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The Drosophila Su(var)3-7 gene is required for oogenesis and female fertility, genetically interacts with piwi and aubergine, but impacts only weakly transposon silencing ...
Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. We present evidences that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
The Drosophila jing gene is a downstream target in the Trachealess/Tango tracheal pathway.
Primary branching in the Drosophila trachea is regulated by the Trachealess (Trh) and Tango (Tgo) basic helix-loop-helix-PAS (bHLH-PAS) heterodimers, the POU protein Drifter (Dfr)/Ventral Veinless (Vvl), and the Pointed (Pnt) ETS transcription factor. The jing gene encodes a zinc finger protein also required for tracheal development. Three Trh/Tgo DNA-binding sites, known as CNS midline elements, in 1.5 kb of jing 5′ cis-regulatory sequence (jing1.5) previously suggested a downstream role for jing in the pathway. Here, we show that jing is a direct downstream target of Trh/Tgo and that Vvl and Pnt are also involved in jing tracheal activation. In vivo lacZ enhancer detection assays were used to identify cis-regulatory elements mediating embryonic expression patterns of jing. A 2.8-kb jing enhancer (jing2.8) drove lacZ expression in all tracheal cell lineages, the CNS midline and Engrailed-positive segmental stripes, mimicking endogenous jing expression. A 1.3-kb element within jing2.8 drove expression that was restricted to Engrailed-positive CNS midline cells and segmental ectodermal stripes. Surprisingly, jing1.5-lacZ expression was restricted to tracheal fusion cells despite the presence of consensus DNA-binding sites for bHLH-PAS, ETS, and POU domain transcription factors. Given the absence of Trh/Tgo DNA-binding sites in the jing1.3 enhancer, these results are consistent with previous observations suggesting a combinatorial basis to Trh-/Tgo-mediated transcriptional regulation in the trachea.
Document Type:

Reference

Product Catalog Number:

17-371
Product Catalog Name:

EZ-ChIP™
Novel Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex-associated Protein (HOAP) Repeat Motif in HP1/HOAP Interactions and Chromocenter Associations
Document Type:

Reference

Product Catalog Number:

12-430
Product Catalog Name:

Dimethyl-Histone H3 (Lys9) Peptide, biotin conjugate
The Drosophila pumilio gene encodes two functional protein isoforms that play multiple roles in germline development, gonadogenesis, oogenesis and embryogenesis.
The pumilio (pum) gene plays an essential role in embryonic patterning and germline stem cell (GSC) maintenance during oogenesis in Drosophila. Here we report on a phenotypic analysis using pum(ovarette) mutations, which reveals multiple functions of pum in primordial germ cell proliferation, larval ovary formation, GSC division, and subsequent oogenic processes, as well as in oviposition. Specifically, by inducing pum(-) GSC clones at the onset of oogenesis, we show that pum is directly involved in GSC division, a function that is distinct from its requirement in primordial germ cells. Furthermore, we show that pum encodes 156- and 130-kD proteins, both of which are functional isoforms. Among pum(ovarette) mutations, pum(1688) specifically eliminates the 156-kD isoform but not the 130-kD isoform, while pum(2003) and pum(4277) specifically affect the 130-kD isoform but not the 156-kD isoform. Normal doses of both isoforms are required for the zygotic function of pum, yet either isoform alone at a normal dose is sufficient for the maternal effect function of pum. A pum cDNA transgene that contains the known open reading frame encodes only the 156-kD isoform and rescues the phenotype of both pum(1688) and pum(2003) mutants. These observations suggest that the 156- and 130-kD isoforms can compensate for each other's function in a dosage-dependent manner. Finally, we present molecular evidence suggesting that the two PUM isoforms share some of their primary structures.
Document Type:

Reference

Product Catalog Number:

03-242
The Drosophila melanogaster CHD1 chromatin remodeling factor modulates global chromosome structure and counteracts HP1a and H3K9me2.
CHD1 is a conserved chromatin remodeling factor that localizes to active genes and functions in nucleosome assembly and positioning as well as histone turnover. Mouse CHD1 is required for the maintenance of stem cell pluripotency while human CHD1 may function as a tumor suppressor. To investigate the action of CHD1 on higher order chromatin structure in differentiated cells, we examined the consequences of loss of CHD1 and over-expression of CHD1 on polytene chromosomes from salivary glands of third instar Drosophila melanogaster larvae. We observed that chromosome structure is sensitive to the amount of this remodeler. Loss of CHD1 resulted in alterations of chromosome structure and an increase in the heterochromatin protein HP1a, while over-expression of CHD1 disrupted higher order chromatin structure and caused a decrease in levels of HP1a. Over-expression of an ATPase inactive form of CHD1 did not result in severe chromosomal defects, suggesting that the ATPase activity is required for this in vivo phenotype. Interestingly, changes in CHD1 protein levels did not correlate with changes in the levels of the euchromatin mark H3K4me3 or elongating RNA Polymerase II. Thus, while CHD1 is localized to transcriptionally active regions of the genome, it can function to alter the levels of HP1a, perhaps through changes in methylation of H3K9.
Document Type:

Reference

Product Catalog Number:

05-745R
Product Catalog Name:

Anti-trimethyl-Histone H3 (Lys4) Antibody, clone 15-10C-E4, rabbit monoclonal
The Drosophila suppressor of sable protein binds to RNA and associates with a subset of polytene chromosome bands.
Mutations of the Drosophila melanogaster suppressor of sable [su(s)] gene, which encodes a 150-kDa nuclear protein [Su(s)], increase the accumulation of specific transcripts in a manner that is not well understood but that appears to involve pre-mRNA processing. Here, we report biochemical analysis of purified, recombinant Su(s) [rSu(s)] expressed in baculovirus and in Escherichia coli as maltose binding protein (MBP) fusions and immunocytochemical analysis of endogenous Su(s). This work has shown that purified, baculovirus-expressed rSu(s) binds to RNA in vitro with a high affinity and limited specificity. Systematic evolution of ligands by exponential enrichment was used to identify preferred RNA targets of rSu(s), and a large proportion of RNAs isolated contain a full or partial match to the consensus sequence UCAGUAGUCU, which was confirmed to be a high-affinity rSu(s) binding site. An MBP-Su(s) fusion protein containing the N-terminal third of Su(s) binds RNAs containing this sequence with a higher specificity than full-length, baculovirus-expressed rSu(s). The consensus sequence resembles both a cryptic 5' splice site and a sequence that is found near the 5' end of some Drosophila transcripts. Immunolocalization studies showed that endogenous Su(s) is distributed in a reticulated pattern in Drosophila embryo and salivary gland nuclei. In salivary gland cells, Su(s) is found both in the nucleoplasm and in association with a subset of polytene chromosome bands. Considering these and previous results, we propose two models to explain how su(s) mutations affect nuclear pre-mRNA processing.
Document Type:

Reference

Product Catalog Number:

MAB1276
Product Catalog Name:

Anti-Nuclei & Chromosomes Antibody, histone H1 protein, clone 1415-1
The Drosophila RNA-binding protein HOW controls the stability of dgrasp mRNA in the follicular epithelium.
Post-transcriptional regulation of RNA stability and localization underlies a wide array of developmental processes, such as axon guidance and epithelial morphogenesis. In Drosophila, ectopic expression of the classically Golgi peripheral protein dGRASP at the plasma membrane is achieved through its mRNA targeting at key developmental time-points, in a process critical to follicular epithelium integrity. However, the trans-acting factors that tightly regulate the spatio-temporal dynamics of dgrasp are unknown. Using an in silico approach, we identified two putative HOW Response Elements (HRE1 and HRE2) within the dgrasp open reading frame for binding to Held Out Wings (HOW), a member of the Signal Transduction and Activation of RNA family of RNA-binding proteins. Using RNA immunoprecipitations, we confirmed this by showing that the short cytoplasmic isoform of HOW binds directly to dgrasp HRE1. Furthermore, HOW loss of function in vivo leads to a significant decrease in dgrasp mRNA levels. We demonstrate that HRE1 protects dgrasp mRNA from cytoplasmic degradation, but does not mediate its targeting. We propose that this binding event promotes the formation of ribonucleoprotein particles that ensure dgrasp stability during transport to the basal plasma membrane, thus enabling the local translation of dgrasp for its roles at non-Golgi locations.
Document Type:

Reference

Product Catalog Number:

17-700
Product Catalog Name:

Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit
The Drosophila serine protease homologue Scarface regulates JNK signalling in a negative-feedback loop during epithelial morphogenesis.
In Drosophila melanogaster, dorsal closure is a model of tissue morphogenesis leading to the dorsal migration and sealing of the embryonic ectoderm. The activation of the JNK signal transduction pathway, specifically in the leading edge cells, is essential to this process. In a genome-wide microarray screen, we identified new JNK target genes during dorsal closure. One of them is the gene scarface (scaf), which belongs to the large family of trypsin-like serine proteases. Some proteins of this family, like Scaf, bear an inactive catalytic site, representing a subgroup of serine protease homologues (SPH) whose functions are poorly understood. Here, we show that scaf is a general transcriptional target of the JNK pathway coding for a secreted SPH. scaf loss-of-function induces defects in JNK-controlled morphogenetic events such as embryonic dorsal closure and adult male terminalia rotation. Live imaging of the latter process reveals that, like for dorsal closure, JNK directs the dorsal fusion of two epithelial layers in the pupal genital disc. Genetic data show that scaf loss-of-function mimics JNK over-activity. Moreover, scaf ectopic expression aggravates the effect of the JNK negative regulator puc on male genitalia rotation. We finally demonstrate that scaf acts as an antagonist by negatively regulating JNK activity. Overall, our results identify the SPH-encoding gene scaf as a new transcriptional target of JNK signalling and reveal the first secreted regulator of the JNK pathway acting in a negative-feedback loop during epithelial morphogenesis.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
The Drosophila basic helix-loop-helix protein DIMMED directly activates PHM, a gene encoding a neuropeptide-amidating enzyme.
The basic helix-loop-helix (bHLH) protein DIMMED (DIMM) supports the differentiation of secretory properties in numerous peptidergic cells of Drosophila melanogaster. DIMM is coexpressed with diverse amidated neuropeptides and with the amidating enzyme peptidylglycine alpha-hydroxylating monooxygenase (PHM) in approximately 300 cells of the late embryo. Here we confirm that DIMM has transcription factor activity in transfected HEK 293 cells and that the PHM gene is a direct target. The mammalian DIMM orthologue MIST1 also transactivated the PHM gene. DIMM activity was dependent on the basic region of the protein and on the sequences of three E-box sites within PHM's first intron; the sites make different contributions to the total activity. These data suggest a model whereby the three E boxes interact cooperatively and independently to produce high PHM transcriptional activation. This DIMM-controlled PHM regulatory region displayed similar properties in vivo. Spatially, its expression mirrored that of the DIMM protein, and its activity was largely dependent on dimm. Further, in vivo expression was highly dependent on the sequences of the same three E boxes. This study supports the hypothesis that DIMM is a master regulator of a peptidergic cell fate in Drosophila and provides a detailed transcriptional mechanism of DIMM action on a defined target gene.
Document Type:

Reference

Product Catalog Number:

AB3080
Product Catalog Name:

Anti-Green Fluorescent Protein Antibody
Drosophila STAT is required for directly maintaining HP1 localization and heterochromatin stability.
STAT (Signal transducer and activator of transcription) is a potent transcription factor and its aberrant activation by phosphorylation is associated with human cancers. We have shown previously that overactivation of JAK, which phosphorylates STAT, disrupts heterochromatin formation globally in Drosophila melanogaster. However, it remains unclear how this effect is mediated and whether STAT is involved. Here, we demonstrate that Drosophila STAT (STAT92E) is involved in controlling heterochromatin protein 1 (HP1) distribution and heterochromatin stability. We found, unexpectedly, that loss of STAT92E, had the same effects as overactivation of JAK in disrupting heterochromatin formation and heterochromatic gene silencing, whereas overexpression of STAT92E had the opposite effects. We have further shown that the unphosphorylated or 'transcriptionally inactive' form of STAT92E is localized on heterochromatin in association with HP1, and is required for stabilizing HP1 localization and histone H3 Lys 9 methylation (H3mK9) . However, activation by phosphorylation reduces heterochromatin-associated STAT92E, causing HP1 displacement and heterochromatin destabilization. Thus, reducing levels of unphosphorylated STAT92E, either by loss of STAT92E or increased phosphorylation, causes heterochromatin instability. These results suggest that activation of STAT by phosphorylation controls both access to chromatin and activity of the transcription machinery.
Document Type:

Reference

Product Catalog Number:

07-212
Product Catalog Name:

Anti-dimethyl-Histone H3 (Lys9) Antibody