A distal single nucleotide polymorphism alters long-range regulation of the PU.1 gene in acute myeloid leukemia

Abstract

Targeted disruption of a highly conserved distal enhancer reduces expression of the PU.1 transcription factor by 80% and leads to acute myeloid leukemia (AML) with frequent cytogenetic aberrations in mice. Here we identify a SNP within this element in humans that is more frequent in AML with a complex karyotype, leads to decreased enhancer activity, and reduces PU.1 expression in myeloid progenitors in a development-dependent manner. This SNP inhibits binding of the chromatin-remodeling transcriptional regulator special AT-rich sequence binding protein 1 (SATB1). Overexpression of SATB1 increased PU.1 expression, and siRNA inhibition of SATB1 downregulated PU.1 expression. Targeted disruption of the distal enhancer led to a loss of regulation of PU.1 by SATB1. Interestingly, disruption of SATB1 in mice led to a selective decrease of PU.1 RNA in specific progenitor types (granulocyte-macrophage and megakaryocyte-erythrocyte progenitors) and a similar effect was observed in AML samples harboring this SNP. Thus we have identified a SNP within a distal enhancer that is associated with a subtype of leukemia and exerts a deleterious effect through remote transcriptional dysregulation in specific progenitor subtypes.

Figure 1. Genomic analysis of distal URE of PU.1.

(A) Schematics of genomic locus of human PU.1 gene including its 5 exons (white box) and –16-kb URE consisting of 2 highly conserved homology regions (gray boxes). Localization of the probes for fluorescence in situ hybridization (RP11-17G12 and RP11-379M04) is indicated by lines with filled circles at the ends. The long (q) and short (p) arms of chromosome 11 and the band 11.2 are indicated. (B) Fluorescence in situ hybridization with probe RP11-379M04 covering the URE locus. Left: Metaphase FISH showing 2 signals on each chromosome 11 (arrows). Right: Interphase FISH of 1 representative of 80 patients with 2 signals per cell. (C) Direct sequencing identifies SNP in the first homology region of PU.1 URE. Representative sequencing traces of patients with wild-type site (WT hom), heterozygous site (het), and homozygous SNP (SNP hom) shown. (D) Identification of a SNP in the second homology region of URE. Representative sequencing graphs of patients with wild-type site, heterozygous site, and homozygous SNP shown. (E) Higher abundance of the homozygous SNP in first homology region in patients with AML with complex karyotypes. Bar diagram shows SNP status of normal controls and AML with normal karyotype, with aberrant noncomplex karyotype, and with complex karyotype. *P = 0.027 (χ²) and P = 0.018 (Fisher’s exact); odds ratio, 2.9; odds ratio 95% confidence interval, 1.22–6.83. (F) Frequency of SNP in the second homology region of URE is not different between normal controls or AML.

Figure 2. The SNP in the first homology region of the URE of PU.1 leads to reduced enhancer activity.

(A) Schematics of the reporter constructs utilized for stable transfections of U937 myeloid cells. Top: The proximal promoter of PU.1 in the pXP2 luciferase vector. Middle: The wild-type URE plus the proximal promoter of PU.1. Bottom: The SNP URE plus the proximal promoter of PU.1. The point mutation representing the SNP is indicated by a star. (B and C) Luciferase reporter assays after stable transfection of the above-described constructs into U937 cells shows reduced enhancer activity of the point-mutated URE. (B) The mean luciferase activity of 3 independent clones is displayed. Error bars indicate SD. (C) The mean luciferase activity of 3 independent cell pools is shown. Luciferase activity was normalized to transgene copy number as determined by Southern blotting. Error bars indicate SD. *P < 0.001.

Figure 3. The SNP in the first homology region of the URE of PU.1 diminishes binding of SATB1.

(A) Chromatin immunoprecipitation shows SATB1 binding to the URE in U937 and HL60 cells. The genomic region of the putative SATB1 binding site was PCR amplified after reverse crosslink of the immunoprecipitates. An input control and precipitates utilizing a SATB1 antibody, no antibody, or a nonspecific control antibody are shown. PCR products were verified by sequencing. (B) EMSA utilizing nuclear extracts of U937 cells and gel-purified probes (WT probe, ³²P, and SNP probe, ³²P) covering the SATB1 binding site are shown. The wild-type probe (WT oligo) and a previously described SATB1 binding probe (SATB1 IgH site) were used for competition. A SATB1 antibody was used for supershift. A labeled Sp1 binding probe served as a loading control in 2 lanes. The ³²P-labeled SATB1 IgH site served as positive control. The presence (+) or absence (–) of the respective reagents is indicated for each lane. The probes are shown below the gel.

Figure 4. SATB1 is a URE-dependent positive regulator of PU.1 in myeloid cells.

(A) SATB1-directed siRNA-expressing construct PU.1 mRNA was significantly reduced upon siRNA-mediated knockdown of SATB1 (U937 siSATB1). n = 3. (B) Western blotting shows diminished PU.1 protein level after siRNA-mediated downregulation of SATB1 protein in stably transfected U937 cells. β-actin protein served as control. (C) Lentiviral overexpression of SATB1 in U937 cells leads to increased PU.1 expression. We utilized IRES-GFP-SATB1 lentivirus to transduce U937 cells. GFP⁺ cells were FACS sorted and subjected to mRNA expression analysis. Sorted GFP^– cells and cells infected with empty IRES-GFP lentivirus served as control. Gene expression data normalized to GAPDH. n = 3. (D) SATB1 overexpression in the absence of URE does not lead to PU.1 upregulation. Cells derived from URE^–/– mice were treated with SATB1-expressing lentivirus, GFP⁺ and GFP^– cells FACS sorted, and SATB1 and PU.1 expression measured. While SATB1 expression was significantly increased in IRES-GFP-SATB1–infected cells (SATB1 GFP⁺), there was no upregulation of PU.1 expression in URE^–/– cells. n = 3. (E and F) Lentiviral overexpression of SATB1 in sorted Lin^–Kit⁺ progenitors from wild-type littermates and URE-knockout mice. Lin^–Kit⁺ cells were FACS sorted and infected with empty control virus or IRES-GFP-SATB1 virus. GFP⁺ cells were sorted and subjected to quantitative RT-PCR. (E) Sorted wild-type progenitors. n = 3. (F) Sorted URE^–/– progenitors. n = 3. (G) Neomycin resistance SATB1 siRNA expression construct was stably transfected into URE^–/– cells and SATB1 and PU.1 expression levels determined. An empty construct served as control. Mean ± SD shown.

Figure 5. SATB1- and SNP-dependent downregulation of PU.1 in distinct myeloid progenitor subsets in vivo.

(A) wbc from fetal livers of SATB1-knockout mice were harvested and KLS cells (Lin^–c-kit⁺Sca1⁺), CMPs (Lin^–c-kit⁺Sca1^–CD34^lowF_cγII/IIIR^low), GMPs (Lin^–c-kit⁺Sca1^–CD34⁺F_cγII/IIIR⁺), and MEPs (Lin^–c-kit⁺Sca1^–CD34^–F_cγII/IIIR^–) were separated by multicolor FACS sorting. PU.1 expression was determined by quantitative RT-PCR. GAPDH served as a control. SD is indicated by error bars (n = 3). While there are no significant changes in total wbc, KLS cells, or CMPs, PU.1 expression is markedly decreased in GMPs and MEPs of SATB1^–/– mice in comparison with wild-type littermates. (B) Total BM of WT/WT (n = 16) and SNP/SNP (n = 5) patients was examined by quantitative RT-PCR. GAPDH expression served as a control. Averages and standard deviations (error bars) are shown. (C) Lin^–CD34⁺CD38^–Thy1^low HSCs of WT/WT (n = 8) and SNP/SNP (n = 4) patients were separated by multicolor FACS and PU. 1 expression was determined by quantitative RT-PCR. (D) Lin^–CD34⁺CD38⁺CD123⁺CD45RA⁺ GMPs of WT/WT (n = 7) and SNP/SNP patients (n = 3) were FACS sorted and PU.1 expression was measured by quantitative RT-PCR. Expression of GAPDH was used as a control. (E) Lin^–CD34⁺CD38⁺CD123^–CD45RA^– MEPs of WT/WT (n = 7) and SNP/SNP patients (n = 3) were FACS sorted and PU.1 expression was measured by quantitative RT-PCR. Error bars represent standard deviation. Statistical significance is indicated by asterisks. P < 0.01.