Figure 6.
PML/RARα transactivates GFI1 through chromatin conformation at the super-enhancer. (A) Chromatin conformation of the PML/RARα-bound super-enhancer region on the GFI1 locus in NB4 cells. Genome browser tracks (top-left panel) show 3 PML/RARα binding peaks corresponding to the promoter (assigned by peak 1), the intronic enhancer (peak 2), and the distal enhancer (peak 3) at the super-enhancer region of GFI1, with black bars indicating the cutting sites of HindIII. The interactions among 3 peaks were determined by the 3C-PCR experiments, with results shown in bottom-left panels and the schematic illustration in bottom-right panels. The anchor primers (ie, H1 and H9) are indicated by red bars. The positive and negative interactions between the anchor primer and the paired 3C primer are indicated by green and black bars, respectively. The curve line represents the interaction between the indicated peaks. Also shown are the sequencing results of the cut and religated sequences of the 3C-PCR products (top-right panel), confirming the interactions between peak 1 (H9) and peak 2 (H7), peak 2 (H7) and peak 3 (H1), and peak 1 (H9) and peak 3 (H1). (B) PML/RARα determined the interaction between the intronic enhancer and the promoter of GFI1. The interactions between each pair of 3 cis elements were determined by 3C-PCR with and without PML/RARα protein induced in the PML/RARα-inducible cell line (U937-PR9). (C) PML/RARα-mediated chromatin conformation resulted in the activation of GFI1. The trans activity was determined by the dual luciferase reporter assays in NB4 cells. Peak 1 represents the promoter and peak 2 represents the intronic enhancer as illustrated in Figure 6A. **P < .001. (D-E) PML/RARα oligomerization was required for the chromatin conformation regulation at the GFI1 locus and the subsequent activation of GFI1. The interactions between peaks at the GFI1 locus were examined by 3C-PCR (D) and relative expression of GFI1 detected by reverse transcription (RT)-qPCR (E) in U937 cells transfected with wild-type PML/RARα or PML/RARα mutant (PML/RARα-F158E) for which oligomerization was impaired due to the mutation in the RBCC domain. **P < .001. (F) Schematic illustration of the CRISPR-dCas9-KRAB-mediated interruption of PML/RARα binding and regulation on the super-enhancer of GFI1. sg-GFI1e, targeting the PML/RARα-enriched loci on the GFI1 intronic enhancer (peak 2), was transduced into dCas9-stably expressing NB4 cells. The right panel shows the decrease of PML/RARα binding on the super-enhancer of GFI1 determined by ChIP-qPCR. *P < .01. (G) Direct interruption of PML/RARα binding and regulation on the enhancer region of GFI1 decreased GFI1 expression (left panel) and subsequently induced granulocytic differentiation of NB4 cells (right panel). Relative expression of GFI1 was detected by quantitative reverse transcription qPCR. The percentage of CD11b-positive cells was detected 3 days after sgRNA transduction and determined by flow cytometry. Data represent the mean of 3 replicates ± SD. **P < .001. (H) Direct interruption of PML/RARα binding and regulation on the enhancer region of GFI1 interfered with the occurrence of APL in vivo. The sgRNA-transduced dCas9-expressing NB4 cells targeting GFI1 intronic enhancer were injected into NOD/SCID mice. The Kaplan-Meier analysis was used to estimate the survival of mice. Statistical significance was calculated by the log-rank (Mantel-Cox) test. **P < .001.

PML/RARα transactivates GFI1 through chromatin conformation at the super-enhancer. (A) Chromatin conformation of the PML/RARα-bound super-enhancer region on the GFI1 locus in NB4 cells. Genome browser tracks (top-left panel) show 3 PML/RARα binding peaks corresponding to the promoter (assigned by peak 1), the intronic enhancer (peak 2), and the distal enhancer (peak 3) at the super-enhancer region of GFI1, with black bars indicating the cutting sites of HindIII. The interactions among 3 peaks were determined by the 3C-PCR experiments, with results shown in bottom-left panels and the schematic illustration in bottom-right panels. The anchor primers (ie, H1 and H9) are indicated by red bars. The positive and negative interactions between the anchor primer and the paired 3C primer are indicated by green and black bars, respectively. The curve line represents the interaction between the indicated peaks. Also shown are the sequencing results of the cut and religated sequences of the 3C-PCR products (top-right panel), confirming the interactions between peak 1 (H9) and peak 2 (H7), peak 2 (H7) and peak 3 (H1), and peak 1 (H9) and peak 3 (H1). (B) PML/RARα determined the interaction between the intronic enhancer and the promoter of GFI1. The interactions between each pair of 3 cis elements were determined by 3C-PCR with and without PML/RARα protein induced in the PML/RARα-inducible cell line (U937-PR9). (C) PML/RARα-mediated chromatin conformation resulted in the activation of GFI1. The trans activity was determined by the dual luciferase reporter assays in NB4 cells. Peak 1 represents the promoter and peak 2 represents the intronic enhancer as illustrated in Figure 6A. **P < .001. (D-E) PML/RARα oligomerization was required for the chromatin conformation regulation at the GFI1 locus and the subsequent activation of GFI1. The interactions between peaks at the GFI1 locus were examined by 3C-PCR (D) and relative expression of GFI1 detected by reverse transcription (RT)-qPCR (E) in U937 cells transfected with wild-type PML/RARα or PML/RARα mutant (PML/RARα-F158E) for which oligomerization was impaired due to the mutation in the RBCC domain. **P < .001. (F) Schematic illustration of the CRISPR-dCas9-KRAB-mediated interruption of PML/RARα binding and regulation on the super-enhancer of GFI1. sg-GFI1e, targeting the PML/RARα-enriched loci on the GFI1 intronic enhancer (peak 2), was transduced into dCas9-stably expressing NB4 cells. The right panel shows the decrease of PML/RARα binding on the super-enhancer of GFI1 determined by ChIP-qPCR. *P < .01. (G) Direct interruption of PML/RARα binding and regulation on the enhancer region of GFI1 decreased GFI1 expression (left panel) and subsequently induced granulocytic differentiation of NB4 cells (right panel). Relative expression of GFI1 was detected by quantitative reverse transcription qPCR. The percentage of CD11b-positive cells was detected 3 days after sgRNA transduction and determined by flow cytometry. Data represent the mean of 3 replicates ± SD. **P < .001. (H) Direct interruption of PML/RARα binding and regulation on the enhancer region of GFI1 interfered with the occurrence of APL in vivo. The sgRNA-transduced dCas9-expressing NB4 cells targeting GFI1 intronic enhancer were injected into NOD/SCID mice. The Kaplan-Meier analysis was used to estimate the survival of mice. Statistical significance was calculated by the log-rank (Mantel-Cox) test. **P < .001.

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