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. 2016 Jun;30(6):1375-87.
doi: 10.1038/leu.2016.27. Epub 2016 Mar 2.

PU.1 Cooperates With IRF4 and IRF8 to Suppress pre-B-cell Leukemia

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Free PMC article

PU.1 Cooperates With IRF4 and IRF8 to Suppress pre-B-cell Leukemia

S H M Pang et al. Leukemia. .
Free PMC article

Abstract

The Ets family transcription factor PU.1 and the interferon regulatory factor (IRF)4 and IRF8 regulate gene expression by binding to composite DNA sequences known as Ets/interferon consensus elements. Although all three factors are expressed from the onset of B-cell development, single deficiency of these factors in B-cell progenitors only mildly impacts on bone marrow B lymphopoiesis. Here we tested whether PU.1 cooperates with IRF factors in regulating early B-cell development. Lack of PU.1 and IRF4 resulted in a partial block in development the pre-B-cell stage. The combined deletion of PU.1 and IRF8 reduced recirculating B-cell numbers. Strikingly, all PU.1/IRF4 and ~50% of PU.1/IRF8 double deficient mice developed pre-B-cell acute lymphoblastic leukemia (B-ALL) associated with reduced expression of the established B-lineage tumor suppressor genes, Ikaros and Spi-B. These genes are directly regulated by PU.1/IRF4/IRF8, and restoration of Ikaros or Spi-B expression inhibited leukemic cell growth. In summary, we demonstrate that PU.1, IRF4 and IRF8 cooperate to regulate early B-cell development and to prevent pre-B-ALL formation.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Overlapping binding sites between PU.1, IRF4 and IRF8. A) Co-localization of IRF4, IRF8 and PU.1 peaks in pro-B cells were determined by multiple overlap analysis of the respective ChIP-seq data. The number of binding sites for each overlap is shown. B) The average density of IRF4− (green), IRF8− (blue) and PU.1-binding (red) in pro-B cells are shown for a 5 kb region centered on the summits of the IRF4 peaks. C) The densities of IRF4−, IRF8− and PU.1-binding in pro-B cells are displayed as a heat map, sorted by position of the highest IRF4 peak density, from upstream to downstream. D) Overlap of IRF4− and PU.1-binding in pro-B cells. The number of binding sites is indicated together with the top-ranked motif identified with the indicated E-value. For IRF4-only binding sites no significant motif could be found. E) Multiple overlap of differentially regulated genes of Irf4, Irf8 and PU.1 single KO pro-B cells. The number of differentially regulated genes is shown for the respective overlap.
Figure 2
Figure 2
Analysis of B cell development in the absence of PU.1 and IRF4. (AB) BM cells were isolated from mice of the indicated genotypes and analyzed for CD19, B220 and IgM expression. Representative flow cytometric plots show the gates used to calculate the number of (A) total CD19+B220+ B cells and (B) CD19+ BM compartments based on B220 and IgM expression used to define pro/pre-B cells immature, transitional and recirculating B cells. (CG) Total cell number of each cell population were calculated from the gating shown in (A) and (B). A simplified genotype nomenclature is shown below the graphs with symbols representing the existence of two (+), one (+/−) or no (−) functional alleles for the indicated genes. The genotypes are presented in the same order as in (A). Data are mean ± SD from 3 to 13 mice per genotype. p values compare the indicated groups using a paired t test (two tailed). * p <0.05, ** p<0.005, *** p<0.0005.
Figure 3
Figure 3
Analysis of the pro- and pre-B cell compartments in the absence of PU.1 and IRF4. BM cells were isolated from mice of the indicated genotypes were analyzed for the frequency of (A) CD19+B220+IgMc-Kit+ pro-B and CD19+B220+IgMc-Kit pre-B cells. (B) Representative flow cytometric plots of CD25 expression on CD19+ cells. Box indicates the location of pre-B cells. (C) ChIP-seq mapping of IRF4, IRF8 and PU.1 binding plus the indicated histone modifications and DNase I hypersensitive sites (DHS) at the regulatory regions of Il2ra (encoding CD25) in pro-B cells. Gray boxes highlight the IRF4 binding peaks. Arrow shows the direction of Il2ra transcription. Bars below the ChIP-seq tracks indicate transcription factor-binding regions identified by MACS peak calling. (DE) Fold change (normalized to the wild-type value set as 1) in the total number of each cell population from each genotype were quantified from the gating shown in (AB). A simplified genotype nomenclature is shown below the graphs with symbols representing the existence of two (+), one (+/−) or no (−) functional alleles for the indicated genes. The full genotypes are presented in the same order as in (A). The data are mean ± SD from 3 to 13 mice per genotype. p values compare the indicated groups using a paired t test (two tailed). * p <0.05.
Figure 4
Figure 4
Enhanced IL-7 dependent proliferation and pre-BCR expression in the absence of PU.1, IRF4 and IRF8. (A) The BM cells of the indicated genotypes were isolated and enriched for B220+ cells. The cells were cultured in the presence of IL-7, and reseeded at the point when the cells of each indicated genotype were confluent. The cumulative frequencies of cells of each genotype were calculated based on the total number of cells on that day and the dilution factor used. (B) Expression levels of IL-7Rα on CD19+B220+IgM+ B cells, and small pre-B and large pre-B cells gated as in (Figure 3A). (C) BM cells of each genotype were isolated and analysed on the pro/pre-B cells (gated as in Figure 3A) for pre-BCR expression levels using an antibody that detects a conformational epitope of the pre-BCR but not with surrogate light chain, λ5, or VpreB in the absence of IgH. A simplified genotype nomenclature is shown below the graphs with symbols representing the existence of two (+), one (+/−) or no (−) functional alleles for the indicated genes. The full genotypes are presented in the same order as in Figure 2A. The data in (A) and (C) are mean ± SD between 3 to 8 mice per genotype. p values compare the indicated groups using a paired t test (two tailed). * p <0.05, ** p<0.005, *** p<0.0005. ns, not significant. The cross symbol indicates the death of the cells growing in culture.
Figure 5
Figure 5
Development of leukemia in mice lacking PU.1, IRF4 and/or IRF8. (AB) Kaplan-Meier survival plot for aging mice of the indicated genotypes. n, indicates the number of mice recorded for each genotype. (C) Spleens from moribund mice of the indicated genotypes. (D) Histological section of the spleens from a moribund mouse of indicated genotype. Enlarged spleens showed infiltration of lymphocytes into the red pulp and destruction of the original organ architecture, when compared to the WT control. Magnification is indicated. Lower magnification images are shown in Supplementary Figure 4.
Figure 6
Figure 6
Development of pre-B acute lymphoblastic leukemia in PU.1, IRF4 or IRF8 compound mutant mice. (A) Representative plots of CD19 and B220 expression from isolated splenocytes of moribund mice (upper panel), and cultured leukemic cells (lower panel) of the indicated genotypes. Data are compared to normal control wild-type splenocytes (WT). (B) Expression of IL-7Rα, IgM and Igκ on the gated CD19+B220 leukemic cells and CD19+B220+ WT B cells from (A). Gray histograms show unstained controls. (C) DNA was extracted from leukemias of indicated genotype and subjected to PCR for Igh VDJ rearrangements with sets of primers recognizing various VH gene families. The amplified samples were run on an agarose gel and the alleles assessed and shown. (D) Leukemic cells of the indicated genotypes were lysed and subjected to western blotting for IgM and Igκ. Actin serves as a control for equal protein loading. WT pre-B cells served as a positive control. Note the Igκ in WT pre-B cells is intracellular. Mouse tumor numbers are indicated. (E) Cultured leukemic cells of the indicated genotypes were lysed and subjected to western blotting for PU.1, IRF4, IRF8 and Pax5. Actin serves as a control for equal protein loading. Symbols in (D) and (E) represent the existence of two (+), one (+/−) or no (−) functional alleles for the indicated genes.
Figure 7
Figure 7
Spib and Ikzf1 are transcriptionally activated by PU.1 and IRF proteins. (AC) RNA samples from healthy pre-B cells (identified as B220+CD19+cKitIgM) and leukemias of the indicated genotypes were subjected to RT-qPCR to measure the relative expression of steady-state mRNA transcripts for Blnk (A), Ikzf1 (B) and Spib (C). Transcript frequencies were normalized to Hprt transcript levels. The data are mean ± SD between 2 to 10 samples per genotype. p values compare the indicated groups using a paired t test (two tailed). Lower panels, ChIP-seq analysis of PU.1, IRF4, and IRF8 binding and the indicated histone modifications and DNase I hypersensitive sites (DHS) at the regulatory regions of Blnk (A), Ikzf1 (B) and Spib (C) in short-term cultured pro-B cells. Gray boxes highlight the PU.1-IRF binding peaks. Arrows indicate the direction of gene transcription. Bars below the ChIP-seq tracks indicate transcription factor-binding regions identified by MACS peak calling. Symbols in (AC) represent the existence of two (+), one (+/−) or no (−) functional alleles for the indicated genes. p values compare the indicated groups using a paired t test (two tailed). * p <0.05, ** p<0.005, *** p<0.0005.
Figure 8
Figure 8
Ectopic expression of Spi-B and Ikaros oppose IL-7 dependent proliferation of pre-B-ALL. Leukemic cells from mice of the indicated genotypes were infected with Ikaros (A) or Spi-B (B)-expressing, or control MIGR1 (GFP) retroviral vectors. The estrogen analogue 4-hydroxytamoxifen (4-OH Tx) (0.5μM) was added at the start of infection to activate the expressed Ikaros-ERT2 fusion protein. Flow cytometry was used to determine the percentage of GFP+ cells over the indicated time course. Lower panel indicates the relative expression of mRNA transcript for Ikzf1 and Spib from infected cells from A and B. Data are the mean ± SD of 3 to 4 independent B-ALL samples per genotype and were normalized for relative frequency of GFP expression at day 1 (set as 1). p values compare the indicated data point to the MIGR1 control using a paired t test (two tailed* p <0.05, ** p<0.005, *** p<0.0005. (C) Gene expression data for IRF4, IRF8, SPIB and SPI1 (PU.1) from B-ALL patient samples and control BM. B-ALL cohorts: 1. Hyperdiploid (n=112); 2. TCF3/PBX1 t(1;19) (n=40); 3. ETV6/RUNX1 t(12;21) (n=100); 4. MLL (n=29), 5. BCR/ABL t(9;22) (n=22); 6. Hypodiploid (n=9); 7. Other B-ALL (n=174); 8. Normal CD19+CD10+ BM pre-B cells (n=4) and 9. Normal CD34+ hematopoietic progenitors (n=4). Note that IRF4 and SPIB are not expressed in early hematopoietic CD34+ progenitors in contrast to CD19+CD10+ pre-B cells of healthy individuals.

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