Panoramix SUMOylation on chromatin connects the piRNA pathway to the cellular heterochromatin machinery.

Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors on binding to nascent target RNAs are poorly understood. Here, we explain the mechanism by which the PIWI-interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. We find that Panoramix, a corepressor required for piRNA-guided heterochromatin formation, is SUMOylated on chromatin in a Piwi-dependent manner. SUMOylation, together with an amphipathic LxxLL motif in Panoramix's intrinsically disordered repressor domain, are necessary and sufficient to recruit Small ovary (Sov), a multi-zinc-finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that eliminate the Panoramix-Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, Piwi engages the heterochromatin machinery specifically at transposon loci by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation.

A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary.

Argonaute proteins of the PIWI clade complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example, nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.

A Heterochromatin-Specific RNA Export Pathway Facilitates piRNA Production.

PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30 nt piRNAs are processed in the cytoplasm from long non-coding RNAs that often lack RNA processing hallmarks of export-competent transcripts. By studying how these transcripts achieve nuclear export, we uncover an RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1 and accumulates together with Bootlegger in peri-nuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. These findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to export unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.

A heterochromatin-dependent transcription machinery drives piRNA expression.

Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced is not understood. Here we show that, in Drosophila, transcription of PIWI-interacting RNA (piRNA) clusters-small RNA source loci in animal gonads-is enforced through RNA polymerase II pre-initiation complex formation within repressive heterochromatin. This is accomplished through Moonshiner, a paralogue of a basal transcription factor IIA (TFIIA) subunit, which is recruited to piRNA clusters via the heterochromatin protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA-box binding protein (TBP)-related factor TRF2, an animal TFIID core variant. Thus, transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that we argue is a recurring theme in evolution.

Automatic Segmentation of Drosophila Neural Compartments Using GAL4 Expression Data Reveals Novel Visual Pathways.

Identifying distinct anatomical structures within the brain and developing genetic tools to target them are fundamental steps for understanding brain function. We hypothesize that enhancer expression patterns can be used to automatically identify functional units such as neuropils and fiber tracts. We used two recent, genome-scale Drosophila GAL4 libraries and associated confocal image datasets to segment large brain regions into smaller subvolumes. Our results (available at https://strawlab.org/braincode) support this hypothesis because regions with well-known anatomy, namely the antennal lobes and central complex, were automatically segmented into familiar compartments. The basis for the structural assignment is clustering of voxels based on patterns of enhancer expression. These initial clusters are agglomerated to make hierarchical predictions of structure. We applied the algorithm to central brain regions receiving input from the optic lobes. Based on the automated segmentation and manual validation, we can identify and provide promising driver lines for 11 previously identified and 14 novel types of visual projection neurons and their associated optic glomeruli. The same strategy can be used in other brain regions and likely other species, including vertebrates.

Adaptive and Background-Aware GAL4 Expression Enhancement of Co-registered Confocal Microscopy Images.

GAL4 gene expression imaging using confocal microscopy is a common and powerful technique used to study the nervous system of a model organism such as Drosophila melanogaster. Recent research projects focused on high throughput screenings of thousands of different driver lines, resulting in large image databases. The amount of data generated makes manual assessment tedious or even impossible. The first and most important step in any automatic image processing and data extraction pipeline is to enhance areas with relevant signal. However, data acquired via high throughput imaging tends to be less then ideal for this task, often showing high amounts of background signal. Furthermore, neuronal structures and in particular thin and elongated projections with a weak staining signal are easily lost. In this paper we present a method for enhancing the relevant signal by utilizing a Hessian-based filter to augment thin and weak tube-like structures in the image. To get optimal results, we present a novel adaptive background-aware enhancement filter parametrized with the local background intensity, which is estimated based on a common background model. We also integrate recent research on adaptive image enhancement into our approach, allowing us to propose an effective solution for known problems present in confocal microscopy images. We provide an evaluation based on annotated image data and compare our results against current state-of-the-art algorithms. The results show that our algorithm clearly outperforms the existing solutions.

Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery.

The repression of transposable elements in eukaryotes often involves their transcriptional silencing via targeted chromatin modifications. In animal gonads, nuclear Argonaute proteins of the PIWI clade complexed with small guide RNAs (piRNAs) serve as sequence specificity determinants in this process. How binding of nuclear PIWI-piRNA complexes to nascent transcripts orchestrates heterochromatin formation and transcriptional silencing is unknown. Here, we characterize CG9754/Silencio as an essential piRNA pathway factor that is required for Piwi-mediated transcriptional silencing in Drosophila. Ectopic targeting of Silencio to RNA or DNA is sufficient to elicit silencing independently of Piwi and known piRNA pathway factors. Instead, Silencio requires the H3K9 methyltransferase Eggless/SetDB1 for its silencing ability. In agreement with this, SetDB1, but not Su(var)3-9, is required for Piwi-mediated transcriptional silencing genome-wide. Due to its interaction with the target-engaged Piwi-piRNA complex, we suggest that Silencio acts as linker between the sequence specificity factor Piwi and the cellular heterochromatin machinery.

Structure-based neuron retrieval across Drosophila brains.

Comparing local neural structures across large sets of examples is crucial when studying gene functions, and their effect in the Drosophila brain. The current practice of aligning brain volume data to a joint reference frame is based on the neuropil. However, even after alignment neurons exhibit residual location and shape variability that, together with image noise, hamper direct quantitative comparison and retrieval of similar structures on an intensity basis. In this paper, we propose and evaluate an image-based retrieval method for neurons, relying on local appearance, which can cope with spatial variability across the population. For an object of interest marked in a query case, the method ranks cases drawn from a large data set based on local neuron appearance in confocal microscopy data. The approach is based on capturing the orientation of neurons based on structure tensors and expanding this field via Gradient Vector Flow. During retrieval, the algorithm compares fields across cases, and calculates a corresponding ranking of most similar cases with regard to the local structure of interest. Experimental results demonstrate that the similarity measure and ranking mechanisms yield high precision and recall in realistic search scenarios.

Cellular and behavioral functions of fruitless isoforms in Drosophila courtship.

BACKGROUND: Male-specific products of the fruitless (fru) gene control the development and function of neuronal circuits that underlie male-specific behaviors in Drosophila, including courtship. Alternative splicing generates at least three distinct Fru isoforms, each containing a different zinc-finger domain. Here, we examine the expression and function of each of these isoforms. RESULTS: We show that most fru(+) cells express all three isoforms, yet each isoform has a distinct function in the elaboration of sexually dimorphic circuitry and behavior. The strongest impairment in courtship behavior is observed in fru(C) mutants, which fail to copulate, lack sine song, and do not generate courtship song in the absence of visual stimuli. Cellular dimorphisms in the fru circuit are dependent on Fru(C) rather than other single Fru isoforms. Removal of Fru(C) from the neuronal classes vAB3 or aSP4 leads to cell-autonomous feminization of arborizations and loss of courtship in the dark. CONCLUSIONS: These data map specific aspects of courtship behavior to the level of single fru isoforms and fru(+) cell types-an important step toward elucidating the chain of causality from gene to circuit to behavior.

Long persistence of importin-beta explains extended survival of cells and zygotes that lack the encoding gene.

Importin-beta is an essential component of nuclear protein import, spindle formation and nuclear envelope assembly. Formerly, the function of the Drosophila Ketel gene, which encodes importin-beta and is essential for the survival to adulthood, seemed to be required only in the mitotically active cells. We report here that importin-beta function is required in every cell and that this protein possesses an exceptionally long life span. Mosaic analysis, using gynanders, indicated that zygotic function of the Ketel gene is essential in a large group of cells in the embryos. Expression of a UAS-Ketel transgene by different tissue specific Gal4 drivers on ketel(null)/- hemizygous background revealed the requirement of Ketel gene function in the ectoderm. Elimination of the Ketel gene function using a UAS-Ketel-RNAi transgene driven by different Gal4 drivers confirmed the indispensability of the Ketel gene in the ectoderm. Using GFP-tagged importin-beta (encoded by a ketel(GFP) allele) we revealed that the maternally provided GFP-importin-beta molecules persist up to larval life. The zygotic Ketel gene is expressed in every cell during early gastrulation. Although the gene is then turned off in the non-dividing cells, the produced importin-beta molecules persist long and carry out nuclear protein import throughout the subsequent stages of development. In the continuously dividing diploid cells, the Ketel gene is constitutively expressed to fulfill all three functions of importin-beta.

Tubulin polymerization promoting proteins (TPPPs): members of a new family with distinct structures and functions.

TPPP/p25 is a brain-specific protein, which induces tubulin polymerization and microtubule (MT) bundling and is enriched in Lewy bodies characteristic of Parkinson's disease [Tirián et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 13976-13981]. We identified two human gene sequences, CG1-38 and p25beta, which encoded homologous proteins, that we termed p20 and p18, respectively. These homologous proteins display 60% identity with tubulin polymerization promoting protein/p25 (TPPP/p25); however, the N-terminal segment of TPPP/p25 is missing. They could be clustered into three subfamilies present in mammals and other vertebrates. We cloned, isolated, and characterized the structural and functional properties of the recombinant human proteins at molecular, ultrastructural, and cellular levels using a number of tools. These data revealed that, while p20 behaved as a disorganized protein similarly to TPPP/p25, which was described as a flexible and inherently dynamic protein with a long unstructured N-terminal tail, p18 was featured in more ordered fashion. TPPP/p25 and p20 specifically attached to MTs causing MT bundling both in vitro and in vivo; p18 protein did not cross-link MTs, and it distributed homogeneously within the cytosol of the transfected HeLa cells. These data indicate that the two shorter homologues display distinct structural features that determine their associations to MTs. The properties of p20 resemble TPPP/p25. The bundling activity of these two proteins results in the stabilization of the microtubular network, which is likely related to their physiological functions.

Neural circuitry that governs Drosophila male courtship behavior.

Male-specific fruitless (fru) products (Fru(M)) are both necessary and sufficient to "hardwire" the potential for male courtship behavior into the Drosophila nervous system. Fru(M) is expressed in approximately 2% of neurons in the male nervous system, but not in the female. We have targeted the insertion of GAL4 into the fru locus, allowing us to visualize and manipulate the Fru(M)-expressing neurons in the male as well as their counterparts in the female. We present evidence that these neurons are directly and specifically involved in male courtship behavior and that at least some of them are interconnected in a circuit. This circuit includes olfactory neurons required for the behavioral response to sex pheromones. Anatomical differences in this circuit that might account for the dramatic differences in male and female sexual behavior are not apparent.

Dynamic targeting of microtubules by TPPP/p25 affects cell survival.

Recently we identified TPPP/p25 (tubulin polymerization promoting protein/p25) as a brain-specific unstructured protein that induced aberrant microtubule assemblies and ultrastructure in vitro and as a new marker for Parkinson's disease and other synucleopathies. In this paper the structural and functional consequences of TPPP/p25 are characterized to elucidate the relationship between the in vitro and the pathological phenomena. We show that at low expression levels EGFP-TPPP/p25 specifically colocalizes with the microtubule network of HeLa and NRK cells. We found that the colocalization was dynamic (tg=5 seconds by fluorescence recovery after photobleaching) and changed during the phases of mitosis. Time-lapse and immunofluorescence experiments revealed that high levels of EGFP-TPPP/p25 inhibited cell division and promoted cell death. At high expression levels or in the presence of proteosome inhibitor, green fusion protein accumulated around centrosomes forming an aggresome-like structure protruding into the nucleus or a filamentous cage of microtubules surrounding the nucleus. These structures showed high resistance to vinblastin. We propose that a potential function of TPPP/p25 is the stabilization of physiological microtubular ultrastructures, however, its upregulation may directly or indirectly initiate the formation of aberrant protein aggregates such as pathological inclusions.

Natively unfolded tubulin polymerization promoting protein TPPP/p25 is a common marker of alpha-synucleinopathies.

The novel basic, heat-stable tubulin polymerization promoting protein TPPP/p25 is associated with microtubules in vitro and can induce the formation of aberrant microtubule assemblies. We show by 1H-NMR spectroscopy that TPPP/p25 is natively unfolded. Antisera against peptide 186GKGKAGRVDLVDESG200NH2 (186-200) are highly specific to TPPP/p25. Immunohistochemistry and confocal microscopy demonstrates that TPPP/p25 is enriched in filamentous alpha-synuclein bearing Lewy bodies of Parkinson's (PD) and diffuse Lewy body disease (DLBD), as well as glial inclusions of multiple system atrophy (MSA). There is a correlation between TPPP/p25 and alpha-synuclein immunoreactivity in Western blot. In contrast, TPPP/p25 is not associated with abnormally phosphorylated tau in various inclusions of Pick's disease (PiD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). However, electron microscopy confirms clusters of TPPP/p25 immunoreactivity along filaments of unstructured but not compact neurofibrillary tangles in Alzheimer's disease (AD). TPPP/p25 seems to be a novel marker of alpha-synucleinopathies.

P446L-importin-beta inhibits nuclear envelope assembly by sequestering nuclear envelope assembly factors to the microtubules.

The P446L mutant Drosophila importin-beta (P446L-imp-beta) has been reported to prohibit--in dominant negative fashion--nuclear envelope (NE) assembly. Along elucidating the mode of action of P446L-imp-beta we studied in vitro NE assembly on Sepharose beads. While Drosophila embryo extracts support NE assembly over Sepharose beads coated with Ran, NE assembly does not take place in extracts supplied with exogenous P446L-imp-beta. A NE also forms over importin-beta-coated beads. Surprisingly, when immobilized to Sepharose beads P446L-imp-beta as efficiently recruits NE vesicles as normal importin-beta. The discrepancy in behavior of cytoplasmic and bead-bound P446L-imp-beta appears to be related to icreased--as compared to normal importin-beta--microtubule (MT) binding ability of P446L-imp-beta. While wild-type importin-beta is able to bind MTs and the binding decreases upon RanGTP interaction, P446L-imp-beta cannot be removed from the MTs by RanGTP. P446L-imp-beta, like normal importin-beta, binds some types of the nucleoporins that have been known to be required for NE assembly at the end of mitosis. It appears that the inhibitory effect of P446L-imp-beta on NE assembly is caused by sequestering some of the nucleoporins required for NE assembly to the MTs.

The importin-beta P446L dominant-negative mutant protein loses RanGTP binding ability and blocks the formation of intact nuclear envelope.

Three of the four independently induced Ketel(D) dominantnegative female sterile mutations that identify the Drosophila importin-beta gene, originated from a C4114--> T transition and the concurrent replacement of Pro446 by Leu (P446L). CD spectroscopy of representative peptides with Pro or Leu in the crucial position revealed that upon the Pro-->Leu exchange the P446L mutant protein loses flexibility and attains most likely an open conformation. The P446L mutation abolishes RanGTP binding of the P446L mutant form of importin-beta protein and results in increased RanGDP binding ability. Notably, the P446L mutant importin-beta does not exert its dominant-negative effect on nuclear protein import and has no effect on mitotic spindle-related functions and chromosome segregation. However, it interferes with nuclear envelope formation during mitosis-to-interphase transition, revealing a novel function of importin-beta.