Prep1 (pKnox1) Regulates Mouse Embryonic HSC Cycling and Self-Renewal Affecting the Stat1-Sca1 IFN-Dependent Pathway

<div><p>A hypomorphic <i>Prep1</i> mutation results in embryonic lethality at late gestation with a pleiotropic embryonic phenotype that includes defects in all hematopoietic lineages. Reduced functionality of the hematopoietic stem cells (HSCs) compartment might be responsible for the hematopoietic phenotype observed at mid-gestation. In this paper we demonstrate that Prep1 regulates the number of HSCs in fetal livers (FLs), their clonogenic potential and their ability to <i>de novo</i> generate the hematopoietic system in ablated hosts. Furthermore, we show that Prep1 controls the self-renewal ability of the FL HSC compartment as demonstrated by serial transplantation experiments. The premature exhaustion of Prep1 mutant HSCs correlates with the reduced quiescent stem cell pool thus suggesting that Prep1 regulates the self-renewal ability by controlling the quiescence/proliferation balance. Finally, we show that in FL HSCs Prep1 absence induces the interferon signaling pathway leading to premature cycling and exhaustion of fetal HSCs.</p></div

In vitro characterization of <i>Prep1^i/i</i> and WT leukemic cells.

<p>(<b>A</b>) FACS plots show c-Kit expression on GFP-positive cells in leukemic WT or <i>Prep1^i/i</i> BM cells (right side). Bar graph represents the percent of c-Kit positive cells on GFP-positive population on BM cells from different BMTs (left side). Error bars indicate SD. *p-value <0.001. (<b>B</b>) FACS profiles show c-Kit and Gr-1 expressions on BM from mice with AML induced by transplantation of <i>Prep1^i/i</i> or WT cells overexpressing <i>Meis1-HoxA9</i> (left side). Bar graph shows the percent of cells co-expressing c-Kit and GR-1 on BM from serially transplanted mice. Error bars represent SD. *p-value <0.001. The number of the mice analyzed for each group is shown on the graph.</p

Limited proteolysis analysis of recombinant PREP1 and PBX1.

<p>Full length PREP1 and PBX1 were subjected to limited proteolysis with trypsin. The reactions (total volume 100 μl) were performed at room temperature, 10 μl volumes were taken out at the indicated time points, supplemented with sample buffer and boiled prior to loading onto SDS PAGE. The gels were Coomassie stained. <b>A. Limited proteolysis of PREP1.</b> Lane M, Bio-Rad size standard; two bands of ~40 kDa (1) and ~28 kDa (2) were chosen for subsequent N-terminal sequencing. <b>B. Limited proteolysis of PBX1 with trypsin.</b> Lane M, Bio-Rad size standard; band 3 is the proteolysis fragment chosen for mass spectrometry analysis. <b>C and D. Identification of PREP1 and PBX1 fragments by MALDI-TOF mass spectrometry analysis.</b> Peptides of PREP1 and PBX1 were identified by MALDI-TOF analysis after digestion of fragments 1–3 with trypsin. Fragment 1 contained PREP1 and the matching peptides (red) covered 52.5% starting from the N-terminus and ending at residue 344. Fragment 2 contains the N-terminal part of PREP1 excluding the homeodomain. Fragment 3 contains PBX1, and the peptides (blue) covered 40.2% of the sequence, from residue 7 to 308.</p

Prep1 controls the number and the functionality of HSCs in E14.5 FLs.

<p>(A) 50, 100 or 200 HSCs sorted from <i>Prep1^+/+</i> and <i>Prep1^i/i</i> FLs were transplanted into lethally irradiated mice in competition with 2×10⁵ CD45.1 BM cells. Mice showing more than 2% CD45.2⁺ cells in the PB were considered as positively repopulated. HSCs are identified as CD45.2⁺ L⁻S⁺K⁺CD150⁺CD48⁻CD41⁻cells. The graph represents the percentage of positively repopulated mice 16 weeks after transplantation (n = 4 for each genotype) (B) The graph indicates the mean chimerism shown by transplanted mice at each cell dosage in the PB 16 weeks after transplantation. (C) 2000 LSK cells purified form <i>Prep1^+/+</i> or <i>Prep1^i/i</i> FLs were transplanted in competition with 1×10⁶ BM cells into lethally irradiated CD45.1 recipients. (D) PB analyses to detect donor-derived (CD45.2⁺) cells performed at 7, 12,16 and 20 weeks after transplantation. In the graph, black bars represent the mean of CD45.2⁺ cells in the PB of <i>Prep1^+/+</i> or <i>Prep1^i/i</i> reconstituted mice (n = 4 for each genotype; p<0.001). (E–F) RUs (on the left) were calculated on FACS data (on the right) obtained from BMs of repopulated mice 20 weeks after transplantation. (E) Mean of RUs in Prep1^+/+ or Prep1^i/i-transplanted mice; p = 0.05. (F) Mean of HSCs RUs in <i>Prep1^+/+</i> or <i>Prep1^i/i</i>-transplanted mice; p = 0.01. HSCs are identified as CD45.2⁺ L⁻S⁺K⁺CD150⁺CD48⁻CD41⁻cells.</p

Prep1 influences HSC quiescent pool rather than HSCs apoptosis.

<p>(A) Representative FACS contour plots to identify apoptotic <i>Prep1^+/+</i> and <i>Prep1^i/i</i> HSCs are shown on the left. The represented plots refer to L⁻S⁺K⁺CD150⁺ gate and numbers in the FACS plots indicate the percentage of cells in parental gates. On the right, the graph represents the mean of apoptotic HSCs (Annexin⁺ DAPI⁻) (n = 3; p = not significant). (B) Representative FACS contour plots to identify the cell cycle distribution of <i>Prep1^+/+</i> and <i>Prep1^i/i</i> HSCs are shown on the left. The represented plots refer to L⁻S⁺K⁺CD150⁺ gate and numbers in the FACS plots indicate the percentage of cells in parental gates. On the right, the graph represents the mean of G0 (Ki67⁻Hoechst^l°^w), G1 (Ki67⁺Hoechst^l°^w) and S/G2/M (Ki67⁺Hoechst^hi) HSCs (n = 3; p = not significant) (n = 3; G0 p = 0.02; G1 and S/G2/M p = not significant).</p

Prep1 does not affect Smads expression but modulates their phosphorylation in the EML1 progenitor cell line.

<p>(A) Prep1 knock-down (KD) was assessed by Immuno-blotting analysis. Actin was used as loading control. (B–C) Prep1 KD or control EML1 cells were incubated 4 h with (+) or without (−) TGFβ (10 ng/ml). (B) Histograms show fold induction of the indicated transcripts in Prep1 KD or control EML1 cells as measured by qRT-PCR. The data represent 2 independent experiments. (C) Smads and their phosphorylated forms (as indicated) were detected by Western blot analyses. Vinculin was used as loading control. The data were reproduced in 2 independent experiments.</p

Prep1^i/i HSCs undergo exhaustion faster than wt in serial transplantation assay.

<p>(A) 2×10⁶ unfractionated BM cells from primary recipients were injected into secondary hosts. Primary recipients had received 1×10⁶ unfractionated CD45.2⁺ FL cells together with 1×10⁶ unfractionated CD45.1⁺ wt BM cells (chimerism shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107916#pone.0107916.s004" target="_blank">FigureS4</a>). (B) PB analyses of secondary recipients were performed 8 and 20 weeks after transplantation to detect CD45.2⁺ cells. Each recipient is depicted with a separate symbol, blue and red colors indicating <i>Prep1^+/+</i> and <i>Prep1^i/i</i> reconstituted mice, respectively. Black bars represent the mean values in <i>Prep1^+/+</i> or <i>Prep1^i/i</i> reconstituted mice (n = 8 for each genotype at both time points; p-value<0.001 at both time points). (C) 20 weeks after transplantation, PB of secondary hosts was analysed for donor-derived myeloid cells (CD45.2⁺Gr1⁺Mac1⁺; p = 0.000017), B lymphoid cells (CD45.2⁺B220⁺; p = 0.000007) and T lymphoid cells (CD45.2⁺CD3⁺; p = 0.000017). Black bars represent mean values (n = 8 for each genotype). (D) Tertiary transplantations were performed injecting 2×10⁶ BM cells from secondary recipients into tertiary hosts. (E) PB of tertiary hosts was analyzed 20 weeks after transplantation to detect CD45.2⁺ cells. Black bars represent the mean values (n = 10 for <i>Prep1^+/+</i> and n = 8 for <i>Prep1^i/i</i>; p-value = 0.007). On the right, representative FACS histograms obtained for CD45.2⁺ cells of control and mutant tertiary recipients. (F) The ratio between the number of positively repopulated mice (CD45.2⁺ cells >2%) and transplanted mice is represented for primary, secondary and tertiary transplantations for both genotypes (primary transplantation n = 10 for each genotype; secondary transplantation n = 8 for each genotype; tertiary transplantation n = 10 for <i>Prep1^+/+</i> and n = 8 for <i>Prep1^i/i</i>).</p

Prep1 affects stem and progenitors compartments in E14.5 FLs.

<p>(A–D) Representative FACS analyses of <i>Prep1^+/+</i> and <i>Prep1^i/i</i> FLs are shown on the left. Graphs describing percentage and absolute numbers are reported on the right. Numbers in the FACS plots indicate the percentage of cells in parental gates. (A) Lin⁻Sca1⁺cKit⁺ population is shown. FACS plots are referred to Lineage⁻ gate (n = 8 for each genotype; % L⁻S⁺K⁺ p = 0.01; # L⁻S⁺K⁺ p = 0.01). (B) HSC population is identified as L⁻S⁺K⁺CD150⁺ CD41⁻CD48⁻. FACS plots are referred to L⁻S⁺K⁺ gate (n = 8 for each genotype; % HSCs p = 0.0000056; # HSCs p = not significant). (C) CLPs are identified as Lin⁻Il7R⁺Sca1^intckit^int. FACS plots are referred to Lin⁻Il7R⁺ gate. (n = 4 for each genotype; % CLPs p = 0.00014; # CLPs p = 0.00023). (D) FACS plots regarding myeloid progenitors refer to Lin⁻Sca1⁻ckit^hi gate. GMPs are FcγR⁺CD34⁺ (n = 4 for each genotype; % GMPs p = 0.05; # GMPs p = 0.07); CMPs are FcγR^intCD34^+/l°^w (n = 4 for each genotype; % CMPs p = 0.005; # CMPs p = 0.007); MEPs are FcγR⁻CD34⁻ (n = 4 for each genotype; % MEPs p = not significant; # MEPs p = not significant).</p

Analysis of expression of Pbx1NT transgene, Prep1 and Pbx2 proteins and Prep1-Pbx DNA-binding activity in Pbx1NT TG mice.

<p>(A) Western blot analysis of Pbx1NT expression in the thymus of <i>Pbx1NT TG mice</i>. Cytoplasmic extracts (30 µg) of total thymus of Pbx1NT TG mice (TG) and their wild-type controls (WT) were blotted onto PVDF membranes and incubated with anti-Pbx1 antibody. Anti-β-actin antibody was used as loading control. (B) RT-PCR analysis of Pbx1NT expression in sorted DN1, DN3 as well as in unsorted (TOTAL) thymocytes of <i>Pbx1NT TG mice</i>. Total RNA was isolated from the DN1, DN3 and total thymocytes and reverse transcribed. PCR amplification was performed using Pbx1NT-specific primers for 30 cycles and β-actin primers for 25 cycles. (C) Nuclear (Nuc) and cytoplasmic (Cyt) extracts of Pbx1NT TG mice (TG) and their wild-type controls (WT) were blotted onto PVDF membranes and incubated with anti-Prep1 and anti-Pbx2 antibodies. Anti-β-actin and anti-HMG1 antibodies were used as loading controls for cytoplasmic and nuclear fractions. (D) Total RNA was isolated from thymocytes of two Pbx1NT TG mice (TG) and their wild-type controls (WT) and reverse transcribed. PCR amplification was performed using Prep1 and Pbx2 -specific primers for 28 cycles and β-actin primers for 25 cycles. (E) Electrophoretic mobility shift assays were performed with the radiolabeled O1 double-stranded oligonucleotide, incubated with nuclear extracts from the thymi of wild-type (WT) or Pbx1NT TG (TG) mice without (−) or with antibodies against Prep1, Pbx1, Pbx2, Pbx3, Pbx4, Meis1 or Meis2. Pbx2-Prep1 arrow indicates the migration of complexes formed between Prep1 and Pbx2. SS, supershift.</p

Primers used for cloning of PREP1 and PBX1 constructs into PGEX-6p-2rbs.

<p>Primers used for cloning of PREP1 and PBX1 constructs into PGEX-6p-2rbs.</p