The Notch signaling pathway regulates gene expression programs to influence the specification of cell fate in diverse tissues. In response to ligand binding, the intracellular domain of the Notch receptor is cleaved by the ␥-secretase complex and then translocates to the nucleus. There, it binds the transcriptional repressor CSL, triggering its conversion to an activator of Notch target gene expression. The events that control this conversion are poorly understood. We show that the transcriptional corepressor, MTG16, interacts with both CSL and the intracellular domains of Notch receptors, suggesting a pivotal role in regulation of the Notch transcription complex. The Notch1 intracellular domain disrupts the MTG16-CSL interaction. Ex vivo fate specification in response to Notch signal activation is impaired in Mtg16 ؊/؊ hematopoietic progenitors, and restored by MTG16 expression. An MTG16 derivative lacking the binding site for the intracellular domain of Notch1 fails to restore Notch-dependent cell fate. These data suggest that MTG16 interfaces with critical components of the Notch transcription complex to affect Notch-dependent lineage allocation in hematopoiesis.
The myeloid translocation gene (MTG) family of transcriptional corepressors consists of three highly conserved members: MTG8, MTG16, and MTGR1, each evolutionarily related to the Drosophila protein NERVY and with orthologs across the mammalian hierarchy. By coordinating coincident interactions between DNA binding proteins, other corepressors, and epigenetic effectors, MTG proteins occupy a critical nexus in transcriptional control complexes to profoundly impact the specification of cell fate. MTG family members are most conserved within nervy homology regions (NHRs) 1-4, with each region fulfilling functions common to the family. Studies of functional differences between MTG proteins require carefully qualified immunologic reagents specific to each family member. We have developed a group of a-MTG16 antibodies and carefully characterized their specificity for MTG16. These tools reveal that MTG16 is concentrated in the cytoplasm of erythroleukemia cell lines from human and mouse. Using the chromosome region maintenance 1 (CRM1) antagonist leptomycin B, we show that MTG16 levels rise in the nucleus of murine erythroleukemia cells and decline in the cytoplasm. Together, these data indicate bidirectional movement of MTG16 between cytoplasmic and nuclear compartments. Our work reveals an unrecognized feature of MTG16 regulation that may impact cell fate specification and provides reagents to address important questions regarding MTG16 functions in vivo.
257 The Notch signaling pathway regulates gene expression programs to control the specification of cell fate in diverse tissues. In response to ligand binding, the intracellular domain of Notch receptors is cleaved by the γ-secretase complex and then translocates to the nucleus. There it binds the transcriptional repressor CSL, triggering its conversion to an activator of Notch target gene expression. The events that control this conversion are poorly understood. We show that the transcriptional co-repressor, MTG16, interacts with both CSL and the intracellular domains of Notch receptors. The MTG16 NHR3 domain contributes to CSL binding, while N-ICD binding sites lie within the PST1 and PST2 domains. The Notch intracellular domain disrupts the MTG16—CSL interaction, suggesting a pivotal role in regulating the Notch transcription complex. Using co-culture of Lin-/Sca-1+/c-Kit+ (LSK) cells on OP9-DL1 stromal cells, we show that ex vivo fate specification in response to Notch signal activation is altered in Mtg16 (−/−) hematopoietic progenitors. While Notch signal activation specifies lymphoid fate in Mtg16 (+/+) LSK cells, Mtg16 (−/−) LSK cells display cell surface marker expression reminiscent of myeloid differentiation. We used this lineage allocation assay to assess the contribution of MTG16 to cell fate determination. Lymphoid fate specification is restored by MTG16WT expression in Mtg16 (−/−) LSK cells. However, an MTG16 mutant deficient in N1-ICD binding is defective in this assay, suggesting this region is important to Notch-dependent lineage allocation. These data suggest that MTG family proteins interface with critical components of the Notch transcription complex and intimate a functional relationship between MTG proteins and Notch signaling in normal and malignant hematopoiesis. Disclosures: No relevant conflicts of interest to declare.
The myeloid translocation genes (MTG) are preferred targets of chromosomal translocations with the Runx1 gene in acute myeloid leukemia (AML) pathogenesis. The resulting fusion proteins, RUNX1-MTG8 and RUNX1-MTG16, influence gene expression programs that control self-renewal and differentiation in hematopoiesis. How they engage the machinery of self-renewal to alter hematopoietic development is poorly understood. To gain additional insights into this question, we investigated structure–function relationships between RUNX1-MTG fusion proteins and core components of the Notch transcription complex, a critical determinant of cell fate specification in diverse tissues. The Notch transcription complex is composed minimally of the transcriptional repressor CSL, the intracellular domain of Notch receptors (N-ICD) and the transcriptional co-activator Mastermind (MAML). We show that RUNX1-MTG fusion proteins transactivate a Notch-responsive element from the Hes1 promoter. Using MTG16 as a model protein, we show that N-ICDs interact with a discrete domain in the MTG N-terminus. We translated these findings to the fusion proteins and found N-ICD binding was retained by RUNX1-MTGs and a RUNX1-MTG16 mutant that lacks the N1-ICD binding site. In parallel experiments, we defined the binding region for MTG proteins and their fusion protein derivatives on the N1-ICD. Informed by these structural relationships, we utilized RUNX1-MTG proteins or mutants that lack N1-ICD binding sites in transcriptional reporter assays. Our findings suggest that RUNX1-MTG fusion proteins alter transcription of the Notch target gene, Hes1 and intimate a structure–function relationship between RUNX1-MTG fusion proteins and Notch signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3905.
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