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. 2012 Aug 14;22(2):209-21.
doi: 10.1016/j.ccr.2012.06.007.

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

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Free PMC article

Core Transcriptional Regulatory Circuit Controlled by the TAL1 Complex in Human T Cell Acute Lymphoblastic Leukemia

Takaomi Sanda et al. Cancer Cell. .
Free PMC article

Abstract

The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3, and RUNX1. We show that TAL1 forms a positive interconnected autoregulatory loop with GATA3 and RUNX1 and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.

Figures

Figure 1
Figure 1. TAL1 occupancy is highly consistent across T-ALL cell lines and primagraft samples
(A) Gene tracks represent binding of TAL1 in T-ALL primagrafts (Prima 2 and Prima 5) and cell lines (CCRF-CEM and Jurkat) at the CD69, TCRA and NKX3-1 loci. The x-axis indicates the linear sequence of genomic DNA, and the y-axis the total number of mapped reads. The black horizontal bar above each gene example indicates the genomic scale in kilobases (kb). Black boxes in the gene map represent exons, and arrows indicate the location and direction of the transcriptional start site. Arrowheads denote regions bound by TAL1. (B) Pairwise comparison of the TAL1-bound regions found in T-ALL primagrafts and cell lines. Fractions of the top 200 TAL1-bound regions in each cell type (rows) that were occupied by TAL1 in the other cell types (columns) are shown as a matrix. Regions occupied by the unrelated transcription factor NRSF in Jurkat served as a negative control. (C) Distances from the center of the TAL1-bound region (top 200) in Prima 5 to the center of the nearest bound region of the indicated transcription factors were determined and grouped into bins (x-axis); the heights of bars represent the sum of the bound regions in each bin (y-axis). (D) Each region bound by TAL1 was mapped to the closest Refseq gene: distal (blue) and proximal (red) promoters, exon (green), intron (violet) and intergenic regions (light blue) more than 10 kb from the gene. (E) The representative position weight matrix for each motif enriched in TAL1 by ChIP-seq and the range of E-values found across different T-ALL cell types are shown. See also Figure S1 and Table S1.
Figure 2
Figure 2. TAL1 co-occupies genomic sites with HEB, E2A, LMO1/2, GATA3 and RUNX1 in T-ALL cells
(A) Gene tracks represent binding of TAL1, HEB, E2A, LMO1 or LMO2, GATA3 and RUNX1 at the TCRA locus in human T-ALL cell lines. See Figure 1A legend for details. (B) Pairwise comparison of transcription factor-bound regions in Jurkat cells. Fraction of the top 200 bound regions for each transcription factor (rows) co-occupied by the all bound regions of the other transcription factors (columns) are shown as a matrix. (C) Distributions of TAL1, HEB, E2A, LMO1, GATA3 and RUNX1 in regions bound by these transcription factors in Jurkat cells: Group 1 (bound by TAL1, HEB, E2A, LMO1, GATA3 or RUNX1), Group 2 (bound by GATA3 alone) or Group 3 (bound by RUNX1 alone). For each region (y-axis), the sequence density centered at 0 indicates overlapping bound regions. (D) DREME-based motif analysis of the regions shown in Figure 2C. The results are presented as position-weight matrices with the E-value significance and the name of the motif family within searched sequences. See also Figure S2 and Tables S2–4.
Figure 3
Figure 3. TAL1, GATA3 and RUNX1 form a positive interconnected auto-regulatory loop
(A) Gene tracks represent binding of TAL1, GATA3, RUNX1 and CBP at the TAL1 (right), RUNX1 (middle) and GATA3 (left) loci in Jurkat cells. See Figure 1A legend for details. (B) Co-occupancy by TAL1, GATA3 and RUNX1 at the TAL1, RUNX1 and GATA3 gene loci in multiple T-ALL cells. Enrichment of regions indicated in panel A (TAL1 enhancer, RUNX1 +23 and GATA3 +280) in four T-ALL cell samples (Jurkat, CCRF-CEM, Prima 2 and Prima 5) was analyzed by ChIP-PCR. The NANOG promoter was used as a negative control. The error bars represent the standard deviation (SD) of the fold enrichment. The red line represents the two-fold enrichment detection for the negative control. (C) mRNA (left) and protein (right) levels of TAL1, GATA3 and RUNX1 after knockdown (KD) of each of these factors in Jurkat cells. The data are means ± SD of duplicate experiments; *p<0.05, **p<0.01 and ***p<0.001 by two sample, two-tailed t-test. (D) Growth inhibition and apoptosis induction after knockdown of TAL1, GATA3 and RUNX1 in Jurkat cells. Cell viability was measured after 3 and 7 days of lentivirus infection. The growth rate (day7/day3) is reported as means ± SD percentage of that for control shRNAs (GFP and Luc) in triplicate experiments. Apoptosis was analyzed at day 4 after lentiviral infection by flow cytometric analysis of cells stained with AnnexinV-FITC. The values are means ± SD of duplicate experiments. Asterisks denote p-values as described for panel C. (E) Positive interconnected auto-regulatory loop formed by TAL1, GATA3 and RUNX1. Genes are presented as rectangles and proteins as ovals. See also Figure S3 and Table S5.
Figure 4
Figure 4. Expression of high-confidence TAL1 target genes classify T-ALL subtypes
(A) Gene set enrichment analysis (GSEA) to determine the correlation of DNA binding with gene expression change upon knockdown (KD) of TAL1. GSEA plot indicates the degree to which TAL1 targets are overrepresented at the extreme left (downregulated by knockdown) or right (upregulated by knockdown) of the entire ranked list. Solid bars represent bound genes. (B) Heatmap images representing the relative expression levels of high-confidence TAL1 targets in Jurkat cells with or without knockdown of TAL1. Two independent shRNAs targeting TAL1 as well as two control shRNAs (GFP and Luc) were transduced in Jurkat cells. Each row corresponds to a gene and is normalized across the row. (C) TAL1 binding to high-confidence target genes (n=302) in four T-ALL cell samples (Jurkat, CCRF-CEM, Prima 2 and Prima 5). The percentages of high-confidence TAL1 targets bound in Jurkat only (n=1) or multiple T-ALL samples (n=2–4) are indicated. (D) Gene expression changes of TAL1 targets upon TAL1 knockdown in CCRF-CEM and RPMI-8402 cells. Relative expression values (TAL1, TOX, STAT5A, ALDH1A2, NKX3-1, ARID5B, ETV6 and MYB) compared to GAPDH were calculated, normalized for each row and shown as a heatmap. For all genes, the changes were significant at p<0.05 by two sample, two-tailed t-test. (E) Principal component analysis of 75 primary T-ALL samples and seven bone marrow (BM) samples based on the expression profile of high-confidence TAL1 target genes. Primary T-ALL samples were classified into three groups (TAL/LMO, TLX or HOXA) based on the genetic alterations reported in the original article (Homminga et al., 2011). The analysis was performed with a set of 238 genes that are bound by TAL1 and significantly downregulated after TAL1 knockdown (see Figure 4B, left). See also Figure S4, Tables S6 and S7.
Figure 5
Figure 5. TAL1 positively regulates target genes with GATA3 and RUNX1 in Jurkat cells
(A, B) GSEA to determine the correlation of DNA binding with gene expression changes upon knockdown (KD) of GATA3 or RUNX1, respectively. See Figure 4A legend for details. (C) Heatmap images representing the relative expression levels of high-confidence TAL1 targets in Jurkat cells with or without knockdown of TAL1 (left), GATA3 (middle) or RUNX1 (right). See Figure 4B legend for details. (D, E) GSEA of expression changes of high-confidence TAL1 targets upon knockdown of GATA3 or RUNX1, respectively. TAL1 target genes (n=238) that were significantly downregulated by TAL1 knockdown (Figure 4B, left) were used as a gene set.
Figure 6
Figure 6. TAL1 positively regulates the MYB oncogene, which coordinately regulates TAL1 target genes
(A) Gene tracks represent binding of TAL1, HEB, E2A, LMO1, GATA3, RUNX1 and CBP at the MYB gene locus in Jurkat cells. See Figure 1A legend for details. (B) Co-occupancy by the TAL1 complex at the MYB gene in multiple TALL cell samples by ChIP-PCR. See Figure 3B legend for details. (C) mRNA (top) and protein (bottom) levels of MYB after knockdown (KD) of TAL1, GATA3 and RUNX1 in Jurkat cells. The data are means ± SD of duplicate experiments; *p<0.05 and **p<0.01 by two sample, two-tailed t-test. (D) GSEA of expression changes of high-confidence TAL1 targets upon MYB knockdown. TAL1 target genes (n=238) that were significantly downregulated by TAL1 knockdown were used as a gene set. See Figure 4A legend for details. (E) Positive feed-forward loop formed by the TAL1 complex and MYB that controls the gene expression program of T-ALL cells. MYB is bound and activated by the TAL1 complex and, in turn, regulates the same set of genes. See also Figure S5.
Figure 7
Figure 7. TAL1 positively regulates the expression of a specific subset of genes that are negatively regulated by HEB and E2A
(A, B) GSEA to determine the correlation of DNA binding with gene expression changes upon knockdown (KD) of HEB or E2A, respectively. See Figure 4A legend for details. (C, D) GSEA of expression changes of high-confidence TAL1 targets upon knockdown of HEB or E2A, respectively. TAL1 target genes (n=238) that were significantly downregulated by TAL1 knockdown (Figure 4B, left) were used as a gene set. (E) Heatmap image representing expression levels of the responsive TAL1 targets in Jurkat cells upon knockdown of TAL1, HEB or E2A. Relative gene expression levels normalized for each gene are illustrated. See also Figure S6 and Table S8.
Figure 8
Figure 8. TRIB2 gene is required for the survival of T-ALL cells
(A) Gene tracks represent binding of TAL1, HEB, E2A, LMO1, GATA3, RUNX1 and CBP at the TRIB2 gene locus in Jurkat cells. See Figure 1A legend for details. (B) Comparison of mRNA expression of TRIB2 gene in three TAL1-positive T-ALL cell lines transduced with shRNAs targeting TAL1, E2A or control shRNAs and analyzed by qRT-PCR. Mean fold-changes (knockdown/controls, log2) are shown. (C) shRNA screen with 12,500 inducible shRNAs that target 1,050 genes, performed on two TAL1-positive T-ALL cell lines (Jurkat and CCRF-CEM). Depletion of TRIB2 shRNAs from the cell population was calculated as uninduced/induced, log2, and shown as the mean ± standard error of the mean of four independent experiments. (D) Growth inhibition by TRIB2 knockdown in Jurkat cells. Cell viability was measured after 3, 5, 7 and 9 days of lentivirus infection with control (GFP) and TRIB2 shRNA. The growth rate (fold-change) compared to day 3 is indicated. Values are means ± SD of triplicate experiments. (E) cDNA containing the wild-type TRIB2 coding region was transduced by retroviral infection of Jurkat cells, followed by lentivirus-mediated transduction of infected cells with control GFP or TRIB2 shRNA. The growth rate (day7/day3) after lentivirus infection was assessed for TRIB2 shRNA relative to GFP shRNA and is shown as the means ± SD of triplicate experiments; ** p<0.01 and *** p<0.001 by two sample, two-tailed t-test. (F) Growth rate (day 7/day 3) was assessed for each TRIB2 shRNA relative to control GFP shRNA in each cell line (Jurkat, RPMI-8402, PF-382 or MOLT-4) and is reported as the means ± SD of triplicate experiments. (G) Apoptosis was measured in four T–ALL cell lines after 4 days of lentiviral infection by flow cytometric analysis of cells stained with AnnexinV-FITC. The values are means ± SD of triplicate experiments. (H) Model of differential regulation of E-protein (HEB and E2A) targets in normal versus malignant T-cells. TRIB2 that is repressed by HEB and E2A in normal cells (left) is upregulated by the TAL1 complex in T-ALL (right). See also Figure S7 and Table S9.

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