Ramanuj Dasgupta, Ph.D.

A Systematic Screen for Micro-RNAs Regulating the Canonical Wnt Pathway
Anton, Roman; Chatterjee, Sujash S; Simundza, Julia; Cowin, Pamela; Dasgupta, Ramanuj
2011 ;6(10):e26257-e26257, PLoS ONE
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
— id: 140537, year: 2011, vol: 6, page: e26257, stat: Journal Article,

Feature Article: From the Cover: An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway
Gonsalves, Foster C; Klein, Keren; Carson, Brittany B; Katz, Shauna; Ekas, Laura A; Evans, Steve; Nagourney, Robert; Cardozo, Timothy; Brown, Anthony M C; Dasgupta, Ramanuj
2011 Apr 12;108(15):5954-5963, Proceedings of the National Academy of Sciences of the United States of America
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
— id: 130910, year: 2011, vol: 108, page: 5954, stat: Journal Article,

Yan, an ETS-domain transcription factor, negatively modulates the Wingless pathway in the Drosophila eye
Olson, Emily R; Pancratov, Raluca; Chatterjee, Sujash S; Changkakoty, Binita; Pervaiz, Zeeshan; Dasgupta, Ramanuj
2011 ;12(10):1047-1054, EMBO reports
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
— id: 138112, year: 2011, vol: 12, page: 1047, stat: Journal Article,

Postgenomic technologies targeting the Wnt signaling network
Pancratov, Raluca; Dasgupta, Ramanuj
2011 Nov;3(6):649-665, Wiley interdisciplinary reviews. Systems biology & medicine
The recent development of high-throughput sequencing technologies and the availability of whole genome sequences of a variety of living organisms, including that of humans, have led to an enormous push in the quest for a comprehensive inquiry for the function of each and every gene discovered in different model organisms. A major conclusion from the sequencing projects was that while forward genetics had been extremely successful in identifying key genes/components of many biological processes, such as signal transduction cascades, the function(s) of the majority of genes in the genome remains a mystery. In this article, we discuss the use of a variety of high-throughput postgenomic tools, including functional genomics, proteomics, and chemical genetics that are being implemented in an exhaustive molecular dissection of a key evolutionarily conserved signal transduction pathway, namely the Wnt/wingless (wg) pathway and its associated signaling network. WIREs Syst Biol Med 2011 3 649-665 DOI: 10.1002/wsbm.140 For further resources related to this article, please visit the WIREs website
— id: 138103, year: 2011, vol: 3, page: 649, stat: Journal Article,

Functional genomic approaches targeting the wnt signaling network
Dasgupta, Ramanuj
2009 Jul;10(7):620-631, Current drug targets
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
— id: 100675, year: 2009, vol: 10, page: 620, stat: Journal Article,

Bili inhibits Wnt/beta-catenin signaling by regulating the recruitment of axin to LRP6
Kategaya, Lorna S; Changkakoty, Binita; Biechele, Travis; Conrad, William H; Kaykas, Ajamete; Dasgupta, Ramanuj; Moon, Randall T
2009 ;4(7):e6129-e6129, PLoS ONE
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
— id: 101871, year: 2009, vol: 4, page: e6129, stat: Journal Article,

High-throughput RNAi screen in Drosophila
DasGupta, Ramanuj; Gonsalves, Foster C
2008 ;469:163-184, Methods in molecular biology
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
— id: 92186, year: 2008, vol: 469, page: 163, stat: Journal Article,

Function of the wingless signaling pathway in Drosophila
Gonsalves, Foster C; DasGupta, Ramanuj
2008 ;469:115-125, Methods in molecular biology
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
— id: 92184, year: 2008, vol: 469, page: 115, stat: Journal Article,

A case study of the reproducibility of transcriptional reporter cell-based RNAi screens in Drosophila
DasGupta, Ramanuj; Nybakken, Kent; Booker, Matthew; Mathey-Prevot, Bernard; Gonsalves, Foster; Changkakoty, Binita; Perrimon, Norbert
2007 Sep 28;8(9):R203-R203, Genome biology
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
— id: 74273, year: 2007, vol: 8, page: R203, stat: Journal Article,

Functional genomics reveals genes involved in protein secretion and Golgi organization
Bard, Frederic; Casano, Laetitia; Mallabiabarrena, Arrate; Wallace, Erin; Saito, Kota; Kitayama, Hitoshi; Guizzunti, Gianni; Hu, Yue; Wendler, Franz; Dasgupta, Ramanuj; Perrimon, Norbert; Malhotra, Vivek
2006 Feb 2;439(7076):604-607, Nature
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
— id: 63618, year: 2006, vol: 439, page: 604, stat: Journal Article,

Drosophila Wnt/Fz pathways
DasGupta, Ramanuj; Boutros, Michael; Perrimon, Norbert
2005 May 10;2005(283):cm5-cm5, Science's STKE
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
— id: 59510, year: 2005, vol: 2005, page: cm5, stat: Journal Article,

Functional genomic analysis of the Wnt-wingless signaling pathway
DasGupta, Ramanuj; Kaykas, Ajamete; Moon, Randall T; Perrimon, Norbert
2005 May 6;308(5723):826-833, Science
The Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50% of the genes identified in the screen have human orthologs, of which approximately 18% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease
— id: 59511, year: 2005, vol: 308, page: 826, stat: Journal Article,

Notch modulates Wnt signalling by associating with Armadillo/beta-catenin and regulating its transcriptional activity
Hayward, Penny; Brennan, Keith; Sanders, Phil; Balayo, Tina; DasGupta, Ramanuj; Perrimon, Norbert; Martinez Arias, Alfonso
2005 Apr;132(8):1819-1830, Development
The establishment and stability of cell fates during development depend on the integration of multiple signals, which ultimately modulate specific patterns of gene expression. While there is ample evidence for this integration at the level of gene regulatory sequences, little is known about its operation at other levels of cellular activity. Wnt and Notch signalling are important elements of the circuitry that regulates gene expression in development and disease. Genetic analysis has suggested that in addition to convergence on the transcription of specific genes, there are modulatory cross-regulatory interactions between these signalling pathways. We report that the nodal point of these interactions is an activity of Notch that regulates the activity and the amount of the active/oncogenic form of Armadillo/beta-catenin. This activity of Notch is independent of that induced upon cleavage of its intracellular domain and which mediates transcription through Su(H)/CBF1. The modulatory function of Notch described here, contributes to the establishment of a robust threshold for Wnt signalling which is likely to play important roles in both normal and pathological situations
— id: 59512, year: 2005, vol: 132, page: 1819, stat: Journal Article,

The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors
Baeg, Gyeong-Hun; Selva, Erica M; Goodman, Robyn M; Dasgupta, Ramanuj; Perrimon, Norbert
2004 Dec 1;276(1):89-100, Developmental biology (Orlando)
We have examined the respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, we find that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. Our studies, together with others [Kirkpatrick, C.A., Dimitroff, B.D., Rawson, J.M., Selleck, S.B., 2004. Spatial regulation of Wingless morphogen distribution and signalling by Dally-like protein. Dev. Cell (in press)], reveals that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, our analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient
— id: 59513, year: 2004, vol: 276, page: 89, stat: Journal Article,

Using RNAi to catch Drosophila genes in a web of interactions: insights into cancer research
Dasgupta, Ramanuj; Perrimon, Norbert
2004 Nov 1;23(51):8359-8365, Oncogene
The completion of whole-genome sequencing of various model organisms and the recent explosion of new technologies in the field of Functional Genomics and Proteomics is poised to revolutionize the way scientists identify and characterize gene function. One of the most significant advances in recent years has been the application of RNA interference (RNAi) as a means of assaying gene function. In the post-genomic era, advances in the field of cancer biology will rely upon the rapid identification and characterization of genes that regulate cell growth, proliferation, and apoptosis. Significant efforts are being directed towards cancer therapy and devising efficient means of selectively delivering drugs to cancerous cells. In this review, we discuss the promise of integrating genome-wide RNAi screens with proteomic approaches and small-molecule chemical genetic screens, towards improving our ability to understand and treat cancer
— id: 59514, year: 2004, vol: 23, page: 8359, stat: Journal Article,

Links between signal transduction, transcription and adhesion in epithelial bud development
Jamora, Colin; DasGupta, Ramanuj; Kocieniewski, Pawel; Fuchs, Elaine
2003 Mar 20;422(6929):317-322, Nature
The morphogenesis of organs as diverse as lungs, teeth and hair follicles is initiated by a downgrowth from a layer of epithelial stem cells. During follicular morphogenesis, stem cells form this bud structure by changing their polarity and cell-cell contacts. Here we show that this process is achieved through simultaneous receipt of two external signals: a Wnt protein to stabilize beta-catenin, and a bone morphogenetic protein (BMP) inhibitor to produce Lef1. Beta-catenin then binds to, and activates, Lef1 transcription complexes that appear to act uncharacteristically by downregulating the gene encoding E-cadherin, an important component of polarity and intercellular adhesion. When either signal is missing, functional Lef1 complexes are not made, and E-cadherin downregulation and follicle morphogenesis are impaired. In Drosophila, E-cadherin can influence the plane of cell division and cytoskeletal dynamics. Consistent with this notion, we show that forced elevation of E-cadherin levels block invagination and follicle production. Our findings reveal an intricate molecular programme that links two extracellular signalling pathways to the formation of a nuclear transcription factor that acts on target genes to remodel cellular junctions and permit follicle formation
— id: 59515, year: 2003, vol: 422, page: 317, stat: Journal Article,

A developmental conundrum: a stabilized form of beta-catenin lacking the transcriptional activation domain triggers features of hair cell fate in epidermal cells and epidermal cell fate in hair follicle cells
DasGupta, Ramanuj; Rhee, Horace; Fuchs, Elaine
2002 Jul 22;158(2):331-344, Journal of cell biology
Wnt signaling orchestrates morphogenetic processes in which changes in gene expression are associated with dramatic changes in cell organization within developing tissue/organs. Upon signaling, excess beta-catenin not utilized at cell-cell junctions becomes stabilized, where it can provide the transcriptional activating domain for Lef/Tcf DNA binding proteins. In skin epithelium, forced stabilization of beta-catenin in epidermis promotes hair follicle morphogenesis, whereas conditional removal of beta-catenin in hair progenitor cells specifies an epidermal fate. We now report that a single protein, a stabilized version of beta-catenin lacking the COOH-terminal transactivation domain, acts in epidermis to promote hair fates and in hair cells to promote epidermal fate. This reveals fundamental differences in ways that epidermal and hair cells naturally respond to beta-catenin signaling. In exploring the phenotype, we uncovered mechanistic insights into the complexities of Lef1/Tcf/beta-catenin signaling. Importantly, how a cell will respond to the transgene product, where it will be localized, and whether it can lead to activation of endogenous beta-catenin/Tcf/Lef complexes is specifically tailored to skin stem cells, their particular lineage and their relative stage of differentiation. Finally, by varying the level of beta-catenin signaling during a cell fate program, the skin cell appears to be pliable, switching fates multiple times
— id: 59516, year: 2002, vol: 158, page: 331, stat: Journal Article,