Drosophila genome-wide RNAi screens: are they delivering the promise?

The emergence of RNA interference (RNAi) on the heels of the successful completion of the Drosophila genome project was seen by many as the ace in functional genomics: Its application would quickly assign a function to all genes in this organism and help delineate the complex web of interactions or networks linking them at the systemic level. A few years wiser and a number of genome-wide Drosophila RNAi screens later, we reflect on the state of high-throughput RNAi screens in Drosophila and ask whether the initial promise was fulfilled. We review the impact that this approach has had in the field of Drosophila research and chart out strategies to extract maximal benefit from the application of RNAi to gene discovery and pursuit of systems biology.

Hydrophobic residues Phe751 and Leu753 are essential for STAT5 transcriptional activity.

One facet of cytokine signaling is relayed to the nucleus by the activation, through tyrosine phosphorylation, of latent cytoplasmic signal transducers and activators of transcription (STAT) family members. It has been demonstrated that the C termini of STATs contain the transactivation domain and are essential for the transactivation of target genes. To better understand the function of the STAT C terminus, we have generated a series of C-terminal mutants in STAT5a and examined their effects on transactivation, tyrosine phosphorylation, and DNA binding. Using GAL4 chimerae with the C terminus of STAT5, we have defined a 12-amino acid region essential for STAT5 transactivation. Surprisingly, deletion of these 12 amino acids in the context of the native STAT5 backbone preserved the overall transcriptional activity of the protein. Further analysis revealed that deletion of this region resulted in hyper-DNA binding activity, thus compensating for the weakened transactivation domain. Using site-directed mutagenesis, we show that within this 12-amino acid region the acidic residues were non-essential for transactivation. In contrast, the non-acidic residues were crucial for transactivation. Mutating either Phe(751) or Leu(753) to alanine abolished transactivation suggesting that these residues were essential for connecting STAT5 to the basal transcriptional machinery.

Rapid selection of tetracycline-controlled inducible cell lines using a green fluorescent-transactivator fusion protein.

We describe a modification of the tetracycline-controlled expression system that facilitates the rapid identification of tetracycline-sensitive clones. The TetR/VP16 transactivator protein was tagged with the green fluorescent protein (GFP) at its N-terminus. This results in a functional transactivator which allows cells expressing high levels of the modified transactivator to be selected by fluorescent-activated cell sorting. After expansion, single cell clones that maintain a high level of GFP fluorescence can be tested for their ability to transactivate a luciferase gene under control of the Tet operator, leading to the rapid identification of clones with strong inducibility.

Interleukin-3-induced activation of the JAK/STAT pathway is prolonged by proteasome inhibitors.

One facet of cytokine receptor signaling involves the activation of signal transducers and activators of transcription (STATs). STATs are rapidly activated via tyrosine phosphorylation by Janus kinase (JAK) family members and subsequently inactivated within a short period. We investigated the effect of proteasome inhibition on interleukin-3 (IL-3) activation of the JAK/STAT pathway following stimulation of Ba/F3 cells. Treatment of Ba/F3 cells with the proteasome inhibitor, N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL), led to stable tyrosine phosphorylation of the IL-3 receptor, beta common (betac), and STAT5 following stimulation. The effects of LLnL were not restricted to the JAK/STAT pathway, as Shc and mitogen-activated protein kinase (MAPK) phosphorylation were also prolonged in LLnL-treated cells. Further investigation showed these stable phosphorylation events were the result of prolonged activation of JAK2 and JAK1. These observations were confirmed using pharmacologic inhibitors. In the presence of LLnL, stable phosphorylation of STAT5 and betac was abrogated if the tyrosine kinase inhibitor, staurosporine, was added. The effect of staurosporine on STAT5 phosphorylation could be overcome if the phosphatase inhibitor, vanadate, was also added, suggesting phosphorylated STAT5 could be stabilized by phosphatase, but not by proteasome inhibition per se. These observations are consistent with the hypothesis that proteasome-mediated protein degradation can modulate the activity of the JAK/STAT pathway by regulating the deactivation of JAK.

JAK2 is required for induction of the murine DUB-1 gene.

Cytokine receptors activate multiple signal transduction pathways, resulting in the induction of specific target genes. We have recently identified a hematopoietic cell-specific immediate-early gene, DUB-1, that encodes a growth-regulatory deubiquitinating enzyme. The DUB-1 gene contains a 112-bp enhancer element that is specifically induced by the beta c subunit of the interleukin-3 (IL-3) receptor. To investigate the mechanism of DUB-1 induction, we examined the effects of dominant-negative forms of JAK kinases, STAT transcription factors, and Raf-1 in transient transfection assays. In Ba/F3 cells, IL-3 induced a dose-dependent activation of DUB-1-luciferase (luc) and GAS-luc reporter constructs. A dominant-negative form of JAK2 (truncated at amino acid 829) inhibited the induction of DUB-1-luc and GAS-luc by IL-3. A dominant-negative form of STAT5 (truncated at amino acid 650) inhibited the induction of GAS-luc but not DUB-1-luc. A dominant-negative form of Raf-1 inhibited the induction of DUB-1-luc but had no effect on the induction of GAS-luc by IL-3. The requirement for JAK2 in the stimulation of the DUB-1 enhancer was further supported by the suppression of DUB-1 induction in Ba/F3 cells stably expressing the dominant-negative JAK2 polypeptide. We hypothesize that IL-3 activates a JAK2/Raf-1 signaling pathway that is required for DUB-1 induction and is independent of STAT5.

Receptors that induce erythroid differentiation of Ba/F3 cells: structural requirements and effect on STAT5 binding.

Ectopic expression of the erythropoietin receptor (EpoR) in the interleukin-3 (IL-3)-dependent cell line Ba/F3 results in growth and partial erythroid differentiation in Epo. In contrast, introduction and activation of the interleukin-5 receptor (IL-5R) or of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) results in proliferation only. As this effect is specific to the EpoR, the role of its extracellular or cytoplasmic domain in differentiation was tested after construction of two chimeric receptors. One receptor contained the extracellular domain of EpoR fused to the endodomain of IL-3R beta-chain (E/beta), while the other contained the EpoR cytoplasmic region fused to the extracellular domain of GM-CSFR alpha-chain (GMER). Surprisingly, both receptors induced differentiation ruling out a strict specificity of the extracellular or cytoplasmic region of EpoR in this process. Instead the ability to signal differentiation correlated with structural features shared by the EpoR, GMER, and E/beta receptors. Dimerization of all three receptors results in the pairing of two signal transducing chains in the cytoplasm, in contrast to the mitogenic receptors IL-3R, IL-5R, GM-CSFR, which assemble as alphabeta heterodimers. Two new chimeric receptors that fulfilled the structural requirement exemplified by EpoR, but lacked any part of EpoR, were designed to consolidate this model. They consisted of the ectodomains of the GMR-alpha and IL-5R alpha, respectively, fused to the endodomain of IL-3R beta-chain. Both receptors were as effective as EpoR in signaling differentiation in response to their cognate ligand. Another property of receptors fulfilling these structural requirements is that they cause a marked delay in signal transducers and activators of transcription 5 (STAT5) activation on ligand stimulation. Taken together our studies show that structural assembly of receptors dictates their potential to signal erythroid differentiation in Ba/F3 cells, that differentiation can take place in the absence of Epo and that a delay in STAT5 activation is highly predictive of this process.

The murine DUB-1 gene is specifically induced by the betac subunit of interleukin-3 receptor.

Cytokines regulate cell growth and differentiation by inducing the expression of specific target genes. We have recently isolated a cytokine-inducible, immediate-early cDNA, DUB-1, that encodes a deubiquitinating enzyme. The DUB-1 mRNA was specifically induced by the receptors for interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-5, suggesting a role for the beta common (betac subunit known to be shared by these receptors. In order to identify the mechanism of cytokine induction, we isolated a murine genomic clone for DUB-1 containing a functional promoter region. The DUB-1 gene contains two exons, and the nucleotide sequence of its coding region is identical to the sequence of DUB-1 cDNA. Various regions of the 5' flanking region of the DUB-1 gene were assayed for cytokine-inducible activity. An enhancer region that retains the beta c-specific inducible activity of the DUB-1 gene was identified. Enhancer activity was localized to a 112-bp fragment located 1.4 kb upstream from the ATG start codon. Gel mobility shift assays revealed two specific protein complexes that bound to this minimal enhancer region. One complex was induced by betac signaling, while the other was noninducible. Finally, the membrane-proximal region of human betac was required for DUB-1 induction. In conclusion, DUB-1 is the first example of an immediate-early gene that is induced by a specific subunit of a cytokine receptor. Further analysis of the DUB-1 enhancer element may reveal specific determinants of a betac-specific signaling pathway.

Cooperation between core binding factor and adjacent promoter elements contributes to the tissue-specific expression of interleukin-3.

Tissue-specific expression of interleukin-3 (IL-3) is mediated via cis-acting elements located within 315 base pairs of the transcription start. This is achieved in part through the positive activities of the AP-1 and Elf-1 sites in the IL-3 promoter. The contribution to T cell-specific expression by other promoter sites was assessed in a transient expression assay with IL-3 promoter constructs linked to a luciferase gene, focusing initially on the core binding factor (CBF) site, which is footprinted in vivo upon T cell activation. Activity of the CBF site is shown to be critically dependent on the adjacent activator site Act-1. Together the Act-1 and CBF sites form a functional unit (AC unit) with dual activity. The AC unit is demonstrated to enhance basal activity of promoters both in fibroblasts and T cells. This activity is further inducible in activated T cells, but not in fibroblasts. In addition to the already identified NIP repressor site, evidence is presented for a second repressor region that restricts promoter activity in fibroblasts. Finally, a novel positive regulatory element has been mapped in the IL-3 promoter between nucleotide -180 and -210 that leads to increased expression in T cells. Together these results demonstrate that T cell expression of IL-3 is not specified by the activity of a single tissue-specific element, but instead involves multiple interacting elements that provide both specific positive regulation in T cells and specific negative regulation in fibroblasts.

Multiple proteins interact with the nuclear inhibitory protein repressor element in the human interleukin-3 promoter.

T cell expression of interleukin 3 (IL-3) is directed by positive and negative cis-acting DNA elements clustered within 300 base pairs of the transcriptional start site. A strong repressor element, termed nuclear inhibitory protein (NIP), was previously mapped to a segment of the IL-3 promoter between nucleotides -271 and -250. Functional characterization of this element demonstrates that it can mediate repression when linked in cis to a heterologous promoter. DNA binding experiments were carried out to characterize the repressor activity. Using varying conditions, three distinct complexes were shown to interact specifically with the NIP region, although only one correlates with repressor activity. Complex 1 results from binding of a ubiquitous polypeptide that recognizes the 3' portion of this sequence and is not required for repression. Complex 2 corresponds to binding of transcription factor (upstream stimulatory factor) to an E-box motif in the 5' portion of the NIP region. DNA binding specificity of complex 3 overlaps with that of upstream stimulatory factor but is clearly distinct. To determine which of the latter two complexes represents NIP activity, we incorporated small alterations into the NIP site of an IL-3 promoter-linked reporter construct and examined their effects on NIP-mediated repression. Functional specificity for repression matches the DNA binding specificity of complex 3; both repressor activity and complex 3 binding require the consensus sequence CTCACNTNC.

Multiple transcription start sites and 5' alternate splicing of murine IL-3 receptor beta-chain transcripts.

The murine interleukin-3 receptor beta-chain genes, IL-3R beta IL-3 and IL-3R beta c, encode the signal transducing chains of the high affinity receptors for IL-3 and IL-3, GM-CSF and IL-5 respectively. Little is known about the regulation of their expression. To enable the study of the promotors of IL-3R beta IL-3 and IL-3R beta c, we have characterized their respective 5' untranslated regions using a modified 5' RACE protocol. Four classes of alternatively spliced transcripts were isolated that initiate in a 400 nt region upstream from a previously reported start site(1). The initially reported partial IL-3R beta IL-3 clone belongs to the first class of transcripts(2). The second class starts in the middle of an intron as defined by the first class. The 3rd and 4th class establish 2 novel splice donor sites. These results were confirmed by RNAse-protection assay. The complex organization as evident from our data establishes an experimental framework for future experiments aimed at the study of the promoters for the murine IL-3R beta genes.

Differentiation domains of the erythropoietin receptor.

Ectopic expression of the erythropoietin receptor (Epo-R) in Ba/F3, an interleukin-3 (IL-3)-dependent progenitor cell line, confers both Epo-dependent cell growth and Epo-dependent induction of beta-globin mRNA. We have used this system of limited erythroid differentiation to characterize the role of the Epo-R in differentiation. In particular, we have been interested in identifying a differentiation domain of the Epo-R. We have studied three chimeras encoding regions of the extracellular region of the Epo-R and the intracellular region of the IL-3R beta IL-3. After transfection into Ba/F3 cells, all three chimeras conferred Epo-dependent growth and induced the expression of beta-globin, suggesting that the extracellular region of the Epo-R plays a critical role in differentiation. However, a truncated Epo-R containing only the extracellular region of the Epo-R and a 15 amino acid cytoplasmic tail does not induced beta-globin expression, although it is processed to the cell surface and binds Epo. These experiments show that the extracytoplasmic region of the Epo-R is necessary but not sufficient to induce erythroid-specific differentiation in this system.

Identification of a critical regulatory site in the human interleukin-3 promoter by in vivo footprinting.

Interleukin-3 (IL-3) is involved in proliferation and differentiation of hematopoietic progenitor cells. Its expression is subject to precise, tissue-specific regulation, and has been studied extensively in the gibbon T-cell line MLA 144 by a combination of functional assays and DNA binding experiments. To extend these studies, the gibbon IL-3 promoter was cloned and in vivo footprinting of the gibbon and human IL-3 proximal promoters was performed. The gibbon IL-3 promoter was found to be highly homologous to its human counterpart and both promoters yielded identical in vivo footprints after induction of IL-3 synthesis. In particular, we observed specific protection of three guanines over a core sequence TGTGGTTT (IF-1IL3) that had not been recognized in previous studies. IF-1IL3 is not found in other cytokine promoters, but it is conserved in the IL-3 promoter of several species and is similar to a recurring motif in viral and T-cell-specific cellular enhancers. IF-1IL3 binds a specific complex in MLA 144 and Jurkat nuclear extracts in vitro, which shares the same specificity as the complex bound by the polyoma virus and T-cell receptor delta enhancers. Mutation of the three guanines in IF-1IL3 core sequence disrupts binding in vitro and abrogates the ability of the IL-3 promoter to mediate inducible expression in T cells. Although IF-1IL3 is necessary for IL-3 expression, it is not sufficient: a truncated IL-3 promoter with an intact IF-1IL3 site but no other activator sites is transcriptionally silent. These studies describe a new regulatory element within the IL-3 promoter that is essential for expression and conserved between species.

Erythropoietin receptor signals both proliferation and erythroid-specific differentiation.

Ectopic expression of the erythropoietin receptor (EPO-R) in Ba/F3, an interleukin 3-dependent progenitor cell line, confers EPO-dependent cell growth. To examine whether the introduced EPO-R could affect differentiation, we isolated Ba/F3-EPO-R subclones in interleukin 3 and assayed for the induction of beta-globin mRNA synthesis after exposure to EPO. Detection of beta-globin mRNA was observed within 3 days of EPO treatment, with peak levels accumulating after 10 days. When EPO was withdrawn, expression of beta-globin mRNA persisted in most clones, suggesting that commitment to erythroid differentiation had occurred. Although EPO-R expression also supports EPO-dependent proliferation of CTLL-2, a mature T-cell line, those cells did not produce globin transcripts, presumably because they lack requisite cellular factors involved in erythrocyte differentiation. We conclude that the EPO-R transmits signals important for both proliferation and differentiation along the erythroid lineage.

Interleukin-3 expression by activated T cells involves an inducible, T-cell-specific factor and an octamer binding protein.

Interleukin-3 (IL-3) is exclusively expressed by activated T and natural killer cells, a function that is tightly controlled both in a lineage-specific and in a stimulation-dependent manner. We have investigated the protein binding characteristics and functional importance of the ACT-1-activating region of the IL-3 promoter. This region binds an inducible, T-cell-specific factor over its 5' end, a site that is necessary for the expression of IL-3 in the absence of other upstream elements. Over its 3' end, it binds a factor that is ubiquitously and constitutively expressed. This factor is Oct-1 or an immunologically related octamer-binding protein, and it plays a role in coordinating the activity of several regulatory elements. These characteristics make the ACT-1 site analogous to the activating ARRE-1 site in the IL-2 promoter. Furthermore, and despite a lack of sequence homology, the promoters of IL-3 and IL-2 share an organizational pattern of regulatory elements that is likely to be important for the T-cell-specific expression of these genes.

Enhanced expression of interleukin-3 and granulocyte-macrophage colony-stimulating factor receptor subunits in murine hematopoietic cells stimulated with hematopoietic growth factors.

AIC2A and AIC2B are closely related genes encoding components of the receptors for murine interleukin-3 (IL-3) (AIC2A) and granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5 (AIC2B). We have studied the parallel regulation of expression of these genes in erythroid and myeloid progenitor cell lines. AIC2A and AIC2B transcription was transiently induced in these cells in response to a variety of hematopoietic growth factors, including erythropoietin (EPO), monocyte-CSF, IL-3, GM-CSF, and stem cell factor (SCF or kit ligand). Run-on assays established that the increase occurred mainly at the transcriptional level. Immunoprecipitation experiments confirmed that the increase in messenger RNA expression resulted in augmented synthesis of both AIC2A and AIC2B proteins, and binding studies further showed these proteins to be functional. We observed a fourfold increase in low-affinity IL-3 sites in an erythroid precursor cell line stimulated with EPO, and a threefold increase in GM-CSF high-affinity sites in a myeloid cell line stimulated with IL-3. In addition, we showed that the increase in the IL-3 receptor chain AIC2A in the erythroid precursor cell line correlated with the ability of IL-3 to exert a cooperative effect with EPO in the induction of beta-globin in these cells.

The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus-forming virus gp55 glycoprotein.

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated by binding either erythropoietin (EPO) or gp55, the Friend spleen focus-forming virus glycoprotein. The highly specific interaction between gp55 and EPO-R triggers cell proliferation and thereby causes the first stage of Friend virus-induced erythroleukemia. We have generated functional chimeric receptors containing regions of the EPO-R and the interleukin-3 receptor (AIC2A polypeptide), a related cytokine receptor which does not interact with gp55. All chimeric receptors were expressed at similar levels, had similar binding affinities for EPO, and conferred EPO-dependent cell growth. Only those chimeric receptors which contained the EPO-R transmembrane region were activated by gp55. These results demonstrate that the transmembrane region of the EPO-R is critical for activation by gp55. In addition, analysis of a soluble, secreted EPO-R and cysteine point mutants of the EPO-R show that the extracytoplasmic region of the EPO-R specifically interacts with gp55.

A functional isoform of the human granulocyte/macrophage colony-stimulating factor receptor has an unusual cytoplasmic domain.

The granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (GMR) transduces a signal that results in the proliferation, differentiation, and functional activation of hematopoietic cells. This study sought to determine whether functional isoforms of the receptor exist that may be important in generating this diversity of cellular response. We have isolated a cDNA encoding an isoform of the low-affinity human GMR that is a product of alternative splicing of the GMR gene and results in a predicted 410-amino acid protein with a cytoplasmic domain that is rich in serine residues, a feature of regions critical in signal transduction for other receptors of the hematopoietin receptor superfamily. This receptor bound ligand and was functionally active when introduced into a murine factor-dependent cell line; mRNA transcripts representative of this isoform were coexpressed with those for a previously isolated 400-amino acid isoform of the GMR in normal hematopoietic and leukemic cells. In view of the recent isolation of a cDNA, designated GM-CSF R beta, that confers high-affinity binding of GM-CSF in cotransfection experiments with the low-affinity receptor, we suggest that the previously isolated low-affinity receptor be designated GM-CSF R alpha 1 and the one described in this report be designated GM-CSF R alpha 2.

Preneoplastic lesions induced by myc and src oncogenes in a heterotopic rat colon.

With the use of viral vectors harboring myc and src oncogenes, we have assessed the potential contribution of these different elements to colonic neoplasia using a transplantation technique resulting in the formation of a heterotopic colon in Wistar Furth rats. While myc alone induced atypia and some dysplasia, src induced focal dysplastic lesions throughout the colon mucosa with evidence of metaplasia. In contrast, lesions induced by myc and src acting cooperatively, were highly dysplastic with evidence of tumor formation after protracted periods. These results indicated the formation of histologically distinct preneoplastic lesions elicited by the action of a single oncogene in colon implants with the production of adenocarcinomas when such oncogenic elements act cooperatively. This model provides an opportunity for studies of the action of different oncogenes, acting singly or in combination, in the multi-step progression to colon tumor formation.