PC1 Localization - Genome Wide Screen

Primary Investigator: Michael J Caplan, MD, PhD

  Autosomal dominant polycystic kidney disease (ADPKD) is a relatively common (~1:1,000), inherited condition characterized by the progressive development of fluid-filled cysts that are derived from renal tubule epithelial cells. The proliferation and expansion of these cysts gradually compresses the surrounding normal kidney tissue and compromises renal function. Mutations in either of two genes lead to ADPKD. The PKD1 gene encodes polycystin-1, an extremely large (4,302 amino acid) membrane protein that is predicted to possess an extracellular N-terminal domain comprised of ~3,000 amino acid residues, 11 membrane spanning domains, and a short C-terminal tail facing the cytoplasm. PKD2 encodes polycystin-2, which is a member of the transient receptor potential (TRP) family of cation channels. The polycystin-1 and 2 proteins participate in a large number of signaling pathways and exhibit complex patterns of subcellular localization. Polycystin-1 and 2 can interact with each other and with numerous other proteins that may modulate their trafficking or their involvement in signal transduction. Both polycystin-1 and 2 localize to the primary cilium, and in addition polycystin-1 is found in the cell surface membranes of renal epithelial cells, while a large pool of polycystin-2 is associated with the membranes of the endoplasmic reticulum. Co-expression of polycystin-2 is required in order for polycystin-1 to traffic to the cell surface. The cytoplasmic C-terminus of polycystin-1 is released by a proteolytic cleavage, and the resultant fragment travels to the nucleus where it influences transcription. Little is known about the mechanisms that govern these complex trafficking and cleavage processes.
  We designed a screen to identify proteins that participate in determining the trafficking properties of polycystin-1, and that regulate the cleavage and nuclear translocation of its C-terminus. The screen made use of a construct encoding the polycystin-1 protein carrying a FLAG epitope tag at its extracellular N-terminus and a HA epitope tag attached to its cytosolic C-terminus. This construct was stably co-expressed with a cDNA encoding polycystin-2 in HEK293 cells. The pool of polycystin-1 present at the cell surface was detected and measured by performing a surface immunofluorescence assay, in which unfixed, unpermeabilized cells chilled to 4o C were incubated first with anti-FLAG antibody and subsequently with a fluorescent dye-conjugated secondary antibody. Cells were then fixed and permeabilized, and immunofluorescence was performed using an antibody directed against the HA epitope followed by a secondary antibody conjugated to a different fluorescent dye. Nuclei were labeled with a third fluorescent dye. Quantitative data were collected using the Opera Confocal platform, and images were analyzed to determine the ratio of the FLAG (surface) fluorescence to the HA fluorescence. This value reports on the fraction of the polycystin-1 protein present in the cells that is expressed at the plasma membrane. In addition, images were analyzed to determine the fraction of the cytosolic HA fluorescence that was localized to nuclear versus non-nuclear compartments. This value was taken as an indication of the extent of cleavage and nuclear translocation of the polycystin-1 C-terminal tail. Thus, the screen allowed us to search simultaneously for proteins whose expression modulates either or both of two physiologically important end points: trafficking of polycystin-1 and/or cleavage and nuclear translocation of its C-terminal tail

Mitochondria Dynamics - Genome Wide Screen

Primary Investigator: Gerald S Shadel, PhD , Ph.D.

Under construction

Natural Product Toxicity Rescue - Genome Wide Screen

Primary Investigator: Craig M Crews, PhD

  Ascertaining the mechanism of action of therapeutically interesting natural products is a considerable challenge. Our group has extensive experience approaching this problem from a biochemical perspective, using affinity reagent derivatives of natural products to isolate binding proteins. However, this technique is not fail-safe, and we were keen to apply a functional genomic approach to mechanism of action to complement our biochemical work. Genomewide siRNA screens of human cells provide an appealing platform for this kind of study, akin to synthetic lethality studies in model organisms: siRNA’s that enhance or suppress the phenotype induced by a compound are likely to knock down genes in pathways important for the action of the compound.
  We undertook a genome-wide siRNA screen to gain insight into the mechanism of cytotoxic action of a natural product chemical of interest to our group. We explored a number of different assay readouts for the pro-apoptotic effect of this compound, and settled on proliferation – as measured by automated cell counting of stained HeLa cell nuclei – as the most robust for our purposes. Impressively, a substantial number of siRNAs were able to partially rescue the anti-proliferative effect of this compound. Fairly stringent hit selection criteria generated a list of 267 hits (out of over 21,000 genes screened). Pathway and network analyses assigned these hits to a variety of functional classes, including some expected (xenobiotic metabolism, apoptosis) and some unexpected.
  We are currently in the process of validating a handpicked selection of 100 of these hits in follow-up assays. The proteins encoded by validated hits can then be tested for direct interactions with the compound in our biochemical protocols. In addition, if small molecule interactors are known for any hit proteins, we can investigate synergistic effects with our molecule of interest. Ultimately, our goal is to understand the direct interactions our compound makes with cellular components, uncover novel signaling pathways, and to better design analogs that might have therapeutic potential.

Differentiation in Glioma Stem Cells - Genome Wide Screen

Primary Investigator: W. Mark Saltzman

  Glioblastoma multiforme (GBM) is highly resistant to surgical, pharmacological, and radiation therapies and poses an important public health problem in the US. Recent studies suggest that one of the reasons for failure is the inability to target glioma stem cells (GSCs), which are the most treatment–resistant cell population within tumors. Evidence indicates that GSCs are cells of origin in glioma development, as they have the exclusive ability to drive tumor formation and a high capacity to promote angiogenesis. Unfortunately, standard regimens for treating gliomas are not able to effectively eliminate GSCs. However, GSC research is still at the stage of infancy: the genes/singling pathways essential for GSC proliferation, self-renewal and differentiation are largely unknown. It has been reported that several pathways, such as Notch, Sonic hedgehog and PI3K/Akt, and their related genes are important for GSCs. However, other signaling pathways and genes are probably also essential. To identify new pathways and genes, a genome-wide siRNA screening is the most promising approach.
  For this purpose, we developed a reverse transfection protocol for efficient delivery of siRNA to GSCs. Several days after transfection, cells were assessed for changes in proliferation, self-renewal and differentiation, which were performed through immunoflourescent staining of the cells using Hoescht 33342 dye and specific surface markers for GSCs. We demonstrated that this protocol could be used for large scale siRNA screening. So far we have completed the initial screening, through which we have identified several candidate genes, knockdown of which produced significant ability to inhibit GSC proliferation and self-renewal,or induce differentiation. Currently, we are in the process of validating selected hits in follow-up assays. We expect to identify new candidate signaling pathways and genes for GSCs, which can be explored for improved treatment of GBM.

HPV Infectivity - Genome Wide Screen

Primary Investigator: Daniel C. DiMaio, MD, PhD

  Viruses are responsible for approximately 15% of all human cancer worldwide. One of the major tumor viruses is human papillomavirus (HPV) type 16, which initiates most cancer of the uterine cervix and a variety of other cancers, including an increasing proportion of head-and-neck cancer. Although prophylactic vaccines have been developed that prevent HPV16 infection, these vaccines have no clear efficacy in women who are already infected and are likely to be poorly efficacious in AIDS patients and other immunosuppressed individuals, who are commonly infected with this virus. HPV16 has a small double-stranded DNA genome and encodes only about 10 proteins, and its ability to affect cell proliferation has been studied intensively. These studies have identified the viral E6 and E7 genes as the primary viral oncogenes and showed that continuous expression of these genes is required to maintain the proliferative state of cervical cancer cells. However, because HPVs are difficult to grow in the laboratory, little is known about the process of HPV16 infection. Understanding the molecular mechanism of HPV16 entry will provide new insights into the process of virus binding and entry and will suggest new anti-viral approaches that may be useful in treating and preventing HPV infections and the cancers they cause. It is now possible to incorporate genes encoding reporter proteins, such as green fluorescent protein (GFP), into non-pathogenic HPV16 pseudoviruses composed of the HPV16 L1 and L2 capsid proteins. These pseudoviruses can be used in simple, high-throughput fluorescence screens to assess the ability of interfering RNAs and small molecules to inhibit the early steps of HPV16 infection.
  We designed a screen to identify cellular genes required for HPV16 infection. Acute infection of cervical cancer cells with HPV16/GFP pseudovirus causes the vast majority of cells to fluoresce. We used an arrayed siRNA library and HPV16/GFP to conduct a robotized genome-wide screen for siRNAs that inhibited (or enhanced) GFP fluorescence in infected cervical cancer cells. Quantitative analysis of the data revealed that the screen was robust, and that it readily and reproducibly detected siRNAs that affected GFP fluorescence. Numerous active siRNA pools were deconvoluted into individual siRNAs and retested. These experiments validated the anti-HPV16 effect of a number of these siRNAs, including several that targeted genes reported by others to be important for HPV16 infection. Bioinformatics analysis revealed that several molecular pathways not previously implicated in HPV infection may be important for the early steps of this process. These experiments have identified a number of cellular genes that are required for the early steps in the HPV16 lifecycle, and suggest that the products of these genes should be explored as potential targets for novel anti-viral approaches.

Multiplexed Analysis of Protein Tyrosine Phosphatase Superfamily

Primary Investigator: Anton M. Bennett, PhD

Under construction

Sigma Antibody Project

In collaboration with Sigma, we are testing their collection of approximately 8,000 antibodies. This will generate antibodies for use in future assay development and cut screening costs significantly

Please see the following link: Sigma