A B Lin-B220+CD43+ C CD24-BP-1- thymus
thymus 1 - 6- preproB proB 15- BP aEnh(f/f) aEnh(f/f) ff;Mx1-Cre 4- 10-
1 CD24
- % CD3+ %
BP hCD4- hCD4+ Cells 1E7 x Cells 2- 5- aEnh(f/f) hCD4- hCD4+
Mx1-Cre CD19 total T E3 E4 E5 hCD4 CD93 D Cebpa Enh/prom-hCD4 preproB hCD4- MFI, no IR (n=6) hCD4+ hCD4-: 903 +/- 170 NS
hCD4+: 1100 +/- 340 H2AX
- +IR
g 1:1 mix -IR:IR -IR
E2 E3 E4 E preproB preproB preB hCD4- hCD4+ preB DNA (ng): 3 .6 .12 3 .6 .12 3 .6 3 .6 3 .6 1.6 kb-
1 kb- DJ1 (1033 bp) 600 bp- DJ2 (716 bp) 400 bp- DJ3 (333 bp) 200 bp-
FIGURE S1. (A) Mononuclear cells per thymus and the percent of thymic cells expressing CD3 from Cebpa Enh(f/f) or Enh(f/f);Mx1-Cre mice exposed to pIpC (mean and SD from three determinations). (B) Representative FC for BP-1;CD93 for preproB from Enh(f/f) and Enh(f/f);Mx-Cre mice post-pIpC. (C) Representative FC for sorting hCD4- and hCD4+ populations within preproB and proB from Cebpa Enh-Prom-hCD4 mice. (D) PreproB cells isolated from Cebpa Enh/Prom-hCD4 mice were analyzed for g-H2AX, gating on their hCD4- and hCD4+ subsets, with representative FC shown (top). Marrow from the same Cebpa Enh/Prom-hCD4 mouse was irradiated to 2 Gy and analyzed 1 hr later after mixing 1:1 with marrow that had not received ionizing radiation (IR, center). Mean fluorescence intensity (MFI) for g-H2AX in hCD4- versus hCD4+ mice, with irradiation (bottom). (E) Semi-quantitative PCR analysis for heavy chain DJ recombination in preproB and preB from a WT mouse (left) and the hCD4- and hCD4+ preproB subsets and preB from a Cebpa Enh/Prom-hCD4 mouse (right). B early preB preB WT preproB CLP proB
A 1
-
Kit
-
SSC SSC
preproB c
BP B220 1.0- Pu.1 Pu.1 kd/kd
0.5- ** CD43 CD24 Sca-1 IL7Ra Flt3
Relative Expression Relative aEnh(f/f) ff;Mx1-Cre WT 4- BM 2- 50-
x1E6 Pu.1(kd/kd) 3- 2- 1- 25-
legs+hips 1-
Percent Flt3+ Percent Cells/leg 1E5 Cells/leg x
Cells/ pre proB early preB CLP CLP proB preB
FIGURE S2. (A) Pu.1 mRNA relative to mS16, with expression in Enh(f/f) preproB set to 1.0 (mean And SD from three determinations). (B) Representative FC for preB, early preB, proB, preproB, CLP and Flt3+ CLP from WT vs Pu.1(kd/kd) mice (top). Enumeration of these subsets from three WT and two Pu.1(kd/kd) mice (bottom).
Table SI. RNAs UP in Top Ten Ingenuity Pathways in Cebpa Enh/prom-hCD4 preproB hCD4+ or hCD4- Cells*
Pathways with Affected Genes UP in Cebpa Enh/Prom-hCD4 preproB hCD4+ Cell Cycle Control of Chromosomal Replication (-logP, R: 19.9, 0.486) LIG1,MCM5,MCM6,CDT1,POLA1,CDC6,ORC6,RPA1,MCM4,RPA2,MCM3,PCNA,PRIM1,MCM2,TOP2A,PRIM2,ORC1,MCM7 Cell Cycle: G2/M DNA Damage Checkpoint Regulation (-logP, R: 11.8, 0.286) CDC25C,CKS2,WEE1,CCNB2,PLK1,AURKA,CDK1,SKP2,CCNB1,CDC25B,TOP2A,CKS1B,PKMYT1,BORA Mitotic Roles of Polo-Like Kinase (-logP, R: 11.2, 0.234) KIF23,CDC25C,ESPL1,CDC20,WEE1,PRC1,CCNB2,PLK1,CDK1,CCNB1,CDC25B,PKMYT1,FBXO5,KIF11,RAD21 Role of BRCA1 in DNA Damage Response (-logP, R: 7.83, 0.167) RAD51,FANCB,RFC4,TOPBP1,RBBP8,BRCA2,PLK1,RPA1,BRIP1,RBL1,RFC5,BLM,E2F2 Mismatch Repair in Eukaryotes (-logP, R: 6.13, 0.375) PCNA,RFC4,FEN1,RPA1,RFC5,EXO1 Role of CHK Proteins in Cell Cycle Checkpoint Control (-logP, R: 5.67, 0.17) PCNA,CDC25C,RFC4,CLSPN,PLK1,RPA1,RFC5,CDK1,E2F2 Cyclins and Cell Cycle Regulation (-logP, R: 5.24, 0.133) CCNA2,CCNE1,CCND3,WEE1,CCNB2,CDKN2C,CDK1,E2F2,SKP2,CCNB1 DNA Double-Strand Break Repair by Homologous Recombination (-logP, R: 5.06, 0.357) RAD51,LIG1,POLA1,BRCA2,RPA1 ATM Signaling (-logP, R: 4.99, 0.125) RAD51,CDC25C,SMC2,TOPBP1,H2AFX,RBBP8,CCNB2,BLM,CDK1,CCNB1 Estrogen-mediated S-phase Entry (-logP, R: 4.97, 0.25) CCNA2,CCNE1,RBL1,CDK1,E2F2,SKP2
Pathways with Affected Genes UP in Cebpa Enh/Prom-hCD4 preproB hCD4- B Cell Development (-logP, R: 4.53, 0.148) PTPRC,HLA-DOA,IGHM,HLA-DRB5 Antigen Presentation Pathway (-logP, R: 3.07, 0.107) HLA-DOA,CD74,HLA-DRB5 Protein Kinase A Signaling (-logP, R: 3.05, 0.0243) PTPRC,PPP1R14C,AKAP13,PXN,CREBBP,PTPRS,PTPN5,ELK1,GNG10 Role of IL-17A in Psoriasis (-logP, R: 2.92, 0.25) S100A8,S100A9 Primary Immunodeficiency Signaling (-logP, R: 2.65, 0.077) PTPRC,IGHA,IGHM FXR/RXR Activation (-logP, R: 2.13, 0.035) APOE,SLC10A1,CREBBP,PLTP Rac Signaling (-logP, R: 2.13, 0.035) MCF2L,CYBB,ELK1,ELK4 Production of Nitric Oxide and Reactive Oxygen Species in Macrophages (-logP, R: 2.12, 0.028) PPP1R14C,APOE,CREBBP,CYBB,S100A8 PI3K Signaling in B Lymphocytes (-logP, R: 1.99, 0.032) PTPRC,CD180,PIK3AP1,ELK1 Cardiac β-adrenergic Signaling (-logP, R: 1.94, 0.031) PPP1R14C,AKAP13,CACNA1E,GNG10 *Top pathways from analysis of RNAs with >2-fold change on average in 2 experiments, with affected genes listed and ordered by -logP values. The -logP values and R values for each pathway are shown in parenthesis. R values indicate the proportion of genes in each pathway affected.
Table SII. RNAs UP in Top Ten Ingenuity Pathways Affected By Cebpa Enhancer Deletion in preproB Cells* Top Pathways with Affected Genes UP in WT compared with +37 kb Cebpa Enhancer-Deleted preproB cells Cell Cycle Control of Chromosomal Replication (-logP, R: 14.6, 0.396) MCM5,LIG1,MCM6,CDT1,POLA1,CDC6,POLE,DNA2,ORC6,CDK1,MCM4,RPA2,MCM3,PCNA,PRIM1,MCM2,TOP2A,PRIM2 CHEK2,MCM7,ORC1 Estrogen-mediated S phase Entry (-logP, R: 10.9, 0.50) TFDP1,E2F3,RBL1,CDK1,SKP2,MYC,CCNA2,CCNE1,E2F7,ESR1,E2F8,E2F2,CDC25A Mitotic Roles of Polo-Like Kinase (-logP, R: 8.81, 0.0266) KIF23,CDC25C,ESPL1,CDC20,WEE1,PRC1,CCNB2,PLK1,CDK1,CCNB1,CDC25B,PLK4,FBXO5,PKMYT1,CHEK2,KIF11, CDC25A G2/M DNA Damage Checkpoint Regulation (-logP, R: 8.79, 0.306) CDC25C,CKS2,WEE1,CCNB2,PLK1,AURKA,CDK1,SKP2,CCNB1,CDC25B,TOP2A,CKS1B,PKMYT1,BORA,CHEK2 Role of BRCA1 in DNA Damage Response (-logP, R: 7.23, 0.212) TOPBP1,BARD1,PLK1,RBL1,E2F3,RAD51,FANCB,RFC4,MRE11,E2F7,BRCA2,BRIP1,BLM,CHEK2,E2F8,E2F2,FANCA Role of CHK Proteins in Checkpoint Control (-logP, R: 6.24, 0.236) CDC25C,PLK1,E2F3,CDK1,PCNA,RFC4,MRE11,E2F7,CLSPN,CHEK2,E2F8,E2F2,CDC25A Cyclins and Cell Cycle Regulation (-logP, R: 5.21, 0.182) TFDP1,WEE1,CCNB2,E2F3,CDK1,CCNB1,SKP2,CCNA2,CCNE1,CCND3,E2F7,E2F8,E2F2,CDC25A DNA Double-Strand Break Repair by Homologous Recombination (-logP, R: 4.79, 0.429) RAD51,LIG1,GEN1,POLA1,MRE11,BRCA2 G1/S Checkpoint Regulation (-logP, R: 4.05, 0.175) MYC,CCNE1,TFDP1,CCND3,E2F7,E2F3,RBL1,E2F2,E2F8,CDC25A,SKP2 DNA Methylation and Transcriptional Repression (-logP, R: 3.22, 0.206) HIST1H4F,HIST1H4A,HIST1H4C,HIST4H4,HIST1H4K,HIST1H4B,HIST1H4H
Top Pathways with Affected Genes UP in preproB after Cebpa +37 kb Enhancer Deletion OX40 Signaling Pathway (-logP, R: 14.9, 0.288) H2-T9,H2-T10,H2-T3,HLA-A,CD4,NFKBIE,H2-K2/H2-Q9,HLA-DQA1,H2-M11,HLA-DQB1,CD3D,CD3G,NFKBID,H2-M2, H2-Eb2,HLA-DMB,HLA-F,HLA-DRB5,H2-Q8 Th1 and Th2 Activation Pathway (-logP, R: 11.7, 0.163) SOCS3,SOCS1,IL12RB1,IL1RL1,HLA-A,CD4,TGFBR3,HLA-DQA1,ACVR2B,HLA-DQB1,HLA-DMB,CD274,IL2RB,IL2RG, DLL1,PRKCQ,NOTCH3,FGFR1,CD3D,CD3G,LTA,H2-Eb2,IL10RA,IL2RA,GATA3,HLA-DRB5 Th2 Pathway (-logP, R: 11.5, 0.273) SOCS3,DLL1,IL2RG,NOTCH3,PRKCQ,IL12RB1,HLA-A,IL1RL1,CD4,FGFR1,TGFBR3,HLA-DQA1,ACVR2B,HLA-DQB1, CD3D,CD3G,H2-Eb2,HLA-DMB,IL2RA,GATA3,HLA-DRB5,IL2RB Allograft Rejection Signaling (-logP, R: 10.7, 0.164) H2-T9,H2-T10,H2-T3,HLA-A,H2-K2/H2-Q9,HLA-DQA1,H2-M11,HLA-DQB1,H2-M2,H2-Eb2,HLA-DMB,HLA-F,TNF, HLA-DRB5,H2-Q8 Th1 Pathway (-logP, R: 9.31, 0.155) SOCS1,SOCS3,DLL1,NOTCH3,PRKCQ,IL12RB1,HLA-A,CD4,FGFR1,HLA-DQA1,HLA-DQB1,CD3D,CD3G,H2Eb2,LTA, HLA-DMB,IL10RA,CD274,GATA3,HLA-DRB5 iCOS-iCOSL Signaling in T Helper Cells (-logP, R: 7.53, 0.147) IL2RG,PRKCQ,HLA-A,CD4,FGFR1,NFKBIE,TRAT1,HLA-DQA1,HLA-DQB1,CD3D,CD3G,NFKBID,CAMK2A,H2Eb2, HLA-DMB,IL2RA,HLA-DRB5,IL2RB Type I Diabetes Mellitus Signaling (-logP, R: 7.42, 0.126) SOCS1,SOCS3,HLA-A,NFKBIE,HLA-DQA1,HLA-DQB1,CD3D,CD3G,NFKBID,H2-Eb2,LTA,HLA-DMB,HLA-F,TNF, HLA-DRB5 Cdc42 Signaling (-logP, R: 6.97, 0.174) VAV2,H2-T9,H2-T10,H2-T3,HLA-A,H2-K2/H2-Q9,HLA-DQA1,H2-M11,HLA-DQB1,CD3D,CD3G,H2-M2,H2-Eb2, HLA-DMB,HLA-F,HLA-DRB5,H2-Q8 T Helper Cell Differentiation (-logP, R: 6.84, 0.19) IL2RG,IL12RB1,HLA-A,H2-Eb2,HLA-DMB,IL10RA,HLA-DQA1,IL2RA,HLA-DQB1,GATA3,TNF,HLA-DRB5 Crosstalk between Dendritic Cells and NK Cells (-logP, R: 6.59, 0.286) CSF2RB,IL2RG,CAMK2A,HLA-A,FSCN1,LTA,TLR7,LTB,HLA-F,TNF,HLA-DRB5,IL2RB *Top pathways from analysis of RNAs with >1.4-fold change on average in 2 experiments, with affected genes listed and ordered by -logP values. The -logP values and R values for each pathway are shown in parenthesis. R values indicate the proportion of genes in each pathway affected.