Figure 1.
IKAROS binds to the promoter of the BCL2L1 (BCL-XL) gene and suppresses BCL-XL expression. (A) IKAROS binding sites were identified by ChIP-seq in the promoter of BCL2L1 (BCL-XL) in the Nalm6 B-ALL cell line (left) and in a B-ALL patient sample (right). The ChIP-seq data for Nalm6 is analyzed with reference genome MRCh38; the custom tracks are shown on UCSC Genome Browser. The ChIP-seq data for patient 9 is analyzed with reference genome MRCh37 and the custom tracks are shown on CisGenome Browser. (B) The qChIP data confirming IKAROS binding at the BCL2L1 promoter in primary B-ALL cells with wild-type IKZF1 (patients 2-4) but not in IKZF1 haploinsufficiency (patient 1). ChIP enrichments are normalized to input. (C) Activity of the BCL2L1 promoter (located at −1 to −700 bp upstream of transcription start site) was assessed by luciferase reporter assay following transfection with the indicated amount of IKZF1 plasmids (pcDNA-IK) or control vector in 293T cells. (D) IKAROS effect on BCL2L1 promoter activity was assessed by luciferase reporter assay using different regions of the promoter. (E) Nalm6 and 697 B-ALL cell lines were transduced to express IKZF1 (Mig-IK) or with empty vector (MIG-CTL). Relative expression of IKZF1 (left) and BCL-XL (right) assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) is graphed. BCL-XL protein levels in Nalm6 and 697 cells were accessed by western blot with anti-BCL-XL specific antibodies (bottom). (F) Nalm6 and 697 B-ALL cell lines were treated with IKZF1 shRNA (shIK) or scramble shRNA control (shCTL). The relative expressions of IKZF1 (left) and BCL2L1 (right) assessed by qRT-PCR are graphed. BCL-XL protein levels in Nalm6 and 697 cells were accessed by western blot with anti-BCL-XL specific antibodies (bottom). (E-F), cells were treated for 3 days. Graphed in panel B is the mean ± SD of 3 independent experiments. Graphed in panels C-F is the mean ± SD of triplicates representative of 1 of 3 independent experiments. **P < .01, ****P < .0001. ns, not significant; pcDNA, plasmid cytomegalovirus DNA; SD, standard deviation.

IKAROS binds to the promoter of the BCL2L1 (BCL-XL) gene and suppresses BCL-XL expression. (A) IKAROS binding sites were identified by ChIP-seq in the promoter of BCL2L1 (BCL-XL) in the Nalm6 B-ALL cell line (left) and in a B-ALL patient sample (right). The ChIP-seq data for Nalm6 is analyzed with reference genome MRCh38; the custom tracks are shown on UCSC Genome Browser. The ChIP-seq data for patient 9 is analyzed with reference genome MRCh37 and the custom tracks are shown on CisGenome Browser. (B) The qChIP data confirming IKAROS binding at the BCL2L1 promoter in primary B-ALL cells with wild-type IKZF1 (patients 2-4) but not in IKZF1 haploinsufficiency (patient 1). ChIP enrichments are normalized to input. (C) Activity of the BCL2L1 promoter (located at −1 to −700 bp upstream of transcription start site) was assessed by luciferase reporter assay following transfection with the indicated amount of IKZF1 plasmids (pcDNA-IK) or control vector in 293T cells. (D) IKAROS effect on BCL2L1 promoter activity was assessed by luciferase reporter assay using different regions of the promoter. (E) Nalm6 and 697 B-ALL cell lines were transduced to express IKZF1 (Mig-IK) or with empty vector (MIG-CTL). Relative expression of IKZF1 (left) and BCL-XL (right) assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) is graphed. BCL-XL protein levels in Nalm6 and 697 cells were accessed by western blot with anti-BCL-XL specific antibodies (bottom). (F) Nalm6 and 697 B-ALL cell lines were treated with IKZF1 shRNA (shIK) or scramble shRNA control (shCTL). The relative expressions of IKZF1 (left) and BCL2L1 (right) assessed by qRT-PCR are graphed. BCL-XL protein levels in Nalm6 and 697 cells were accessed by western blot with anti-BCL-XL specific antibodies (bottom). (E-F), cells were treated for 3 days. Graphed in panel B is the mean ± SD of 3 independent experiments. Graphed in panels C-F is the mean ± SD of triplicates representative of 1 of 3 independent experiments. **P < .01, ****P < .0001. ns, not significant; pcDNA, plasmid cytomegalovirus DNA; SD, standard deviation.

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