SupplementaryMethods

ImmunophenotypingbyFACS

Cells were stained with the following antibodies or the appropriate isotype controls:

cKit‐APC‐eFluor780 (clone 2B8, eBioscience), Sca‐1‐PE‐Cy7 (clone D7, eBioscience),

CD34‐AlexaFluor660 (clone RAM34, eBioscience), FcγRII/III‐PE (clone 2.4G2, BD

Biosciences), B220‐PE (clone RA3‐6B2, eBioscience), CD11b‐PE (clone M1/70, BD

Biosciences),Gr‐1‐APC‐eFluor780 (cloneRB6‐8C5,eBioscience),CD3ε‐APC (clone145‐

2C11, eBioscience), IL7Rα‐PE (clone A7R34, eBioscience), biotinylated mouse lineage

cocktail(Miltenyi),Biotin‐VioBlue(Miltenyi).

Microscopy

The following antibodies were used for immunofluorescence: mouse anti‐α−tubulin

(DM1a,Sigma),goatanti‐GFP,28donkeyanti‐goatconjugatedtoAlexa488,anddonkey

anti‐mouseconjugatedtoAlexa594(Invitrogen).Cellsoncoverslipswerefixedwith4%

PFAandblockedwith0.2% fishskingelatin (Sigma) inPBS.Coverslipsweremounted

withProLongGoldwithDAPI(Invitrogen).ImageswereacquiredusingaDeltavisionRT

imaging system (Applied Precision) (Olympus IX71) equipped with a CCD camera

(CoolSNAPHQ, Roper Scientific), in 0.15 µm serial Z‐sections using anOlympus 100x

1.40 NA PlanApo objective.Data sets were deconvolved using Softworx (Applied

Precision)software.

Microarray

Lin‐ cells from 4 independent Runx1Δ/Δ mice were transduced with Pcgf1 or control

shRNA.After36hofpuromycinselection,totalRNAwasisolatedusingtheRNeasyKit

(Qiagen). RNA quantity and quality was assessed with a NanoDrop 2000

Spectrophotometer (Thermo Scientific) and by capillary gel electrophoresis using an

AgilentRNA6000NanoKit andBioanalyzer (Agilent). 400ngof totalRNAper sample

was labeled with the One‐Cycle Target Labeling and Control Reagent Package

(Affimetrix) as described in the manufacturer’s instructions and hybridized to 4x44K

Agilent Whole Genome GEX IC Mouse arrays (Agilent). Data was analyzed with

GeneSpring Software (Agilent). To identify differentially expressed genes, a two‐way

ANOVAwasperformedfollowedbypost‐hocTukey'stests.Expressionofeachgenewas

assumed to be dependent on two factors: 'treatment' (Pcgf1 or control shRNA) and

'mouse'(4 individuals).Probesshowingasignificantdifference(p<0.01) in 'treatment'

effectswereselectedforfurtheranalysis.Forvisualizationpurposes,expressionvalues

were corrected for differences between individuals by substracting effects associated

with the 'mouse' factor. Original data can be accessed at GEO (accession number

GSE33280).

Proteomics

Immunoprecipitation was carried out with lysates from Pcgf1‐eGFP ES‐cells and

subjectedtoshotgunmassspectrometryasdescribedpreviously.31Proteinlevelswere

comparedtoothersamplesrunatthesametime.Thez‐scorewascalculatedbasedon

the standard deviation of the protein amount in the sample compared to the mean

standarddeviationofthesameprotein inthewholedataset.Data ispresentedasthe

meanofthez‐scoreof3replicates.

Westernblotting

LysateswereseparatedonaNuPAGENovex4‐12%Bis‐Trisgel(Invitrogen)andblotted

onto nitrocellulosemembrane (Millipore). After blocking in 4 % skimmilk, Pcgf123 (a

kind gift from Vivian Bardwell, University of Minnesota, 1:500) or tubulin antibody

(Sigma‐Aldrich, 1:50000)was applied.Antibodies specific forBCOR,Ring1aandRNF2

werepurchasedfromAbcam.SecondaryHRP‐coupledantibodieswerepurchasedfrom

BioRad.BlotsweredevelopedwithECLonAmershamHyperfilm(GEHealthcare).

ChromatinIP

For ChIP assays proteins were crosslinked with 1% (v/v) formaldehyde for 10min at

roomtemperatureandformaldehydewastheninactivatedbytheadditionof125mM

glycine. Cell extracts were sonicated into approximately 500‐nt chromatin fragments

using a Branson 450‐D sonicator. Chromatin extracts containing DNA fragmentswere

immunoprecipitatedusingantibodiesagainstubiquityl‐HistoneH2A(05‐678,Millipore),

and a polyclonal goat anti‐GFP antibody (MPI‐CBG, protein facility). Specificity of the

goat anti‐GFP antibody in immunoprecipitation assays was extensively validated.1

Immunoprecipiated protein‐DNA complexes were elutated from protein G sepharose

(Fastflow GE healthcare) using 200 µl elution buffer (1% SDS, 0.1M NaHCO3), and

incubatedat65°Covernighttoreversethecrosslinks.ElutionsweretreatedwithRNase

AandproteinaseK,andpurifiedwithaPCRDNApurificationkit (Qiagen).ForallChIP

experiments,qPCRswereperformedwiththeSybrgreenqPCRkit (Abgene)onanMX

P3000 qPCR machine (Stratagene). Relative proportions of immunoprecipitated gene

fragments were determined on the basis of the threshold cycle (Ct) for each PCR

product. For every gene fragment analyzed, each sample was quantified in duplicate

fromatleastthreeindependentChIPs.FoldenrichmentswerequantifiedbyqPCRwith

primerslistedinSupplementaryTable2.

GenerationofPcgf1‐eGPFmice

Pcgf1‐eGFPmice were generated by stably transfecting R1/Emouse embryonic stem

cells with the BAC clone RP24‐253F2 obtained from the BACPAC Resource Center

(http://bacpac.chori.org), including the LAP cassette26 fused to the last exonofpcgf1.

Correct protein size and localization was verified by Western blot and immuno‐

fluorescence staining as described previously.26 Chimeric mice were obtained by

injectionofmouseembryonicstemcellsthatcarriedthepcgf1‐eGFPgeneintoC57BL/6

OlaHsdblastocysts.Micewithgermline‐transmittedpcgf1‐eGFPweredetectedbyPCR

utilizingtheprimersfwd5’‐GTCCCGCTGGTTCGGCAAG‐3’andrev5’‐CCTCGCCCTTGCTCA‐

CCAT‐3’.

IdentificationofretroviralintegrationsitesbyLM‐PCR(ligation‐mediatedPCR)

Toanalyzevectorintegrationsites,genomicDNA(1µg)wasdigestedwitheitherPst1or

Csp61 (Fermentas), denatured,and then subjected to second‐strand DNA synthesis

using a biotinylated primer (LTR‐I) specific for the vector LTR. DNA fragments with

vector integration siteswere enriched by pulldown with strepadivin‐

coupledDynabeads®M‑280(DynalBiotechASA),andthensubjectedtoligasemediated

(LM)‐PCRbyligationtoannealedlinkeroligonucleotidesandnestedPCR(Schmidtetal,

HumanGeneTherapy.May2001,12(7):743‐749.).Primers:LTR15’‐TCTGGGGACCATC‐

TGTTCTTGGCCC‐3’;LC15’‐GACCCGGGAGATCTGAATTC‐3’;rvLTRII5’‐CCTGACCTTGATCT‐

GAACTT‐3’;Linker15’‐GACCCGGGAGATCTGAATTCAGTGGCACAGCAGTTAGG‐3’;Linker2

5’‐CCTAACTGCTGTGCCACTGAATTCAGATCTCCCG‐3’;

SouthernBlotanalysis

ClonalityoftheretroviralintegrationsiteswereanalyzedbySouthernBlotanalysisusing

standard procedures. Briefly, genomic DNA(10µg) was digested with the indicated

restriction enzyme, size‐separated on a 0.8% agarose gel, and transferred to a nylon

membrane.Thenylonmembranewashybrizedwitha32P‐dCTPlabeledDNAfragment,

washed,andvisualizedbyautoradiography.

Supplementary Figure 1. Replating capacity and cell numbers of retrovirally

transducedLin‐bonemarrowcellsinmethylcellulose.(A)Lin‐bonemarrowcellswere

transducedwitha retroviral construct tooverexpressRunx1/ETO.Themethylcellulose

replatingcapacityofLin‐cellsisolatedfromRunx1fl/fl(lightgrey)andRunx1∆/∆(darkgrey)

mice retrovirally transducedwithmock,orRunx1/ETO is shown. (B)Results from two

individual serial replating experiments. Lineage‐negative cells isolated from the bone

marrowofRunx1∆/∆miceweretransducedwithanshRNAtargetingPcgf1(shPcgf1)ora

scrambledshRNA(shCtrl).Cellswereweeklyextractedfrommethylcellulose,and10‐60

cells/µlwerereplatedas longascellswere formingcolonies.Resultsareshownfrom

two independent experiments (exp. #1 and #2). (C) Cell numbers from different

replating rounds. Cell counts after each replating were devided by the number of

seededcells.Numbersareindicatedaslog2.

Supplementary Figure 2. Characterization of Pcgf1‐eGFPmice. (A) Lin‐bonemarrow

fromaPcgf1‐eGFPmousestainedwithanantibodyrecognizingeGFP.DAPIwasusedto

stainDNA.Arrowspoint to cellswithdifferent levelsofeGFP. Scalebar:10µm. (B,C)

ImmunoprecipitationfromPcgf1‐eGFPES‐cellsorcontrol(Ctrl)cellsnotexpressingeGFP

usinganeGFP‐specificantibody.Themolecularweight is indicated inKilodalton(kDa).

(B) Silver‐stained SDS‐gel. Specific bands not found in the eGFP‐negative control are

markedwithanasterisk.(C)Co‐IPofPcgf1interactingproteins.SpecificbindingofBCOR,

Ring1aandRnf2 toPcgf1‐eGFPwasevaluatedbydecorating the SDS‐gelwith specific

antibodies.Asterisksmarkbandsspecificallyboundbythenantibody.

Supplementary Figure 3. Replating capacity and colonymorphology ofRunx1∆/∆Lin‐

cells transduced depleted of BCORL1. (A)Runx1∆/∆Lin‐cells transducedwithdifferent

BCORL1 shRNAs (shBcorl1 #1‐3) and grown in methylcellulose in parallel to control

vector transduced (shCtrl) or Pcgf1 shRNA‐transduced (shPcgf1) cells are shown (B).

Quantification of knockdown by indicated BCORL1 shRNAs in comparison to control

(shCrtl)evaluatedbyqRT‐PCR.Errorbarsshowthestandarddeviationof3independent

experiments. (C)Quantificationof colonynumbers in the replatingassay for indicated

treatments.

SupplementaryFigure4.Gatingstrategy forFigure4D.Stemcells(LSK): lineage(lin)‐,

cKit+, Sca‐1+; myeloid progenitor cells (MPC): Lin‐, cKit+, Sca‐1‐; common myeloid

progenitors (CMP): Lin‐, cKit+, Sca‐1‐, CD34+, FcγRII/III‐; granulocyte‐macrophage

progenitors (GMP): Lin‐, cKit+, Sca‐1‐, CD34+, FcγRII/III+, megakaryocyte‐erythrocyte

progenitors (MEP): Lin‐, cKit+, Sca‐1‐, CD34‐, FcγRII/III‐; common lymphoid progenitors

(CLP):Lin‐,IL7Rα+,cKitlo,Sca‐1lo.

Supplementary Figure 5. Immunophenotyping of lineage‐positive cells from Pcgf1‐

eGFPmice.(A)BonemarrowcellsofPcgf1‐eGFPmicewerestainedwiththeantibodies

indicated in the figure and analyzed by FACS. (B and C) Histograms compare eGFP‐

expression in eGFP‐Pcgf1mice (clear graph) compared to eGFP‐negative controlmice

(greygraph)fromcellsisolatedfrombonemarroworfromperipheralbloodasindicated.

(D)PercentagesofeGFP‐positivecellsamongthedifferentcellpopulationsindicatedin

the figure were determined, mean values and standard deviations from 4 different

animalsare shown. Ter119+:erythrocytes;B220+:B‐cells;CD11b+:macrophages;Gr‐

1+:granulocytes;

Supplementary Figure 6. Hierarchical cluster analysis of the 150 most differentially

expressed genes upon Pcgf1 knockdown. (A) Surfacemarkerprofiles are comparable

between lineage‐negative cells transduced with control (shCtrl) or Pcgf1 (shPcgf1)

shRNA.(B)Microarrayanalysisof4independentreplicatesofPcgf1shRNA(shPcgf1)or

control shRNA (shCtrl) transduced Runx1∆/∆ samples is shown. Normalized gene

expressionofthe150mostsignificantlyup‐ordownregulatedgenesinsampleswitha

Pcgf1 knockdown versus control samples in log2 scale is displayed in a hierarchical

clusteranalysis.Pcgf1andHoxAgenesaremarkedbyredblocks.

Supplementary Figure 7.Runx1∆/∆bonemarrow cellswith a knockdown in Pcgf1 do

not show long term engraftment in transplanted mice. (A‐D) Lineage‐negative cells

isolatedfromRunx1∆/∆orRunx1fl/flcontrolanimalsweretransducedwithPcgf1(shPcgf1)

orcontrolshRNA(shCtrl).Tomonitortransducedcells,eGFPwasexpressedonthesame

vector as the shRNAs. 3 different strategies were applied to test engraftment:

Transplantation of 1x106 Lin‐ cells together with 3.5x105 spleen cells into lethally

irradiatedC57BL/6mice(bluedots,n=4),transplantationof1x106Lin‐cellsintoRag2‐/‐γc‐

/‐KitW/Wwmice(greendots,n=2),ortransplantationof2x105sortedeGFP+Lin‐cellsinto

Rag2‐/‐γc‐/‐KitW/Wwmice (purple dots, n=2). (A) Transduction efficiencies of cells before

transplantationwere assessed by FACS analysis of eGFP expression. (B) Frequency of

eGFP+ cells in the bone marrow 2‐6 months after transplantation. (C) Frequency of

eGFP+cellsinperipheralblood5‐7weeksposttransplantation.(D)FrequencyofeGFP+

cells in peripheral blood 2‐6 months post transplantation. Each dot represents one

recipientmouse,themedianisdepictedasadashforeachexperimentalgroup.

SupplementaryFigure8.TransplantationofLin‐cells transducedwithaPcgf1shRNA

into immunocompromised recipients. 1x 106 lin‐ cells transduced with Pcgf1 shRNA

were transplanted into Rag2‐/‐γc‐/‐KitW/Wwmice.After8weekspost transduction,mice

wereanalyzedfortheexpressionofeGFPinbonemarrowandintheperipheralblood.

CellpopulationsweredistinguishedbyFACS.(A)Foranalysisofbonemarrow,thesame

antibodiesasindicatedforfigures4Dwereused.(B)Forbloodanalysis,cellpopulations

weredefinedas:Gr‐1+CD11b+B220‐:myeloidcells,B220+CD11b‐Gr‐1‐CD3‐:B‐cells,

CD3+B220‐:T‐cells.

Supplementary Table 1. Primer sequences for quantitative real‐time RT‐PCR. All

primersusedforqRT‐PCRlistedbelowamplifyregionsthatareseparatedbyanintronin

genomicDNA.MeltingcurveanalysisverifiedthepresenceofonesinglePCRproduct.

Supplementary Table 2.Primer sequences for quantitative real‐timeRT‐PCR of ChIP

samples.

SupplementaryTable3.MassspectrometricanalysisofinteractionpartnersofPcgf1.

R1/EES‐cellswerestablytransducedwithaBACconstructencodingPcgf1C‐terminally

fused to eGFP under the endogenous Pcgf1 promoter. Cells were subjected to

immunoprecipitation using an α‐eGFP antibody. Binding partners were identified by

quantitativemassspectrometry.SD=standarddeviation.n=3.