Fig 1.
TAL1 gene codes for 6 mRNA isoforms that translate to 2 protein isoforms.
(A) Schematic representation of TAL1 mRNA isoforms. Rectangles: exons, black lines: introns; arrow: transcription initiation site, coordinates corresponds to genome build hg19. (B) Schematic representation of TAL1 protein isoforms; grey rectangles represent the protein domain bHLH. (C) CutLL1, MOLT4, CCRF-CEM, Jurkat, and K562 whole cell lysate was extracted and subjected to western blot analysis using TAL1 antibody recognizing both isoforms and an endogenous control. (D) ChIP-seq tracks for H3K27ac and H3K4me3 at the TAL1 locus in the indicated cell lines (genome build hg19). Underlying data can be found in S1 Raw Images.
Fig 2.
TAL1 enhancers promote the expression of specific isoforms.
(A and B) RNA was extracted from Jurkat NLO cells, which express TAL1 exogenously and from enhancer mutated cells, Jurkat Del-12 and analyzed by real-time PCR for total mRNA amount of TAL1 relative to CycloA and hTBP reference genes (A) and for ΔEx3 relative to endogenous TAL1 total mRNA amount. The PSI represents the amount of mRNA molecules that include exon 3 relative to the total number of mRNA molecules that include or exclude this exon. PSI was calculated according to the formula PSI = ΔCq(inclusion isoform)/ΔCq(total mRNA) (B). Plots represent the mean of 3 independent experiments (**P < 0.01, Student t test). (C and D) RNA was extracted from HEK293T cells and CTCF mutated cells, HEK293T ΔCTCF, and analyzed by real-time PCR for total mRNA amount of TAL1 relative to CycloA and hTBP reference genes (C) and for ΔEx3 relative to TAL1 total mRNA amount. PSI was calculated by ΔEx3 relative to TAL1 total mRNA (D). Plots represent the mean of 3 independent experiments (****P < 0.0001, Student t test). (E-G) HEK293T cells were transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with 4 gRNAs targeted to the TAL1 −60 enhancer or TAL1 exon 3 for 30 h. Total RNA was extracted and analyzed by real-time PCR for total mRNA amount of TAL1 relative to CycloA and hTBP reference genes (E) and for ΔEx3 relative to TAL1 total mRNA amount. PSI was calculated by ΔEx3 relative to TAL1 total mRNA (F). ChIP was performed of H3 pan-acetylated at the −60 enhancer and TAL1 exon 3 (G). Values are expressed as dCas9-p300 core (WT) relative to dCas9-p300 core (mut) and horizontal broken lines indicate no change between dCas9-p300 core (WT) relative to dCas9-p300 core (mut). Plots represent the mean of 3 independent experiments (**P < 0.01, ***P < 0.001, ****P < 0.0001 Student t test). (H) Jurkat cells were transfected with siKMT2B or a negative control siRNA (siGFP), and RNA was extracted 72 h posttransfection. Real-time PCR was performed for ΔEx3 relative to TAL1 total mRNA amount. PSI was calculated by ΔEx3 relative to TAL1 total mRNA. Plots represent the mean of 5 independent experiments (**P < 0.01, Student t test). Underyling data can be found in S1 Data.
Fig 3.
TAL1-short promotes transcription from TSS1-3.
(A) HEK293T cells were cotransfected with TAL1 promoters: promoters 1–3, 4, 5, and exon 4 as a negative control. The second plasmid was an empty vector, TAL1-short or TAL1-long. After 30 h, luciferase activity was calculated relative to renilla. Horizontal broken lines indicate transfection with the empty luciferase. The mean was calculated from 3 independent biological experiments, each performed with 3 technical replicates (**P < 0.01, Student t test). (B) HEK293T cells were cotransfected with TAL1 5′ UTRs: TSS 1, 2, 4, and 5 and empty vector as negative control. After 30 h, luciferase activity was calculated relative to renilla. The mean was calculated from 3 independent biological experiments, each performed with 3 technical replicates (*P < 0.05, Student t test) are shown. Underyling data can be found in S1 Data.
Fig 4.
TAL1-short promotes transcription of apoptotic genes and is a stronger transcription factor compared to TAL1-long. (A-H) Jurkat cells were infected with empty vector, FLAG-TAL1-short or FLAG-TAL1-long. FLAG was immunoprecipitated and known TAL1 interacting proteins were detected with indicated antibodies (A). (B-H) FLAG-TAL1-short, TAL1-long, and empty vector’s chromatin were immunoprecipitated using FLAG antibody. In addition, TAL1-total ChIP-seq data were analyzed (see methods). Heatmap showing signal enrichment over 5,876 TAL1-total binding sites (±1,000 kb from the center). Heatmap is sorted by strength of TAL1-short signal centered on ChIP-seq summits. A color scale indicating the relative signal intensity plotted on each heatmap is shown (B). ChIP-seq average signal for TAL1-total, TAL1-short, and TAL1-long and empty vector as a function of distance from TAL1-total peaks (C). Most abundant DNA sequence motifs were identified in TAL1-total, TAL1-short, and TAL1-long ChIP-seq peaks. Percentage of loci was calculated by dividing % of target sequences with motifs by % of background sequences with the motif for ETS (D), GATA (E), RUNX (F), MAF (G), and E-box (H). (I-L) Jurkat cells were infected with inducible shRNA against the 3′ UTR of TAL1. In addition, the cells were infected with either empty vector, TAL1-short or TAL1-long. Following induction with tetracycline for 72 h, RNA was extracted and subjected to sequencing. In addition, analysis was performed on available data of TAL1-total (see methods). Venn diagram representing the overlap of TAL1-short, TAL1-long, and TAL-total target genes (I). Gene Set Enrichment Analysis (GSEA) for apoptotic pathway for TAL1-short and TAL1-long (J). Cells were seeded and counted every day following labeling by trypan blue. Live cells are plotted in (K); trypan blue-labeled cells are plotted in (L). Plots represents the mean of 3 independent experiments (*P < 0.05, ***P < 0.001, and ****P < 0.0001 Student t test). Underyling data can be found in S1 Data and S1 Raw Images.
Fig 5.
TAL1-short but not TAL1-long leads to hematopoietic stem cell exhaustion.
(A) Schematic illustration of the mixed bone marrow chimera experiments. Equal numbers of bone marrow cells from 5FU-treated CD45.1 wild-type mice were transduced with retroviruses expressing either TAL1-short-GFP or with TAL1-long-dtTomato. A mixture of 1:1 ratio of the transduced bone marrow cells was then transplanted into lethally irradiated CD45.2 wild-type recipient mice. Blood samples were taken at different time points to monitor the progression of the bone marrow cell reconstitution using flow cytometry analysis. All mice were killed 14 weeks after BMT, and the spleen and bone marrow were harvested and analyzed. (B) Representative dot plots of Thy1.2 vs. CD19 staining in untransduced (left), dtTomato+ (middle), or GFP+ (right) splenocytes from the recipient mice (C) Bar graph summarizing results shown in (B). (D) Representative flow cytometry histograms of CD11b staining gated on GFP−/dtTomato− (untransduced, left panel), dtTomato+ (Tal1-long, middle panel), or GFP+ (Tal1-short, right panel) splenocytes. (E) Bar graph summarizing results shown in (D). (F) Representative dot plots of Tal1-short GFP vs. Tal1-long dtTomato staining in the blood of the recipient mice at different time points over the course of 13 weeks. (G) Bar graph summarizing results in (F). Each mouse is presented with a different shade. In (C), two-tailed paired t test, *< 0.05, **< 0.01, sd. (n = 6). Underyling data can be found in S1 Data.
Fig 6.
TAL1-short is a stronger differentiation agent.
(A-D) K562 cells were infected with inducible shRNA against the 3′ UTR of TAL1. In addition, the cells were infected with MIGR1 plasmid with GFP at the C-terminal followed by an IRES element. Infection was with empty vector, TAL1-short, or TAL1-long. Following induction with tetracycline for 72 h, cells were treated with 20 μM hemin to promote erythroid differentiation for the indicated time points. RNA was extracted and analyzed by real-time PCR for total mRNA amount of α-, β-, and γ-globin and SLCA4 relative to CycloA and hTBP reference genes without hemin (A) and with treatment (inducing differentiation) for 48 h (B). Cells were seeded and counted every day following labeling by trypan blue. Live cells are plotted in (C) and trypan blue-labeled cells are plotted in (D). Plots represents the mean of 3 independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, Student t test). Underyling data can be found in S1 Data.