Figure 3
Identification of driver mutations associated with loss of Trp53 by mitotic recombination or with the retention of a wild-type copy of Trp53. (a) SYBR Green quantitative real-time PCR (ABI, Carlsbad, CA, USA) was performed on tumour genomic DNA to quantify the relative proportions of Trp53 wild-type and Trp53Tyr alleles in genomic DNA extracted from the leukaemias/lymphomas and data were normalised to the single-copy genes β-Actin and Gapdh (primers are detailed in Supplementary Table 3). Red triangles represent tumours from Trp53−/− mice, blue squares represent tumours from Trp53+/− mice and black circles represent tumours from Trp53+/+ mice. Of the tumours from Trp53+/− mice: open squares are those that have retained a wild-type copy of Trp53, closed dark blue squares are those that have lost the wild-type Trp53 allele by mitotic recombination (MR) and closed light blue squares are those with a mixture of Trp53+/− and Trp53−/− cells and thus were excluded from further analyses. (b). Common insertion sites (CISs) were identified in tumours from Trp53+/− mice that had retained a wild-type copy of Trp53 (dotted blue circle) and those that had lost the wild-type copy (solid blue circle) as described previously.19, 20 CISS were called using a genome wide cut-off of P<0.05. Asterisk indicates the CIS was also found in the other genotype/circle, but below the P<0.05 cut-off. Double asterisk indicates the CISS were in intergenic regions (that is, not located within ±150K base pairs of a gene and were given the label ‘CIS’ followed by the chromosome and the peak location of the Gaussian kernel; there were two regions for ‘tumours retaining a wild-type copy of Trp53’: CIS7:37317163_15k and CIS5:75854217_15k, and one for ‘tumours without a wild-type copy of Trp53’: CIS7:37322632_15k) (c). Location and orientation of the transposon insertions (blue triangles) associated with the Jdp2 CIS (the exons of Jdp2 are represented as boxes). One tumour was found to harbour multiple independent transposon insertion events (indicated with dotted lines). (d). Quantitative PCR (qPCR) was performed on five tumours containing insertions in Jdp2 and nine randomly selected Trp53+/− T-cell tumours (without insertions in Jdp2). RNA was extracted using the RNeasy Minikit (Qiagen), DNAse-treated (Turbo DNase, Ambion, Warrington, UK) and reverse transcribed (RNA to cDNA EcoDry Random Hexamers, Clontech, Mountain View, CA, USA) according to the manufacturer's instructions. Quantitative PCR was performed in triplicate using SYBR Green PCR MasterMix (Applied Biosystems, Carlsbad, CA, USA) and the CT for Trp53 and Jdp2 were normalized to the ‘control’ (average of five housekeeping genes: Gapdh, β-Actin, Hprt1, Rpl32 and Rpl13a) using the 2–CT method.42 Primers used for qPCR are given in Supplementary Table 4. (e). Transient overexpression of JDP2 in NIH3T3 cells resulted in a significant repression of Trp53 proximal promoter activity. The 375 bp mouse Trp53 proximal promoter (Trp53-Luc) was PCR amplified from tail genomic DNA (using primers: F: 5′-AAAAAAAAGGTACCGGTCCACTTACGATAAAAAC-3′ and R: 5′-AAAAAAAAAAGATCTGGTCCCAATGAACTGAAGCT-3′) and cloned into the pGL3-BASIC vector (Promega, Southhampton, UK). The mutated mouse Trp53 proximal promoter in which the 7 bp PF-1 site (5′-TGACTCT-3′) was removed (mutTrp53-Luc) was synthesized (GeneArt-Invitrogen, Paisley, UK) and cloned into the pGL3-BASIC vector. A full-length human JDP2 cDNA was obtained from Origene (Rockville, MD, USA). NIH3T3 cells grown in 96-well plates were transfected with (i) either 100 ng Trp53-Luc (black lines) or mutTrp53-Luc (grey lines), (ii) 20 ng pRL-SV40 (an internal control reporter; Promega) and (iii) either 50 ng JDP2 cDNA or empty vector according to the manufacturer's instructions (Lipofectamine 2000; Invitrogen). Firefly and Renilla luciferase were measured 50 h later using the Dual-Luciferase Reporter Assay System according to the manufacturer's instructions (Promega). The firefly light units were normalised to the Renilla light units. All data were normalised to the average value of the ‘control’ transfection (Luc vector plus empty vector) and were presented as fold-change relative to the control. Experiments were performed in triplicate on at least three independent occasions and the data analysed by two-tailed Student's t-test. (f). Transient overexpression of JDP2 in HEK293T cells represses TRP53 expression. HEK293T cells (Gryphon Eco, Allele Biotechnology, San Diego, CA, USA) were seeded in 12-well plates and transfected with 2 μg Myc-DDK-tagged ORF clone of JDP2 (pCMV6Entry; Origene) or empty vector, according to the manufacturers' instructions (Lipofectamine 2000, Invitrogen). Experiments were performed in triplicate. RNA was extracted 48 h post-transfection and reverse transcribed as described above. Quantitative PCR was performed in triplicate using SYBR Green PCR MasterMix (Applied Biosystems) and the CT for TP53 and JDP2 were normalized as described above. Primers used for qPCR are given in Supplementary Table 4.
