Fig 1: JDP2 is a common integration site in murine insertional mutagenesis studies of T-ALL and is aberrantly expressed in some patients with T-ALL. (A) Number of insertions identified from multiple murine retroviral insertional screens for T-ALL, collated on the RTCG database (Akagi et al., 2004). Gray bars are genes not yet implicated in human T-ALL. (B) JDP2 mRNA expression as determined by qPCR from 34 diagnostic adult T-ALL cases from the UKALL14 trial (black circles) and directly compared with normal thymic subsets sorted by FACS (blue circles). Thymocyte subsets were pooled from five individual donors to reduce intersample variation. qPCR experiments were performed in triplicate from two independent experiments. TN, triple-negative; DP, double-positive; SP, single-positive. Data points represent the mean ± standard error of the mean. (C) JDP2 expression as determined by Affymetrix gene expression array data for 40 pediatric T-ALL patients treated on the COG P9404 trial, separated according to ETP status (Gutierrez et al., 2010). JDP2 expression as determined by Illumina bead-chip array for 53 adult T-ALL patients treated on the ECOG E2993 trial, comparing patients with immature versus cortical/mature phenotypes (Van Vlierberghe et al., 2013). P values were calculated using the two-tailed Student’s t test. (D) Heatmap showing Affymetrix gene expression data for T-ALL patients treated on the COG P9404 trial, together with the mutational status of recurrently mutated genes. Yellow boxes denote wild-type genes, and black boxes, the presence of a genetic lesion. (E) Kaplan–Meier curves showing overall survival for pediatric T-ALL patients treated on the COG P9404 trial, stratified by JDP2 and ETP status. Patients were considered JDP2-positive using a probe presence score of ≥30; survival differences were calculated using the log-rank test. (F) JDP2 gene expression in 21 human T-ALL cell lines as determined by Affymetrix gene expression array. Lower panel shows JDP2 protein expression in a selection of T-ALL cell lines as determined by Western blot analysis. Representative Western blot from two independent experiments is shown.
Fig 2: Jdp2collaborates with Myc and can initiate T-ALL in a zebrafish model. (A) Schematic showing the coinjection strategy used to identify Myc collaborating genes in first generation mosaic transgenic zebrafish. (B) Kaplan–Meier curves showing tumor onset in first-generation mosaic zebrafish coinjected with constructs expressing rag2:Myc + rag2:jdp2 + rag2:mCherry (red line), versus rag2:Myc + rag2:mCherry versus (black line), versus rag2:mCherry alone (blue line). (C) Representative fluorescent images of thymuses from 8-wk-old stable transgenic zebrafish expressing Tg(rag2:mCherry) and Tg(rag2:jdp2). Bars, 1 mm. (D) Quantification of thymus size from Tg(rag2:mCherry) and Tg(rag2:jdp2) assessed by fluorescent microscopy biweekly from 4 to 14 wk of age. To account for intervariation in fish size, thymus size was normalized to head size. * P<0.05; ** P<0.005; *** P<0.0005. Data points represent the mean ± standard error of the mean. (E) Representative fluorescent microscopy images from a 40-wk-old Tg(rag2:mCherry), and three Tg(rag2:jdp2) zebrafish. Bars, 1 mm. (F) Kaplan–Meier curves showing tumor onset in stable Mendelian transgenic zebrafish expressing Tg(rag2:mCherry; red line) and Tg(rag2:jdp2; blue line).
Fig 3: JDP2 prevents apoptosis by directly regulating MCL1. (A) Waterfall plot showing genes most closely correlated with JDP2, using Pearson correlation analysis of gene expression data from 165 T-ALL patients from the MILE study (Haferlach et al., 2010). The y axis represents the Pearson correlation coefficient (R value) positively and negatively associated with JDP2 expression, adjusted for multiple testing using the false discovery rate, and P < 0.001. Genes are distributed across the x-axis, with position of MCL1 highlighted. (B) Direct correlation between JDP2 and MCL1, from Fig. 4 A, is shown in detail together with line of best fit. (C) ChIP-seq tracks for JDP2 and H3K27ac at the MCL1 locus in Loucy cells. (D) Relative JDP2, MCL1, and BCL2 mRNA levels over time after the addition of doxycycline to Loucy cells (left) and KOPT-K1 cells (right) stably expressing doxycycline-inducible JDP2 shRNA#2. qPCR experiments were performed in triplicate and verified in two independent experiments. (E) Western blots showing kinetics of MCL1 depletion and PARP1 cleavage after the addition of doxycycline in Loucy and KOPT-K1 cells expressing a doxycycline-inducible JDP2 shRNA. Representative Western blot from two separate experiments is shown. (F) Doxycycline-inducible JDP2 shRNA expressing Loucy and KOPT-K1 cells were stably transduced to express either GFP (control) or MCL1, and then treated with or without doxycycline, and viable cell number was measured by Cell Titer Glo at 24 h. Bottom: Western blots showing MCL1 and JDP2 expression in the transduced cell lines. P values were calculated using two-tailed Student’s t test applied to triplicate experiments performed twice. Data points represent the mean ± standard error of the mean.
Fig 4: JDP2 regulates the expression of Nrf2 target genes. (a) qPCR analysis of Nrf2 target genes in WT and Jdp2 KO MEFs. The expression levels determined are relative to the level of GAPDH. Error bars denote the S.D. from duplicate reactions by real-time PCR as described in Materials and Methods. The primer sequences of each Nrf2 target gene are listed in Supplementary Table SIII. *P<0.05; **P<0.01. (b–e) The ChIP–qPCR analyses were performed with chromatin extracts from WT and Jdp2 KO MEFs using specific antibodies (Ab) for Jdp2, Nrf2, MafK and IgG (as a negative control). (b and d) Schematic representations indicate the positions of primer motifs in HO-1 (b) and NQO1 promoter regions (d). Extracts from TPA-treated (24 h) or -untreated MEFs (5 × 108 cells) from WT and Jdp2 KO mice were subjected to ChIP assays with the indicated antibodies. Precipitated DNA and input DNA (1/20-fold) were analyzed using PCR with primers specific for E1 and E2 of the HO-1 (c) and ARE of the NQO1 (e) promoters. The nonspecific sequence motifs (NS) were used as a negative control (Supplementary Table SII). The data represent the mean±S.D. (n=3) with P-values from Student's unpaired t-tests: *P<0.05; **P<0.01
Fig 5: JDP2 occupies both promoters and enhancers and can both activate and repress target genes. (A) The preferred binding sequence for JDP2 is the palindromic TRE motif as calculated using Analysis of Motif Enrichment from the MEME suite. The adjusted P value is shown. Mean JDP2 occupancy from the TSS to TES at gene bodies, as analyzed from genome-wide binding by ChIP-seq in Loucy cells. The y axis shows ChIP-seq read concentration normalized to number of base pairs in the given region type. Right: Relative genome-wide occupancy of JDP2 at gene promoters (green), at enhancers (red), and within the gene body (white). (B) Volcano plot showing log2 fold change (FC) in gene expression 20 h after JDP2 knockdown in Loucy (left) and KOPT-K1 (right) cells stably expressing a doxycycline-inducible JDP2 shRNA. The y axis shows adjusted P value. Genes represented in blue are significantly down-regulated, those in red are significantly up-regulated, and those in black are not differentially expressed. RNA-seq was performed in biological triplicate. (C) Graph illustrating the number of genes in Loucy cells that are differentially expressed (by RNA-seq) after JDP2 knockdown and either bound by JDP2 (by ChIP-seq; direct target genes) or not bound by JDP2 (genes regulated indirectly after JDP2 knockdown).
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