Regulation of the Flt3 Gene in Haematopoietic Stem and Early Progenitor Cells - PubMed (original) (raw)
Regulation of the Flt3 Gene in Haematopoietic Stem and Early Progenitor Cells
Giacomo Volpe et al. PLoS One. 2015.
Abstract
The MYB transcription factor plays critical roles in normal and malignant haematopoiesis. We previously showed that MYB was a direct activator of FLT3 expression within the context of acute myeloid leukaemia. During normal haematopoiesis, increasing levels of FLT3 expression determine a strict hierarchy within the haematopoietic stem and early progenitor compartment, which associates with lymphoid and myeloid commitment potential. We use the conditional deletion of the Myb gene to investigate the influence of MYB in Flt3 transcriptional regulation within the haematopoietic stem cell (HSC) hierarchy. In accordance with previous report, in vivo deletion of Myb resulted in rapid biased differentiation of HSC with concomitant loss of proliferation capacity. We find that loss of MYB activity also coincided with decreased FLT3 expression. At the chromatin level, the Flt3 promoter is primed in immature HSC, but occupancy of further intronic elements determines expression. Binding to these locations, MYB and C/EBPα need functional cooperation to activate transcription of the locus. This cooperation is cell context dependent and indicates that MYB and C/EBPα activities are inter-dependent in controlling Flt3 expression to influence lineage commitment of multipotential progenitors.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Fig 1. MYB deletion affects KSL cell number and differentiation.
(A) Flow cytometric analysis of bone marrow from Myb F/F:MxCre mutant mice and Myb +/+:MxCre control animals, 24 or 48 hours following in vivo induction of the Myb F/F allele deletion by p(I:C) injection. The representative two-dimensional plots show the gating on lineage negative cells and the typical KIT+/SCA-1+/LIN- (KSL) staining. Histograms represent the percentage of KSL cells within the total bone marrow population, with numbers presented as mean ± SEM, determined from 3 independent experiments (***, p<0.0001). (B) Control and _Myb_-deleted KSL were sorted and seeded in methylcellulose. Colony numbers and size were assessed after 6 days. The right histograms represent total colony numbers and the relative proportions of colonies based on size respectively. Pictures of the counted colonies are presented in the left panels. (C) Sorted KSL from control and _Myb_-deleted animals 24 hours post injection of poly(I:C) were cultured into liquid medium containing SCF, FL and TPO. After culturing for 2 days, the cells were re-stained and analysed by flow cytometry for expression of CD11 and CD41 (left panel). Cells from the Myb F/F:MxCre bone marrow were sorted on the basis of CD11 and CD41. The sorted cells were spun onto glass slides and stained with Diff-Quick (upper right panel). The images show representative cells demonstrating monocytic (CD11b+) and megakaryocytic (CD11b+CD41+ and CD11b-CD41+) morphologies.
Fig 2. Induced Myb ablation results in FLT3 down-regulation in a population highly enriched in LMPP.
(A) Representative histogram and two-dimensional flow cytometric plot analysis of cells in the KSL compartment of the bone marrow of the cKO and control mice 24 hours post poly(I:C) injection. (B) The cells were further analysed for surface expression of FIT3 and the proportion of different KSL sub-fractions were assessed based on the surface expression of VCAM-1 and CD62L. Regrouping 7 experiments, the right panel shows their relative contribution in percentage of the gated population, indicated as plot header. (C) Serial gating defined a population highly enriched in LMPP (KSL/CD62L+/VCAM-) for which the percentage of FIT3hi cells was measured. (D) Quantitative PCR analysis of Myb, Flt3, _Il7r_α, Dntt and Notch1 RNA expression in KSL cells isolated from the bone marrow of poly(I:C)-induced Myb cKOs and litter-mate controls 24 hours post injection. Expression is normalised to gapdh and standardised the Myb +/+:MxCre control samples, set as 1. Error bars represent the standard error of the mean. Plots are representative of 7 independent experiments. Numbers are plotted as mean ± SEM (***, p<0.0001 and **, p<0.01).
Fig 3. Flt3 intronic element hypersensitivity coincides with FLT3 expression in primary haematopoietic stem/progenitor cells.
Upper panel gives a schematic representation of the murine Flt3 promoter and first intron (filled box indicates exon1) and shows the level of cross-species sequence conservation and the position of repeated sequences. The underlined regions A B and C indicate the general location of the regions of hypersensitivity to DNAseI digestion in HPC7 cells. Lower panel plots show the assessment of DNaseI digestion sensitivity in the HPC7 cell line and in primary KSL FIT3+, KSL FITt3- cells, CMP (LIN-/KIT+/SCA-1-/ CD34-/CD16/32+), GMP (LIN-/KIT+/SCA-1-/CD34+/CD16/32+) and MEP (LIN-/KIT+/SCA-1-/CD34-/CD16/32-). For primary cells, the analysis are restricted to the digestion sites observed in HCP7 at -1.45 kb (HS-A), 0.15kb (HS-B) and +7.5kb (HS-C) from the murine Flt3 initiation codon.
Fig 4. Flt3 regulatory regions enable the recruitment of a combination of master regulators of haematopoiesis.
Detection of in vivo transcription factor binding at the sites of hypersensitivity to nuclease digestion was achieved by ChIP. Relative enrichments from X-ChIP material were determined against the IgG control material by Q-PCR at the location of the hypersensitive regions and normalised against two internal control regions (located at -3.5kb and -0.27kb from the ATG). Error bars represent the standard error of the mean. All plots are representative of a minimum of three independent experiments.
Fig 5. Genetic manipulation and reporter assay studies in HPC7 cells.
Quantitative PCR analysis of gene expression in HPC7 cells following shRNA-mediated silencing and ectopic expression of Myb (A) and Cebpa (B). (C) Analysis of Flt3 transcript expression in HPC7 cells co-transfected with Myb and Cebpa expression vectors or control plasmids. Determined by Q-PCR, Myb, Cebpa and Flt3 RNA levels were normalised against Gapdh abundance and standardised against the corresponding control transfections. (D) Schematic representation of the reporter constructs in which Flt3 regulatory regions were placed upstream (HS-A and HS-B) or downstream (HS-C) the luciferase gene. (E) Transactivation assays in HPC7 cells transfected with vector encoding for MYB and C/EBPα proteins. Results were standardised to 100 and bars represent the average proportions across 4 experiments. All results are representative of 3 or 4 independent experiments. P values representations: ***<0.001 and **<0.01.
Fig 6. Myb and Cebpa knockdown in primary KIT+ enriched bone marrow cells.
(A) Two-dimensional flow cytometry dot plot showing the analysis of the KSL compartment of a KIT+ enriched population transfected with siRNA control or undergoing siRNA-mediated silencing of Myb or Cebpa for 20 hours. (B) Grouping four independent experiments, boxplots depict the variations in percentage of FLT3+ cells within the KSL populations. (C) Quantitative PCR analysis of Myb and Cebpa RNA expression in sorted transfected KSL cells 20 hours post transfection. Expression is normalised to β 2 -microglobulin and standardised to the control samples. Error bars represent the standard error of the mean. Plots are representative of 4 independent experiments. Numbers are plotted as mean ± SEM (**, p<0.001 and *, p<0.05).
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