Abstract
Genetic gain- and loss-of-function studies have traditionally been used to study transcriptional networks regulated by the Notch signaling pathway; however these techniques lack the ability to resolve primary and secondary transcriptional events. In contrast, the γ-secretase inhibitor (GSI) washout assay takes advantage of the reversibility of GSI, a pharmacological inhibitor of Notch signaling, along with the ability of cycloheximide to prevent secondary transcriptional effects to identify direct Notch pathway targets. Here we review this technique and the technical considerations for adapting this assay to a cell type of choice.
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References
Iso T, Kedes L, Hamamori Y (2003) HES and HERP families: multiple effectors of the Notch signaling pathway. J Cell Physiol 194:237–255
Leimeister C, Dale K, Fischer A et al (2000) Oscillating expression of c-Hey2 in the presomitic mesoderm suggests that the segmentation clock may use combinatorial signaling through multiple interacting bHLH factors. Dev Biol 227:91–103
Leimeister C, Schumacher N, Steidl C et al (2000) Analysis of HeyL expression in wild-type and Notch pathway mutant mouse embryos. Mech Dev 98:175–178
Leimeister C, Externbrink A, Klamt B et al (1999) Hey genes: a novel subfamily of hairy- and Enhancer of split related genes specifically expressed during mouse embryogenesis. Mech Dev 85:173–177
Aster JC, Pear WS, Blacklow SC (2008) Notch signaling in leukemia. Annu Rev Pathol 3: 587–613
Liu Z, Turkoz A, Jackson EN et al (2011) Notch1 loss of heterozygosity causes vascular tumors and lethal hemorrhage in mice. J Clin Invest 121:800–808
Nicolas M, Wolfer A, Raj K et al (2003) Notch1 functions as a tumor suppressor in mouse skin. Nat Genet 33:416–421
Sriuranpong V, Borges MW, Ravi RK et al (2001) Notch signaling induces cell cycle arrest in small cell lung cancer cells. Cancer Res 61:3200–3205
Ikawa T, Kawamoto H, Goldrath AW et al (2006) E proteins and Notch signaling cooperate to promote T cell lineage specification and commitment. J Exp Med 203:1329–1342
Lieber T, Kidd S, Alcamo E et al (1993) Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev 7:1949–1965
Gho M, Lecourtois M, Geraud G et al (1996) Subcellular localization of suppressor of hairless in drosophila sense organ cells during Notch signalling. Development 122:1673–1682
Bailey AM, Posakony JW (1995) Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity. Genes Dev 9:2609–2622
Heitzler P, Bourouis M, Ruel L et al (1996) Genes of the Enhancer of split and achaete-scute complexes are required for a regulatory loop between Notch and Delta during lateral signalling in Drosophila. Development 122:161–171
Artavanis-Tsakonas S, Rand MD, Lake RJ (1999) Notch signaling: cell fate control and signal integration in development. Science 284:770–776
Wang H, Zou J, Zhao B et al (2011) Genome-wide analysis reveals conserved and divergent features of Notch1/RBPJ binding in human and murine T-lymphoblastic leukemia cells. Proc Natl Acad Sci U S A 108: 14908–14913
Esler WP, Kimberly WT, Ostaszewski BL et al (2000) Transition-state analogue inhibitors of gamma-secretase bind directly to presenilin-1. Nat Cell Biol 2:428–434
Zhang Z, Nadeau P, Song W et al (2000) Presenilins are required for gamma-secretase cleavage of beta-APP and transmembrane cleavage of Notch-1. Nat Cell Biol 2: 463–465
De Strooper B, Annaert W, Cupers P et al (1999) A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain. Nature 398:518–522
Tsai JY, Wolfe MS, Xia W (2002) The search for gamma-secretase and development of inhibitors. Curr Med Chem 9:1087–1106
De Strooper B, Vassar R, Golde T (2010) The secretases: enzymes with therapeutic potential in Alzheimer disease. Nat Rev Neurol 6: 99–107
Hadland BK, Manley NR, Su D et al (2001) Gamma -secretase inhibitors repress thymocyte development. Proc Natl Acad Sci U S A 98:7487–7491
Geling A, Steiner H, Willem M et al (2002) A gamma-secretase inhibitor blocks Notch signaling in vivo and causes a severe neurogenic phenotype in zebrafish. EMBO Rep 3: 688–694
Kopan R, Ilagan MX (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233
Gordon WR, Arnett KL, Blacklow SC (2008) The molecular logic of Notch signaling–a structural and biochemical perspective. J Cell Sci 121:3109–3119
Weng AP, Ferrando AA, Lee W et al (2004) Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306:269–271
Aster JC, Blacklow SC, Pear WS (2011) Notch signalling in T-cell lymphoblastic leukaemia/lymphoma and other haematological malignancies. J Pathol 223:262–273
Weng AP, Millholland JM, Yashiro-Ohtani Y et al (2006) c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma. Genes Dev 20:2096–2109
Pear WS, Aster JC, Scott ML et al (1996) Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles. J Exp Med 183: 2283–2291
Bailis W, Yashiro-Ohtani Y, Fang TC et al (2013) Notch simultaneously orchestrates multiple helper T cell programs independently of cytokine signals. Immunity 39: 148–159
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Bailis, W., Yashiro-Ohtani, Y., Pear, W.S. (2014). Identifying Direct Notch Transcriptional Targets Using the GSI-Washout Assay. In: Bellen, H., Yamamoto, S. (eds) Notch Signaling. Methods in Molecular Biology, vol 1187. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1139-4_19
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DOI: https://doi.org/10.1007/978-1-4939-1139-4_19
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