TLX3 directly regulates miR-125b-2 expression in T-ALL cells. (A) TLX3 ChIP-seq analysis performed for 1 newly diagnosed primary T-ALL. Gene tracks represent binding at the MIR-125B-2 locus on chromosome 21. Y-axis values show the number of sequence reads. Positions of 2 major binding peaks are indicated relative to the TSS of the shorter transcript of LINC00478, which overlaps with the MIR-99a/LET7C/MIR125B-2 locus. Two TLX3-binding regions are also enriched in H3K4me1 and H3K4me3, marking promoter/transcriptional initiation sites and H3K27ac marking active regulatory elements in the DND41 cell line (ENCODE). (B) TLX3 ChIP-Q-PCR analysis of DND41 and HPB-ALL TLX3–expressing cell lines and of 6 primary TLX3 T-ALLs. Shown are fold enrichment values at +1.3-kb and +29-kb binding regions of LINC00478 locus relative to the IGX1A ORF-free intergenic region containing no TSS. All ChIP experiments were performed at least twice. (C) Histone H3 acetylation at the +1.3-kb and +29-kb chromatin regions in TLX3⁺ and TLX3⁻ cell lines and T-ALLs assessed by ChIP-Q-PCR analysis. Fold enrichment values relative to the IGX1 locus is shown. (D) Luciferase reporter assay in 293T cells. The empty pGL3 promoter plasmid and nonbinding 980-bp fragment immediately downstream of TLX3 binding “+1.3-kb” region served as negative controls. Relative luminescence units (RLU) were determined by normalizing firefly to Renilla luciferase activity (n = 4). (E-H) Relative expression of 2 LINC00478 transcripts normalized to ABL1 (2^−ΔCt × 100): (E-F) in TLX3⁻ (N = 26) and TLX3⁺ (N = 19) T-ALLs and (G-H) in TLX3/OE and CTRL HPC-derived T cells at day 42 of differentiation. (B-H) Data are presented as mean ± SEM of values: ***P < .001; **P < .01; *P < .05: Mann-Whitney U test. (I) A simplified view of TLX3-miR-125b relations in T-ALL and involvement of miR-125b target genes in the blockage of T-cell development.