β-Catenin induces T-cell transformation by promoting genomic instability

Abstract

Deregulated activation of β-catenin in cancer has been correlated with genomic instability. During thymocyte development, β-catenin activates transcription in partnership with T-cell-specific transcription factor 1 (Tcf-1). We previously reported that targeted activation of β-catenin in thymocytes (CAT mice) induces lymphomas that depend on recombination activating gene (RAG) and myelocytomatosis oncogene (Myc) activities. Here we show that these lymphomas have recurring Tcra/Myc translocations that resulted from illegitimate RAG recombination events and resembled oncogenic translocations previously described in human T-ALL. We therefore used the CAT animal model to obtain mechanistic insights into the transformation process. ChIP-seq analysis uncovered a link between Tcf-1 and RAG2 showing that the two proteins shared binding sites marked by trimethylated histone-3 lysine-4 (H3K4me3) throughout the genome, including near the translocation sites. Pretransformed CAT thymocytes had increased DNA damage at the translocating loci and showed altered repair of RAG-induced DNA double strand breaks. These cells were able to survive despite DNA damage because activated β-catenin promoted an antiapoptosis gene expression profile. Thus, activated β-catenin promotes genomic instability that leads to T-cell lymphomas as a consequence of altered double strand break repair and increased survival of thymocytes with damaged DNA.

Keywords: Ctnnb1; DNA recombination Tcf7; beta-catenin/Tcf-1.

Fig. 1.

β-Catenin activation causes genomic instability. Representative images of (A) SKY metaphase from a CAT lymphoma with Tcra/Myc translocations (see Table S1 for full karyotypes) and (B) FISH analysis. (C) Cartoon of the translocation as predicted by FISH and the presence of a fusion transcript (Fig. S1B). BACs used for FISH analysis are indicated. Not drawn to scale.

Fig. 2.

Trimethylated Tcf-1 sites predict RAG2 binding. ChIP-seq analyses of WT thymocytes. (A) Similar genome-wide binding patterns of RAG2, Tcf-1, and H3K4me3. Tag density is plotted for a representative area on Chr6. (B) Similarity analysis using Ward’s clustering of chromatin occupancy by Tcf-1 and RAG2 (described in ref. 53). (C) Normalized tag density distribution centered on shared trimethylated Tcf-1 sites. Solid lines, ChIP; dotted lines, control. Lower right shows overlay of ChIP signal for Tcf-1, RAG2, and H3k4me3. (D) Percentage of peaks that RAG2 shares (■) or that are shared with RAG2 (□) for the indicated subsets. (E) Log2 expression of all genes expressed in DP thymocytes or stratified by the presence of Tcf-1, RAG2, and H3K4me3 (me3) at the gene promoter.

Fig. 3.

Trimethylated Tcf-1 sites are preserved in CAT thymocytes. ChIP-seq analyses of WT and CAT thymocytes. (A) Tcf-1 signal at sites that overlap between WT and CAT plotted as a percentage of maximum. (B) Ward’s hierarchical clustering of tag density for similarity analysis as in Fig. 2. me3, H3K4me3. (C) Absolute number of WT and CAT Tcf-1 sites that overlap with H3K4me3 and are shared between WT and CAT (black) and overlap with H3K4me3 but are not shared between WT and CAT (gray) and do not overlap with H3K4me3 (white).

Fig. 4.

CAT thymocytes have increased DNA damage at translocating loci and altered repair of DSBs. (A) Cooccupancy of Tcf-1 and RAG2 at the translocating Myc/Pvt1 locus. (B–D) Three-dimensional FISH of DP thymocytes. (B) Tcra and Myc loci at <1 μm (N_WT = 304, N_CAT = 317). (C) γ-H2AX association on Tcra/Myc pairs either exclusively on Tcra (Upper) or on both alleles (Lower). (D) γ-H2AX association on both loci in the same cell, (D, i) irrespective of pairing status or (D, ii) in Tcra/Myc pairs, as percentage of total cells (D, i) or of cells with ≥1 Tcra/Myc pair (D, ii). Asterisks indicate statistical significance. P values: (D, i) 1.1 × 10⁻³ and (D, ii) 1.9 × 10⁻². nd, none detected. (Scale bars, 1 μm.) (E) Exemplary CJ sequences along germ-line sequence (top) to illustrate P (blue) and N nucleotides (orange), deletions, and insertions identified in the data set (see Table S3 for all sequences). (F) CJ sequencing results from three mice per genotype were pooled and subjected to statistical analysis (Materials and Methods).

Fig. 5.

β-catenin activation does not impair cell cycle checkpoints. (A) Cell cycle profiles of WT and CAT DP thymocytes from mice injected with BrdU 3 h before analysis. (B) Histograms summarize data from two mice per genotype. (C) Western blot analysis of sorted DP cells in G1 or S/G2/M states of cell cycle. Thymi from newborn mice were pooled for this experiment.

Fig. 6.

β-Catenin confers a survival profile upon thymocytes. (A) Heat map depicting average fold change of expression between CAT and WT for the indicated genes in WT and CAT DP thymocytes (biological replicates in columns). (B) β-catenin stabilization increases Bcl-X_L protein levels. Densitometry was performed on Western blot images, and density is expressed relative to β-Actin (Top). Statistical significance was assessed using an unpaired, two-tailed t test (P = 10⁻⁵, n = 6 per group). (C–E) Freshly isolated WT and CAT thymocytes were cultured as follows, and cell viability was assessed by FACS. (C) Spontaneous cell death with DMSO. (D) Specific cell death (i.e., relative to the survival of mock treated cells) upon γ-irradiation and 20 h of culture with ABT263 or DMSO (mock). (E) Specific cell death upon treatment with etoposide, γ-irradiation, or Brefeldin A.