly understood. Here we use Drosophila and human neuronal cell cultures to show how PANK deficiency leads to abnormalities in F-actin organization. Cells with reduced PANK activity are characterized by abnormally high levels of phosphorylated cofilin, a conserved actin filament severing protein. The increased levels of phospho-cofilin coincide with morphological changes of PANK-deficient Drosophila S2 cells and human neuronal SHSY-5Y cells. The latter exhibit also markedly reduced ability to form neurites in culture - a process that is strongly dependent on actin remodeling. Our results reveal a novel and conserved link between a metabolic biosynthesis pathway, and regulation of cellular actin dynamics.

Drosophila cell lines are valuable tools to study a number of cellular processes, including DNA damage responses and cell cycle checkpoint control. Using an in vitro system instead of a whole organism has two main advantages: it saves time and simple and effective molecular techniques are available. It has been shown that Drosophila cells, similarly to mammalian cells, display cell cycle checkpoint pathways required to survive DNA damaging events (de Vries et al. 2005, Journal of Cell Science 118, 1833-1842; Bae et al. 1995, Experimental Cell Research 217, 541-545). Moreover, a number of proteins involved in checkpoint and cell cycle control in mammals are highly conserved among different species, including Drosophila (de Vries et al. 2005, Journal of Cell Science 118, 1833-1842; Bae et al. 1995, Experimental Cell Research 217, 541-545; LaRocque et al. 2007, Genetics 175, 1023-1033; Sibon et al. 1999, Current Biology 9, 302-312; Purdy et al. 2005, Journal of Cell Science 118, 3305-3315). Because of straightforward and highly efficient methods to downregulate specific transcripts in Drosophila cells, these cells are an excellent system for genome-wide RNA interference (RNAi) screens. Thus, the following methods, assays and techniques: Drosophila cell culture, RNAi, introducing DNA damaging events, determination of cell cycle arrest, and determination of cell cycle distributions described here may well be applied to identifying new players in checkpoint mechanisms and will be helpful to investigate the function of these new players in detail. Results obtained with studies using in vitro systems can subsequently be extended to studies in the complete organism as described in the chapters provided by the Su laboratory and the Takada laboratory.

Drosophila cell lines are valuable tools to study a number of cellular processes, including DNA damage responses and cell cycle checkpoint control. Using an in vitro system instead of a whole organism has two main advantages: it saves time and simple and effective molecular techniques are available. It has been shown that Drosophila cells, similarly to mammalian cells, display cell cycle checkpoint pathways required to survive DNA damaging events (de Vries et al. 2005, Journal of Cell Science 118, 1833-1842; Bae et al. 1995, Experimental Cell Research 217, 541-545). Moreover, a number of proteins involved in checkpoint and cell cycle control in mammals are highly conserved among different species, including Drosophila (de Vries et al. 2005, Journal of Cell Science 118, 1833-1842; Bae et al. 1995, Experimental Cell Research 217, 541-545; LaRocque et al. 2007, Genetics 175, 1023-1033; Sibon et al. 1999, Current Biology 9, 302-312; Purdy et al. 2005, Journal of Cell Science 118, 3305-3315). Because of straightforward and highly efficient methods to downregulate specific transcripts in Drosophila cells, these cells are an excellent system for genome-wide RNA interference (RNAi) screens. Thus, the following methods, assays and techniques: Drosophila cell culture, RNAi, introducing DNA damaging events, determination of cell cycle arrest, and determination of cell cycle distributions described here may well be applied to identifying new players in checkpoint mechanisms and will be helpful to investigate the function of these new players in detail. Results obtained with studies using in vitro systems can subsequently be extended to studies in the complete organism as described in the chapters provided by the Su laboratory and the Takada laboratory.

Pantothenate kinase–associated neurodegeneration (PKAN), a progressive neurodegenerative disorder, is associated with impairment of pantothenate kinase function. Pantothenate kinase is the first enzyme required for de novo synthesis of CoA, an essential metabolic cofactor. The pathophysiology of PKAN is not understood, and there is no cure to halt or reverse the symptoms of this devastating disease. Recently, we and others presented a PKAN Drosophila model, and we demonstrated that impaired function of pantothenate kinase induces a neurodegenerative phenotype and a reduced lifespan. We have explored this Drosophila model further and have demonstrated that impairment of pantothenate kinase is associated with decreased levels of CoA, mitochondrial dysfunction, and increased protein oxidation. Furthermore, we searched for compounds that can rescue pertinent phenotypes of the Drosophila PKAN model and identified pantethine. Pantethine feeding restores CoA levels, improves mitochondrial function, rescues brain degeneration, enhances locomotor abilities, and increases lifespan. We show evidence for the presence of a de novo CoA biosynthesis pathway in which pantethine is used as a precursor compound. Importantly, this pathway is effective in the presence of disrupted pantothenate kinase function. Our data suggest that pantethine may serve as a starting point to develop a possible treatment for PKAN.

Iron and heme are essential nutrients for most pathogenic microorganisms and play a pivotal role in microbial pathogenesis. To survive within the iron-limited environment of the host, bacteria utilize iron-siderophore complexes, iron-binding proteins (transferrin, lactoferrin), free heme and heme bound to hemoproteins (hemoglobin, haptoglobin, hemopexin). A mechanism of iron and heme transport depends on the structures of Gram-negative bacterial membranes. Siderophores, hemophores and outer membrane receptors take part in iron or heme binding. The transport of these ligands across the outer membrane involves outer membrane receptors. The energy for this transport is delivered from the inner membrane by a TonB-ExbB-ExbD complex. The transport across the cytoplasmic membrane involves periplasmic and inner membrane proteins comprising the ABC systems, which utilize the energy derived from ATP hydrolysis. The major regulatory role in iron homeostasis plays a Fur-Fe2+ repressor.