High-resolution analysis of Merkel Cell Polyomavirus in Merkel Cell Carcinoma reveals distinct integration patterns and suggests NHEJ and MMBIR as underlying mechanisms.

Merkel Cell Polyomavirus (MCPyV) is the etiological agent of the majority of Merkel Cell Carcinomas (MCC). MCPyV positive MCCs harbor integrated, defective viral genomes that constitutively express viral oncogenes. Which molecular mechanisms promote viral integration, if distinct integration patterns exist, and if integration occurs preferentially at loci with specific chromatin states is unknown. We here combined short and long-read (nanopore) next-generation sequencing and present the first high-resolution analysis of integration site structure in MCC cell lines as well as primary tumor material. We find two main types of integration site structure: Linear patterns with chromosomal breakpoints that map closely together, and complex integration loci that exhibit local amplification of genomic sequences flanking the viral DNA. Sequence analysis suggests that linear patterns are produced during viral replication by integration of defective/linear genomes into host DNA double strand breaks via non-homologous end joining, NHEJ. In contrast, our data strongly suggest that complex integration patterns are mediated by microhomology-mediated break-induced replication, MMBIR. Furthermore, we show by ChIP-Seq and RNA-Seq analysis that MCPyV preferably integrates in open chromatin and provide evidence that viral oncogene expression is driven by the viral promoter region, rather than transcription from juxtaposed host promoters. Taken together, our data explain the characteristics of MCPyV integration and may also provide a model for integration of other oncogenic DNA viruses such as papillomaviruses.

The MAP kinase p38 is part of Drosophila melanogaster's circadian clock.

All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways.

Mutational Landscape of Virus- and UV-Associated Merkel Cell Carcinoma Cell Lines Is Comparable to Tumor Tissue.

Merkel cell carcinoma (MCC) is a rare, highly aggressive cutaneous malignancy that is either associated with the integration of the Merkel cell polyomavirus or chronic UV exposure. These two types of carcinogenesis are reflected in characteristic mutational features present in MCC tumor lesions. However, the genomic characteristics of MCC cell lines used as preclinical models are not well established. Thus, we analyzed the exomes of three virus-negative and six virus-positive MCC cell lines, all showing a classical neuroendocrine growth pattern. Virus-negative cell lines are characterized by a high tumor mutational burden (TMB), UV-light-induced DNA damage, functionally relevant coding mutations, e.g., in RB1 and TP53, and large amounts of copy number variations (CNVs). In contrast, virus-positive cell lines have a low TMB with few coding mutations and lack prominent mutational signatures, but harbor characteristic CNVs. One of the virus-negative cell lines has a local MYC amplification associated with high MYC mRNA expression. In conclusion, virus-positive and -negative MCC cell lines with a neuroendocrine growth pattern resemble mutational features observed in MCC tissue samples, which strengthens their utility for functional studies.

The neuropeptide pigment-dispersing factor adjusts period and phase of Drosophila's clock.

The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock of Drosophila melanogaster. PDF is necessary for robust activity rhythms and is thought to couple the circadian oscillations of the clock neurons. However, little is known about the action of PDF on individual clock neurons. Here, we combined the period-luciferase reporter system with immunolabeling of clock proteins in wild-type and Pdf(01) mutants to dissect the effects of PDF on specific subgroups of clock neurons. Additionally, PDF levels were elevated to higher than normal levels using specific neural mutants, and a correlation analysis of locomotor activity and clock protein staining served to determine the periods of specific clock cells. We found that PDF has multiple effects on the clock neurons: In some groups of clock neurons, PDF was required for maintaining the oscillations of individual cells, and in others, PDF was required for synchronous cycling of the individual members. Other clock neurons cycled with high amplitude in absence of PDF, but PDF affected their intrinsic clock speed. Sometimes PDF shortened and sometimes PDF lengthened period. Our observations indicate that PDF is crucial for adjusting cycling amplitude, period, and phase of the different players in the circadian clock. Under natural conditions PDF may be required for adapting Drosophila's clock to varying photoperiods. Indeed, we show here that Pdf(01) mutants are not able to adapt their activity to long photoperiods in a wild-type manner.

Period gene expression in four neurons is sufficient for rhythmic activity of Drosophila melanogaster under dim light conditions.

The clock gene expressing lateral neurons (LN) is crucial for Drosophila 's rhythmic locomotor activity under constant conditions. Among the LN, the PDF expressing small ventral lateral neurons (s-LN(v)) are thought to control the morning activity of the fly (M oscillators) and to drive rhythmic activity under constant darkness. In contrast, a 5th PDF-negative s-LN( v) and the dorsal lateral neurons (LN(d)) appeared to control the fly's evening activity (E oscillators) and to drive rhythmic activity under constant light. Here, the authors restricted period gene expression to 4 LN-the 5th s-LN(v) and 3 LN(d)- that are all thought to belong to the E oscillators and tested them in low light conditions. Interestingly, such flies showed rather normal bimodal activity patterns under light moonlight and constant moonlight conditions, except that the phase of M and E peaks was different. This suggests that these 4 neurons behave as ''M'' and ''E'' cells in these conditions. Indeed, they found by PER and TIM immunohistochemistry that 2 LN(d) advanced their phase upon moonlight as predicted for M oscillators, whereas the 5th s-LN(v) and 1 LN(d) delayed their activity upon moonlight as predicted for E oscillators. Their results suggest that the M or E characteristic of clock neurons is rather flexible. M and E oscillator function may not be restricted to certain anatomically defined groups of clock neurons but instead depends on the environmental conditions.

Pigment-dispersing factor (PDF) has different effects on Drosophila's circadian clocks in the accessory medulla and in the dorsal brain.

The neuropeptide pigment-dispersing factor (PDF) is a key transmitter in the circadian clock of Drosophila melanogaster. Here we studied the rhythmic behavior of neural mutants with modified arborizations of the large PDF neurons. In sine oculis(1) (so(1)) mutants we found a higher density of PDF fibers in the fly's pacemaker center, the accessory medulla. These flies exhibited a significantly longer period (24.6 h) than control flies. When PDF levels were elevated to very high levels in the dorsal brain as true for so(mda) mutants and small optic lobes;so(1) double mutants (sol(1);so( 1)), a short-period component split off the long period in behavioral rhythmicity. The short period became shorter the higher the amount of PDF in this brain region and reached a value of approximately 21 h. The period alterations were clearly dependent on PDF, because so(1);Pdf 01 and so(mda);Pdf 01 double mutants showed a single free-running component with a period similar to Pdf 01 mutants (approximately 22.5 h) and significantly longer than the short period of so(mda) mutants. These observations indicate that PDF feeds back on the clock neurons and changes their period. Obviously, PDF lengthens the period of some clock neurons and shortens that of others.

Gene duplication at the achaete-scute complex and morphological complexity of the peripheral nervous system in Diptera.

The number of achaete-scute genes increased during insect evolution, particularly in the Diptera lineage. Sequence comparison indicates that the four achaete-scute genes of Drosophila result from three independent duplication events. After duplication, the new genes acquired individual expression patterns but, in Drosophila, their products can compensate for one another, which raises the question: why retain all four genes? The complexity of the spatial expression of these genes on the notum increased in the lineage leading to the higher Diptera, allowing the development of stereotyped bristle patterns. This probably coincided in time with gene duplication events, raising the possibility that an increase in gene copy number might have provided the flexibility necessary for more complex transcriptional regulation.

Development and morphology of the clock-gene-expressing lateral neurons of Drosophila melanogaster.

The clock-gene-expressing lateral neurons are essential for the locomotor activity rhythm of Drosophila melanogaster. Traditionally, these neurons are divided into three groups: the dorsal lateral neurons (LN(d)), the large ventral lateral neurons (l-LN(v)), and the small ventral lateral neurons (s-LN(v)), whereby the latter group consists of four neurons that express the neuropeptide pigment-dispersing factor (PDF) and a fifth PDF-negative neuron. So far, only the l-LN(v) and the PDF-positive s-LN(v) have been shown to project into the accessory medulla, a small neuropil that contains the circadian pacemaker center in several insects. We show here that the other lateral neurons also arborize in the accessory medulla, predominantly forming postsynaptic sites. Both the l-LN(v) and LN(d) are anatomically well suited to connect the accessory medullae. Whereas the l-LN(v) may receive ipsilateral photic input from the Hofbauer-Buchner eyelet, the LN(d) invade mainly the contralateral accessory medulla and thus may receive photic input from the contralateral side. Both the LN(d) and the l-LN(v) differentiate during midmetamorphosis. They do so in close proximity to one another and the fifth PDF-negative s-LN(v), suggesting that these cell groups may derive from common precursors.

RNA in situ hybridizations on Drosophila whole mounts.

RNA in situ hybridization is a commonly used technique to achieve spatiotemporal detection of transcripts in tissues. This chapter gives an overview of novel techniques using fluorescent dyes, signal amplification methods, and confocal microscopy in regard to chronobiological applications on Drosophila adult brains.

The novel Drosophila tim(blind) mutation affects behavioral rhythms but not periodic eclosion.

Circadian clock function depends on the tightly regulated exclusion or presence of clock proteins within the nucleus. A newly induced long-period timeless mutant, tim(blind), encodes a constitutively hypophosphorylated TIM protein. The mutant protein is not properly degraded by light, and tim(blind) flies show abnormal behavioral responses to light pulses. This is probably caused by impaired nuclear accumulation of TIM(BLIND) protein, which we observed in brain pacemaker neurons and photoreceptor cells of the compound eye. tim(blind) encodes two closely spaced amino acid changes compared to the wild-type TIM protein; one of them is within a putative nuclear export signal of TIM. Under constant conditions, tim(blind) flies exhibit 26-hr free-running locomotor rhythms, which are not correlated with a period lengthening of eclosion rhythms and period-luciferase reporter-gene oscillations. Therefore it seems possible that TIM--in addition to its well-established role as core clock factor--functions as a clock output factor, involved in determining the period length of adult locomotor rhythms.

Identification of circadian-clock-regulated enhancers and genes of Drosophila melanogaster by transposon mobilization and luciferase reporting of cyclical gene expression.

A new way was developed to isolate rhythmically expressed genes in Drosophila by modifying the classic enhancer-trap method. We constructed a P element containing sequences that encode firefly luciferase as a reporter for oscillating gene expression in live flies. After generation of 1176 autosomal insertion lines, bioluminescence screening revealed rhythmic reporter-gene activity in 6% of these strains. Rhythmically fluctuating reporter levels were shown to be altered by clock mutations in genes that specify various circadian transcription factors or repressors. Intriguingly, rhythmic luminescence in certain lines was affected by only a subset of the pacemaker mutations. By isolating genes near 13 of the transposon insertions and determining their temporal mRNA expression pattern, we found that four of the loci adjacent to the trapped enhancers are rhythmically expressed. Therefore, this approach is suitable for identifying genetic loci regulated by the circadian clock. One transposon insert caused a mutation in the rhythmically expressed gene numb. This novel numb allele, as well as previously described ones, was shown to affect the fly's rhythm of locomotor activity. In addition to its known role in cell fate determination, this gene and the phosphotyrosine-binding protein it encodes are likely to function in the circadian system.

Two zebrafish homologues of the Drosophila neurogenic gene groucho and their pattern of transcription during early embryogenesis.

GROUCHO is a Drosophila nuclear protein with structural similarity to the transcriptional repressor TUP1. Drosophila GROUCHO forms complexes with bHLH proteins of the HAIRY-ENHANCER OF SPLIT [-E(SPL)] family, that then act as repressors, for example downstream of the NOTCH signalling pathway. We describe the isolation and sequence of two zebrafish GROUCHO homologues and the pattern of transcript distribution during embryogenesis. Both GRO1 and GRO2 exhibit all sequence features characteristic of the GROUCHO family and, with 79% sequence similarity at the DNA level, can be considered as orthologues of the human GROUCHO homologue TLE3. RNA in situ hybridization shows a distinct pattern of transcript distribution for both genes during embryogenesis suggestive of their participation in neurogenesis and somitogenesis.

Unique self-sustaining circadian oscillators within the brain of Drosophila melanogaster.

In Drosophila circadian rhythms persist in constant darkness (DD). The small ventral Lateral Neurons (s-LNv) mainly control the behavioral circadian rhythm in consortium with the large ventral Lateral Neurons (l-LNv) and dorsal Lateral Neurons (LNd). It is believed that the molecular oscillations of clock genes are the source of this persistent behavior. Indeed the s-LNv, LNd, Dorsal Neurons (DN)-DN2 and DN3 displayed self-sustained molecular oscillations in DD both at RNA and protein levels, except the DN2 oscillates in anti-phase. In contrast, the l-LNv and DN1 displayed self-sustained oscillations at the RNA level, but protein oscillations quickly dampened. Having self-sustained and dampened molecular oscillators together in the DN groups suggested that they play different roles. However, all DN groups seemed to contribute together to the light-dark (LD) behavioral rhythm. The LD entrainment of LN oscillators is achieved through Rhodopsin (RH) and Cryptochrome (CRY). CRY's expression in all DN groups implicates also its role in LD entrainment of DN, like in DN1. However, mutations in cry and glass that did not inflict LD synchronization of the DN2, DN3 oscillator implicate the existence of a novel photoreceptor at least in DN3.

Drosophila timeless2 is required for chromosome stability and circadian photoreception.

In Drosophila, there are two timeless paralogs, timeless1 (tim1) and timeless2 (tim2, or timeout). Phylogenetic analyses suggest that tim1 originated as a duplication of tim2 around the time of the Cambrian explosion. The function of tim1 as a canonical circadian component is well established, but the role of tim2 in the fly is poorly understood. Many organisms possess a single tim2-like gene that has been implicated in DNA synthesis and, in the case of mammals, somewhat controversially, in circadian rhythmicity. Here we analyze the structure and the functional role of fly tim2. tim2 is a large locus (approximately 75 kb) that harbors several transcribed intronic sequences. Using insertional mutations and tissue-specific RNA interference-mediated downregulation, we find that tim2 is an essential gene required for normal DNA metabolism and chromosome integrity. Moreover, tim2 is involved in light entrainment of the adult circadian clock, via its expression in the T1 basket cells of the optic lobes. tim2's residual role in light entrainment thus provides an evolutionary link that may explain why its derived paralog, tim1, came to play such a major role in both circadian photosensitivity and core clock function.

Blocking endocytosis in Drosophila's circadian pacemaker neurons interferes with the endogenous clock in a PDF-dependent way.

The neuropeptide pigment-dispersing factor (PDF) plays an essential role in the circadian clock of the fruit fly Drosophila melanogaster, but many details of PDF signaling in the clock network are still unknown. We tried to interfere with PDF signaling by blocking the GTPase Shibire in PDF neurons. Shibire is an ortholog of the mammalian Dynamins and is essential for endocytosis of clathrin-coated vesicles at the plasma membrane. Such endocytosis is used for neurotransmitter reuptake by presynaptic neurons, which is a prerequisite of synaptic vesicle recycling, and receptor-mediated endocytosis in the postsynaptic neuron, which leads to signal termination. By blocking Shibire function via overexpression of a dominant negative mutant form of Shibire in PDF neurons, we slowed down the behavioral rhythm by 3 h. This effect was absent in PDF receptor null mutants, indicating that we interfered with PDF receptor-mediated endocytosis. Because we obtained similar behavioral phenotypes by increasing the PDF level in regions close to PDF neurons, we conclude that blocking Shibire did prolong PDF signaling in the neurons that respond to PDF. Obviously, terminating the PDF signaling via receptor-mediated endocytosis is a crucial step in determining the period of behavioral rhythms.

Cryptochrome is present in the compound eyes and a subset of Drosophila's clock neurons.

Cryptochrome (CRY) is intimately associated with the circadian clock of many organisms. In the fruit fly Drosophila melanogaster, CRY seems to be involved in photoreception as well as in the core clockwork. In spite of the critical role of CRY for the clock of Drosophila, it was not quite clear whether CRY is expressed in every clock cell. With the help of a new antibody and a mutant that lacks CRY, we show here that CRY is expressed in specific subsets of Drosophila's pacemaker neurons and in the photoreceptor cells of the compound eyes. In the pacemaker neurons, CRY levels and kinetics under light-dark cycles are quite different from each other. High-amplitude oscillations are observed in only three groups of clock neurons, suggesting that these three groups are strongly receptive to light. The different CRY kinetics may account for phase differences in oscillations of the clock proteins observed in these three groups in earlier studies. The molecular clock of the neurons that contain lower CRY levels or are completely CRY negative can still be synchronized by light, probably via intercellular communication with the CRY-positive neurons as well as via external photoreceptors.

Moonlight shifts the endogenous clock of Drosophila melanogaster.

The ability to be synchronized by light-dark cycles is a fundamental property of circadian clocks. Although there are indications that circadian clocks are extremely light-sensitive and that they can be set by the low irradiances that occur at dawn and dusk, this has not been shown on the cellular level. Here, we demonstrate that a subset of Drosophila's pacemaker neurons responds to nocturnal dim light. At a nighttime illumination comparable to quarter-moonlight intensity, the flies increase activity levels and shift their typical morning and evening activity peaks into the night. In parallel, clock protein levels are reduced, and clock protein rhythms shift in opposed direction in subsets of the previously identified morning and evening pacemaker cells. No effect was observed on the peripheral clock in the eye. Our results demonstrate that the neurons driving rhythmic behavior are extremely light-sensitive and capable of shifting activity in response to the very low light intensities that regularly occur in nature. This sensitivity may be instrumental in adaptation to different photoperiods, as was proposed by the morning and evening oscillator model of Pittendrigh and Daan. We also show that this adaptation depends on retinal input but is independent of cryptochrome.

The expression of pannier and achaete-scute homologues in a mosquito suggests an ancient role of pannier as a selector gene in the regulation of the dorsal body pattern.

The Drosophila gene pannier (pnr) has recently been assigned to a new class of selector genes (Calleja, M., Herranz, H., Estella, C., Casal, J., Lawrence, P., Simpson, P. and Morata, G. (2000). Development 127, 3971-3980; (Mann, R. S. and Morata, G. (2000). ANNU: Rev. Cell Dev. Biol. 16, 243-271). It specifies pattern in the dorsal body. On the dorsal notum it is expressed in a broad medial domain and directly regulates transcription of the achaete-scute (ac-sc) genes driving their expression in small discrete clusters within this domain at the sites of each future bristle. This spatial resolution is achieved through modulation of Pnr activity by specific co-factors and by a number of discrete cis-regulatory enhancers in the ac-sc gene complex. We have isolated homologues of pnr and ac-sc in Anopheles gambiae, a basal species of Diptera that diverged from Drosophila melanogaster (Dm) about 200 million years ago, and examined their expression patterns. We found that an ac-sc homologue of Anopheles, Ag-ASH, is expressed on the dorsal medial notum at the sites where sensory organs emerge in several domains that are identical to those of the pnr homologue, Ag-pnr. This suggests that activation of Ag-ASH by Ag-Pnr has been conserved. Indeed, when expressed in Drosophila, Ag-pnr is able to mimic the effects of ectopic expression of Dm-pnr and induce ectopic bristles. These results are discussed in the context of the gene duplication events and the acquisition of a modular promoter, that may have occurred at different times in the lineage leading to derived species such as Drosophila. The bristle pattern of Anopheles correlates in a novel fashion with the expression domains of Ag-pnr/Ag-ASH. While precursors for the sensory scales can arise anywhere within the expression domains, bristle precursors arise exclusively along the borders. This points to the existence of specific positional information along the borders, and suggests that Ag-pnr specifies pattern in the medial, dorsal notum, as in Drosophila, but via a different mechanism.