oi:1.138/nture1233 1.2 P=.7 P=.38 P=.44 e G1+G7 Single CD34 -Flk2 - LSK ells olonies Cre: (mt) + (pt) P=.18 Normlize expression.8.4 P=.4 LoxP: 36 p WT: 296 p G1+G l: 1.9 kp Cre: (mt) + (pt) Gtm Ospl H13 Smt2 Pon3 :38 p Mting strtegy l/l Mx1/Sl re l/+ pipc/tx p p Δ/+ Cre+ n Cre- mt pt l/l Mx1/Sl re l/+ X m X mt X mt pt pipc/tx m Δ/+ Cre+ n Cre- Ig2 mt pt pt g h (mt) (pt) (mt) (pt) PIPC l*:1.9 kp : 38 p l/+ mδ /+ m Δ/+ LoxP LoxP Peripherl Bloo l 4 / 4 / 4 4 / 4 / 4 / 2 2 / 2 / 2 2 / 2 / 24 24 / 24 / 24 24 / 24 LoxP:36 p WT:296 p (1 x1 3 ) Asolute numer lous 36p G1 G7 G - Δ.4k 1.6k 2. k 2 1 1 6 weeks m l/+ Lox P TAX (mt) (pt) (mt) (pt) l *: 1.9 kp : 38 p PIPC G1+G G1+G7 LoxP:36 p WT:296 p Supplementry Figure 1. Illustrtion o the struture o the -Ig2 lous n inserting sites o Lox; n mesurement o knokout eieny., qrt-pcr nlysis. Dt s men ± s.e.m o n=3., Mting strtegy or llele speii onitionl eletion o -. - LoxP sites. - unmethylte - n - methylte -. Dshe rrow inites ntiipte expression o Ig2 n in mδ/+., Arrows represent the genotyping primers. Big open ox represents 2k ; Δ (1.6k); smll open ox represents remining unelete portion o -., Iniviul mouse FACS plot quntittion o solute numer ter eletion o - rom the mternl llele n their ontrol littermtes. e,, Colonies erive rom Cre- ontrol n Cre+ mternl or pternl-erive l/+ lleles genotype or eletion o LoxP n the loxe region. * By this PCR l n Wt lleles re inistinguishle n the llele is preerentilly mpliie ompre to the Wt llele. Representtive t shown or 4 iniviul olonies rom eh genotype. g, Summry o genotyping results or (g,h) ove (numer o olonies positive or inite n / totl numer teste). Genotyping in peripherl loo- Mternl or pternl erive l/+ n /+ lleles were genotype eore n ter re inution. Primer pirs (see ig1)g1 n G mpliy the l, WT n lleles; G1 n G7 mpliy LoxP o the l llele n Wt llele. * By this PCR, l n Wt lleles were inistinguishle n the llele is preerentilly mpliie ompre to the WT llele. h, Upper pnel- Representtive gel imge eore (-) n ter (+) re inution using pipc or Mx-1 re moel. Lower pnel- Representtive gel imge eore (-) n ter (+) re inution using tmoxien or Sl-re moel. WWW.NATURE.COM/NATURE 1
RESEARCH SUPPLEMENTARY INFORMATION Flk2 Asolute numer (1x1 6 ) e 3 2 1. 1 ±.2 28 ±.3.26±.4 CD34.6±.1 p l/+.24±. p l/+.6±.1 Totl one mrrow p l/+ p /+ 6 weeks 6 months 6 weeks. 6 months.32±.4 p /+ 14 ±.2.2±.1 28 ±.8 Ki67 1 3 Ater Fu p /+.6±.1.6±.4 Asolute numer (1x1 3 ) Asolute numer (1x1 3 ) 2 2 1 1 4 3 2 1 66.6% 9.69% 1 4 m l/+ 1 2 1 1 G1 G Dpi S/G2/M 23.7%.% 1 2K 4K 6K p l/+ p /+ LSK p l/+ p /+ LSK 8.1% 17.4% 1 4 G1 S/G2/M 1 3 1 2 1 1 1 G 2.43%.9% 2K 4K 6K Supplementry Figure 2 Phenotypi nlysis o HSC ter eletion o -. FACS plot with requenies n solute numer o s, s n s in p /+ with their ontrol littermtes. &, ter 6 weeks eletion (n=4). &, ter 6 months eletion (n=4). e, Asolute numer o totl one mrrow., Representtive s plot imge o ell yle nlysis post u. 2 WWW.NATURE.COM/NATURE
RESEARCH Asolute numer (1x1 4 ells) 2 1 1 Bone mrrow - 6 months post inution p=.2 Progenitors p=.3 p=.22 m l/+ p=.33 Asolute numer (1x1 6 ells) 3 2 1 p=.1 Myeloi m l/+ CMP GMP MEP CLP Gr1/M1 Ter 119 CD41 Asolute numer (1x1 ells) 2 1 1 p=.27 T Lymphoi m l/+ p=.2 Asolute numer (1x1 4 ells) 2 1 1 B Lymphoi p=.42 m l/+ CD3 CD4 CD8 Immture Mture Pre/ProB Supplementry Figure 3 Linege nlysis ter mternl eletion o -. Quntiition y FACS nlysis o, erly progenitors, myeloi, T lymphoi n, B lymphoi in one mrrow o m /+ n their ontrol littermtes ter 6 months o pipc inution (n=3). Error r represents s.e.m. WWW.NATURE.COM/NATURE 3
RESEARCH SUPPLEMENTARY INFORMATION Mting strtegy or resue experiments l/l X +/- l/l Ig1r l/+ X MC Ig1r MC +/- m l/ + Ig1r l/l PIPC m /+ Ig1r -/- Cre+ n Cre - % Donor engrtment 4 3 2 1 16-2 weeks post-trnsplnttion Control Ig1r -/- Ig1r -/- p=.29 H13 Copg2 Tss4 Dhr7 Trpp9 Impt Atp1 Ppp1r9 Sl2218 Nn Gtl2 Zrsr1 Ckn1 Mts2 Dlk1 Klr1 Mkrn1 Cmh Htr2 Nnt T112 Mkrn3 D Begin Smt2 Ig2 Qpt Rin F7 Np1l Asl2 Xlr3 Peg3 Mst1r Trp73 Control Ig1r -/- Ig1r -/- 6.7 7.6 Gns Sh Xist 3.4 Gtm Prim2 Ue3 Impt Mts2 Dn Tpi2 F7 Plgl1 Asl2 Qpt Tsix Npl1 Gr3 Mst1r Xlr3 Gpr1 Ig2s 3.8 H13 C81 Comm1 Sh Npl14 Dlk1 Prim2 Dhr7 Blp Cmh Trpp9 Impt Inpp Sge Zp97 Zrsr1 Peg12 Xlr4 Nn Begin Ckn1 Mkrn3 D Tnrs23 Qpt Dn Mest Rin Tpi2 Xlr3 Peg3 Gpr1 8.9 4. Supplementry Figure 4. Blokge o Ig2 signling pthwy prtilly resues the phenotype in m- mutnt., Resue experiment mting strtegy., Perent onor engrtment 16-2 weeks post trnsplnttion. Hierrhil lustering o imprinte genes., Hetmps or expression o imprinte genes in 3 popultions, log2(+1). Genes hve een selete or reovery eet, whih is 2-ol loss rom WT to m /+ ollowe y n inrese in m /+ Ig1r-/-. 4 WWW.NATURE.COM/NATURE
RESEARCH Chr7 Begin Dlk1 mrpl23 Ig2 Ins2 Th Asl2 Tss4 C81 Knq1 Knq1ot1 Ckn1 Sl2218 Phl2 Np1l4 Crs Tnrs23 Osl Meg3 Rtl1 Rin Rin Ppp2r Dio3 Atp1 Ue3 Ipw Snor161 Snur Nn Mgel2 Peg12 luster Chr7 -Ig2 luster Knq1 luster Chr12 mirna luster Dlk1-Dio3 luster Supplementry Figure Perturtion o imprinting gene lusters ter eletion o - rom the mternl llele. UCSC genome rowser epition o luster, -Ig2, Knq1 luster in hromosome 7n Dlk1-Dio3 in hromosome 12. WWW.NATURE.COM/NATURE
RESEARCH SUPPLEMENTARY INFORMATION Control, Ig1r -/-, m /+ Ig1r -/- 1 1 1 e 1.4 17. 16.1 7.71 8. 8.6... Supplementry Figure 6. Genewise hierrhil lustering o ontrol n mutnts s, s n s, Log2(+1). All genes hve mx () 1. Hetmps -, genes with ol-hnge etween ny two smples, Eulien istne. N=26,4688,344 respetively. Color spe hs een ppe t 1 or visiility. Hetmps -, present/sent genes, using inry istne to show presene/sene trens. N=32,742,2 respetively. 6 WWW.NATURE.COM/NATURE
RESEARCH 1. mir-1982 12.84.8 Control Ig1r -/- Ig1r -/- mir-1931 snor-2.6.4 snor-17 snor-78 7.7 snor-24.2 snor-27. Ap2 Gpr63 Dusp26 P2rx2 Kt1 Chrn7 U8 2.67 4 8 3 6 2 4 1 2 Lepr Fm167 Fgr4 Cir1 Gp1 Cl4 Itg3 Il6 Gn Mpk 11 e Gene Control Ig1r -/- Ig1r -/- mir-686 6637 3274 3132 4241 mir-1839 11 1 7 mir-196-1 662 847 let7 146 371 mir-762 42 927 mir-3113 689 1631 mir-1949 41 29 mir-1198 44 mir-19 44 mir-27 1927 Gene mir-671 Control 1 1 Ig1r -/- 26 Ig1r -/- mir-1893 278 mir-19 1191 mir-374-1 321 Supplementry Figure 7. RNA sequening nlysis o sorte LT n ST HSCs rom ierent mutnts. Genes not ete in single mutnt Ig1r-/-:, n, Hetmp o mirna n snornas not ete in single mutnt Ig1r-/-. Genes not ete in single mutnt m /+:, n,. e, mirnas reovere in oule mutnt y Ig1r-/-., mirnas ete minly in single mutnts., Frgments Per Kilose o trnsript per Million mppe res. Tles e, roune up to nerest integer. WWW.NATURE.COM/NATURE 7