Shane Scoggin

Identification of NF-κB-regulated genes induced by TNFα utilizing expression profiling and RNA interference

Tumor necrosis factor α (TNFα) is a proinflammatory cytokine with important roles in regulating inflammatory responses as well as cell cycle proliferation and apoptosis. Although TNFα stimulates apoptosis, it also activates the transcription factor NF-κB, and studies have shown that inhibition of NF-κB potentiates the cytotoxicity of TNFα. Since several chemotherapy agents act like TNFα to both promote apoptosis and activate NF-κB, understanding the role of NF-κB in suppressing apoptosis may have significant clinical applications. To understand the effects of stimulation with TNFα and the role of NF-κB in regulating this response, a 23k human cDNA microarray was used to screen TNFα-inducible genes in HeLa cells. Real-time PCR verified expression changes in 16 of these genes and revealed three distinct temporal patterns of expression after TNFα stimulation. Using RNA interference to disrupt expression of the p65 subunit of NF-κB, all but two of the genes were shown to depend on this transcription factor for their expression, which correlated well with the existence of NF-κB binding sites in most of their promoters. Inflammatory, proapoptotic, and antiapoptotic genes were all shown to be regulated by NF-κB, demonstrating the wide variety of targets activated by NF-κB signaling and the necessity of differentiating among these genes for therapeutic purposes.

Differential Gene Expression Analysis Reveals Generation of an Autocrine Loop by a Mutant Epidermal Growth Factor Receptor in Glioma Cells

The epidermal growth factor receptor (EGFR) gene is commonly amplified and rearranged in glioblastoma multiforme leading to overexpression of wild-type and mutant EGFRs. Expression of wild-type EGFR ligands, such as transforming growth factor-alpha (TGF-alpha) or heparin-binding EGF (HB-EGF), is also often increased in gliomas resulting in an autocrine loop that contributes to the growth autonomy of glioma cells. Glioblastoma multiformes express a characteristic EGFR mutant (EGFRvIII, de 2-7) that does not bind ligand, signals constitutively, and is more tumorigenic than the wild-type receptor. However, the downstream signals that mediate this increased tumorigenicity are not well understood. We hypothesized that signals induced specifically by EGFRvIII and not the wild-type receptor are more likely to mediate its increased tumorigenic activity and examined the gene expression profiles resulting from inducible expression of comparable levels of either wild-type EGFR or EGFRvIII in a U251-MG glioma cell line. Expression of EGFRvIII resulted in specific up-regulation of a small group of genes. Remarkably, all these genes, which include TGFA, HB-EGF, EPHA2, IL8, MAP4K4, FOSL1, EMP1, and DUSP6, influence signaling pathways known to play a key role in oncogenesis and function in interconnected networks. Increased expression of EGFRvIII-induced genes was validated by real-time PCR. The mutant receptor does not bind ligand, and EGFRvIII-induced expression of TGF-alpha and HB-EGF suggests that EGFRvIII plays a role in generating an autocrine loop using the wild-type EGFR in glioma. It also raises the possibility that EGFRvIII may signal, at least in part, through the wild-type receptor. Indeed, we show that inhibiting the activity of HB-EGF, a potent mitogen, with neutralizing antibodies reduces cell proliferation induced by expression of EGFRvIII. This suggests that the EGFRvIII-HB-EGF-wild-type EGFR autocrine loop plays an important role in signal transduction by EGFRvIII in glioma cells. We also show by immunohistochemistry that HB-EGF expression correlates with the presence of EGFRvIII in glioblastoma multiforme. Thus, our study provides a new insight into oncogenic signaling by EGFRvIII and improves our understanding of how autocrine loops are generated in glioma.

Systematic Discovery of Rab GTPases with Synaptic Functions in Drosophila

BACKGROUND Neurons require highly specialized intracellular membrane trafficking, especially at synapses. Rab GTPases are considered master regulators of membrane trafficking in all cells, and only very few Rabs have known neuron-specific functions. Here, we present the first systematic characterization of neuronal expression, subcellular localization, and function of Rab GTPases in an organism with a brain. RESULTS We report the surprising discovery that half of all Drosophila Rabs function specifically or predominantly in distinct subsets of neurons in the brain. Furthermore, functional profiling of the GTP/GDP-bound states reveals that these neuronal Rabs are almost exclusively active at synapses and the majority of these synaptic Rabs specifically mark synaptic recycling endosomal compartments. Our profiling strategy is based on Gal4 knockins in large genomic fragments that are additionally designed to generate mutants by ends-out homologous recombination. We generated 36 large genomic targeting vectors and transgenic rab-Gal4 fly strains for 25 rab genes. Proof-of-principle knockout of the synaptic rab27 reveals a sleep phenotype that matches its cell-specific expression. CONCLUSIONS Our findings suggest that up to half of all Drosophila Rabs exert specialized synaptic functions. The tools presented here allow systematic functional studies of these Rabs and provide a method that is applicable to any large gene family in Drosophila.

The Wnt pathway limits BMP signaling outside of the germline stem cell niche in Drosophila ovaries

The mechanisms that modulate and limit the signaling output of adult stem cell niches remain poorly understood. To gain further insights into how these microenvironments are regulated in vivo, we performed a candidate gene screen designed to identify factors that restrict BMP signal production to the cap cells that comprise the germline stem cell (GSC) niche of Drosophila ovaries. Through these efforts, we found that disruption of Wnt4 and components of the canonical Wnt pathway results in a complex germ cell phenotype marked by an expansion of GSC-like cells, pre-cystoblasts and cystoblasts in young females. This phenotype correlates with an increase of decapentaplegic (dpp) mRNA levels within escort cells and varying levels of BMP responsiveness in the germline. Further genetic experiments show that Wnt4, which exhibits graded expression in somatic cells of germaria, activates the Wnt pathway in posteriorly positioned escort cells. The activation of the Wnt pathway appears to be limited by the BMP pathway itself, as loss of Mad in escort cells results in the expansion of Wnt pathway activation. Wnt pathway activity changes within germaria during the course of aging, coincident with changes in dpp production. These data suggest that mutual antagonism between the BMP and Wnt pathways in somatic cells helps to regulate germ cell differentiation.

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Combining recombineering and ends-out homologous recombination to systematically characterize Drosophila gene families: Rab GTPases as a case study

Evaluating how an individual gene contributes to a particular biological process benefits greatly from a comprehensive understanding of all members of its gene family. Such knowledge is ideally obtained using multicellular model organisms, which provide rapid and decisive platforms for determining gene function. We recently established a novel transgenesis platform in Drosophila to systematically knock out all members of the Rab small GTPase family of membrane regulators. This platform combines BAC transgenesis/recombineering with ends-out homologous recombinations and GatewayTM technologies and provides a new rapid and scalable method that eases the manipulation of endogenous loci. This method not only allows for the generation of molecularly defined lesions, but also the precise replacement or tagging of genes in their endogenous loci. Using this method, we found that up to half of all Rab GTPases exhibit enriched expression at synapses in the nervous system. Here we provide critical details about the underlying recombineering and transgenesis method, new cassettes for tagging endogenous loci and information on important parameters that will allow Drosophila researchers to target members of other gene families.

Systematic discovery of genetic modulation by Jumonji histone demethylases in Drosophila

Jumonji (JmjC) domain proteins influence gene expression and chromatin organization by way of histone demethylation, which provides a means to regulate the activity of genes across the genome. JmjC proteins have been associated with many human diseases including various cancers, developmental and neurological disorders, however, the shared biology and possible common contribution to organismal development and tissue homeostasis of all JmjC proteins remains unclear. Here, we systematically tested the function of all 13 Drosophila JmjC genes. Generation of molecularly defined null mutants revealed that loss of 8 out of 13 JmjC genes modify position effect variegation (PEV) phenotypes, consistent with their ascribed role in regulating chromatin organization. However, most JmjC genes do not critically regulate development, as 10 members are viable and fertile with no obvious developmental defects. Rather, we find that different JmjC mutants specifically alter the phenotypic outcomes in various sensitized genetic backgrounds. Our data demonstrate that, rather than controlling essential gene expression programs, Drosophila JmjC proteins generally act to “fine-tune” different biological processes.

Drosophila CG2469 Encodes a Homolog of Human CTR9 and Is Essential for Development.

Conserved from yeast to humans, the Paf1 complex participates in a number of diverse processes including transcriptional initiation and polyadenylation. This complex typically includes five proteins: Paf1, Rtf1, Cdc73, Leo1, and Ctr9. Previous efforts identified clear Drosophila homologs of Paf1, Rtf1, and Cdc73 based on sequence similarity. Further work showed that these proteins help to regulate gene expression and are required for viability. To date, a Drosophila homolog of Ctr9 has remained uncharacterized. Here, we show that the gene CG2469 encodes a functional Drosophila Ctr9 homolog. Both human and Drosophila Ctr9 localize to the nuclei of Drosophila cells and appear enriched in histone locus bodies. RNAi knockdown of Drosophila Ctr9 results in a germline stem cell loss phenotype marked by defects in the morphology of germ cell nuclei. A molecular null mutation of Drosophila Ctr9 results in lethality and a human cDNA CTR9 transgene rescues this phenotype. Clonal analysis in the ovary using this null allele reveals that loss of Drosophila Ctr9 results in a reduction of global levels of histone H3 trimethylation of lysine 4 (H3K4me3), but does not compromise the maintenance of stem cells in ovaries. Given the differences between the null mutant and RNAi knockdown phenotypes, the germ cell defects caused by RNAi likely result from the combined loss of Drosophila Ctr9 and other unidentified genes. These data provide further evidence that the function of this Paf1 complex component is conserved across species.

Erratum: Identification of NF-κB-regulated genes induced by TNFα utilizing expression profiling and RNA interference (Oncogene (2003) 22 (2054-2064) doi:10.1038/sj.onc.1206262)

Correction to: Oncogene (2003) 22, 2054–2064. doi: 10.1038/sj.onc.1206262 Since the publication of the above manuscript, the authors have identified an error in the sequence of the p65 siRNA oligonucleotide as listed in the study. The error is on page 2062, under Materials and methods, SiRNAi transfection and RNA preparation.