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1 doi:1.138/nature11496 Cl. 8 Cl. E93 Rag1 -/- 3H9 + BM Rag1 -/- BM CD CD c-kit c-kit c-kit wt Spleen c-kit B22 B22 IgM IgM IgM Supplementary Figure 1. FACS analysis of single-cell-derived pre-b cell clones. Single cell clones (B6/Cast) were analyzed by FACS for the presence or absence of CD, IgM, B22 and c-kit expression. Pre-B cells isolated from Rag1 -/- 3H9 + bone marrow (BM), early pro-b cells isolated from Rag1 -/- BM, and splenic B cells isolated from wt mice, were used as controls. 1

2 a b ~15xV H 13xD H J H Vκ Deg. + Jκ1 Jκ4 J H 2 J H 3 V H Q52-J H 4 Rearrangement..5 J H 4. V H Gam3.8-J H 4 J H 1 J H 2 J H 3 Non specific wt IgM + wt IgM+ cl. B3 cl. 8 cl. X Spleen BM J H 4 Spleen Cl. 8 Cl. B3 Cl. B52 Cl. E93 c Fold Enricment Vκ-Cκ Vκ-Jκ Hrs Supplementary Figure 2. Molecular analysis of pre-b cell clones. (a) DNA from single cell clones (hcκ/mcκ clone B3; B6/Cast clones 8, B52 and E93) was analyzed for heavy chain rearrangement by PCR using a reverse primer downstream of J H 4 and different forward primers for different V H families (as indicated in diagram). (b) DNA from wt splenocytes and single cell pre-b clones (hcκ/mcκ clones B3 and X; B6/Cast clone 8) was analyzed for Vκ-Jκ rearrangement using quantitative PCR. (c) RNA and DNA were extracted at 12 hr intervals from a pre-b cell clone (B3) induced to differentiate following IL-7 withdrawal, and were subjected to real-time PCR analyses for rearrangement. Prior to PCR analysis RNA was reverse transcribed. 2

3 a Fold Enrichment 1 5 Pck-1 Jκ H3Ac Fold Enrichment 1 5 Hbb Jκ Mrps5 H3K4me3 b HhaI Jκ BccI Cκ HhaI Jκ H3Ac H3Ac H3K4me3 Supplementary Figure 3. Ig monoallelic chromatin structure in pre-b cells. (a) ChIP analysis on chromatin derived from a B6/Cast single-cell-derived pre-b clone (Clone 8) using antibodies to H3Ac or H3K4me3. qpcr using specific primers for the 3'J (left) and J 5 (right) regions was carried out. The fold enrichment was calculated as Bound/Input (see Methods). Pck-1 or -globin (Hbb) was used as negative controls to which results were normalized. Mrps5 was a positive control. (b) Preferential enrichment of H3Ac or H3K4me3 (as indicated) was determined by digesting the chromatin input and bound fractions with HhaI or BccI (shown in Fig. 2b). 3

4 a Fold enrichment Jκ Hbb Mrps5 Fold enrichment Cryaa Pax5 Jκ CD19 b Clone 8 AhdI HhaI Clone non specific Pax5 c HhaI Clone E BccI Clone E Pax5 d 1 Clone G11 Cast Clone G72 Cast Bound (%) 5 B6 B6 B6 Supplementary Figure 4. and Pax5 are preferentially recruited to one! allele in pre-b cells. (a) ChIP analysis on chromatin derived from B6/Cast pre-b clone (Clone 8) with antibodies to (left) or Pax5 (right), using primers specific for the 3'J! region. The degree of enrichment was calculated as Bound/Input. "-crystallin (Cryaa) or #-globin (Hbb) was used as negative controls to which results were normalized. Mrps5 and CD19 were used as positive controls. (b, c) Preferential enrichment of or Pax5 in clone 8 (b) and E93 (c) was determined by amplifying the 3 J! region followed by digesting the input and bound chromatin fractions with the indicated restriction enzymes (d) is preferentially recruited to one! allele in B6/Cast single cell clones (G11 and G72), as described in b and c. Quantification of representative results is shown in the histograms. 4

5 Cκ Rosa26 Supplementary Figure 5. The replication time mode for each allele in ES cells is not maintained in a clonal manner. Single cells derived from an ES clone carrying the imog marked Rosa26 transgene were assayed by FISH using the Cκ (red) and imog probe (green) for the Rosa26 transgene. As was previously shown, the Rosa26 site is situated in close proximity with the endogenous κ locus. Cκ single/doubles were then scored for the location of Rosa26 transgene. In 5% of the cells, the single was associated with the Rosa26-carrying chromosome 6 (left) and in 5% the single was not associated with the ROSA26 carrying chromosome 6 (right). 5

6 a HSC m h HSC - Mouse 1 b HSC hcκ mcκ +GFP - GFP Mouse 2 hcκ mcκ Mouse 1 +GFP GFP Mouse 4 hcκ mcκ Mouse 2 +GFP GFP Mouse 1 c MPPs - Mouse 1 hcκ mcκ d CLPs m h CLPs - Mouse 1 hcκ mcκ CLPs - Mouse 2 hcκ mcκ Mouse 2 Supplementary Figure 6. Establishment of! allelic choice in HSCs, MPPs and CLPs. (a) HSCs were isolated from hc!/mc! mouse bone marrow and infected with lentiviral vector expressing GFP. SCID mice were reconstituted with GFP expressing cells, spleens were extracted 2-3 months later and hc! and mc! expressing B cells were separated by FACS. Viral integration sites of mouse 1 were detected through LAM-PCR 1, and gels were then quantified as described in Methods. DNA from mice 2 and 4 was subjected to Southern blot analyses as described in Figure 4a and gels were then quantified as described in Methods. (b) To validate the LAM-PCR results, SCID mice were reconstituted with DS-Red-infected HSCs extracted from GFP +/ female mice in which the GFP construct has been inserted into the X chromosome. Splenic B cells were sorted for GFP + and GFP cells and viral integration sites were analyzed by LAM-PCR (top graph). DNA from mice 1 and 2 was subjected to Southern blot analyses as described in Figure 4b and gels were then quantified as described in (a). (c, d) MPPs (c) or CLPs (d) were isolated from the bone marrow of hc!/mc! mice and treated as in (a). SCID mice were reconstituted with GFP expressing cells and spleens were extracted 2 months later. FACS-sorted hc! and mc! expressing B cells were analyzed for viral integration through LAM-PCR. 1. Schmidt, M. et al. High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nature Methods 4, (27) 6

7 Pro-B cells m h Pro-B cells m h Pro-B cells - Mouse 1 Pro-B cells - Mouse 2 hck mck hck mck Supplementary Figure 7. Establishment of! allelic choice in pro-b cells. Pro-B cells were isolated from hc!/mc! mouse bone marrow and infected with lentiviral vector expressing GFP. SCID mice were reconstituted with GFP expressing cells, spleens were extracted 2-3 months later and hc! and mc! expressing B cells were separated by FACS. Viral integration sites were detected through LAM-PCR 1, and gels were then quantified as described in Methods. 1. Schmidt, M. et al. High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR). Nature Methods 4, (27) 7

8 a Rag1 -/- cl. G1 CD B22 c-kit IgM b Fold enrichment H3Ac Bound (%) 1 5 mck hck c Fold Enrichment 1 5 H3K4me3 Bound (%) 1 5 mck hck Cryaa Cκ Actb H3Ac Hbb Jκ Mrps5 H3K4me3 d Fold enrichment Bound (%) 1 5 mck hck Hbb Jκ Mrps5 Supplementary Figure 8. Differential chromatin marking of the κ locus in pro-b cells. (a) FACS analysis of a mcκ/hcκ single-cell-derived pro-b cell clone (Rag1 -/- cl.g1) for the presence or absence of c-kit, B22, CD and IgM. (b, c and d) ChIP analysis on chromatin derived from these cells with antibodies to H3Ac (b) or H3K4me3 (c) or (d) using primers specific for the indicated κ regions (left). The degree of enrichment was calculated as Bound/Input following semi-quantitative (b) or q-pcr analyses (c,d). Negative controls as well as positive controls are shown to the left and right of the κ region, respectively. In order to differentiate between the alleles input and bound fractions were amplified with primers specific for the mcκ and hcκ regions. The histograms represent quantification of the normalized data (described in Methods) of the semi-quantitative PCR analyses (right). 8

9 Supplementary Figure 9. Plasticity versus clonality of asynchronous replication patterns in the hematopoietic system HSC, MPP, and CLP single cell clones originating from mice carrying a large deletion on the Tcrβ paternal allele gene were assayed by FISH for asynchronous DNA replication by counting the number of Cκ single/doubles, using the Cκ probe. FISH examples; in one cell the early replicating Igκ (green, double dot) is on the Tcrβ wt allele (red); in the other the unreplicated Igκ allele (green, single dot) is associated with this allele. 9

10 Supplementary Table 1. Primers for ChIP enrichment -TaqMan Gene 5 Primer Taqman probe 3 Primer Jk5 AGTGTGAAAGCTGAGCGAAA CCTGCCTGTGAAGCCAGTCCA CACAGTGAGGACTATGACATGC Hbb GCTGCTGGTTGTCTACCCTT TCCAAAGCTATCAAAGTACCGCTGGG GCAGAGGCAGAGGATAGGTC Mrps5 GCTGACGACGAACTTGTGAC TCCTCACCGCCTGCAGCAAC AGCTACACAATGCAGGGAGA Primers for ChIP enrichment-sybr Gene Primer Name Primer Sequence Pck-1 Pck L AAC ACA CCC TCG GTC AAC A Pck R TGA AAT GAC CCT GCC TAC CT Cryaa acrystalin L CCA TCA GCC CCT ACT ACC GC acrystalin R CTG AGC AGC TAG GAG GAA CC CD19 CD19 L GAT TTG GAA GAG TGC CTA CA CD19 R GCC TGC CTC CTA CTA AGG TA Primers for ChIP-allelic determination and DNA replication Gene Primer Name Primer Sequence Ck Ck BCCI L CAC CCT CAC GTT GAC CAA G Ck BCCI R CAC TCA TTC CTG TTG AAG CTC Jk Jk HhaI L AAA TGG ATG TGG GAG CAA AC Jk HhaI R CAG AGC TCT AGG CCC CTC TT Jk Jk AhdI alt L GGC CAA CGT TTT GTA AGA CA Jk AhdI L GGC CAC GGT TTT GTA AGA CA Jk AhdI R CAG GGT GAA CGC CAA ATG hck hck L CCT CCA ATC GGG TAA CTC C hck R GAC TTC GCA GGC GTA GAC TT mck mck L ATC TTC CCA CCA TCC AGT GA mck R TGC CAT CAA TCT TCC ACT TG Primers for Chip-Seq Gene Primer Name Primer Sequence Jk Jk seq L AGC CTG CCC TAG ACA AAC CT Jk seq R TTT AGC CTG AAA ATC ATT CCA A Primers for DNA and RNA rearrangement Gene Primer Name Primer Sequence Vk VkDeg5' GTC CCT GCC AGG TT(C/T) AGT GGC AGT GG(A/G) TC(A/T) (A/G)GG AC Vk Vk4-74L GCA CTG CCA GCT CAA GTG TA Vk Vk4-78L CTC ACC CAG TCT CCA GCA AT Jk Jk1 R CGT TTG ATT TCC AGC TTG GT Jk Jk4 R GCC AGG AAT GGC TCA TTT AG UBC UBC L CAG CCG TAT ATC TTC CCA GAC T UBCR CTC AGA GGG ATG CCA GTA ATC TA GAPDH GAPDH L CCT GGA GAA ACC TGC CAA G GAPDH R CAA CCT GGT CCT CAG TGT AGC Primers for reconstitution Vk Vk Deg 1 GCG AAG CTT CCC TGA TCG CTT CAC AGG CAG TGG Vk Vk Deg 2 GCG AAG CTT CCC(AT)GC TCG CTT CAG TGG CAG TGG Vk Vk Deg 2 GCG AAG CTT CCC A(GT)(AC) CAG GTT CAG TGG CAG TGG mck mck R1 ACG CCA TTT TGT CGT TCA CTG CCA hck hck R1 GAG TTA CCC GAT TGG AGG GCG TTA Ck Ck BCCI R CAC TCA TTC CTG TTG AAG CTC Primers for LAM-PCR Primer Name Primer Sequence Modification 3' LTR-I up B GAG CTC TCT GGC TAA CTA GG 5' Biotin 3' LTR-I down B GAA CCC ACT GCT TAA GCC TCA 5' Biotin LTR-II B AGC TTG CCT TGA GTG CTT CA 5' Biotin LTR-III AGT AGT GTG TGC CCG TCT GT Unmodified LC-I GAC CCG GGA GAT CTG AAT TC Unmodified LC-II AGT GGC ACA GCA GTT AGG Unmodified 1

11 Supplementary Table 2. Clone Region H3AC H3K4me3 % Pref. % Non Pref. % Pref. % Non Pref. hc /mc 1 C NA NA 7 C NA NA X C NA NA B3 C NA NA E1 C NA NA B6/Cast 4 J C J C

12 Supplementary Table 3. Asynchronous replication is not heritable in ES cells. Fibroblasts* Clone Maternal early Paternal early Pool 4A ES cells** Clone Maternal early Paternal early I G E *Pooled embryonic fibroblasts and individual ear fibroblast subclones originating from mice carrying the neo-lacz marked maternal Rosa26 transgene were assayed by FISH for asynchronous DNA replication by counting the number of Cκ single/doubles, using the Cκ and neo-lacz probes. The number of paternal early or maternal early nuclei among cells showing asynchronous replication pattern was assayed (data was taken from Mostoslavsky et al.). **The same analysis was performed on single cell ES clones harboring the IiMOG transgene in the Rosa26 locus. Cκ and IiMOG probes were used for FISH analysis. 1. Mostoslavsky, R. et al. Asynchronous replication and allelic exclusion in the immune system. Nature 414, (21) 12

13 Supplementary Table 4. C OlfR Nanog Clone Paternal Maternal Paternal Maternal Paternal Maternal HSC H HSC H MPP H CLP G CLP B HSC, MPP, and CLP single cell clones originating from mice carrying a large deletion on the Tcr paternal allele gene were assayed by FISH for asynchronous DNA replication by counting the number of single/doubles using the C olfactory receptor (OlfR) and Nanog 1 probes. The number of paternal early or maternal early nuclei was determined by reference to a TCR probe. 1. Miyanari Y, Yorres-Padilla ME. Nature. 212 Feb 12:483(739):