ETO, but not leukemogenic fusion protein AML1/ETO, augments RBP-Jkappa/SHARP-mediated repression of notch target genes

Department of Cellular and Molecular Immunology, Max Planck Institute of Immunobiology, Stübeweg 51, 79108 Freiburg, Germany.
Molecular and Cellular Biology (Impact Factor: 4.78). 06/2008; 28(10):3502-12. DOI: 10.1128/MCB.01966-07
Source: PubMed


Notch is a transmembrane receptor that determines cell fates and pattern formation in all animal species. After specific ligand
binding, the intracellular part of Notch is cleaved off and translocates to the nucleus, where it targets the DNA binding
protein RBP-Jκ. In the absence of Notch, RBP-Jκ represses Notch target genes by recruiting a corepressor complex. We and others
have previously identified SHARP as one component of this complex. Here, we show that the corepressor ETO as well as the leukemogenic
fusion protein AML1/ETO directly interacts with SHARP, that ETO is part of the endogenous RBP-Jκ-containing corepressor complex,
and that ETO is found at Notch target gene promoters. In functional assays, corepressor ETO, but not AML1/ETO, augments SHARP-mediated
repression in an histone deacetylase-dependent manner. Furthermore, either the knockdown of ETO or the overexpression of AML1/ETO
activates Notch target genes. Therefore, we propose that AML1/ETO can disturb the normal, repressive function of ETO at Notch
target genes. This activating (or derepressing) effect of AML1/ETO may contribute to its oncogenic potential in myeloid leukemia.

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Available from: Robert Liefke, Aug 14, 2014
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    • "Like other ETO homologue proteins, MTG16 only binds DNA in cooperation with site-specific transcription factors [7] competing out coactivators and causing repressed chromatin conformation by the action of recruited histone deacetylases (HDACs). MTG16 links a number of transcription factors to its chromatin silencing machinery [8], [9], [10], [11], [12]. Among the members of its family, MTG16 is the most highly expressed in primary hematopoietic cells confined especially to stem/progenitor, erythroid, megakaryocytic and B cells [13], [14]. "
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    ABSTRACT: The myeloid translocation gene 16 product MTG16 is found in multiple transcription factor-containing complexes as a regulator of gene expression implicated in development and tumorigenesis. A stable Tet-On system for doxycycline-dependent expression of MTG16 was established in B-lymphoblastoid Raji cells to unravel its molecular functions in transformed cells. A noticeable finding was that expression of certain genes involved in tumor cell metabolism including 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 and 4 (PFKFB3 and PFKFB4), and pyruvate dehydrogenase kinase isoenzyme 1 (PDK1) was rapidly diminished when MTG16 was expressed. Furthermore, hypoxia-stimulated production of PFKFB3, PFKFB4 and PDK1 was inhibited by MTG16 expression. The genes in question encode key regulators of glycolysis and its coupling to mitochondrial metabolism and are commonly found to be overexpressed in transformed cells. The MTG16 Nervy Homology Region 2 (NHR2) oligomerization domain and the NHR3 protein-protein interaction domain were required intact for inhibition of PFKFB3, PFKFB4 and PDK1 expression to occur. Expression of MTG16 reduced glycolytic metabolism while mitochondrial respiration and formation of reactive oxygen species increased. The metabolic changes were paralleled by increased phosphorylation of mitogen-activated protein kinases, reduced levels of amino acids and inhibition of proliferation with a decreased fraction of cells in S-phase. Overall, our findings show that MTG16 can serve as a brake on glycolysis, a stimulator of mitochondrial respiration and an inhibitor of cell proliferation. Hence, elevation of MTG16 might have anti-tumor effect.
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    • "Many of these factors are dedicated transcriptional repressors or corepressors implicated in various human disorders, including leukemia, lymphoma and neurodegeneration. Intriguingly, several of these SMRT/N-CoR associating factors are also functionally connected with the Notch pathway (Kao et al., 1998; Doroquez et al., 2007; Salat et al., 2008; Tong et al., 2011), raising the possibility that SMRT, N-CoR and some of their associating factors may converge in regulating the Notch pathway. The Notch signaling pathway is well-conserved in metazoans, and is fundamental for pattern formation, cell fate specification and cell growth in multiple vertebrate and invertebrate tissues (Artavanis-Tsakonas et al., 1999; Lai, 2004; Schweisguth, 2004; Bray, 2006; Fortini, 2009; Kopan and Ilagan, 2009). "
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    ABSTRACT: SMRTER (SMRT-related and ecdysone receptor interacting factor) is the Drosophila homologue of the vertebrate proteins SMRT and N-CoR, and forms with them a well-conserved family of transcriptional corepressors. Molecular characterization of SMRT-family proteins in cultured cells has implicated them in a wide range of transcriptional regulatory pathways. However, little is currently known about how this conserved class of transcriptional corepressors regulates the development of particular tissues via specific pathways. In this study, through our characterization of multiple Smrter (Smr) mutant lines, mosaic analysis of a loss-of-function Smr allele, and studies of two independent Smr RNAi fly lines, we report that SMRTER is required for the development of both ovarian follicle cells and the wing. In these two tissues, SMRTER inhibits not only the ecdysone pathway, but also the Notch pathway. We differentiate SMRTER's influence on these two signaling pathways by showing that SMRTER inhibits the Notch pathway, but not the ecdysone pathway, in a spatiotemporally restricted manner. We further confirm the likely involvement of SMRTER in the Notch pathway by demonstrating a direct interaction between SMRTER and Suppressor of Hairless [Su(H)], a DNA-binding transcription factor pivotal in the Notch pathway, and the colocalization of both proteins at many chromosomal regions in salivary glands. Based on our results, we propose that SMRTER regulates the Notch pathway through its association with Su(H), and that overcoming a SMRTER-mediated transcriptional repression barrier may represent a key mechanism used by the Notch pathway to control the precise timing of events and the formation of sharp boundaries between cells in multiple tissues during development.
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    • "of Wnt and Notch signalling (TCF4 and CSL) (Melnick et al, 2000; McGhee et al, 2003; Chevallier et al, 2004; Zhang et al, 2004; Schuh et al, 2005; Goardon et al, 2006; Moore et al, 2008; Salat et al, 2008; Engel et al, 2010). To begin to address which of these pathways might be critical to Mtg16-depen- dent stem cell functions, we re-expressed Mtg16, or a point mutant of Mtg16 that fails to suppress E protein-dependent transcription (F210A; R220A is a control mutation) (Hunt et al, 2011) in null BM cells and performed LTC-IC assays (Figure 5C; see Supplementary Figure S5 for expression controls). "
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