Supporting InformationOhno et al. 10.1073/pnas.1011054107SI Materials and MethodsMice. Animal experiments were done by using C57BL6 mice andmice deficient in Rae28 with a congenic genetic background. Thegeneration of mice lacking Rae28 has been described previously(1). FLCs were subjected to the genetic complementation assaybecause Rae−/− mice were lethal at the neonatal period.

Real-Time PCR. Total cellular RNA was extracted from cells withthe Mini RNA Isolation Kit (ZYMO Research) and reverse-transcribed using TaqMan Reverse Transcription Reagents(Applied Biosystems-Life Technologies). The product was sub-jected to real-time quantitative PCR analysis by using TaqManGene Expression Assays and an ABI Prism 7700 sequence de-tection system (Applied Biosystems-Life Technologies).

Transfection Experiments. cDNAs were subcloned into the down-stream of the CMV promoter of the pcDNA expression vector(Invitrogen-Life Technologies). The plasmid DNAs were trans-fected into HEK-293, grown in DMEM (Invitrogen-Life Tech-nologies) supplemented with 10% FBS (HyClone-ThermoFisher),by the calcium phosphate coprecipitation method, and the re-sultant transfectants were subjected to further analyses. To exam-ine E2F activity, reporter plasmids were transfected into HEK-293by the calcium phosphate coprecipitation method. Twelve hoursafter transfection, the cells were washed and then serum-deprivedfor 24 h. Next, the cells were lysed and subjected to a luciferaseassay with the Dual-Glo luciferase assay system (Promega) ac-cording to the manufacturer’s protocol. Firefly luciferase activitywas normalized by Renilla luciferase activities, which was used asan internal control to monitor transfection efficiency. All assayswere performed at least three times.

Yeast Two-Hybrid Analyses.A LexA-Gal4-based two-hybrid systemwas used to examine protein–protein interaction. pBTM116 wasused to express the bait protein fused to the DNA-binding do-main of LexA, and pACT was used to express the prey proteinfused to the transactivation domain of Gal4. These were co-transformed to the Saccharomyces cerevisiae L40 strain by treat-ment with lithium acetate, and the resulting yeast colonies weresubjected to the β-galactosidase assay (2).

siRNA Experiments. HEK-293 were transfected with the followingdouble-stranded (ds)RNAs (Dharmacon-ThermoFisher) at 40nM by using Lipofectamine RNAiMAX (Invitrogen-Life Tech-nologies): Cul4a, GAACUUCCGAGACAGACCU; Geminin,a mixture of the following four ds RNAs—GGAGUCAUUUG-AUCUUAUG, GAGACUGAAUGGUGAACCU, AGAAGU-AGCAGAACAUGUA, and UUGAAUCACUGGAUAAUCA.siPerfect Negative Control (Sigma) was transfected at the sameconcentration as a nontarget negative control. On the otherhand, mouse BMCs were cultured in DMEM supplemented with15% FBS, 100 ng/mL mouse stem cell factor (SCF) (R&D Sys-tems), 100 ng/mL human thrombopoietin (TPO) (R&D Systems),and 100 ng/mL mouse Flt3 ligand (R&D Systems) for 24 h. Cells(5 × 105) were harvested and resuspended in 1 mL Accell siRNAdelivery media (Dharmacon-ThermoFisher) supplemented with100 ng/mL mouse SCF, 100 ng/mL human TPO, and 100 ng/mLmouse Flt3 ligand and cultured with 0.5 μMAccell SMART poolof siRNA for mouse Geminin (a mixture of the following fourds RNAs: CUUUGAUCUUAUAAGUAAA, UCACUAUGU-UAAAAUACAG, GUGUCAGAUAUAAACUGUA, and UU-UGAAUCACCGGAUAGUC) or with the negative control

(Accell nontargeting pool) (Dharmacon-ThermoFisher) for 72 h.The cells were subjected to further analyses.

Immunoprecipitation and Immunoblot Analysis. Cell extracts wereobtained by resuspending cell pellets in RIPA buffer consisting of10% glycerol, 0.5%TritonX-100, 20mMHepes (pH8.0), 150mMNaCl, 1 mM EDTA, 1.5 mM MgCl2, and a protease inhibitormixture, Complete Mini (Roche Molecular Biochemicals), soni-cated for 30 s on ice, and centrifuged for 15 min at 15,000 × g. Thesupernatant of the lysate was subjected to immunoprecipitationexperiments, and the lysate to immunoprecipitation with Gam-maBindGSepharose (GEHealthcare). Proteinswere separatedbySDS/PAGE, transferred to Immobilon-P (Millipore), immuno-blotted with primary antibodies, and visualized with horseradishperoxidase-conjugated anti-rabbit IgG and SuperSignal WestFemto Maximum Sensitivity Substrate (Pierce Biotechnology-ThermoFisher). To examine ubiquitination and protein stability invivo, cells were treatedwithMG132 (20 μM) (Peptide Institute) for6 h. For the pulse-chase labeling experiment, Geminin was labeledwith [35S]methionine, immunoprecipitated, and detected by auto-radiography as described previously (3).

Chromatin Fractionation. Cells were lysed for 10 min on ice incytoskeleton buffer containing 0.1% Triton X-100, 10 mM Pipes(pH 6.8), 10 mM NaCl, 1.5 mM MgCl2, 300 mM sucrose, 10 mMNaF, and 1 mM ATP and centrifuged for 4 min at 2,000 × g. Thepellet was treated with RNase-free DNase I (1 unit/μL) (RocheMolecular Biochemicals) for 45 min at 25 °C and placed on ice in300 mM NaCl for 5 min. After centrifugation for 15 min at15,000 × g, the supernatant was subjected to further analyses (4).

Retrovirus-Mediated Gene Transduction. MEP or MPI was cotrans-fected with gag, pol, and VSV-G envelope expression plasmids(kindly provided by T. Friedmann, University of California, SanDiego, CA) into HEK-293 with Lipofectamine 2000 (Invitrogen-Life Technologies). Ecotropic packaging cell line PlatE, kindlyprovided by T. Kitamura (Institute forMedical Science, Universityof Tokyo), was infectedmore than three times with a virus, and thesupernatants were concentrated by centrifugation at 6,000 × g for16 h to produce a high-titer helper-free retrovirus. FLCs, extractedfrom 15.5-d post-coitum. embryos, and BMCs were cultured for48 h in DMEM supplemented with 10% FBS, 100 ng/mL mouseSCF, 100 ng/mL human TPO, and 100 ng/mL mouse Flt3 ligandand in 32D in RPMI medium 1640 (Invitrogen-Life Technologies)supplemented with 10% FBS and 10 units/mL mouse IL-3 (R&DSystems). The cells were then culturedwith retrovirus in retronectin-coated dishes (Takara) for 48 h in the same medium with theaddition of 5 μg/mL protamine sulfate (Sigma-Aldrich). Retro-virally transduced cells were detected with FACSCalibur andFACSAria (BD Biosciences Immunocytometry Systems) on thebasis of their EYFP expression (4).

Analysis of Cell Cycle, Apoptosis, Geminin Protein Expression, andHematopoiesis. Cell cycle was analyzed by using APc-BrdU flowkit (BD pharmingen) according to the manufacturer’s instruction.Additional staining for Geminin was done by using a rabbitpolyclonal antibody raised against GST-Geminin and an anti-rabbit IgG-PE antibody as primary and secondary antibodiesrespectively. Apoptosis was examined as described previously.Cell sorting analysis was performed on FACSCalibur andFACSCanto II (BD Biosciences Immunocytometry Systems) (4).Hematopoiesis was assessed through clonogenic, replating, andLTC-IC and LTR activities. Clonogenic and replating activities

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were assayed as follows. Mouse BMCs were cultured in DMEMsupplemented with 15% FBS, 100 ng/mL mouse SCF, 100 ng/mLhuman TPO, and 100 ng/mL mouse Flt3 ligand for 24 h. Cellswere then infected with retroviruses. After washing in MEMα-medium (Invitrogen-Life Technologies), cells (1 × 104) wereresuspended in methylcellulose semisolid medium (MethocultM3231) (Stem Cell Technologies) and were plated in 35-mmculture dishes in the presence of 0.5% FBS, 10 ng/mL mouseSCF, 10 ng/mL mouse GM-CSF (R&D Systems), 10 ng/mLmouse IL-3, and 3 units/mL human erythropoietin (EPO) (ChugaiPharmaceutical). Colonies were counted 7–9 d after culture underan inverted microscope. All of the cells were harvested frommethylcellulose medium, washed with 1× PBS, and then replatedin the same medium (1 × 104 cells/35-mm dish). Colony scoringand replating were repeated every 7 d until no colonies were ob-served in the cultures. LTC-IC activity was examined by detectingmyeloid colony forming units in culture (CFU-Cs) in FLCs, whichwere cultured on S17 feeder layer in the myeloid long-term culturemedium (Methocult M5300) (Stem Cell Technologies) containing10−6 M hydrocortisone sodium hemisuccinate (Sigma-Aldrich);half of the medium was changed every week for 6 wk. LTR ac-tivities were assayed by injecting retrovirally transduced or super-transduced FLCs (1 × 106) and 4 × 105 competitors into congenicmice lethally irradiated with 9.0 Gy given in a single dose froma 137Cs γ-ray source. The peripheral blood of the recipients wassubjected to cell sorting analysis, and the percentage of EYFP+-multilineage cells were examined after transplantation (5).

Reconstitution of RDCOXB4 in Sf9 and in Vitro Ubiquitination Assay.Sf9 cells were cultured in Grace’s insect cell culture medium(Invitrogen-Life Technologies) supplemented with 10% FBS and0.06% tryptose phosphate broth-Bacto (Difco Laboratories).cDNAs for GST- or His6-tagged Roc1, Ddb1, Cul4a, and Flag-Hoxb4 were inserted into pV-IKS and pVL1392, respectively, andthe recombinant vectors were cotransfected into Sf9 cells witha linearized BaculoGold baculovirus DNA (BD Pharmingen) forviral particle formation by means of Cellfectin (Invitrogen-LifeTechnologies). Recombinant baculoviruses were amplified by

repeated infection. Sf9 were then infected with high-titer viruses,and 72 h postinfection, the cells were washed with cold PBS andsuspended in homogenizing buffer [20 mM Tris (pH 7.9), 4 mMMgCl2, 500 mMNaCl, 0.4 mMEDTA, 2 mMDTT, 20% glycerol,0.1%Nonidet P-40, and 1mMZnCl2 with CompleteMini (RocheMolecular Biochemicals)]. The suspension was then homoge-nized and centrifuged at 15, 000 × g for 10 min, after which thesupernatant was subjected to glutathione affinity column chro-matography (glutathione Sepharose 4 Fast Flow) (GE Health-care), and the extract in the homogenizing buffer without DTTwas subjected to a metal affinity column chromatography (Co-Agarose) (Wako Pure Chemical) and progressive purificationthrough Superose 6 10/300 GL column chromatography (GEHealthcare). Recombinant myc-Geminin tagged with His6 andmyc in the N- and C-terminal portions, respectively, was producedin Escherichia coli, BL21, and its soluble fraction was subjected toa Ni-NTA affinity chromatography (Ni-NTA Agarose) (Qiagen).The recombinant myc-Geminin was incubated in a 20-μl reactionmixture containing 50 mM Tris·HCl (pH 7.9), 5 mM MgCl2,0.6 mM DTT, 2 mM ATP, 0.1 μg ubiquitin-activating enzyme E1(Wako Pure Chemical), 0.6 μg ubiquitin-conjugating enzymeUbc5c (Wako Pure Chemical), and 1 μg biotin-ubiquitin or 10 μgubiquitin (Boston Biochem) and the purified recombinant com-plex. After incubation at 37 °C for 1 h, reaction was terminatedwith a protein sample buffer, run on SDS/PAGE and subjected toimmunoblot analysis. Ubiquitinated Geminin was immunopre-cipitated or detected with an anti-myc polyclonal or a monoclonalantibody (9E10), respectively. Ubiquitination was confirmed bydetection of biotin-ubiquitin with the Vectastain Elite ABC kit(Vector Laboratories) (4).

Antibodies. Primary and secondary antibodies used are listed inTable S1.

Statistical Analysis. More than three independent experimentswere performed, and the findings were statistically analyzed. Theresults are shown with SEMs (Fig. 2A, Fig. 3 B and C, Fig. 6A,Fig. S1D, Fig. S2 A–E, Fig. S4, Fig. S5 D and F, and Fig. S6A).

1. Takihara Y, et al. (1997) Targeted disruption of the mouse homologue of theDrosophila polyhomeotic gene leads to altered anteroposterior patterning and neuralcrest defects. Development 124:3673–3682.

2. Mitsui K, et al. (1999) A novel human gene encoding HECT domain and RCC1-likerepeats interacts with cyclins and is potentially regulated by the tumor suppressorproteins. Biochem Biophys Res Commun 266:115–122.

3. Ohtsubo M, Okazaki H, Nishimoto T (1989) The RCC1 protein, a regulator for the onsetof chromosome condensation locates in the nucleus and binds to DNA. J Cell Biol 109:1389–1397.

4. Ohtsubo M, et al. (2008) Polycomb-group complex 1 acts as an E3 ubiquitin ligase forGeminin to sustain hematopoietic stem cell activity. Proc Natl Acad Sci USA 105:10396–10401.

5. Ohta H, et al. (2002) Polycomb group gene rae28 is required for sustaining activity ofhematopoietic stem cells. J Exp Med 195:759–770.

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Fig. S1. RDCOXB4 complex formation and the effect on Geminin. (A) The yeast two-hybrid analysis with Hoxb4 as bait and Cul4a as prey. The analysis withmock vectors is shown as negative control. (B–F) 32D were retrovirally transduced with Hoxb4 and subjected to immunoblot and cell sorting analyses.RDCOXB4 complex formation was confirmed by means of immunoprecipitation analysis with an anti-Hoxb4 antibody (B). Even though cell populations in the Sand G2/M phases and mRNA for Geminin were increased by Hoxb4 transduction (C and D), Geminin protein was down-regulated throughout the cell cycle (E).Reduced Geminin protein was also confirmed by immunoblot analysis, whereas Hoxb4 transduction-mediated down-regulation of Geminin protein wassuppressed by the treatment with MG132 (F). (G) The yeast two-hybrid analysis with Geminin as bait and Hoxb4 derivatives as prey. (H) Immunoprecipitationanalysis of the Hoxb4-Geminin interaction. HEK-293 transfected with Flag-Hoxb4 derivatives and Geminin were subjected to immunoprecipitation analysis. Theprecipitates were blotted with anti-Flag and anti-Geminin antibodies, respectively.

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Fig. S2. Effect of Hoxb4 transduction in Rae−/−FLC. (A) Cell cycle. (B) Apoptosis. Percentage of 7AAD− and Annexin V+ cells is shown below each panel. (C)Clonogenic activity. (D) LTC-IC activity. (Lower) Representative colonies observed in the clonogenic and LTC-IC assays. (E) Expression of mRNAs for Geminin,Cdt1, and Cyclin A2, detected by RT-PCR analysis. (F) Expression of Cdt1 and Cyclin A2 detected by immunoblot analysis.

Fig. S3. Effect of Hoxb4 transduction on Geminin protein expression in each subpopulation of hematopoietic cells. BMCs transduced with Hoxb4 was sub-jected to cell sorting analysis, and Geminin protein expression in Lin+, KSL, and CD34−KSL cells was determined.

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Fig. S4. Effect of Geminin restoration in Hoxb4-transduced BMCs. BMCs were transduced with Hoxb4 and Geminin by using MPI and MEP retroviral vectors,respectively, and the effect on hematopoiesis was examined. (A) Clonogenic activity. (B) Replating activity. (C) LTC-IC activity. (Lower in A and C) Represen-tative colonies observed in the clonogenic and LTC-IC assays.

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Fig. S5. Effect of Geminin knockdown on hematopoiesis. BMCs were transfected with siRNA for Geminin, and the effect on the cell cycle, Geminin protein,clonogenic, and replating activities was examined. (A) Cell cycle. (B) Geminin protein expression. The siRNA transfection significantly reduced Geminin proteinthroughout the cell cycle. (C) (Upper) Close-up and (Lower) microscopic views of the colonies. (D) Replating activity. Cells were transfected with the siRNA ineach step of replating. (E) Reduced Geminin protein by siRNA transfection was restored by Geminin transduction, which was confirmed by cell sorting analysis.(F) The clonogenic activity enhanced by Geminin knockdown was markedly suppressed by Geminin restoration.

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Fig. S6. Effect of Hoxb4 or Cul4a on Geminin. HEK-293 were transfected with Flag-Hoxb4 or myc-Cul4a, and the effect on Geminin expression was examined.(A) Effect of Hoxb4 transfection on expression of Geminin mRNA was examined by RT-PCR. (B) Effect of Hoxb4 transfection on the stability of Geminin. (Upper)[35S]-labeled Geminin was traced by means of autoradiography after immunoprecipitation. (Lower) Bands were scanned by using the National Institutes ofHealth Image program. (C) Synergistic effect of Hoxb4 and Cul4a cotransfection on Geminin protein expression. The effect was suppressed by treatment withMG132.

Fig. S7. Ubiquitination of Geminin and destruction box-deleted Geminin (Geminin-DBD) by Hoxb4. (A) Either Geminin or Geminin-DBD was cotransfectedwith myc-Cul4a, Flag-Hoxb4, and HA-ubiquitin (HA-Ub) into HEK-293, and mobility-shifted Geminin or Geminin-DBD bands were detected by immunoblotanalysis with an anti-Geminin polyclonal antibody. (B) Mobility-shifted Geminin bands were confirmed to be ubiquitin-conjugated Geminin by means ofimmunoblot analysis of immunoprecipitated Geminin with an anti-HA antibody, which can detect the molecules conjugated with HA-ubiquitin. The ubiq-uitinated Geminin was increased synergistically by the cotransfection of myc-Cul4a and Flag-Hoxb4.

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Fig. S8. Reconstitution and purification of the RDCOXB4 complex in Sf9 cells and the E3 ubiquitin ligase activity for Geminin. (A) Gel filtration analysis of therecombinant RDCOXB4 complex. I: input affinity-purified recombinant RDCOXB4. The molecular size was examined by means of gel filtration fractionation onSuperose 6 10/300GL, and the presence of each of the components in the complex was confirmed by immunoblot analysis. A peak fraction of the RDCOXB4complex is indicated by a thick line. Each of ubiquitin ligase core components (His6-Roc1, Ddb1, and Cul4a) was detected in the peak fraction of Flag-Hoxb4with a molecular weight similar to that of the recombinant complex consisting of stoichiometrically determined amounts of the components (260 kDa). Al-though stronger Hoxb4 bands were also detected in the fractions corresponding to lower molecular weights, these bands may represent Flag-Hoxb4 thatbecame detached from the complex during gel filtration fractionation. The affinity-purified recombinant RDCOXB4 was subjected to an in vitro ubiquitinationassay with purified bacterially produced recombinant His6- and myc-tagged Geminin (myc-Geminin). This assay system included myc-Geminin, E1, E2, ubiquitin,and RDCOXB4. The reaction product was examined by means of immunoblot analysis using an anti-myc monoclonal antibody. (B) Dosage dependency of theRDCOXB4 complex and time course of the ubiquitination reaction. Geminin, myc-Geminin; −, no reaction; *GST; #overexposure of the same sample as thatshown in the lane indicated by **. (C) Geminin ubiquitination with methyl-ubiquitin. It was hypothesized that mobility-shifted bands for ubiquitinatedGeminin may represent either formation of an elongated ubiquitin chain at a single lysine residue in Geminin or attachment of a single ubiquitin molecule tomultiple lysine residues in Geminin. To determine the validity of these suppositions, we examined Geminin ubiquitination by using methyl-ubiquitin.(D) Ubiquitination of Geminin-DBD. An asterisk indicates unspecified bands. Either Geminin or Geminin-DBD was subjected to the in vitro ubiquitination assay.Poly-ubiquitinated molecules were observed in the reaction product with Geminin-DBD as well as in that with Geminin. (E) Ubiquitination of each of theRDCOXB4 members in the reaction products of the in vitro ubiquitination assay. Poly-ubiquitinated GST-Roc1, Ddb1, Cul4a, and Hoxb4 were detected in thereaction products. However, it remains unclear whether self-ubiquitination of RDCOXB4 members is required for regulating protein stability of the complexthrough UPS or for inducing E3 ubiquitin ligase activity. Similar poly-ubiquitinated molecules were also detected in the reaction products with RDCOXB4(N>A)except for Hoxb4N>A itself, suggesting that an N-to-A mutation in the homeodomain eliminated activity of the E3 ubiquitin ligase for Geminin but not activityof the self-ubiquitination of the ubiquitin ligase core component. Poly-ubiquitinated Hoxb4N>A was barely detectable, which may be one of the reasons whyHoxb4N>A was abundantly detectable in the purified RDCOXB4 complex. Ub, ubiquitinated molecules. Both * and ** indicate unspecified bands.

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Table S1. Antibodies used in the study

Antibody (clone or product number) Species* Manufacturer

Probe-conjugated antibodyAnti-CD34+FITC(RAM34) Mouse (m) eBioscienceAnti-Sca-1/Ly-6A/E+PE-Cy7(D7) Mouse (m) eBioscienceAnti-c-Kit /CD117+APC-Alexa-Fluor750(2B8) Mouse (m) eBioscienceAnti-B220/CD45R+APC(RA3-6B2) Mouse (m) BD BiosciencesAnti-CD3ε+APC(145-2C11) Mouse (m) BD BiosciencesAnti-Gr-1/Ly-6G+APC(RB6-8C5) Mouse (m) eBioscienceAnti-Mac-1α/CD11b+APC(M1/70) Mouse (m) eBioscienceAnti-TER-119+APC(TER-119) Mouse (m) eBioscience

Primary antibodyAnti-Flag(ANTI-FLAG M2) Mouse (m) SigmaAnti-myc(sc-789) Rabbit (p) Santa Cruz BiotechnologyAnti-myc(9E10) Mouse (m) Santa Cruz BiotechnologyAnti-HA(12CA5) Mouse (m) RocheAnti-Geminin Rabbit (p)†

Anti-Cdt1 Rabbit (p)†

Anti-Cdt1(sc-28262) Rabbit (p) Santa Cruz BiotechnologyAnti-Mcm2(sc-9839) Goat (p) Santa Cruz BiotechnologyAnti-Roc1 Rabbit (p) ZymedAnti-Ddb1 Rabbit (p) Santa Cruz BiotechnologyAnti-Cul4a Rabbit (p) BethylAnti-Hoxb4 (sc-2807) Rabbit (p) SantaCruz BiotechnologyAnti-Hoxb4 (sc-15001) Goat (p) SantaCruz BiotechnologyAnti-Gapdh(GAPDH-71.1) Mouse (m) SigmaAnti-β-actin(AC-74) Mouse (m) Sigma

Secondary antibody‡

Anti-rabbit IgG+PE(611-108-122) Goat (p) RocklandAnti-rabbit IgG+HRP(1858415) Goat (p) PierceAnti-mouse IgG+HRP(1858413) Goat (p) PierceAnti-goat IgG+HRP(sc-2020) Donkey (P) Santa Cruz Biotechnology

*Monoclonal antibody and polyclonal antibody are indicated by m or p, respectively.†Polyclonal antibodies against GST-fusion recombinant molecules generated for this study.‡Secondary antibodies conjugated with labels.

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