SUPPLEMENTARY INFORMATION

Transcription

1 Supplementary Figure 1. Defective T and B cell lineage development in the absence of 53BP1. a, Average number of thymocytes in WT, 53BP1 /, p53 /, 53BP1 / / p53, Nbs1 tr735 and 53BP1 / Nbs1 tr735 mice (n 3 mice analyzed per genotype;error bars, s.d.). b, Number of B cells (B220 + ) in mutant mice bone marrow expressed as a percentage relative to littermate controls (n 3 mice analyzed per genotype). c, TCRβ surface expression in freshly isolated thymocytes d, Thy-1 and TCRγδ surface expression in thymocytes and splenocytes from WT and mutant mice. e, TUNEL staining of thymus tissue sections. Quantitation of apoptotic cells revealed an average of 7.14 apoptotic cells/mm 2 2 for WT,, 26.6 cells/mm in 53BP1 / and 18.2 cells/mm 2 in 53BP1 / / p53 thymus sections. 1

2 Supplementary Figure 2. Normal T cell development in the absence of H2AX and MDC1. a, Surface expression of CD4 vs. CD8 in WT, MDC1 / and H2AX / thymocytes determined by flow cytometry. b, TCRβ surface expression in freshly isolated WT, MDC1 /, H2AX / and Atm / thymocytes. c, Frequency at which TCRcα or (TCRvα+ TCRcα) signal is lost from one allele in WT, 53BP1 /, H2AX /, and MDC1 / thymocytes( > 400 cells analyzed per genotype). 2

3 Supplemetary Figure 3. Rescue of thymocyte cellularity by expression of a prerearranged TCR (AND). a, Average number of thymocytes from 53BP1 / RAG2 / AND + and 53BP1 +/+ RAG2 / AND + mice (error bars, s.d., n 3) b, Representative examples of CD4 and CD8 surface expression in 53BP1 -/- RAG2 / AND + and 53BP1 +/+ RAG2 / AND + thymocytes determined by flow cytometry. 3

4 Supplementary Figure 4. Jα α utilization. Amplified TCRα cdna from 53BP1 /, 53BP1 / Nbs1 tr735 or WT thymocytes was hybridized to different Jα gene segment-specific and a Cα-specific oligonucleotide as described 29. The index number reflects the Jα oligonucleotide hybridization relative to WT. 4

5 Supplementary Figure 5. Distance distributions. Histograms show the distribution of spatial distances in WT and 53BP1 / DP thymocytes. Measurements were performed in DP thymocytes using proximal, middle and distal TCRα probe sets as well as probes on mouse Chromosome 1D. The skewness (skew), kurtosis (kurt) and p values for the distance distributions are: proximal (WT: skew=2.3, kurt=10.7; 53BP1 /, skew=3.7, kurt=19.4; p=0.57, two tailed Wilcoxon rank sum test); middle: (WT: skew=1.1, kurt=1.6; 53BP1 /, skew=1.8, kurt=7.5; p<0.0001, two tailed Wilcoxon rank sum test); distal: (WT: skew=1.6, kurt=3.9; 53BP1 /, skew=1.6, kurt=3.7; p<0.0001, two tailed 3 Wilcoxon rank sum test); Chr1: (WT: skew=1.1, kurt=1.7; 53BP1 /, skew=0.75, kurt=0.2; p=0.55, two tailed Wilcoxon rank sum test). 5

6 Supplementary Table 1: Antigen-receptor associated chromosomal aberrations in metaphase spreads analyzed by Fluorescence-in situ-hybridization (FISH). Antigenreceptor (TCRα and IgH) specific, chromosome 14 or 12 paints and telomere probes were used. Lymph node T cells: Total TCRα associated aberrations TCRα-associated breaks TCRα associated translocations 53BP1 -/- 2/60 (3.3%) 1/60 (1.7%) 1/60 (1.7%) 53BP1 -/- 0/60 (0.0%) 0/60 (0.0%) 0/60 (0.0%) 53BP1 -/- 3/78 (3.8%) 2/78 (2.6%) 1/78 (1.3%) 53BP1 -/- 0/46 (0.0%) 0/46 (0.0%) 0/46 (0.0%) 53BP1 -/- 0/55 (0.0%) 0/55 (0.0%) 0/55 (0.0%) 53BP1 -/- 0/60 (0.0%) 0/60 (0.0%) 0/60 (0.0%) 53BP1 -/- / p53 -/- 7/50 (14.0%) 4/50 (8.0%) 3/50 (6.0%) 53BP1 -/- / p53 -/- 4/81 (4.9%) 2/81 (2.5%) 2/81 (1.2%) 53BP1 -/- /Nbs1 tr735 6/60 (10.0%) 1/60 (1.7%) 5/60 (8.3%) 53BP1 -/- /Nbs1 tr735 3/42 (7.1%) 1/42 (2.4%) 2/42 (4.8%) 53BP1 -/- /Nbs1 tr735 6/70 (8.6%) 1/70 (1.4%) 5/70 (7.1%) p53 -/- 0/58 (0.0%) 0/58 (0.0%) 0/58 (0.0%) Nbs1 tr735 0/62 (0.0%) 0/62 (0.0%) 0/62 (0.0%) Nbs1 tr735 0/60 (0.0%) 0/60 (0.0%) 0/60 (0.0%) Nbs tr735 0/20 (0.0%) 0/20 (0.0%) 0/20 (0.0%) WT 0/53 (0.0%) 0/53 (0.0%) 0/53 (0.0%) WT 0/53 (0.0%) 0/53 (0.0%) 0/53 (0.0%) Sum WT 0/150 (0.0%) 0/150 (0.0%) 0/150 (0.0%) Sum 53BP1 -/- 5/359 (1.4%) 3/359 (0.8%) 2/359 (0.6%) Sum p53 -/- 0/58 (0.0%) 0/58 (0.0%) 0/58 (0.0%) Sum 53BP1 -/- / p53 -/- 11/131 (8.4%) 6/131 (4.6%) 4/131 (3.1%) Sum Nbs1 tr735 0/122 (0.0%) 0/122 (0.0%) 0/122 (0.0%) Sum 53BP1 -/- /Nbs1 tr735 15/172 (8.7%) 3/172 (1.7%) 12/172 (7.0%) Splenic B220 + cells (+RP105, d1): Total IgHcα associated aberrations IgHcα-associated breaks IgHcα associated translocations 53BP1 -/- 0/50 (0.0%) 0/50 (0.0%) 0/50 (0.0%) 53BP1 -/- 0/62 (0.0%) 0/62 (0.0%) 0/62 (0.0%) 53BP1 -/- 0/30 (0.0%) 0/30 (0.0%) 0/30 (0.0%) 53BP1 -/- 0/58 (0.0%) 0/58 (0.0%) 0/58 (0.0%) 53BP1 -/- 0/48 (0.0%) 0/48 (0.0%) 0/48 (0.0%) 53BP1 -/- / p53 -/- 1/50 (2.0%) 1/50 (2.0%) 0/50 (0.0%) 53BP1 -/- / p53 -/- 3/53 (5.7%) 3/53 (5.7%) 0/53 (0.0%) 53BP1 -/- / p53 -/- 2/47 (4.3%) 1/47 (2.1%) 1/47 (2.1%) 53BP1 -/- /Nbs1 tr735 1/50 (2.0%) 1/50 (2.0%) 0/50 (0.0%) 53BP1 -/- /Nbs1 tr735 2/50 (4.0%) 2/50 (4.0%) 0/50 (0.0%) 6

7 p53 -/- 0/45 (0.0%) 0/45 (0.0%) 0/45 (0.0%) p53 -/- 0/50 (0.0%) 0/50 (0.0%) 0/50 (0.0%) p53 -/- 0/51 (0.0%) 0/51 (0.0%) 0/51 (0.0%) Nbs1 tr735 0/54 (0.0%) 0/54 (0.0%) 0/54 (0.0%) Nbs1 tr735 0/53 (0.0%) 0/53 (0.0%) 0/53 (0.0%) WT 0/50 (0.0%) 0/50 (0.0%) 0/50 (0.0%) WT 0/54 (0.0%) 0/54 (0.0%) 0/54 (0.0%) WT 0/54 (0.0%) 0/54 (0.0%) 0/54 (0.0%) Sum WT 0/158 (0.0%) 0/158 (0.0%) 0/158 (0.0%) Sum 53BP1 -/- 0/248 (0.0%) 0/248 (0.0%) 0/248 (0.0%) Sum p53 -/- 0/146 (0.0%) 0/146 (0.0%) 0/146 (0.0%) Sum 53BP1 -/- / p53 -/- 6/150 (4.0%) 5/150 (3.3%) 1/150 (0.7%) Sum Nbs1 tr735 0/107 (0.0%) 0/107 (0.0%) 0/107 (0.0%) Sum 53BP1 -/- /Nbs1 tr735 3/100 (3.0%) 3/100 (3.0%) 0/100 (0.0%) Bone marrow B220 + cells (+IL7, d5): Total IgHcα associated aberrations IgHcα-associated breaks IgHcα associated translocations 53BP1 -/- 1/77 (1.3%) 1/77 (1.3%) 0/77 (0.0%) 53BP1 -/- 1/33 (3.0%) 1/33 (3.0%) 0/33 (0.0%) 53BP1 -/- 1/60 (1.7%) 1/60 (1.7%) 0/60 (0.0%) 53BP1 -/- 1/70 (1.4%) 1/70 (1.4%) 0/70 (0.0%) 53BP1 -/- / p53 -/- 3/70 (4.3%) 3/70 (4.3%) 0/70 (0.0%) 53BP1 -/- / p53 -/- 2/55 (3.6%) 1/55 (1.8%) 1/55 (1.8%) 53BP1 -/- /Nbs1 tr735 2/54 (3.7%) 2/54 (3.7%) 0/54 (0.0%) 53BP1 -/- /Nbs1 tr735 1/33 (3.0%) 1/33 (3.0%) 0/33 (0.0%) p53 -/- 0/53 (0.0%) 0/53 (0.0%) 0/53 (0.0%) p53 -/- 0/56 (0.0%) 0/56 (0.0%) 0/56 (0.0%) p53 -/- 0/52 (0.0%) 0/52 (0.0%) 0/52 (0.0%) Nbs1 tr735 0/54 (0.0%) 0/54 (0.0%) 0/54 (0.0%) Nbs1 tr735 0/55 (0.0%) 0/55 (0.0%) 0/55 (0.0%) WT 0/50 (0.0%) 0/50 (0.0%) 0/50 (0.0%) WT 0/55 (0.0%) 0/55 (0.0%) 0/55 (0.0%) WT 0/54 (0.0%) 0/54 (0.0%) 0/54 (0.0%) WT 0/55 (0.0%) 0/55 (0.0%) 0/55 (0.0%) Sum WT 0/214 (0.0%) 0/214 (0.0%) 0/214 (0.0%) Sum 53BP1 -/- 4/240 (1.7%) 4/240 (1.7%) 0/240 (0.0%) Sum p53 -/- 0/161 (0.0%) 0/161 (0.0%) 0/161 (0.0%) Sum 53BP1 -/- / p53 -/- 5/125 (4.0%) 4/125 (3.2%) 1/125 (0.8%) Sum Nbs1 tr735 0/109 (0.0%) 0/109 (0.0%) 0/109 (0.0%) Sum 53BP1 -/- /Nbs1 tr735 3/87 (3.4%) 3/87 (3.4%) 0/87 (0.0%) 7

8 Supplementary Table 2a: Comparison of Coding joints Wildtype 53BP1 -/- Vβ14-Dβ1-Jβ1.1 (n=15) Vβ14-Dβ1-Jβ1.1 (n=24) deletions Vβ bp 1.79 bp additions Vβ14-Dβ bp 1.75 bp deletions Dβ bp 6.17 bp additions Dβ1-Jβ bp 1.79 bp deletions Jβ bp 1.54 bp microhomology 0/15 1/24 Vβ10-Dβ2-Jβ2.1 (n=57) Vβ10-Dβ2-Jβ2.1 (n=52) deletions Vβ bp 3.44 bp additions Vβ10-Dβ bp 2.85 bp deletions Dβ bp 7.21 bp additions Dβ2-Jβ bp 2.79 bp deletions Jβ bp 4.29 bp microhomology 1/57 0/52 Vδ2-Dδ1- Dδ2-Jδ1 (n=17) Vδ2-Dδ1- Dδ2-Jδ1 (n=42) deletions Vδ bp 2.60 bp additions Vδ2-Dδ bp 1.83 bp deletions Dδ bp 7.02 bp additions Dδ1-Dδ bp 2.81 bp deletions Dδ bp 6.71 bp additions Dδ2-Jδ bp 3.62 bp deletions Jδ bp 0.93 bp microhomology 0/17 4/42 Vδ3-Dδ1- Dδ2-Jδ1 (n=44) Vδ3-Dδ1- Dδ2-Jδ1 (n=38) deletions Vδ bp 2.63 bp additions Vδ3-Dδ bp 1.84 bp deletions Dδ bp 6.71 bp additions Dδ1-Dδ bp 2.68 bp deletions Dδ bp 5.95 bp additions Dδ2-Jδ bp 3.61 bp deletions Jδ bp 1.79 bp microhomology 5/44 1/38 Vδ4.1-Dδ1- Dδ2-Jδ1 (n=16) Vδ4.1-Dδ1- Dδ2-Jδ1 (n=18) deletions Vδ bp 2.28 bp additions Vδ4.1-Dδ bp 1.89 bp deletions Dδ bp 6.78 bp additions Dδ1-Dδ bp 2.50 bp deletions Dδ bp 4.94 bp additions Dδ2-Jδ bp 3.11 bp deletions Jδ bp 1.17 bp microhomology 0/16 0/18 Vδ5-Dδ1- Dδ2-Jδ1 (n=15) Vδ5-Dδ1- Dδ2-Jδ1 (n=18) deletions Vd bp 1.67 bp additions Vd5-Dd bp 3.44 bp deletions Dd bp 5.44 bp additions Dd1-Dd bp 1.61 bp deletions Dd bp 5.06 bp additions Dd2-Jd bp 3.83 bp deletions Jd bp 1.67 bp microhomology 0/15 0/18 8

9 Vα- Jα56 (n=37) Vα- Jα56 (n=81) deletions Vα 1.84 bp 1.65 bp additions Vα-Jα bp 4.04 bp deletions Jα bp bp microhomology 2/37 3/81 Supplementary Table 2b: Episomal signal joint insertions in V(D)J junctions from 53BP1 -/- thymocytes. Insert * : AGGCACACAAAATGCCTTCCACACAAAATGGGATCTCTGGTTGTTTTTTGTTTTGTTTTCTGGTTTTGTTTG TGTGTGGGGGGGACTTTTGACAAATTTTGTTTTGTTTTTATGTTTTTATCTTATTAATTTTTATTTTTTTATT TTGATTTTTTTATTGGAGAGAGGTAAGAAGAAAGATCATGAATTCGGGTGGGTA Insert : GCATATCACACAGGTTGAAGTATATTAAACCTCTGTTCAGAAACACTCAGCTGTGACTCCCTGTATAAGT CTTAGCACTGCAAAGAGTCCATTGAGCGCTGTAGGGAAATATGTAAGCTTTATTGACTCTTTTTGTTGTTG CTGTATATTTGGTCTGTATTGTCACTGCAAGAATTTCAGAATAGAAAAGAATATCACCAAAGTCTACAGT GATACAAATCCTAATCACATTCCTCAATATTGATCAAGCGGACTGACAGTGACGGAGCCCCAGTAGACGA GGTCACATGGGGA Insert : CGTATCCCTCCGATATGCCACTTCCCTCCACAGAGGTAAGTGCCTGAGTGGCTGGGCTGGGAGGCTTGGA TCTGCAGGGAACTTTGCTGCATTTTCTTATCCAGCTTAGCGGTTATTTTGTCCTTACTTTTCTCATCTCCTC CTTTCTGTAATGTCACCAAGGAGACGAGG Insert : ACCTTAAGGCTACATGACACATATTAAAGCCTTAACTGGGATGGTCATGAAGCCAAGACCTGATAGTGCT GGCTTTCTGAGACATAGAACTATGTATGTGTGGGTCTACGTTTAAGACAAGCTCTGCCAAGAGAATTTCA TATGTGTACTGTGCAGGAAGGTTTACTATGGAATATGTTGTTCCCACATGCTGCTCAAACATCAACCTCCT GTAAGCTAACCCATCCTTGTACATGATGTTAAGTCATGATGAGCCAGCTGGATGAGTAACACATGCTGTC TTTAAAGGATAGCACCATGAAAGCCCTCTAGCCATGACATCAGAGCAAGAAGAGGATGGGAAACAGCTG CTGAGGTTTTTGGAATGGCCTCAGTAGCTGTGC 9

10 Supplementary Table 3: Distance measurements of probe signals in thymocytes analyzed by 3D-Fluorescence-in situ-hybridization (FISH). DN thymocytes, probe set 334B8 and 255N13 (proximal TCRα) Genotype μm (%) μm (%) μm (%) Sample size Average distance (μm) Wild-type Wild-type BP1 -/ BP1 -/ DN thymocytes, probe set 74E19 and (middle TCRα) Genotype μm (%) μm (%) μm (%) Sample size Average distance (μm) Wild-type Wild-type BP1 -/ BP1 -/ DP thymocytes, probe set 334B8 and 255N13 (proximal TCRα) Genotype μm (%) μm (%) μm (%) Sample size Average distance (μm) Wild-type Wild-type Wild-type BP1 -/ BP1 -/ BP1 -/ DP thymocytes, probe set 74E19 and (middle TCRα) Genotype μm (%) μm (%) μm (%) Sample size Average distance (μm) Wild-type Wild-type Wild-type BP1 -/ BP1 -/ BP1 -/

11 DP thymocytes, probe set 304L21 and 10K20 (distal TCRα) Genotype μm (%) μm (%) μm (%) Sample size Average distance (μm) Wild-type Wild-type BP1 -/ BP1 -/ DP thymocytes, probe set 309A8 and 336F10 (Chr1D) Genotype μm (%) μm (%) μm (%) Sample size Average distance (μm) Wild-type Wild-type BP1 -/ BP1 -/

12 Supplemetary Table 4: Oligonucleotide primers used for standard and real-time PCR. Gene Name Sequence (5 3 ) Real-time PCR: Dδ1 5Dd1SE TGGGATCCTGAGTTTTCGGACTCT Dδ2 5Dd2 GGCGGATCCACATGCAGAAAACACCTG Dδ2 3Dd2R CAGGGATCCTGGGAGACGGTTCTTCACCCT Vδ4 DV104F2 ACTCGCAGCTGGGGGATGCT Vδ5 DV105F3 TGTTCAAGGTCGATTTTCTGTG Jδ1 3Jd1C CGTGGATCCACAGTCACTTGGGTT Jδ2 3Jd1F3 CCGGATCCCCCTCCAAAAAAG Jδ2 3Jd1R6 CCCGGATCCGCTTCTTAGCTTTTC Cα 3CAF1 GTGTGAGCATGGGAGACAGA Cα 3CAR1 TGGAGAGAGACAGGGGAAAG Coding Joint analysis: Vδ2 5Vd2 GTGGATCCTTGGCTATTGACATTTC Vδ3 5Vd3 GCCGGATCCACTTCTCCTTCTGTGA Vδ4 5Vd4 AGCGGATCCGGCCGTTCTTCCTGT Vδ5 5Vd5 GGCGGATCCCTCTGAGCTGGTCAGTG Jδ1 3Jd1 GGCGAATTCACTTGGGTTCCTTGTCC Vα Vα2degenerate CCCAAGCTTAGCAGCAGGTGAGACAAAGT Jα56 Jα56 GGAATTCAGGTGACGGCTTATGTTTAAAA TdT polyadenylation assay: D1 D2 UNIV-BglII T17-UNIV ACGTTGGTGAGAAACGAAGGC GCTCTAGATGGAAAGTCTCTCACACTACCGG GAAGATCTGTAAAACGACGGCCAGT GTAAAACGACGGCCAGTCGACTTTTTTTTTTTTTTTTT 12