ANTI-MTG16 ANTIBODIES REVEAL MTG16 SUBCELLULAR DISTRIBUTION AND NUCLEOCYTOPLASMIC TRANSPORT IN ERYTHROLEUKEMIA CELLS.

The myeloid translocation gene (MTG) family of transcriptional co-repressors 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 co-repressors 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 (NHR) 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 α-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 CRM1 antagonist, leptomycin-B, we show that MTG16 levels rise in the nucleus of MEL 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.

Myeloid translocation gene 16 (MTG16) interacts with Notch transcription complex components to integrate Notch signaling in hematopoietic cell fate specification.

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 gamma-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.