Fig 1: ChIP on factors involved in D–JH recombination. The D–Cµ domain in its unrearranged configuration is shown in the top scheme together with the sense and anti-sense transcripts (black arrows) produced in the domain. The relative position of the primers (colored arrows) used in qPCR is indicated. Degenerate primers were used to amplify DSP segments. The histograms below show the fold enrichment for RNA polymerase II and its phosphorylated forms at Serine 5 (S5) or at Serine 2 (S2), H3K4me3, RAG1 and the BRG1 subunit of the SWI/SNF remodeling complex. Chromatin from Rag2-deficient pro-B cells was immunoprecipitated with the corresponding antibodies. The ß-globin gene was used as a negative control. The histograms show the standard deviation (n = 5). Statistical analysis (t-test). (ns): not significant, (*) significant (P < 0.05), (**) very significant (P < 0.01), (***) and (****): extremely significant (P < 0.001 and P < 0.0001 respectively).
Fig 2: Revised mechanism of DUX4/IGH-driven oncogenic splicing. In B-ALL, chromosome translocation gives rise to DUX4/IGH. The loss of C-terminal domain, together with the potent DNA-binding activity in HD1-HD2, can from a dumbbell-shape trans dimer for the recognition of double tandem DRE-DRE in ERG, CLEC12A and C6orf89. The resulting DUX4/IGH-mediated DNA crosslinking might allow the recruitment of RAG1/2 to the DRE-DRE sites, catalyzing V(D)J-like cleavage/recombination and alternative splicing in leukemia. Abbreviations: DUX4/IGH: Double homeobox 4 fused with immunoglobulin heavy chain; B-ALL: B cell acute lymphoblastic leukemia; HD: Homeobox domain; DRE: DUX4-resposive-element; ERG: E-26 transformation-specific (ETS) family related gene; CLEC12A: C-type lectin domain family 12, member A; C6orf89: Chromosome 6 open reading frame 89
Fig 3: Analysis of DH rearrangements in pro-B cells and DP thymocytes. (A) Schematic of DH gene segments and positions of primers used to amplify DJH recombinations. (B) Genomic DNA prepared from C57BL/6 pro-B cells and DP thymocytes (Figures S3B–D) were used in PCR reactions with a 5' primer located upstream of DFL16.1 (green arrow), or one that hybridizes to all 6 DSP2 gene segments (brown arrows), and a 3' primer located after JH4 (black arrow). Four-fold serially diluted DNAs were used for PCR amplification reaction followed by separation of the products by electrophoresis through 1% agarose gels. PCR analysis was carried out with two independent preparations of pro-B and DP cells and the data shown is one representative example. An amplicon from the ß-globin gene was used as loading control and a no-DNA control is shown in lane 1. (C) DSP2 and DFL16.1 utilization in pro-B cells and DP thymocytes was calculated after band intensity quantitations from two different gels using Gene Tool. Error bars represent standard error of the mean between two independent gel quantitations. (D) Rag1 and Rag2 binding to the IgH locus was evaluated by chromatin immunoprecipitation. Immunoprecipitated genomic DNA and input DNA were used for qPCR and fold enrichment was calculated as described by Ji et al. (23). DP thymocytes were compared to the D345 pro-B cell line as indicated. ?-actin served as positive control for Rag2 but negative control for Rag1. C?3 is used as negative control for both Rag1 and Rag2. TCRJß2 gene (TRBJ2-5) is used as additional positive control for DP thymocytes. For each independent experiment PCR was done in triplicate. Data shown is the mean of two independent experiments. Error bars represent standard error of mean (n = 2). (E) Utilization of DSP2 gene segments in DJH junctions in DP thymocytes from C57BL/6 mice. DSP2-JH1 recombined products were amplified using a forward pan-DSP2 primer and a reverse primer located 3' of JH1. Amplification products were gel purified followed by adapter ligation and sequencing (left panel). The number of reads aligned to each DSP2 gene are shown in Figure S3F. Percentage of reads mapping to indicated DSP2 gene segments are shown (after removal of redundant reads). DSP2-JH1 amplification products were also cloned into pGEM-T vector and 60 clones were sequenced (right panel). Number of clones with each gene segment are shown in the bar graph. Thirty clones were sequenced each time from two different PCR amplification. 50 out of 60 clones that had unique junctional sequences are represented in the bar graph.
Fig 4: SATB1 controls expression of TCR and other adhesion molecule genes.a Important thymic receptors were underexpressed in Satb1 cKO based on RNA-seq data. b Scatter plot indicating positive correlation between gene expression changes and the difference between H3K27ac overinteracting and underinteracting loops in WT compared to Satb1 cKO thymocytes that was normalized to the 1 kbp gene length. The grey zones indicate 95% confidence level interval for predictions from a linear model (red and blue lines for genes present and absent in SATB1-dependent loops, respectively). c Genomic tracks as well as SATB1 and CTCF HiChIP loops at the T cell receptor alpha locus (TCRa). The bottom green genomic tracks of log2 fold change RNA-seq values summarize the overall deregulation of the TCRa locus, with variable regions (Trav genes) being mostly underexpressed, TCRd locus overexpressed and TCRa joining regions (Traj genes) displaying geometrically symmetric deregulation splitting the region into over- and under-expressed in Satb1 cKO cells (magenta arrow). This deregulation was markedly correlated with the presence of SATB1-dependent loops. Note especially the region of joining genes which manifests a deregulation similar to previous reports from Satb1 depleted animals84,85. Both SATB1 and CTCF loops displayed a tendency to connect the TCR enhancer (green arrow) to the regions inside the locus. The presented loops were called with low stringency parameters and with a different set of binding sites compared to the rest of our study due to technical details explained in the methods section. All tracks refer to murine thymocytes. d SATB1-dependent promoter-enhancer regulatory loops control the expression of both Rag1 and Rag2 genes. Legend: th – thymocytes, DP – CD4+CD8+ T cells, RKO – Rad21fl/flCd4-Cre+ and SKO – Satb1fl/flCd4-Cre+. e RNA levels of Rag1 and Rag2 genes in WT and Satb1 cKO (SKO) thymocytes based on three biological replicates of RNA-seq experiments. Data are presented as mean values ± s.d. FDR scores from DESeq2 analysis are provided. f Protein levels of RAG1 and RAG2. The Western blot analysis was performed three times for each protein. g Differential H3K27ac HiChIP loops reflected the deregulation of Traj regions.
Fig 5: The direct interaction between RAG1/2 and DUX4/IGH. (A) Duolink proximity ligation assay (PLA). The two primary antibodies used in this study were generated from rabbit (WT/mutant DUX4/IGHs) and mouse (RAG1/2), respectively. Left panel, the direct interaction between DUX/4/IGH and RAG1/2 in Reh cells is visualized by fluorescently labeled complementary oligonucleotide probes. Right panel, the co-expression of WT/mutant DUX4/IGH and RAG1/2 in Reh cells are monitored by Western blotting (A). (B) Co-immunoprecipitation (co-IP) assay. The endogenous RAG1/2 in Reh cells is pulled down by WT/mutant HA-DUX4/IGHs using antibody against HA. Vice versa, the WT/mutant HA-DUX4/IGHs are pulled down by RAG1/2 using antibodies against human RAG1/2. (C) Structure-based mammalian two-hybrid assay. The relative luciferase activities were used to monitor the interaction between WT/mutant DUX4/IGH and RAG1/2. The binding between WT/mutant DUX4/IGHs and RAG1/2 were all normalized against the pACT vector: pBIND-RAG1+ pBIND-RAG2 interaction (i.e., the binding value of the latter was set to 1). All data are shown as mean ± SD. *, P < 0.05. **, P < 0.01. ***, P < 0.001. All experiments had been repeated at least three times. Abbreviations: RAG1/2: Recombination-activating genes 1/2; DUX4/IGH: Double homeobox 4 fused with immunoglobulin heavy chain; PLA: Proximity ligation assay; WT: Wild type; co-IP: Co-immunoprecipitation; SD: Standard deviation
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