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Stress hematopoiesis is regulated by the Krüppel-like transcription factor ZBP-89 - PubMed

Stress hematopoiesis is regulated by the Krüppel-like transcription factor ZBP-89

Xiangen Li et al. Stem Cells. 2014 Mar.

Abstract

Previous studies have shown that ZBP-89 (Zfp148) plays a critical role in erythroid lineage development, with its loss at the embryonic stage causing lethal anemia and thrombocytopenia. Its role in adult hematopoiesis has not been described. We now show that conditional deletion of ZBP-89 in adult mouse hematopoietic stem/progenitor cells (HSPC) causes anemia and thrombocytopenia that are transient in the steady state, but readily uncovered following chemically induced erythro/megakaryopoietic stress. Unexpectedly, stress induced by bone marrow transplantation of ZBP89(-/-) HSPC also resulted in a myeloid-to-B lymphoid lineage switch in bone marrow recipients. The erythroid and myeloid/B lymphoid lineage anomalies in ZBP89(-/-) HSPC are reproduced in vitro in the ZBP-89-silenced multipotent hematopoietic cell line FDCP-Mix A4, and are associated with the upregulation of PU.1 and downregulation of SCL/Tal1 and GATA-1 in ZBP89-deficient cells. Chromatin immunoprecipitation and luciferase reporter assays show that ZBP-89 is a direct repressor of PU.1 and activator of SCL/Tal1 and GATA-1. These data identify an important role for ZBP-89 in regulating stress hematopoiesis in adult mouse bone marrow.

Keywords: Erythroid progenitors; Hematopoietic stem cells; Stress hematopoiesis; Transcription factors; Transplantation.

© 2013 AlphaMed Press.

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Conflict of interest statement

Disclosure of potential conflicts of interest. The authors indicate no potential conflicts of interest.

Figures

Figure 1
Figure 1. Generation and PB phenotype of ZBP-89 CKO mice

(A) Strategy for inducible inactivation of ZBP89 in HSPC. Schematic of targeted ZBP-89 exons 8 and 9 (in white), with non-coding region of exon 9 in gray. Restriction sites (P, PstI; E, EcoR1; B, BamH1; H, HindIII; X, Xba1), LoxP sites (open arrows) and FRT sites (closed arrows) are indicated. F1, F2, R1 and R2 represent approximate position and orientation of the primers used in PCR. TK, thymidine kinase; NEO, neomycin. Sizes of the left (3.1kb) and right (5.6kb) vector arms are shown. (B) PCR genotyping showing deletion of the floxed segment of ZBP-89 in single CFU-GM stem cell colonies 8 weeks after pIpC treatment. Lanes 1–4, BM colonies from control (ZBP-89fl/fl-MxCre−) mice, and lanes 5–8 are colonies from ZBP-89 CKO (ZBP-89−/−-MxCre+) mice post pIpC. IL2 is included as internal control. (C) PB white blood cells (WBC)-, RBC-, and platelet counts and hemoglobin (Hgb) level 1–8 weeks after pIpC in (C) PB white blood cells (WBC)-, RBC-, and platelet counts and hemoglobin (Hgb) level 1–8 weeks after pIpC in six control mice (filled diamonds, dotted lines) and in seven ZBP-89 CKO mice (open squares, solid lines). Results are shown as mean ± SD, from 2 independent experiments. *P < 0.05, **P < 0.01. (D) CD41+, CD11b+, CD3+ and B220+ cells in PB from normal (open bars) and ZBP89 CKO mice (black bars) 3 weeks after pIpC injections (mean ± SD, n=6 in each group).

Figure 2
Figure 2. Transcription factor profiles of BM HSPC from ZBP-89 CKO and control mice 3 weeks after the last dose of pIpC

(A) Histograms (mean ± SD, n=3) showing the percentage of LT-HSC and MPP, CLP, CMP, GMP and MEP in fractionated BM cells from ZBP-89 CKO and in control mice. (B) RT-PCR analysis of transcription factors in BM progenitors derived from ZBP-89 CKO and control mice (bars are colored as in A). Results are from two independent experiments, each representing pooled samples from two mice in each group. * P<0.05; ** P<0.01.

Figure 3
Figure 3. Effect of ZBP-89 CKO on stress erythropoiesis and thrombopoiesis

(A) Histograms (mean ± SD, n=4) showing the effect of PHZ-induced hemolysis on RBC and platelet (PLT) counts in PB of ZBP-89 CKO mice (white bars here and in C, D) compared to control mice (black bars here and in C, D). (B) Representative FACS analyses of BM proerythroblasts (CD71highTer119low) and basophilic erythroblasts (CD71highTer119high) (left) and in basophilic- and in polychromatic (CD71lowTer119high) erythroblasts in spleen (right) of control and ZBP-89 CKO mice, two weeks after the last pIpC injection and 5 days after PHZ. Numbers for each box reflect percentages of the gated cells, with each representing the mean value from four mice in each group. (C, D) Histograms (mean ± SD, n=4) showing the effect of 5-fluorouracil (5-FU)-induced platelet depletion on circulating platelet-, white blood cells (WBC)- and RBC counts (C), and on surface phenotype, analyzed by FACS from ZBP-89 CKO and control mice, two weeks after the last pIpC treatment and 6 days after 5-FU (D). * P<0.05.

Figure 4
Figure 4

PB cell counts and percentage of immature hematopoietic lineages in 1° BM transplant recipients. (A) Schematic of the experimental design: BM cells (CD45.2+) from ZBP-89 CKO and control mice were mixed with wild type (WT, CD45.1+) BM cells at a 1:1 ratio and the mixture injected into irradiated wild-type (WT) recipients (CD45.1+). pIpC injections started 6 weeks later and over a 2-week period. PB samples were analyzed at 3–32 weeks after the last pIpC dose and BM was examined at 38 weeks. (B) Isolation of lineage-specific CD45.2+CD45.1− PB cells from control recipients of 1° BM at 3 weeks post pIpC. (C) Percentage of PB megakaryocytes (CD41+), myeloid- (CD11b+), T- (CD3+) and B (B220+)-cells in the CD45.2+ population of ZBP-89−/− and control (ZBP-89fl/fl) mice at the indicated times after pIpC injections. (D) Histograms showing percentage of the different immature hematopoietic lineages in the CD45.2+ BM population of ZBP-89−/− (mean ± SD, n=7) and control mice (mean ± SD, n=8) 38 weeks after pIpC treatment. (E) Histograms (mean ± SD, n=4) showing the percentage of Pre-Pro-B cells, Pro-B cells and Pre-B cells in the CD45.2+ BM population of ZBP-89−/− and control mice 38 weeks after pIpC treatment. * P<0.05, **P<0.01.

Figure 5
Figure 5. Hematopoietic lineages and their transcriptional profiles in 2° BM transplant recipients and in ZBP-89-silenced A4 cells

(A) Percentages of the different HSPC lineages in CD45.2+ PB at different times after 2° BM transplantation. Results given are mean ± SD (n=5 mice in each group). (B, C) Histograms (mean ± SD, n=4) showing the percentages of the different BM progenitors in CD45.2+ BM from 2nd BMT recipients at 30 weeks post transplantation from control (black bars) and CKO mice (white bars). * P<0.05, ** P<0.01. (D) Histograms (mean ± SD, n=2 independent experiments) showing gene expression profiles in BM progenitors from ZBP-89-deficient cells 36 weeks after 2° BMT (white bars) relative to that in control mice (black bars). For each experiment, RNAs from two mice in each group were pooled for RT-PCR. * P<0.05; ** P<0.01. (E) Histograms (mean ± SD, n=2 experiments) showing gene expression profiles in control and ZBP-89-silenced A4 progenitors. * P<0.05.

Figure 6
Figure 6. ZBP-89 is a transcriptional regulator of GATA1, PU.1 and SCL

(A) Schematic of a genomic segment containing mouse GATA1 locus, with two cell-type specific first exons (IT and IE), five coding exons (II–VI), and the G1HE region. The other cis-regulatory elements (double GATA, CACCC and GATA repeats) are not shown. Lower panel, ChIP assays showing specific binding of ZBP-89 to the 5′ region of the GATA1 gene hematopoietic enhancer (G1HE)(which allows GATA1 expression in erythroid lineage). (B) Wt and mutant G1HE (G1HE-(124–235)-luc in which one of the G5 string comprising the ZBP-89 core binding motif is deleted) reporter constructs used (see methods). (C) Histograms (mean±SD, n=2) showing luciferase (Luc) activity in ZBP-89-transfected QM7 cells driven by wild-type (WT) or mutant G1HE (where the ZBP-89 site is mutated). Background luciferase activity was obtained using the control (C) promoter-less vector pGL2. (D) Schematic of a genomic segment containing the nontranslated region of PU.1, the minimal promoter (white bar) and the five coding exons (in black). Transcription start site (+1) and direction (horizontal arrow) are shown. Lower panel, ChIP assays showing specific interaction of ZBP-89 with two PCR products in the −15/−14 URE of PU.1. Normal IgG, used as negative control, and input DNA used as a positive control. (E) pXP-214 kb/-0.334 kb/Luc plasmid containing the 3.5kb −15/−14 URE (in black) cloned upstream of the PU.1 minimal 0.5kb promoter (in white) driving Luc reporter (in gray). Position and sequence of the three predicted ZBP-89 binding sites (core motif capitalized) in −15/14 URE are shown. (F) Histograms (mean±SD, n=2) showing Luc activity in WT and ZBP-89-silenced MEL cells driven by −15/14 URE. In C, F, I, relative units represent Luc activity normalized against β-Gal values. (G) Schematic of a genomic segment containing mouse SCL, comprising 7 exons, with noncoding exons in white. Lower panel, ChIP assays showing specific interaction of ZBP-89 with 1a or 1b promoter regions. (H) Schematic of the construct containing promoters la and lb of SCL cloned upstream of Luc in SCL-pGL2 vector. The two predicted ZBP-89 binding sites are shown. Position and nature of substitution of the two central pyrimidines in the ZBP-89 binding motif of la and lb are indicated above and below the respective sequence. Exons 1a and 1b are shown as white boxes. (I) Histograms (mean ± SD, n=2) showing Luc activity driven by −2kb to +1 DNA region of WT SCL or by SCL in which ZBP-89 consensus-binding sites in promoter 1a (SCL-1a) or 1a+1b (SCL-1a/b) were mutated. Background luciferase activity was obtained using the control promoter-less vector pGL2.

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