CRISPR-Cas13 mediated Knock Down in Drosophila cultured cells

Manipulation of gene expression is one of the best approaches for studying gene function in vivo. CRISPR-Cas13 has the potential to be a powerful technique for manipulating RNA expression in diverse animal systems in vivo, including Drosophila melanogaster. Studies using Cas13 in mammalian cell lines for gene knockdown showed increased on-target efficiency and decreased off-targeting relative to RNAi. Moreover, catalytically inactive Cas13 fusions can be used to image RNA molecules, install precise changes to the epitranscriptome, or alter splicing. However, recent studies have suggested that there may be limitations to the deployment of these tools in Drosophila, so further optimization of the system is required. Here, we report a new set of PspCas13b and RfxCas13d expression constructs and use these reagents to successfully knockdown both reporter and endogenous transcripts in Drosophila cells. As toxicity issues have been reported with high level of Cas13, we effectively decreased PspCas13b expression without impairing its function by tuning down translation. Furthermore, we altered the spatial activity of both PspCas13b and RfxCas13d by introducing Nuclear Exportation Sequences (NES) and Nuclear Localization Sequences (NLS) while maintaining activity. Finally, we generated a stable cell line expressing RfxCas13d under the inducible metallothionein promoter, establishing a useful tool for high-throughput genetic screening. Thus, we report new reagents for performing RNA CRISPR-Cas13 experiments in Drosophila, providing additional Cas13 expression constructs that retain activity.

Systematic Screen for Drosophila Transcriptional Regulators Phosphorylated in Response to Insulin/mTOR Pathway

Insulin/insulin-like growth factor signaling (IIS) is a conserved mechanism to regulate animal physiology in response to nutrition. IIS activity controls gene expression, but only a subset of transcriptional regulators (TRs) targeted by the IIS pathway is currently known. Here we report the results of an unbiased screen for Drosophila TRs phosphorylated in an IIS-dependent manner. To conduct the screen, we built a library of 857 V5/Strep-tagged TRs under the control of Copper-inducible metallothionein promoter (pMt). The insulin-induced phosphorylation changes were detected by using Phos-tag SDS-PAGE and Western blotting. Eight proteins were found to display increased phosphorylation after acute insulin treatment. In each case, the insulin-induced phosphorylation was abrogated by mTORC1 inhibitor rapamycin. The hits included two components of the NURF complex (NURF38 and NURF55), bHLHZip transcription factor Max, as well as the Drosophila ortholog of human proliferation-associated 2G4 (dPA2G4). Subsequent experiments revealed that the expression of the dPA2G4 gene was promoted by the mTOR pathway, likely through transcription factor Myc. Furthermore, NURF38 was found to be necessary for growth in larvae, consistent with the role of IIS/mTOR pathway in growth control.

Activation of PLC by an endogenous cytokine (GBP) in Drosophila S3 cells and its application as a model for studying inositol phosphate signalling through ITPK1

Using immortalized [3H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, we determined that dGBP initially mobilized Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activated a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminated mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3→Ins(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117–28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.

NF-κB Target Genes Activation Via Displacement of HDACs Bound to NF-κB p50 Homodimers by C/EBPα, Its AML Oncoproteins or C/EBPβ.

Abstract 3637 The NF-κB transcription factors are involved in inflammation, proliferation, and apoptosis. The p50:p65 heterodimer, the most common transcription activating form of NF-κB, is retained in the cytosol and translocates into the nucleus only upon stimulation. In contrast, the p50 homodimer is the primary nuclear NF-κB species in unstimulated cells, and recruit HDACs to repress NF-κB-target gene expression. Dysregulation of NF-κB frequently occurs in AML and lymphoma. C/EBPα is mutated in approximately 10% of patients with AML, resulting in expression of C/EBPαp30 or C/EBPαLZ oncoproteins. We showed that C/EBPα or its oncoproteins, including leucine zipper variants (C/EBPαLZ) that cannot bind DNA directly, protects hematopoietic cells from apoptosis. Through interaction with NF-κB p50, C/EBPα or its AML mutants activate several anti-apoptotic genes including BCL2 and FLIP. C/EBPβ or its truncated LIP isoform are over-expressed in Hodgkin lymphoma and breast cancer. Using Ba/F3 cell lines carrying an estrogen receptor fusion of C/EBPα or C/EBPαLZ, we now show that activation of C/EBPα isoforms results in a significant 90% reduction in the association of endogenous HDAC1 or HDAC3 with the FLIP promoter, within 5 hours. In contrast, activation of the basic region mutant C/EBPαBR that cannot bind p50 did not have an effect. Accordingly, ChIP analysis demonstrated that on the FLIP promoter, the expression of C/EBPα or C/EBPαLZ resulted in a 2.5 or 4-fold increase in histone H3 or H4 acetylation, respectively. To study the specific displacement of HDACs from p50 we used Eμ-C/EBPα transgenic mice which express C/EBPα in splenic lymphoid cells, in contrast to control littermates. The ectopic presence of C/EBPα resulted in a significant reduction of endogenous HDAC1 or HDAC3 on the FLIP promoter to 17 or 4% of the expression in wild type mice. In contrast, in p50-/- mice, the C/EBPα transgene had a minimal effect on HDAC1 or a modest reduction of HDAC3 occupancy on the FLIP promoter, to 90 or 50% of levels seen in p50-/- controls. These findings suggest that on the FLIP promoter C/EBPα displaces primarily p50-bound HDACs. HDAC1 or HDAC3 co-ip with p50 in transiently transfected 293T cells. The co-expression of C/EBPα, its C/EBPαLZ or C/EBPαp30 AML mutants, or C/EBPβ interrupted interaction of p50 with HDAC1 and to a lesser extent with HDAC3. In striking correlation to our previous findings, the basic region mutant C/EBPαBR does not interrupt this interaction. As a control, we confirmed that C/EBPα or C/EBPβ do not disrupt the NF-κB p50:p65 co-ip. We have shown that interaction with p50 is required for induction of bcl-2 and FLIP by C/EBPα and that p50 binds and induces the CEBPA gene. Using ChIP analysis, we now show that in U937 AML cells endogenous C/EBPα, C/EBPβ and p50 specifically bind the proximal segment of the nfkb1 (p50) promoter. In addition, C/EBPαLZ expressed from the metallothionein promoter in Ba/F3 cells binds a proximal site in the nfkb1 promoter. C/EBPα or C/EBPαLZ induced p50 RNA expression 3.5-fold, while C/EBPαBR was not effective. Treating the cells with the translation inhibitor cycloheximide did not change p50 induction, suggesting direct activation. C/EBPα, C/EBPαLZ but not C/EBPαBR activated to a similar degree luciferase reporters containing 1800 or 250 bp fragments of the nfkb1 promoter. In addition, C/EBPβ or its truncated LIP isoform activated these reporters, although LIP cannot activate a (CEBP)2TK-Luc reporter. The murine nfkb1 promoter contains 2 proximal κB sites that are 100% conserved between mouse and human. p50 but not C/EBP avidly binds either of these sites in a gel shift assay. Disruption of either κB site abrogated 65 to 80% of reporter activation, and mutating both resulted in activation of less than 10% by either C/EBPα or C/EBPαLZ. In summary, we show that C/EBPs displace HDAC from NF-κB p50, providing a mechanism whereby C/EBPα or its AML mutants can derepress NF-κB target genes and contribute to their dysregulated expression. In addition, C/EBPα, a C/EBPαLZ AML variant that cannot bind DNA directly, or C/EBPβ bind and activate the nfkb1 promoter through interaction with NF-κB bound to two proximal, highly conserved κB sites. Together these findings lend support to the idea that disrupting the C/EBP:NF-κB p50 complex may offer a complementary approach for targeting NF-κB activity in the nucleus to favor apoptosis in AML or other malignancies in which C/EBP family members are expressed. Disclosures: No relevant conflicts of interest to declare.

Transient expression in tobacco Bright Yellow 2 cells and pollen grains: A fast, efficient and reliable system for functional promoter analysis of plant genes

Gene expression is mediated by DNA sequences directly upstream from the coding sequences, recruited transcription factors and RNA polymerase in a spatially-defined manner. Understanding promoter strength and regulation would enhance our understanding of gene expression. The goal of this study was to develop a fast, efficient and reliable method for testing basal promoter activity and identifying core sequences within its pollen specific elements. In this paper we examined the functionality of buckwheat metallothionein promoter by a histochemical GUS assay in two transient expression systems: BY2 cells and pollen grains. Strong promoter activity was observed in both systems.

Use of the Metallothionein Promoter-Human Growth Hormone-Releasing Hormone (GHRH) Mouse to Identify Regulatory Pathways that Suppress Pituitary Somatotrope Hyperplasia and Adenoma Formation due to GHRH-Receptor Hyperactivation

Hyperactivation of the GHRH receptor or downstream signaling components is associated with hyperplasia of the pituitary somatotrope population, in which adenomas form relatively late in life, with less than 100% penetrance. Hyperplastic and adenomatous pituitaries of metallothionein promoter-human GHRH transgenic (Tg) mice (4 and > 10 months, respectively) were used to identify mechanisms that may prevent or delay adenoma formation in the presence of excess GHRH. In hyperplastic pituitaries, expression of the late G1/G2 marker Ki67 increased, whereas the proportion of 5-bromo-2′-deoxyuridine-labeled cells (S phase marker) did not differ from age-matched controls. These results indicate cell cycle progression is blocked, with further evidence suggesting that enhanced p27 activity may contribute to this process. For adenomas, formation was associated with loss of p27 activity (nuclear localization and mRNA). Increased endogenous somatostatin (SST) tone may also slow the conversion from hyperplastic to adenomatous state because mRNA levels for SST receptors, sst2 and sst5, were elevated in hyperplastic pituitaries, whereas adenomas were associated with a decline in sst1 and sst5 mRNA. Also, SST-knockout Tg pituitaries were larger and adenomas formed earlier compared with those of SST-intact Tg mice. Unexpectedly, these changes were independent of changes in proliferation rate within the hyperplastic tissue, suggesting that endogenous SST controls GHRH-induced adenoma formation primarily via modulation of apoptotic and/or cellular senescence pathways, consistent with the predicted function of some of the most differentially expressed genes (Casp1, MAP2K1, TNFR2) identified by membrane arrays and confirmed by quantitative real-time RT-PCR.