Faculty Bibliography

We report the identification of yan, an ETS-domain transcription factor belonging to the Drosophila epidermal growth factor receptor (DER) pathway, as an antagonist of the Wingless signalling pathway. We demonstrate that cells lacking yan function in the Drosophila eye show increased Wingless pathway activity, and inhibition of Wingless signalling in yan(-/-) cells rescues the yan mutant phenotype. Biochemical analysis shows that Yan physically associates with Armadillo, a crucial effector of the Wingless pathway, thereby suggesting a direct regulatory mechanism. We conclude that yan represents a new and unsuspected molecular link between the Wingless and DER pathways

Misregulated beta-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 beta-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 beta-catenin. We show that these inhibitors efficiently block Wnt/beta-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

MicroRNAs (miRs) and the canonical Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the canonical Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of a-catenin (beta-cat), miR-1 and miR-613 act upstream of beta-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive beta-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer

BACKGROUND: Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ss-catenin pathway. METHODOLOGY/PRINCIPAL FINDINGS: In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a beta-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/beta-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/beta-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation. CONCLUSIONS: These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/beta-catenin pathway

The sequencing of whole genomes, including those of model organisms, has provided an unprecedented resource to the research community to make sense of the genetic code. However, it is the advent of novel functional genomic technologies that have been truly instrumental in bridging the gap between gene sequence and gene function. The past few years have witnessed a rapid growth in the development and implementation of high-throughput screening (HTS) technologies that researchers are now using to discover 'gene-function' in an unbiased, systematic, time and cost-efficient manner. 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) or short-interfering RNA (siRNA) into cells or whole organisms can effectively suppress endogenous gene expression. The RNAi-based screening technology has made it feasible to query the function of whole genomes in the regulation of conserved cell-signaling pathways and the crosstalk between them in 'signaling networks' that are known to influence important cell biological functions, such as cell proliferation and growth, cell morphology, cell adhesion and cell death. In this review we discuss the application, advantages and limitations of RNAi and other post-genomic technologies in the identification of novel modulators of cell-signaling pathways, with a focus on the Wnt signaling pathway. We also discuss the future scope and utility of designing additional variants of these genome-scale screens

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

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: Off-target effects (OTEs) have been demonstrated to be a major source of false-positives in RNA interference high-throughput screens (RNAi-HTS). In this study, we re-assess the previously published transcriptional reporter-based whole-genome RNAi screens for the Wingless and Hedgehog-signaling pathways using 2nd generation dsRNA libraries. Furthermore, we investigate other factors that may influence the outcome of such screens, including cell-type specificity, robustness of reporters, and assay normalization that determine the efficacy of RNAi-knockdown of target genes

Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum

Wnts [also known as Wingless (Wg)] are a family of conserved signaling molecules involved in a plethora of fundamental developmental and cell biological processes, such as cell proliferation, differentiation, and cell polarity. Dysregulation of the pathway can be detrimental, because several components are tumorigenic when mutated and are associated with hepatic, colorectal, breast, and skin cancers. First identified in the fruit fly Drosophila melanogaster as a gene family responsible for patterning the embryonic epidermis, the Wnt gene family, including Wg, encode secreted glycoproteins that activate receptor-mediated signaling pathways leading to numerous transcriptional and cellular responses. The main function of the canonical Wg pathway is to stabilize the cytoplasmic pool of a key mediator, beta-catenin [beta-catenin, known as Armadillo (Arm) in fruit flies], which is otherwise degraded by the proteasome pathway. Initially identified as a key player in stabilizing cell-cell adherens junctions, Arm is now known to also act as a transcription factor by forming a complex with the lymphoid enhancer factor (LEF)/T cell-specific transcription factor (TCF) family of high mobility group (HMG)-box transcription factors. Upon Wnt/Wg stimulation, stabilized Arm translocates to the nucleus, where, together with LEF/TCF transcription factors, it activates downstream target genes that regulate numerous cell biological processes