Shoko Saito

Aberrant intracellular localization of SET‐CAN fusion protein, associated with a leukemia, disorganizes nuclear export

The SET‐CAN fusion gene is the product of a chromosomal rearrangement found on 9q34 associated with an acute undifferentiated leukemia. SET‐CAN encodes an almost complete SET protein fused to the C‐terminal two‐thirds of CAN. SET is also known as TAF‐Iβ, a histone chaperone and intracellular inhibitor of protein phosphatase 2A, whereas CAN is identical to Nup214, a nucleoporin protein. To obtain insight into the leukemogenic function of SET/TAF‐Iβ‐CAN/Nup214, we have examined its subcellular localization. Immunofluorescence analyses showed that SET/TAF‐Iβ and CAN/Nup214 are found in the nucleus and the nuclear envelope, respectively, whereas the majority of SET/TAF‐Iβ‐CAN/Nup214 is localized in the nucleus. SET/TAF‐Iβ‐CAN/Nup214 interacted with hCRM1, one of the nuclear export factors, and caused aberrant intracellular localization of hCRM1. In cells expressing SET/TAF‐Iβ‐CAN/Nup214, a protein containing a nuclear export signal accumulated in the nucleus. The export of this protein was partially restored by overexpression of hCRM1. These results suggest that aberrantly localized molecules associated with SET/TAF‐Iβ‐CAN/Nup214 may be involved in oncogenesis.

The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

Summary We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3Sci), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3Sci/+ SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3Sci/+ SCN slices. In conclusion, by cloning Zfhx3Sci, we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

Impairment of erythroid and megakaryocytic differentiation by a Leukemia-Associated and t(9;9)-derived fusion gene product, SET/TAF-Iβ-CAN/Nup214†

SET‐CAN associated with the t(9;9) in acute undifferentiated leukemia encodes almost the entire sequence of SET and the C‐terminal two‐third portion of CAN, including the FG repeat region. To clarify a role(s) of SET‐CAN in leukemogenesis, we developed transgenic mice expressing SET‐CAN under the control of the Gata1 gene hematopoietic regulatory domain that is active in distinct sets of hematopoietic cells. SET‐CAN transgenic mice showed anemia, thrombocytopenia, and splenomegaly. A significant number of transgenic mice started dying after 6 months post‐birth, being in good agreement with the fact that red blood cells and platelets decreased. We found that a significant number of c‐kit+ myeloid cells appeared in peripheral blood in transgenic mice. Characterization of the bone marrow cells of transgenic mice indicated impairment in hematopoietic differentiation of erythroid, megakaryocytic, and B cell lineages by SET‐CAN. Transgenic mice, in particular, exhibited a high population of the c‐kit+Sca‐1+Lin− fraction in bone marrow cells compared with that of the control littermates. Our results demonstrate that SET‐CAN blocks the hematopoietic differentiation program—one of the characteristics of acute myeloid leukemia. J. Cell. Physiol. 214: 322–333, 2008.

Interferon-Inducible Antiviral Protein MxA Enhances Cell Death Triggered by Endoplasmic Reticulum Stress

Human myxovirus resistance gene A (MxA) is a type I interferon-inducible protein and exhibits the antiviral activity against a variety of RNA viruses, including influenza virus. Previously, we reported that MxA accelerates cell death of influenza virus-infected cells through caspase-dependent and -independent mechanisms. Similar to other viruses, influenza virus infection induces endoplasmic reticulum (ER) stress, which is one of cell death inducers. Here, we have demonstrated that MxA enhances ER stress signaling in cells infected with influenza virus. ER stress-induced events, such as expression of BiP mRNA and processing of XBP1 mRNA, were upregulated in cells expressing MxA by treatment with an ER stress inducer, tunicamycin (TM), as well as influenza virus infection. TM-induced cell death was also accelerated by MxA. Furthermore, we showed that MxA interacts with BiP and overexpression of BiP reduces MxA-promoted ER stress signaling. Because cell death in virus-infected cells is one of ultimate anti-virus mechanisms, we propose that MxA-enhanced ER stress signaling is a part of the antiviral activity of MxA by accelerating cell death.

Leukemia-Associated Nup214 Fusion Proteins Disturb the XPO1-Mediated Nuclear-Cytoplasmic Transport Pathway and Thereby the NF-κB Signaling Pathway

ABSTRACT Nuclear-cytoplasmic transport through nuclear pore complexes is mediated by nuclear transport receptors. Previous reports have suggested that aberrant nuclear-cytoplasmic transport due to mutations or overexpression of nuclear pore complexes and nuclear transport receptors is closely linked to diseases. Nup214, a component of nuclear pore complexes, has been found as chimeric fusion proteins in leukemia. Among various Nup214 fusion proteins, SET-Nup214 and DEK-Nup214 have been shown to be engaged in tumorigenesis, but their oncogenic mechanisms remain unclear. In this study, we examined the functions of the Nup214 fusion proteins by focusing on their effects on nuclear-cytoplasmic transport. We found that SET-Nup214 and DEK-Nup214 interact with exportin-1 (XPO1)/CRM1 and nuclear RNA export factor 1 (NXF1)/TAP, which mediate leucine-rich nuclear export signal (NES)-dependent protein export and mRNA export, respectively. SET-Nup214 and DEK-Nup214 decreased the XPO1-mediated nuclear export of NES proteins such as cyclin B and proteins involved in the NF-κB signaling pathway by tethering XPO1 onto nuclear dots where Nup214 fusion proteins are localized. We also demonstrated that SET-Nup214 and DEK-Nup214 expression inhibited NF-κB-mediated transcription by abnormal tethering of the complex containing p65 and its inhibitor, IκB, in the nucleus. These results suggest that SET-Nup214 and DEK-Nup214 perturb the regulation of gene expression through alteration of the nuclear-cytoplasmic transport system.

Function of Nup98 subtypes and their fusion proteins, Nup98-TopIIβ and Nup98-SETBP1 in nuclear-cytoplasmic transport

Nup98 is a component of the nuclear pore complex. The nup98-fusion genes derived by chromosome translocations are involved in hematopoietic malignancies. Here, we investigated the functions of Nup98 isoforms and two unexamined Nup98-fusion proteins, Nup98-TopIIβ and Nup98-SETBP1. We first demonstrated that two Nup98 isoforms are expressed in various mouse tissues and similarly localized in the nucleus and the nuclear envelope. We also showed that Nup98-TopIIβ and Nup98-SETBP1 are localized in the nucleus and partially co-localized with full-length Nup98 and a nuclear export receptor XPO1. We demonstrated that Nup98-TopIIβ and Nup98-SETBP1 negatively regulate the XPO1-mediated protein export. Our results will contribute to the understanding of the molecular mechanism by which the Nup98-fusion proteins induce tumorigenesis.