Our research project consists of a “systems-level” molecular and genetic characterization of the Notch signaling network. We will combine large-scale proteomic and genetic approaches to establish and validate a comprehensive protein interaction map of elements involved in Notch signaling (Notch “interactome”). The first step in our project is to generate a protein complex map for ~120 Drosophila Notch related proteins using high throughput co-affinity protein complex purification followed by mass spectrometry analysis. Thereafter, we will take advantage of a Drosophila genetic mutant collection to verify the biological relevance of the interactions in vivo. It is our hope that this systems-level study will provide insights into the Notch genetic circuitry and reveal high-level properties that govern this pathway.
Notch Research Projects
We are using a novel set of knock-in (KI) mice, which we generated in collaboration with Dr Jeff Greve (Exelixis Inc) to examine functional properties of four Notch paralogues in development, adult tissue homeostasis and disease. Four Notch(1-4)CreERT2tg/+ KI strains have Tamoxifen -inducible CRE (CreERT2) inserted into exon1 of respective Notch paralogues and can be used in combination with standard reporter strains such as R26R for conditional labeling of cells expressing individual paralogues. We are currently using these reporter lines to examine lineages and developmental potential of Notch expressing cells in mammary gland, ovary and GI tract. Four Rosa26 N1ICDtg/+ KI strains have Cre-inducible form of activated Notch1,-2,-3 or-4 (”Rosa26-floxSTOPflox-NiICD-IRES-YFP”) knocked-in into Rosa26 locus. These four lines can either be used in conjunction with Notch(1-4)CreERT2tg/+ strains or tissue-specific Cre lines to examine and compare biological impact of constitutive activation of Notch paralogues in different target tissues.
Joseph Arboleda-Velasquez; Debbie Kelly (Tom Walz lab, HMS)
We have previously identified 28 genes co-regulated by Notch and Ras in the Drosophila embryo at two different time-points of development by genome wide gene expression profiling and are currently using genetics, genomics and mammalian cell culture to address whether these genes define the backbone of a conserved network of Notch and Ras signal integration. Ultimately, these analyses may aid in uncovering general paradigms of signal integration and may be crucial to understanding the regulation of normal or pathological biological processes such as stem cell differentiation and cancer.
As we explore the evolution of Notch signaling between invertebrates and higher organisms, we are interested in understanding the functional specificity of the different Notch paralogs in mammals. In particular, we are studying the relationship between Notch 1 and Notch 3. With Debbie Kelly and Tom Waltz at the Harvard Medical School, we are elucidating the structure of the extracellular domain of Notch receptors by molecular electron microscopy. With Joseph Arboleda-Velasquez in our lab, we have developed a sensitive ligand-based Notch signaling assay in MEFs co-cultures that allows us to harness the specificity of normal and pathological Notch signaling. We are applying this assay to elucidating the genotype/phenotype relationship of CADASIL Notch 3 variants. Altogether, these studies will provide insights in the structure/function relationship of Notch receptors.
Aberrant signaling through the Notch receptor is invariably associated with mutant development in model organisms as well as in humans. Relevant to my work is the dominant syndrome CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), which has been associated with specific mutations in Notch 3. However, the functional nature of the mutations remains enigmatic. The association of Notch 3 expression in vascular smooth muscle cells and the fact that the clinical symptoms of CADASIL include ischemic strokes has lead to the suggestion, that Notch 3 function is associated with vascular function.
We adopted an integrated strategy to probe the biology and pathobiology of Notch 3 and CADASIL by generating novel mouse transgenic models that carry specific CADASIL mutations identified in two Colombian populations (C455R and R1031C) for which we have access to relevant clinical information and postmortem tissue through a collaboration with Colombian scientists led by Dr. Francisco Lopera (University of Antioquia). We previously defined a striking susceptibility to ischemic stroke in a Notch 3 knockout animal and we are currently using this model in rescue experiments to probe the functionality of CADASIL mutations using new mouse models. We are also applying proteomic methodologies for the study of CADASIL vessel pathology using laser capture micro-dissection on postmortem human tissue followed by mass spectrometry. These studies allowed us to identify candidate proteins that may have translational potential as biomarkers.
Collaborators: Laboratory of Tomas Kirchhausen (Dept. of Cell Biology, HMS)
We are combining genetic and molecular approaches to examine aspects of trafficking in the regulation of Notch signaling activity. To monitor this trafficking both in vivo and in vitro, we have constructed a roster of fluorescently tagged Notch (receptor) and Delta (ligand) transgenes. In addition, we used phenotypes associated with kurtz and deltex as a basis for a genetic screen. Kurtz, the Drosophila homologue of non-visual β-arrestin and in combination with the ubiquitin ligase deltex, a known modifier of Notch signals, was previously shown to modulate the Notch protein levels and signaling. Based on this observation, we screened the Exelixis collection to define the genetic circuitry that integrates Notch and β-arrestin function. Amongst the 126 insertions that modified a deltex-kurtz double mutant wing phenotype were several that affected loci predicted to be involved in protein trafficking. We are currently using RNAi and genetics to test whether the identified modifiers alter fluorescently tagged Notch and Delta trafficking.
In addition to its many roles during development, aberrant Notch activity has been associated with various forms of cancers, including more than 50% of the cases of T-cell lymphoblastic leukaemia (T-ALL). Although Notch itself is capable of inducing hyperproliferation, it requires the action of additional genes to induce a full-blown oncogenic state. Despite the large number of genes known to affect Notch activity, the gamut of genes capable of cooperating with Notch receptor activation to influence proliferation and oncogenesis remains unknown. To identify genes capable of affecting Notch-dependent proliferation, we have performed a screen of the Exelixis collection to uncover modifiers of a large eye phenotype that is associated with ey-GAL4 directed expression of an activated form of Notch. We have identified ~300 modifier genes, of which 171 have human homologs. In addition to genes that simply enhance or suppress Notch-induced proliferation, we have also identified a particularly interesting subset of modifiers that, in concert with activated Notch, induce an ectopic eye phenotype consistent with metastatic activity. We are currently exploring the role of several of these modifier genes and their relationship with Notch using assays in both Drosophila and mammalian cell culture. The validity of this functional analysis and its potential relevance to oncogenesis will be examined tissue culture models and in vivo tumor xenografts.