Abstract: Misregulated β-catenin responsive transcription (CRT) has been implicated in the genesis of various malignancies, including colorectal carcinomas, and it is a key therapeutic target in combating various cancers. Despite significant effort, successful clinical implementation of CRT inhibitory therapeutics remains a challenging goal. This is, in part, because of the challenge of identifying inhibitory compounds that specifically modulate the nuclear transcriptional activity of β-catenin while not affecting its cytoskeletal function in stabilizing adherens junctions at the cell membrane. Here, we report an RNAi-based modifier screening strategy for the identification of CRT inhibitors. Our data provide support for the specificity of these inhibitory compounds in antagonizing the transcriptional function of nuclear β-catenin. We show that these inhibitors efficiently block Wnt/β-catenin-induced target genes and phenotypes in various mammalian and cancer cell lines. Importantly, these Wnt inhibitors are specifically cytotoxic to human colon tumor biopsy cultures as well as colon cancer cell lines that exhibit deregulated Wnt signaling.
Abstract: Little is known about the molecular mechanisms by which STAT proteins promote tumorigenesis. Drosophila is an ideal system for investigating this issue, as there is a single STAT (Stat92E), and its hyperactivation causes overgrowths resembling human tumors. Here we report the first identification of a dominant-active Stat92E protein, Stat92E(DeltaNDeltaC), which lacks both N- and C-termini. Mis-expression of Stat92E(DeltaNDeltaC)in vivo causes melanotic tumors, while in vitro it transactivates a Stat92E-luciferase reporter in the absence of stimulation. These gain-of-function phenotypes require phosphorylation of Y(711) and dimer formation with full-length Stat92E. Furthermore, a single point mutation, an R(442P) substitution in the DNA-binding domain, abolishes Stat92E function. Recombinant Stat92E(R442P) translocates to the nucleus following activation but fails to function in all assays tested. Interestingly, R(442) is conserved in most STATs in higher organisms, suggesting conservation of function. Modeling of Stat92E indicates that R(442) may contact the minor groove of DNA via invariant TC bases in the consensus binding element bound by all STAT proteins. We conclude that the N- and C- termini function unexpectedly in negatively regulating Stat92E activity, possibly by decreasing dimer dephosphorylation or increasing stability of DNA interaction, and that Stat92E(R442) has a nuclear function by altering dimer:DNA binding.
Abstract: Genetic and biochemical analyses in model systems such as the fruitfly, Drosophila melanogaster, have successfully identified several genes that play key regulatory roles in fundamental cellular and developmental processes. However, the analyses of the complete genome sequences of Drosophila, as well as of humans, now reveal that traditional methods have ascribed functions to only a fraction of the total predicted genes. Thus, the roles for many, as yet unidentified genes, in normal development and cancer remain to be discovered. The challenge presented by the various large-scale genome projects is how to derive biologically relevant information from the raw sequences. The past few years have witnessed a rapid growth in the development and implementation high-throughput screening (HTS) technologies that researchers are now using to discover "gene-function" in an unbiased, systematic, and time-efficient manner. In fact one of the most promising functional genomic approach that has emerged in the past few years is based on RNA-interference (RNAi), in which the introduction of double-stranded RNA (dsRNA) into cells or whole organisms has been shown to be an effective tool to suppress endogenous gene expression. The RNAi technology has made it feasible to query the function of every gene in the genome for their potential function in a given cell-biological process using cell-based assays. This chapter discusses the application, advantages, and limitations of this powerful technology in the identification of novel modulators of cell-signaling pathways as well as its future scope and utility in designing more efficient genome-scale screens.
Abstract: Signaling by the wingless pathway has been shown to govern numerous developmental processes. Much of our current understanding of wingless signaling mechanisms comes from studies conducted in Drosophila melanogaster, which offers superior experimental tractability for genetic and developmental studies. Wingless signaling is highly consequential during normal development and patterning of Drosophila. Its earliest identifiable role during development of Drosophila is in the embryonic segmentation cascade, wherein wingless functions as a segment polarity gene and serves to pattern each individual segment along the antero-posterior axis of the developing embryo. Subsequent developmental roles fulfilled by wingless include patterning the developing wings, legs, eyes, CNS, heart, and muscles. Each of these developmental contexts offers excellent systems to query mechanisms regulating different aspects of wingless signal transduction such as synthesis, secretion, reception, and transcription. This chapter presents a brief overview on the functions of wingless signaling during development of Drosophila melanogaster.
Abstract: Spatiotemporal modulation of the evolutionarily conserved, intercellular Notch signaling pathway is important in the development of many animals. Examples include the regulation of neural-epidermal fate decisions in neurogenic ectoderm of Drosophila and somitogenesis in vertebrate presomitic mesoderm. In both these and most other cases, it appears that Notch-class transmembrane receptors are ubiquitously expressed. Modulation of the pathway is achieved primarily by the localized expression of the activating ligand or by alteration of receptor specificity through a glycosyl transferase. In contrast, we present this report of an instance where the abundance of the Notch-class mRNA itself is dynamically regulated. Taking advantage of the long cell cycle of the two-cell-stage embryo of the leech Helobdella robusta, we show that this regulation is achieved at the levels of both transcript stability and transcription. Moreover, MAPK signaling plays a significant role in regulating accumulation of the transcript by virtue of its effect on Hro-notch mRNA stability. Intracellular injection of heterologous reporter mRNAs shows that the Hro-notch 3' UTR, containing seven AU-rich elements, is key to regulating transcript stability. Thus, we show that regulation of the Notch pathway can occur at a previously underappreciated level, namely that of transcript stability. Given that AU-rich elements occur in the 3' UTR of Notch-class genes in Drosophila, human, and Caenorhabditis elegans, regulation of Notch signaling by modulation of mRNA levels may be operating in other animals as well.
Abstract: Off-target effects have been demonstrated to be a major source of false-positives in RNA interference (RNAi) high-throughput screens. In this study, we re-assess the previously published transcriptional reporter-based whole-genome RNAi screens for the Wingless and Hedgehog signaling pathways using second generation double-stranded RNA libraries. Furthermore, we investigate other factors that may influence the outcome of such screens, including cell-type specificity, robustness of reporters, and assay normalization, which determine the efficacy of RNAi-knockdown of target genes.
Abstract: We describe a developmental analysis of Drosophila Cullin-5 (Cul-5) identified from the genome sequence on the basis of its high degree of homology to vertebrate and worm sequences. The gene is expressed in a restricted manner in ectodermal cells throughout development suggesting pleiotropic functions. We decided to examine the phenotypes of Cul-5 aberrations in two well-studied developmental systems: the neuromuscular junction (NMJ) and the developing sensory organ. Alteration of Cul-5 levels in motoneurons results in an increase in bouton number at the NMJ. The cells of a sensory organ on the adult notum arise from a single progenitor cell by regulated cell division. Aberrations in Cul-5 affect different steps in the lineage consistent with a role in cell fate determination, proliferation, and death. Such phenotypes highlight the multiple cellular processes in which Cul-5 can participate.
Abstract: To understand the evolution of segmentation, we must compare segmentation in all three major groups of eusegmented animals: vertebrates, arthropods, and annelids. The leech Helobdella robusta is an experimentally tractable annelid representative, which makes segments in anteroposterior progression from a posterior growth zone consisting of 10 identified stem cells. In vertebrates and some arthropods, Notch signaling is required for normal segmentation and functions via regulation of hes-class genes. We have previously characterized the expression of an hes-class gene (Hro-hes) during segmentation in Helobdella, and here, we characterize the expression of an H. robusta notch homolog (Hro-notch) during this process. We find that Hro-notch is transcribed in the segmental founder cells (blast cells) and their stem-cell precursors (teloblasts), as well as in other nonsegmental tissues. The mesodermal and ectodermal lineages show clear differences in the levels of Hro-notch expression. Finally, Hro-notch is shown to be inherited by newly born segmental founder cells as well as transcribed by them before their first cell division.
Abstract: We have cloned genes related to hairy and Enhancer of split (hes) from glossiphoniid leeches, Helobdella robusta and Theromyzon rude. In leech, segments arise sequentially in anteroposterior progression from a posterior growth zone that consists of five bilaterally paired embryonic stem cells called teloblasts. Each teloblast gives rise to segmental founder cells (primary blast cells) that contribute iterated sets of definitive progeny in each segment. Thus, in leech, the "segmentation clock," is closely identified with the cell cycle clock of the teloblasts. We have characterized normal expression patterns of mRNA and protein for the H. robusta hes-class gene (Hro-hes). Semiquantitative RT-PCR revealed that Hro-hes mRNA levels peak while the teloblasts are actively producing primary blast cells. RT-PCR, in situ hybridization and immunostaining revealed that Hro-hes is expressed as early as the first zygotic mitosis and throughout early development. Hro-hes is expressed in macromeres, pro-teloblasts, teloblasts and primary blast cells. HRO-HES protein is localized in the nuclei of cells expressing HRO-HES during interphase; nuclear HRO-HES is reduced during mitosis. In contrast, Hro-hes is transcribed during mitosis and its transcripts are associated with mitotic apparatus (MA). Thus, Hro-hes transcription cycles in antiphase to the nuclear localization of HRO-HES protein. These results indicate that Hro-hes expression, and thus possibly its biological activity, is linked to the cell cycle.
