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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2016, Vol. 10 Issue (4) : 410-419     DOI: 10.1007/s11684-016-0489-0
RESEARCH ARTICLE |
Inhibition of the nuclear export of p65 and IQCG in leukemogenesis by NUP98-IQCG
Mengmeng Pan1,Qiyao Zhang1,2,Ping Liu1,Jinyan Huang1,Yueying Wang1(),Saijuan Chen1,2()
1. State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital Affiliated to Shanghai Jiao Tong University (SJTU) School of Medicine, Shanghai 200025, China
2. Institute of Health Sciences, Shanghai Institutes for Biological Sciences and Graduate School, Chinese Academy of Sciences and SJTU School of Medicine, Shanghai 200025, China
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Abstract  

NUP98 fuses with approximately 34 different partner genes via translocation in hematological malignancies. Transgenic or retrovirus-mediated bone marrow transplanted mouse models reveal the leukemogenesis of some NUP98-related fusion genes. We previously reported the fusion protein NUP98-IQ motif containing G (IQCG) in a myeloid/T lymphoid bi-phenoleukemia patient with t(3;11) and confirmed its leukemogenic ability. Herein, we demonstrated the association of NUP98-IQCG with CRM1, and found that NUP98-IQCG expression inhibits the CRM1-mediated nuclear export of p65 and enhances the transcriptional activity of nuclear factor-κB. Moreover, IQCG could be entrapped in the nucleus by NUP98-IQCG, and the fusion protein interacts with calmodulin via the IQ motif in a calcium-independent manner. Therefore, the inhibition of nuclear exports of p65 and IQCG might contribute to the leukemogenesis of NUP98-IQCG.

Keywords NUP98-IQCG      nuclear export      NF-κB      CRM1     
Corresponding Authors: Yueying Wang,Saijuan Chen   
Just Accepted Date: 07 November 2016   Online First Date: 21 November 2016    Issue Date: 01 December 2016
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-016-0489-0     OR     http://academic.hep.com.cn/fmd/EN/Y2016/V10/I4/410
Fig.1  NUP98-IQCG associates with CRM1. HEK293T cells were transfected with either pEGFP-empty (GFP) or NUP98-IQCG (NI)-expressing vector. The cells were immunostained with anti-CRM1 antibody combined with Alexa Fluor 594-conjugated secondary antibody (red). Transfected Cells were treated with 10 nmol/L LMB for 2 h before fixation in LMB group (+ LMB). The nuclei were visualized by Hoechst staining (blue). Bar, 10 mm.
Fig.2  NUP98-IQCG entraps p65 in nucleus in HEK293T cells. (A) Schematics of the pEGFP vector carrying NUP98-IQCG (NI), NUP98, IQCG, NI without the IQCG portion (NIΔIQCG), or NI without the NUP98 portion (NIΔNUP98). (B) HEK293T cells were transfected with either pEGFP empty vector (GFP), or vectors expressing NUP98-IQCG (NI), NUP98, IQCG, NIΔIQCG, or NIΔNUP98. The cells were immunostained with anti-p65 antibody combined with Alexa Fluor 594-conjugated secondary antibody (red). The untransfected cells were treated with 10 nmol/L LMB for 2 h before fixation in LMB group. The nuclei were visualized by Hoechst staining (blue). Bar, 10 mm. (C) Histogram of the relative proportions of cells with varying subcellular localization of p65 in transfected HEK293T cells. Cells were classified into three categories according to the distribution of p65: all concentrated in cytoplasm (C), localized primarily in the cytoplasm (cytoplasm>nucleus, C>N), or equally present in the cytoplasm and in the nucleus (cytoplasm ≈ nucleus, C ≈ N). Error bars represent the mean±SD of three independent experiments. (D) COS-7 cells were transfected with either pEGFP-empty (GFP) or NUP98-IQCG (NI)-expressing vector. The cells were immunostained with anti-p65 antibody combined with Alexa Fluor 594-conjugated secondary antibody (red). The nuclei were visualized by Hoechst staining (blue). Bar, 10 mm. (E) GFP+ bone marrow cells isolated from control and NUP98-IQCG (NI) mice were immunostained with anti-p65 primary antibody and Alexa Fluor 594-conjugated secondary antibody (red). The nuclei were visualized by Hoechst staining (blue). Bar, 20 mm.
Fig.3  NUP98-IQCG has no effect on the subcellular localizations of p50 and IkBa. HEK293T (A and B) and COS-7 cells (C and D) were transfected with either pEGFP-empty (GFP) or NUP98-IQCG (NI)-expressing vector. The cells were immunostained with anti-p50 (A and C) or anti-IkBa (B and D) antibodies combined with Alexa Fluor 594-conjugated secondary antibody (red). The untransfected cells were treated with 10 nmol/L LMB for 2 h before fixation in the LMB group. The nuclei were visualized by Hoechst staining (blue). Bar, 10 mm.
Fig.4  NUP98-IQCG-induced p65 nuclear retention and NF-kB activation are associated with the inhibition of CRM1-mediated nuclear export. (A) HEK293T cells were transfected with pEGFP-tagged CRM1 (CRM1) with (lower panel) or without (upper panel) the LMB treatment. The cells were immunostained with anti-p65 antibody combined with Alexa Fluor 594-conjugated secondary antibody (red). The nuclei were visualized by Hoechst staining (blue). Bar, 10 mm. (B) HEK293T cells were transiently transfected with mCherry-NI (red) combined with pEGFP-empty (GFP) or CRM1-expressing vector (green) with or without LMB treatment. The cells were immunostained with anti-p65 antibody combined with Alexa Fluor 647-conjugated secondary antibody (purple). The nuclei were visualized by Hoechst staining (blue). Bar, 10 mm. (C) The protein levels of p65, p50, and IkBa in HEK293T cells transfected with pEGFP-empty (GFP) or NUP98-IQCG (NI)-expressing vector. b-actin and a-tubulin serve as loading controls. (D) Activation of NF-kB-firefly luciferase by NUP98-IQCG (NI). NUP98-HOXA9 (NH9) was used as a positive control. All transfection experiments were performed in triplicate and repeated thrice. *P<0.05; **P<0.01.
Fig.5  NUP98-IQCG causes nuclear retention of IQCG. (A) Schematics of the mCherry vector carrying NUP98-IQCG (NI), and the pEGFP vector carrying NUP98, IQCG, or NI without the IQCG portion (NIΔIQCG). (B) HEK293T cells were transiently transfected with mCherry-NI (red, left panel) combined with pEGFP vectors expressing NUP98, IQCG, or NIΔIQCG (green). Hoechst staining was used to detect the nucleus (blue). Bar, 10 mm. (C) HEK293T cells were transiently transfected with pEGFP-IQCG (green, left panel) combined with either mCherry-empty (mCherry) or NUP98-IQCG-expressing (mCherry-NI) vector (red). Hoechst staining was used to detect the nucleus (blue). Cells transfected with pEGFP-IQCG (bottom panels) combined with empty mCherry were treated with 10 nmol/L LMB for 2 h before fixation. Bar, 10 mm. (D) Histogram of the relative proportions of cells with varying subcellular localization of IQCG. Cells were classified into three categories according to IQCG distribution: all concentrated in cytoplasm (C), localized primarily in the cytoplasm (cytoplasm>nucleus, C>N), or equally present in the cytoplasm and in the nucleus (cytoplasm ≈ nucleus, C ≈ N). Error bars represent the mean±SD of three independent experiments.
Fig.6  NUP98-IQCG interacts with CaM and the calcium signaling pathway is dysregulated in NUP98-IQCG mice. (A) Ability of NUP98-IQCG (NI) and NUP98-IQCGΔIQ (NIΔIQ) to interact with CaM under varying calcium concentrations, as determined by Co-IP experiments. Anti-Flag M2 magnetic beads were used for immunoprecipitation (IP), and antibodies against CaM or Flag were used for Western blot. (B) GO analysis of downregulated genes via DAVID (FC>2; FDR<0.05). (C) Enrichment plots for gene sets of the calcium signaling pathway identified by GSEA. NES: normalized enrichment score. P: nominal P value. (D) Quantitative RT-PCR of the four genes crucial to the calcium signaling pathway in GFP+ c-Kit+ BM samples of the control (Con) and NUP98-IQCG (NI) mice. Error bars represent mean±SD (n = 3 per group). *P<0.05, **P<0.01, ***P<0.001.
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