Hoxa9 Transduction Induces Hematopoietic Stem and Progenitor Cell Activity through Direct Down-Regulation of Geminin Protein

<div><p>Hoxb4, a 3′-located Hox gene, enhances hematopoietic stem cell (HSC) activity, while a subset of 5′-located Hox genes is involved in hematopoiesis and leukemogenesis, and some of them are common translocation partners for Nucleoporin 98 (Nup98) in patients with leukemia. Although these Hox gene derivatives are believed to act as transcription regulators, the molecular involvement of the Hox gene derivatives in hematopoiesis and leukemogenesis remains largely elusive. Since we previously showed that Hoxb4 forms a complex with a Roc1-Ddb1-Cul4a ubiquitin ligase core component and functions as an E3 ubiquitin ligase activator for Geminin, we here examined the E3 ubiquitin ligase activities of the 5′-located Hox genes, Hoxa9 and Hoxc13, and Nup98-Hoxa9. Hoxa9 formed a similar complex with the Roc1-Ddb1-Cul4a component to induce ubiquitination of Geminin, but the others did not. Retroviral transduction-mediated overexpression or siRNA-mediated knock-down of Hoxa9 respectively down-regulated or up-regulated Geminin in hematopoietic cells. And Hoxa9 transduction-induced repopulating and clonogenic activities were suppressed by Geminin supertransduction. These findings suggest that Hoxa9 and Hoxb4 differ from Hoxc13 and Nup98-Hoxa9 in their molecular role in hematopoiesis, and that Hoxa9 induces the activity of HSCs and hematopoietic progenitors at least in part through direct down-regulation of Geminin.</p> </div

Expression of Hoxa9 in hematopoietic cells, and effect of siRNA-mediated Hoxa9 knock-down on Geminin expression in FL.

<p>(<b>A</b>) Expression of Hoxa9 mRNA in BM and FL. (<b>B</b>) Efficiency of siRNA transfection into FL. Hoxa9siRNA was co-transfected with either non-labeled non-targeting (dotted line) or green fluorescent dye-labeled non-targeting siRNA (green line) as an indicator to monitor the transfection efficiency. High transfection efficiency was confirmed by detecting expression of the indicator by flow cytometry. Rate of a green-fluorescence⁺ or green-fluorescence⁻ subpopulation (%) in the transfectants is shown in the upper part of the panel. (<b>C</b>) Effect of Hoxa9 siRNA transfection on expression of mRNA for Hoxa9, Hoxa10, Hoxb4, Hoxd13, Geminin, Cdt1 and Cyclin A2. Specific knock-down of Hoxa9 mRNA expression was confirmed by real-time PCR analysis. Hoxa9siRNA, siRNA for Hoxa9; C, non-targeting control siRNA. (<b>D</b>) Effect of siRNA transfection on Geminin protein expression in each phase of the cell cycle. Geomean of fluorescence intensity for Geminin protein was shown. (<b>E</b>) Effect of siRNA transfection on cell cycle. (<b>F</b>) Effect of siRNA transfection on apoptosis. The data from three independent experiments were subjected to the statistical analysis. The representative cell sorting data are shown.</p

Effect on clonogenic activity.

<p>(<b>A</b>) Effect of transduction of Hox derivatives on clonogenic activity. Numbers and types of colonies are shown in the upper panel, and close-up photographs of representative colonies in the lower panel. (<b>B</b>) Effect of Geminin supertransduction on Hoxa9 transduction-mediated induction of clonogenic activity. Numbers and types of colonies are shown in the upper panel. Close-up photographs of representative colonies and images obtained with an inverted microscopy are shown in the lower panel. A cell cluster with more than 20 cells was counted as a colony under microscopy. (<b>C</b>) Effect of Geminin supertransduction on Hoxa9 transduction-mediated induction of replating activity. MEP and MPI, empty control vectors.</p

Effects of Hoxb4, Hoxa9, Hoxc13 and Nup98-Hoxa9 transduction on BM.

<p>Retrovirally transduced cells were subjected to flow cytometry analysis as well as to real-time PCR analysis. (<b>A</b>) Cell populations in each phase of the cell cycle were analyzed by cell sorting analysis. (<b>B</b>) Relative expression of mRNA for Geminin. (<b>C</b>) Geminin protein expression in each phase of the cell cycle. Geomean of the fluorescence intensity was shown. MEP, an empty control vector.</p

Immunoprecipitation analysis of Hox derivatives and effect of Cul4a knock-down on Hoxa9-mediated down-regulation of Geminin protein in HEK-293 cells.

<p>(<b>A</b>) Either of Flag-Hoxa9, Flag-Hoxc13 or Flag-Nup98-Hoxa9 was transfected in HEK-293 cells, and the complex formation with endogenous Cul4a, Ddb1 and Roc1 was examined by means of immunoprecipitation analysis using an anti-Flag antibody. (<b>B</b>) Cul4a siRNA was transfected, and the effect on Hoxa9-mediated down-regulation of Geminin protein was examined. Down-regulation of Cul4a by siRNA was confirmed by immunoblot analysis, and the level was restored by transfection of myc-tagged Cul4a. Endogenous Cul4a was also detected in myc-tagged Cul4a-transfected cells even if cells were pre-treated with siRNA for Cul4a probably because exogenously overexpressed mRNA for Cul4a prevented siRNA from affecting endogenous Cul4a.</p

Effect of Geminin supertransduction on Hoxa9 transduction-mediated hematopoietic induction in reconstituted BM.

<p>(<b>A</b>) Repopulating activities of retrovirally transduced BM. Repopulating activities in the reconstituted mice were examined 1 month (1M) and 3 months (3M) after the injection. Since the repopulating activity was low in MEP+MPI-transduced BM, mice injected with 5-fold higher numbers of retrovirally transduced cells were used in subsequent analyses as controls. The number of recipient mice is indicated above each bar. (<b>B</b>) Cell populations in each phase of the cell cycle were analyzed by cell sorting analysis. (<b>C</b>) Geminin protein expression in each phase of the cell cycle. (<b>D</b>) Cell sorting procedure for analyzing the primitive hematopoietic cells. BM were subjected to cell sorting analysis. Primitive hematopoietic cells in EYFP⁺ cells were examined by the immunophenotype analysis. *, cells with a non-specific Sca1 signal due to the spectrum overlap (<b>E</b>) Cell numbers of Lin⁻, progenitor, CD34⁺KSL and CD34⁻KSL subpopulations in BM from the reconstituted mice. The data from three independent mice were subjected to the statistical analysis. MEP and MPI, empty control vectors.</p

Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin

<div><p>Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the <i>Geminin</i> gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin) was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.</p></div

Geminin transduction by means of CP-Geminin

<p>(A) CP-Geminin incorporation into NIH 3T3 cells. FITC-conjugated CP-Geminin and Geminin were added into the medium. One and 12h after the addition, cells were observed under a confocal microscope. The nucleus was stained with Hoechst33342. DIC: differential interference contrast (B) FITC-conjugated CP-Geminin incorporation into leukemic cell lines 24 h after the addition. (C) Dose dependency of CP-Geminin incorporation into NIH 3T3 cells. FITC-conjugated CP-Geminin was added to the medium at the indicated concentration, and 12h after the addition, the cells were examined under a confocal microscope. (D) Withdrawal study of CP-Geminin. FITC-conjugated CP-Geminin was added to the medium and the incorporation was confirmed 12h after the addition. CP-Geminin was then removed by replacing the medium with CP-Geminin with one without CP-Geminin. One and 4h after the removal, the cells were examined under a confocal microscope.</p

Effect of CP-Geminin on chromatin configuration and transcription.

<p>(A) Effect of CP-Geminin on nuclease accessibility of the E2F-R chromatin locus of the <i>Geminin</i> gene. Twenty-four h after transduction of CP-Geminin (1,000 nM), cells were subjected to nuclease accessibility analysis. BSA, control (B) Effect on transcription of the <i>Geminin</i> gene promoter. Twenty four h after transduction of CP-Geminin (1,000 nM), E2F1 (1 μg) and the luciferase reporter plasmids (1.5 μg) were co-transfected, and transcription activity of the <i>Geminin</i> gene promoter was examined with the luciferase reporter assay. (C) Effect of CP-Geminin on mRNA expression of the endogenous genes, <i>Geminin</i>, <i>Mcm7</i>, <i>CcnA2</i> and <i>Actb2</i>. Twenty four h after transduction of CP-Geminin (1,000 nM), E2F1 (1 μg) was transfected, and cells were subjected to TaqMan real-time PCR analysis an additional 24h after the transfection. *: P< 0.01.</p

Molecular interaction of incorporated CP-Geminin with the target substrates.

<p>Expression plasmid vectors for Myc-Cdt1, HA-Brahma-domainII and HA-Brg1-domainII were transfected into NIH 3T3 cells, after which CP-Geminin was added. The cells were then subjected to immunoprecipitation analysis. 1. Control, 2. Flag-Geminin, 3. CP-Geminin including a Flag tag.</p