AbstractMEIS1 is a transcription factor expressed in hematopoietic stem and progenitor cells and in mature megakaryocytes. This biphasic expression of MEIS1 suggests that the function of MEIS1 in stem cells is distinct from its function in lineage committed cells. Mouse models show that Meis1 is required for renewal of stem cells, but the function of MEIS1 in human hematopoietic progenitor cells has not been investigated. We show that two MEIS1 splice variants are expressed in hematopoietic progenitor cells. Constitutive expression of both variants directed human hematopoietic progenitors towards a megakaryocyte-erythrocyte progenitor fate. Ectopic expression of either MEIS1 splice variant in common myeloid progenitor cells, and even in granulocyte-monocyte progenitors, resulted in increased erythroid differentiation at the expense of granulocyte and macrophage differentiation. Conversely, silencing MEIS1 expression in progenitor cells induced a block in erythroid expansion and decreased megakaryocytic colony formation capacity. Gene expression profiling revealed that both MEIS1 splice variants induce a transcriptional program enriched for erythroid and megakaryocytic genes. Our results indicate that MEIS1 expression induces lineage commitment towards a megakaryocyte-erythroid progenitor cell fate in common myeloid progenitor cells through activation of genes that define a megakaryocyte-erythroid-specific gene expression program.
Production of appropriate numbers of distinct blood cell types is dependent on controlling lineage commitment in hematopoietic tissues and terminal differentiation into circulating blood cells. Transcription factors regulate the renewal of hematopoietic stem cells (HSC) and specify distinct lineages.91 By also controlling each other’s expression, transcription factors induce lineage-specific gene expression and repress other differentiation routes. The differentiation of common myeloid progenitors (CMP) towards either granulocyte-monocyte progenitors (GMP) or megakaryocyte-erythrocyte progenitors (MEP), for example, is regulated by transcription factors, among which GATA1 plays a crucial role.1110 However, depletion of Gata1 alone does not impair MEP differentiation or enhance myeloid output, indicating that additional factors regulate the megakaryocyte-erythroid fate.12
The Meis1 (myeloid ecotropoic viral insertion site 1) transcription factor gene was first described as a common viral integration site associated with tumor formation in BXH-2 mice.13 MEIS1 overexpression together with overexpression of HOXA9 sustains leukemic stem cell potential in human acute myeloid leukemia and childhood acute lymphoblastic leukemia.1514
MEIS1 is a homeobox gene of the TALE (three amino acids loop extension) family, encoded on human chromosome 2p15. The MEIS1 amino terminus contains binding domains for its interaction partners, the transcription factors HOXA9 and PBX1, followed by a homeobox DNA binding domain and carboxy-terminal transcriptional activation domains.16 Previous studies have shown that MEIS1 pre-mRNA undergoes alternative splicing in a highly conserved manner resulting in four splice variants of which two, MEIS1B and MEIS1D, are expressed in hematopoietic cells.1713 MEIS1B and MEIS1D proteins differ in their carboxy termini, with MEIS1D being the transcriptionally more active splice variant.18
MEIS1 is expressed in hematopoietic stem and progenitor cells20199 and increases during human megakaryocytic differentiation.21 Meis1 null-mutant mice die at embryonic day 14.5 due to a poorly developed hematopoietic compartment, lack of megakaryocytes and platelets and defective vascularization.2276 Meis1-deficient zebrafish display a lack of megakaryocytes, decreased erythrocyte levels and vascularization defects,23 consistent with the finding in null-mutant mice. Meis1 depletion in murine HSC using lineage-specific Cre-recombinase results in cell cycle entry and loss of quiescence.9
The most prominent murine models used to study Meis1 function to date rely on homologous recombination resulting in Meis1 depletion during embryogenesis. Given the construction of these knockouts it was not possible to compare the function of the different Meis1 splice variants or specifically study MEIS1 in hematopoietic cells in these murine models.76 Here, we investigated for the first time the role of MEIS1 in adult human hematopoietic progenitor cells. Expression of the two MEIS1 splice variants was modulated in sorted hematopoietic stem and progenitor cell subsets using lentiviral knockdown or overexpression of MEIS1 fused to green fluorescent protein (GFP), an approach that had been described before,24 enabling us to delineate the effects of MEIS1 on specific stages of lineage commitment. Furthermore, using transcriptional profiling of MEIS1-overexpressing CD34 hematopoietic stem and progenitor cells, we generated an overview of the transcriptional changes following MEIS1 expression.
Additional information is provided in the Online Supplement.
Human CD34+ cells
Mobilized peripheral blood (MPB) and cord blood (CB) cells were obtained after informed consent and with the approval of the ethical committee of local hospitals. Bone marrow was collected by sternal puncture from patients undergoing cardiac surgery. CD34 cells were isolated from CB or MPB samples within 48 hours after arrival of the material using magnetic cell sorting according to the manufacturer’s specifications (Miltenyi Biotech, Bergisch Gladbach, Germany). All samples contained more than 90% CD34 cells and less than 5% CD41 determined by flow cytometry (LSRII, Becton Dickinson).
Lentiviral transduction, colony formation assays and human primary cell culture
CD34 cells were transduced with MEIS1 expression or shRNA constructs and incubated overnight in CellGro medium (CellGenix, Frankfurt, Germany) with 100 ng/mL each of stem cell factor (SCF, R&D Systems Abingdon, UK), thrombopoietin (TPO; Sanquin PeliKines, Amsterdam, The Netherlands), Fms-like tyrosine kinase3-ligand (Flt3; Miltenyi Biotech) and 20 ng/mL interleukin 6 (IL6; Miltenyi Biotech). Forty-eight hours after transduction, cells were sorted for GFP-expression using the FACSAria II Cell Sorter (BD Biosciences). GFP-positive cells were used in either Colony Gel colony formation assays (Cell Systems, Frankfurt, Germany) or Megacult colony formation assays (Stem Cell Technologies, Grenoble, France), according to the manufacturers’ specifications. Erythroid differentiation in liquid culture was achieved by seeding CD34 cells into CellGro medium (CellGenix) with 100 ng/mL of SCF (R&D Systems), 3 U/mL erythropoietin (EPO; R&D Systems) and 10 ng/mL interleukin 3 (IL-3; R&D Systems). For transduction of committed erythroid progenitor cells, CD34 cells were cultured towards the erythroid lineage in CellGro medium (CellGenix) as described above. After 7 days in culture, expression of CD71 (transferrin receptor) and CD235a (glycophorin A) was measured using flow cytometry (LSRII, Becton Dickinson). Cells were transduced with MEIS1 expression or knockdown constructs and left to differentiate towards erythroid cells for a further 5 days. At the end of the culture cells were counted, labeled with PeCy7-conjugated CD34 (BD Biosciences) and tricolor-conjugated CD235a (Caltag Laboratories, Buckingham, UK) and analyzed using flow cytometry (LSRII, Becton Dickinson).
Hematopoietic stem and progenitor cell subfractions
CD34cells from MPB samples were stained with PE-Cy7-conjugated CD34, PerCP-conjugated CD38, FITC-conjugated CD45RA, V450 Horizon-conjugated CD45, APC-conjugated BAH clone recognizing a MEP-specific epitope and PE-conjugated CD123 (all from BD Biosciences). Cells were sorted into HSC (CD34/CD38), CMP (CD34/CD38/CD123/CD45RA), GMP (CD34/CD38/CD123/CD45RA) and MEP (CD34/CD38/CD123/CD45RA-/BAH1)2625 using a FACS Aria II cell sorter (BD Biosciences). After sorting, subfractions were transduced with virus particles carrying MEIS1B or MEIS1D expression constructs or an empty vector control. Forty-eight hours after transduction, cells were sorted for GFP-expression and 1000 GFP-positive cells were seeded into Colony Gel for the colony formation assay (Cell Systems) according to the manufacturer’s specifications.
Differential expression of MEIS1 splice variants in human CD34+ hematopoietic stem and progenitor cells
To investigate the expression of the MEIS1B and MEIS1D splice variants (Figure 1A) in human primary hematopoietic stem and progenitor cells, we sorted CD34 cells into subfractions to obtain HSC, CMP, GMP and MEP.2625 MEIS1 mRNA was present in all subfractions, with the expression of MEIS1D being 4-fold higher than MEIS1B (Figure 1B). Expression of both MEIS1 splice variants decreased when HSC differentiated towards the more committed CMP, GMP, and MEP (Figure 1B).
When CD34 cells from MPB or CB were allowed to differentiate into megakaryocytes, MEIS1D protein expression decreased while mRNA was still present. MEIS1B mRNA and protein expression increased throughout megakaryopoiesis, with expression being highest in terminally differentiated megakaryocytes (Figure 1C,D). Taken together, our data show that MEIS1B and MEIS1D are present in hematopoietic stem cells and their expression decreases when the cells differentiate into more committed progenitors. Expression of MEIS1 is then reinforced during megakaryopoiesis with MEIS1B being the more abundant splice variant.
MEIS1 expression skews CD34+ cells towards a megakaryocyte-erythrocyte progenitor fate
To investigate the functional role of MEIS1 during lineage commitment of hematopoietic stem and progenitor cells, we ectopically expressed MEIS1 in CD34 cells using lentiviral expression vectors with MEIS1B or MEIS1D full length cDNA fused to GFP at the amino-terminus. The functionality of the fusion protein has already been reported.24 MEIS1B expression increased 60-fold in sorted GFP-positive cells 48 h after transduction, whereas that of MEIS1D increased 15-fold compared to empty vector controls (Figure 2A), expression levels well within the range of physiological MEIS1 fluctuation. Seeding transduced CD34 cells one cell per well in semisolid media promoting erythroid or granulocyte-macrophage differentiation did not alter total colony numbers (empty vector 16±6.5, MEIS1B 24±13.5, MEIS1D 16±13.8) while the phenotypic distribution significantly changed. The numbers of burst-forming unit erythroid (BFU-E) and colony-forming unit erythroid (CFU-E) increased by 3-fold with MEIS1B overexpression and 1.9-fold with MEIS1D compared to empty vector. Colony-forming unit granulocyte-macrophage (CFU-GM) numbers decreased by 3-fold with MEIS1B and 6-fold upon MEIS1D expression compared to empty vector (Figure 2C).
Next, expression of both MEIS1 variants was silenced in CD34 cells with short hairpins targeting either exon 7 (sh72) or the 3′ UTR (sh68), sequences present in both MEIS1 variants (Figure 2B) reducing MEIS1B expression by 60% with sh68 and about 70% with sh72. When transduced CD34 cells were plated out in semisolid media, MEIS1 knockdown resulted in significantly less total colony formation with sh72 (4±1.8, P<0.01) than with either the short hairpin control shc002 (25±2.5) or sh68 (19±2.6). Furthermore, knockdown almost completely abrogated erythroid colony formation with sh68 or sh72 compared to shc002 (Figure 2D). CFU-GM numbers were not affected by MEIS1 knockdown (Figure 2D) suggesting that MEIS1 expression controls erythroid versus granulocytic differentiation. In parallel, transduced CD34 cells were plated in semisolid media promoting megakaryocytic differentiation. MEIS1B and MEIS1D overexpression increased the number of megakaryocytic colonies by 1.6-fold (174±81) and 2-fold (237±57), respectively, compared to the vector control (111 ± 16) (Figure 2E). In addition to increased colony numbers, the colony size was larger (Figure 2F) upon overexpression with either one of the two MEIS1 splice variants. MEIS1 knockdown in CD34 cells reduced megakaryocytic colony formation by 18% with sh68 (152±38) and by 30% with sh72 (145±39) (Figure 2G) and colony size compared to shc002 (174±32) (Figure 2H). These results suggest that MEIS1 expression, especially MEIS1D, induces a MEP fate in hematopoietic stem and progenitor cells.
MEIS1 induces erythroid commitment in myeloid progenitor cells
To determine at which level of hematopoietic commitment the MEIS1 variants exert their effects, MPB-derived CD34 cells were sorted into HSC, CMP and GMP (Figure 3A), transduced with MEIS1B or MEIS1D overexpression vectors, sorted for GFP expression and seeded into semisolid medium. Neither MEIS1B nor MEIS1D overexpression significantly changed the differentiation potential of the HSC-enriched fraction (Figure 3B). Expression of either splice variant in the CMP caused a 2-fold increase in erythroid colonies, accompanied by a significant decrease in CFU-GM (Figure 3C) reflecting the phenotype of total CD34 cells after MEIS1 overexpression (Figure 2C). Strikingly, expression of the MEIS1 splice variants enabled the formation of erythroid colonies in the GMP fraction, a progenitor subset committed to differentiate into granulocytic-monocytic cells. The increase in erythroid colonies was accompanied by a significant reduction in CFU-GM (Figure 3D), suggesting that MEIS1 is able to reprogram GMP towards erythroid commitment. Our findings show that expression of MEIS1B and MEIS1D skews CMP towards a MEP fate and that MEIS1 expression reprograms GMP into the erythroid lineage.
MEIS1 positively regulates erythroid determination
To further examine the role of MEIS1 in erythroid commitment and differentiation, transduced CD34 cells were grown in liquid culture supporting erythroid proliferation and differentiation.
A 2.3-fold increase in cellular yield was observed upon overexpression of MEIS1B and a 2.2-fold increase was found with MEIS1D overexpression compared to empty vector controls (Figure 4A). While cell numbers increased with MEIS1 expression, glycophorin A (CD235a) expression was the same for MEIS1B- or MEIS1D-expressing cells as well as for empty vector cells (Figure 4A) suggesting that MEIS1 induces skewing of CD34 cells towards an erythroid fate, but not erythoid differentiation. Vice versa, MEIS1 knockdown caused a 47% reduction in cell numbers with sh68 and a 60% reduction with sh72 compared to control cells (Figure 4B). The decline in erythroid cells was accompanied by a 25% decrease in CD235a expression with sh68 and a 57% decrease with sh72 (Figure 4B), further underlining that MEIS1 expression is needed to direct CD34 progenitor cells to an erythroid fate.
To determine whether MEIS1 also affects committed erythroblasts, CD34 cells were first cultured for 7 days to establish an erythroid fate, and subsequently transduced with MEIS1 overexpression and knockdown constructs. After an additional 4 days of culture, overexpression of MEIS1B or MEIS1D resulted in 2-fold more cells compared to empty vector (Figure 4C). A trend towards increased CD235a expression was also detected (Figure 4C), but differences did not reach statistical significance. Silencing MEIS1 did not affect the amount of cells (Figure 4D), consistent with a physiological downregulation of MEIS1B and MEIS1D during physiological erythropoiesis (Figure 4E). Together these findings indicate that MEIS1 expression is crucial to establish an erythroid fate in hematopoietic progenitor cells but is not needed for erythoid differentiation.
MEIS1 expression induces a megakaryocyte-erythroid progenitor fate
Elevated MEIS1B or MEIS1D expression increased erythroid colony formation from CMP and GMP, suggesting that MEIS1 induces a MEP gene expression profile in these progenitors. To characterize the transcriptional changes caused by MEIS1, the gene expression profile of CD34 cells from MPB was determined 48 h after transduction with GFP-fused MEIS1B or MEIS1D expression vectors, or the empty vector control. Total RNA from three biologically independent experiments was hybridized to an Illumina Human HT-12 v4 expression bead array. After VSN normalization, gene expression was compared between the three different groups (empty vector, MEIS1B, or MEIS1D) using Benjamini Hochberg FDR values smaller than 0.05 as a cutoff. Sixty-six genes were differentially regulated upon MEIS1B overexpression, 125 genes with MEIS1D overexpression and 69 genes were differentially regulated in both overexpression conditions compared to empty vector controls. Overexpression of either MEIS1B or MEIS1D resulted in similar expression patterns, indicating that both splice variants regulate common targets (Figure 5A, Table 1, Online Supplementary Table S1). The MEIS1-regulated genes in CD34 cells included transcripts whose expression had previously been described to be regulated by MEIS1, such as cyclin D1 (CCND1)249 and cyclin D3 (CCND3)279 (Table 1, Online Supplementary Table S1).
Together with these previously described targets, MEIS1 increased expression of genes associated with megakaryopoiesis and erythropoiesis such as KLF1, HBD, HBG, SLC40A1, THBS1, GPIb, VWA5A and GATA2 (Table 1, Online Supplementary Table S1). Notably, transcripts known to be specific for HSC were downregulated upon MEIS1 expression, including PROM1 (CD133), AK2 and CD34. CD48, a gene marking granulocytic cell commitment, was also downregulated.
To examine the lineage specification induced upon MEIS1 expression further, we compared our expression profiling data with the Haematlas.21 In this study, genes specific for CD4 T helper lymphocytes, CD8 T cytotoxic lymphocytes, CD14 monocytes, CD19 B lymphocytes, CD56 natural killer cells and CD66 granulocytes were identified based on expression profiling. The percentage of common genes was most pronounced between MEIS1-overexpressing CD34 cells and myelo-erythroid lineage cells, resulting in 30% (erythroblasts) and 38% (megakaryocytes) common transcripts (Figure 5B, Table 2 and Online Supplementary Table S2). Vice versa, few gene expression patterns were shared between MEIS1-overexpressing CD34 cells and cells of the lymphoid lineage with 10.8% in CD56 natural killer cells and a maximum of 14.6% genes expressed in CD19 B lymphocytes. The increase in the amount of overlapping expression profiles underlines that MEIS1 overexpression in CD34 cells induces an expression profile characteristic of megakaryocytes and erythroid cells.
To characterize the role of MEIS1 on lineage determination, gene set enrichment analysis was performed. In a previous study, Jaatinen et al. compared the expression profiles of CB-derived CD133 HSC and CD133 progenitor cells.28 When the expression profile of CD34 cells overexpressing MEIS1 was compared with that of CD133 cells, we found an overall negative correlation for HSC-associated genes. The correlation was significant for MEIS1D (Online Supplementary Table S3, Online Supplementary Figure S1). Vice versa, a positive correlation was detected between transcripts downregulated in CD133 cells and transcripts upregulated upon MEIS1 overexpression, and this correlation was significant for MEIS1B (Online Supplementary Figure S1). The positively correlated genes included the erythroid markers KLF1, HBB and RHAG (Online Supplementary Table S3). Taken together, these findings further confirm that MEIS1 expression induces a progenitor fate in HSC with a bias towards the megakaryocyte-erythroid lineage.
While the critical role of Meis1 in maintaining quiescence and self-renewal capacity in HSC has been demonstrated earlier,31299 little is known about the role of this transcription factor in more committed progenitor cells. MEIS1 expression decreases when HSC differentiate to multipotent progenitors, but increases again during megakaryopoiesis. Here, we show that MEIS1 is important for the lineage commitment towards the MEP fate. Erythroid and megakaryocytic colony formation correlated with MEIS1 expression, both in total CD34 cells and in common myeloid progenitors enriched from CD34 cells. To understand the transcriptional changes inducing the observed bias towards a MEP fate, we performed gene expression profiling which indicated that MEIS1 expression increased the expression of multiple megakaryocyte-erythroid-specific genes and downregulated genes controlling myeloid differentiation and stem cell expansion.
While we showed that, in adult human hematopoiesis, MEIS1 exerts its effects at the level of the CMP and GMP, other studies also indicated a role for Meis1 downstream of the CMP in the skewing of MEP towards the megakaryocytic lineage at the expense of erythroid commitment.30 These differences can be explained in several ways. First, the study by Cai et al. was performed in a model system resembling primitive hematopoiesis whereas we investigated adult hematopoiesis. The transcription factor composition and transcription factor complexes that MEIS1 contributes to in these different cell types may therefore differ leading to divergent results. Second, in the study by Cai et al., hematopoietic colonies derived from embryonic stem cells were cultured in semisolid media using a mix of cytokines (TPO, SCF, IL-3, IL-6, VEGF, Flt3 but lacking erythropoietin) which impairs the ability of embryonic stem cells to differentiate into the erythroid lineage and promotes differentiation into the megakaryocytic lineage. This explains the impairment in erythroid progenitor cells that they observed as compared to the enhanced megakaryocyte differentiation. Third, only in CD41 cells did MEIS1 overexpression substantially decrease erythrocyte-specific genes and enhance megakaryocyte-specific genes. This indicates that MEIS1 only suppresses erythroid differentiation in already committed megakaryocyte progenitor cells.
On the other hand, even though the results of our study and that by Cai et al. differ, in both cases a clear effect of MEIS1 on cell proliferation was detected. The effect of MEIS1 on proliferation has also been reported earlier in the context of retina development24 indicating a robust and conserved role for MEIS1 in proliferation that seems to be tissue-independent.
We further investigated the dynamics of the observed bias towards the MEP fate using expression profiling of MEIS1-overexpressing CD34 cells. It has been postulated that HSC can directly differentiate towards megakaryocytes.32 Therefore, MEIS1 could first reestablish a HSC fate from which the cells differentiate into the megakaryocyte-erythroid lineage. However, when CD34 cells were sorted into stem and progenitor cells, overexpression of MEIS1 in the HSC subset did not result in a lineage bias towards a MEP fate. Furthermore, expression profiling showed downregulation of genes associated with a HSC fate, including PROM1 (CD133),28 AK2, and even CD34, indicating that the observed bias towards a MEP fate upon MEIS1 overexpression is not caused by inducing a HSC fate first.
MEIS1 could also inhibit GMP differentiation, which by itself may cause a bias towards MEP. Indeed we showed that ectopic MEIS1 blocked the differentiation into the granulocytic-monocytic lineage and decreased the expression of specific granulocytic genes such as CD48 and elastase (ELANE) in CD34 cells. This hypothesis is supported by the study of Calvo et al., who reported that MEIS1 expression is able to inhibit granulocyte colony-stimulating factor-dependent granulocytic differentiation of immortalized progenitor cells, although this effect was only observed upon co-expression with HOXA9.33 However, we also found genes whose expression was slightly increased upon MEIS1 overexpression in GMP, such as the monocyte-specific gene CD68. Instead, the clear induction of erythroid-megakaryocyte-specific genes strongly suggests that MEIS1 overexpression directly promotes a MEP fate.
Two MEIS1 splice variants, MEIS1B and MEIS1D, are expressed in hematopoietic stem and progenitor cells and their expression decreases from the most immature stem cells to more committed progenitors.1713 Structurally, the two splice variants both contain the N-terminal PBX/HOX-interacting domain, a central DNA-binding homeodomain, and two C-terminal transactivation domains, but MEIS1B has an additional transactivation domain which regulates the transcriptional activity of MEIS1.1816 Upon trimerization with PBX1 and HOXA9, MEIS1 maintains the transcriptional activity of the complex by inhibiting nuclear export and promoting nuclear import.3534 Using separate overexpression of the splice variants, we showed that both induce the same phenotype, a skewing of CMP and GMP towards a MEP fate. Expression profiling further revealed that both splice variants control expression of many common transcripts, strongly indicating overlapping functions. However, in most experiments we observed a stronger phenotype upon over-expression of MEIS1D, suggesting more efficient regulation of gene expression by MEIS1D. Earlier studies also investigated the role of Meis1 splice variants, using a model of primitive hematopoiesis. In this setting, it was found that the expression of Meis1b, which is equivalent to MEIS1D in our study, induced significant skewing of MEP towards the megakaryocytic lineage.30 This further underlines the stronger transcriptional activity of MEIS1D.
While it has been shown that Meis1 depletion in murine HSC results in loss of quiescence and increased cell cycle entry,9 it is less clear whether MEIS1 also affects the cell cycle of more committed progenitor cells. We found that silencing MEIS1 resulted in almost no erythroid colonies, while granulocytic colony numbers were unaltered. This implies an important role for MEIS1 in the expansion of erythroid progenitor cells. These results were confirmed in liquid cultures in which MEIS1 also affected erythroid expansion although at later stages of erythropoiesis, MEIS1 expression declined. Comparative transcriptional profiling showed that MEIS1 induces expression of CCND1 and CCND3 in CD34 cells which are linked to HSC and MEP expansion.3736 In addition, CCND3 was also found to be essential for megakaryopoiesis, promoting progression through the G1-phase of the cell cycle.38 These results strongly suggest that MEIS1 reinforces MEP expansion and megakaryocytic differentiation via induction of CCND1 and CCND3. Of note, Meis1 has also been found to regulate ccnd1 expression during eye development in zebrafish indicating a strong, tissue-independent, regulation of Ccnd1 by Meis1.
In conclusion, we show that in hematopoietic progenitor cells, MEIS1 is critical for the commitment towards the megakaryocytic and erythroid lineages by inducing expression of lineage-specific genes. Expression profiling of CD34 cells after MEIS1 overexpression revealed transcriptional changes in genes with currently unknown functions in lineage commitment or differentiation. Given the biphasic expression of MEIS1 in both hematopoietic stem and progenitor cells and adult megakaryocytes but not erythrocytes, further studies should focus on the effects of MEIS1 and its target genes on early and late megakaryocytic differentiation.
This work was funded by Sanquin Blood Supply, grant PPOC-09-023, and the Netherlands Genomics Initiative (NGI-MEC).
- The online version of this article has a Supplementary Appendix
- Authorship and Disclosures Information on authorship, contributions, and financial & other disclosures was provided by the authors and is available with the online version of this article at www.haematologica.org.
- Received February 25, 2014.
- Accepted August 1, 2014.
- Ichikawa M, Goyama S, Asai T, Kawazu M, Nakagawa M, Takeshita M. AML1/Runx1 negatively regulates quiescent hematopoietic stem cells in adult hematopoiesis. J Immunol. 2008; 180(7):4402-8. PubMedhttps://doi.org/10.4049/jimmunol.180.7.4402Google Scholar
- Tsai FY, Keller G, Kuo FC, Weiss M, Chen J, Rosenblatt M. An early haematopoietic defect in mice lacking the transcription factor GATA-2. Nature. 1994; 371(6494):221-6. PubMedhttps://doi.org/10.1038/371221a0Google Scholar
- Kawada H, Ito T, Pharr PN, Spyropoulos DD, Watson DK, Ogawa M. Defective megakaryopoiesis and abnormal erythroid development in Fli-1 gene-targeted mice. Int J Hematol. 2001; 73(4):463-8. PubMedhttps://doi.org/10.1007/BF02994008Google Scholar
- Kruse EA, Loughran SJ, Baldwin TM, Josefsson EC, Ellis S, Watson DK. Dual requirement for the ETS transcription factors Fli-1 and Erg in hematopoietic stem cells and the megakaryocyte lineage. Proc Natl Acad Sci USA. 2009; 106(33):13814-9. PubMedhttps://doi.org/10.1073/pnas.0906556106Google Scholar
- Souroullas GP, Salmon JM, Sablitzky F, Curtis DJ, Goodell MA. Adult hematopoietic stem and progenitor cells require either Lyl1 or Scl for survival. Cell Stem Cell. 2009; 4(2):180-6. PubMedhttps://doi.org/10.1016/j.stem.2009.01.001Google Scholar
- Hisa T, Spence SE, Rachel RA, Fujita M, Nakamura T, Ward JM. Hematopoietic, angiogenic and eye defects in Meis1 mutant animals. EMBO J. 2004; 23(2):450-9. PubMedhttps://doi.org/10.1038/sj.emboj.7600038Google Scholar
- Azcoitia V, Aracil M, Martinez A, Torres M. The homeodomain protein Meis1 is essential for definitive hematopoiesis and vascular patterning in the mouse embryo. Dev Biol. 2005; 280(2):307-20. PubMedhttps://doi.org/10.1016/j.ydbio.2005.01.004Google Scholar
- Orkin SH, Zon LI. Hematopoiesis: an evolving paradigm for stem cell biology. Cell. 2008; 132(4):631-44. PubMedhttps://doi.org/10.1016/j.cell.2008.01.025Google Scholar
- Ariki R, Morikawa S, Mabuchi Y, Suzuki S, Nakatake M, Yoshioka K. Homeodomain transcription factor meis1 is a critical regulator of adult bone marrow hematopoiesis. PLoS One. 2014; 9(2):e87646. PubMedhttps://doi.org/10.1371/journal.pone.0087646Google Scholar
- Burda P, Laslo P, Stopka T. The role of PU.1 and GATA-1 transcription factors during normal and leukemogenic hematopoiesis. Leukemia. 2010; 24(7):1249-57. PubMedhttps://doi.org/10.1038/leu.2010.104Google Scholar
- Mancini E, Sanjuan-Pla A, Luciani L, Moore S, Grover A, Zay A. FOG-1 and GATA-1 act sequentially to specify definitive megakaryocytic and erythroid progenitors. EMBO J. 2012; 31(2):351-65. PubMedhttps://doi.org/10.1038/emboj.2011.390Google Scholar
- Gutierrez L, Tsukamoto S, Suzuki M, Yamamoto-Mukai H, Yamamoto M, Philipsen S, Ohneda K. Ablation of Gata1 in adult mice results in aplastic crisis, revealing its essential role in steady-state and stress erythropoiesis. Blood. 2008; 111(8):4375-85. PubMedhttps://doi.org/10.1182/blood-2007-09-115121Google Scholar
- Moskow JJ, Bullrich F, Huebner K, Daar IO, Buchberg AM. Meis1, a PBX1-related homeobox gene involved in myeloid leukemia in BXH-2 mice. Mol Cell Biol. 1995; 15(10):5434-43. PubMedGoogle Scholar
- Smith JE, Bollekens JA, Inghirami G, Takeshita K. Cloning and mapping of the MEIS1 gene, the human homolog of a murine leukemogenic gene. Genomics. 1997; 43(1):99-103. PubMedhttps://doi.org/10.1006/geno.1997.4766Google Scholar
- Lawrence HJ, Rozenfeld S, Cruz C, Matsukuma K, Kwong A, Komuves L. Frequent co-expression of the HOXA9 and MEIS1 homeobox genes in human myeloid leukemias. Leukemia. 1999; 13(12):1993-9. PubMedhttps://doi.org/10.1038/sj/leu/2401578Google Scholar
- Longobardi E, Penkov D, Mateos D, De FG, Torres M, Blasi F. Biochemistry of the tale transcription factors PREP, MEIS, and PBX in vertebrates. Dev Dyn. 2014; 243(1):59-75. PubMedhttps://doi.org/10.1002/dvdy.24016Google Scholar
- Geerts D, Revet I, Jorritsma G, Schilderink N, Versteeg R. MEIS homeobox genes in neuroblastoma. Cancer Lett. 2005; 228(1–2):43-50. PubMedhttps://doi.org/10.1016/j.canlet.2005.01.047Google Scholar
- Huang H, Rastegar M, Bodner C, Goh SL, Rambaldi I, Featherstone M. MEIS C termini harbor transcriptional activation domains that respond to cell signaling. J Biol Chem. 2005; 280(11):10119-27. PubMedhttps://doi.org/10.1074/jbc.M413963200Google Scholar
- Hu YL, Fong S, Ferrell C, Largman C, Shen WF. HOXA9 modulates its oncogenic partner Meis1 to influence normal hematopoiesis. Mol Cell Biol. 2009; 29(18):5181-92. PubMedhttps://doi.org/10.1128/MCB.00545-09Google Scholar
- Pineault N, Helgason CD, Lawrence HJ, Humphries RK. Differential expression of Hox, Meis1, and Pbx1 genes in primitive cells throughout murine hematopoietic ontogeny. Exp Hematol. 2002; 30(1):49-57. PubMedhttps://doi.org/10.1016/S0301-472X(01)00757-3Google Scholar
- Watkins NA, Gusnanto A, de Bono B, De S, Miranda-Saavedra D, Hardie DL. A HaemAtlas: characterizing gene expression in differentiated human blood cells. Blood. 2009; 113(19):e1-9. PubMedhttps://doi.org/10.1182/blood-2008-06-162958Google Scholar
- Carramolino L, Fuentes J, Garcia-Andres C, Azcoitia V, Riethmacher D, Torres M. Platelets play an essential role in separating the blood and lymphatic vasculatures during embryonic angiogenesis. Circ Res. 2010; 106(7):1197-201. PubMedhttps://doi.org/10.1161/CIRCRESAHA.110.218073Google Scholar
- Cvejic A, Serbanovic-Canic J, Stemple DL, Ouwehand WH. The role of meis1 in primitive and definitive hematopoiesis during zebrafish development. Haematologica. 2011; 96(2):190-8. PubMedhttps://doi.org/10.3324/haematol.2010.027698Google Scholar
- Bessa J, Tavares MJ, Santos J, Kikuta H, Laplante M, Becker TS. meis1 regulates cyclin D1 and c-myc expression, and controls the proliferation of the multipotent cells in the early developing zebrafish eye. Development. 2008; 135(5):799-803. PubMedhttps://doi.org/10.1242/dev.011932Google Scholar
- Edvardsson L, Dykes J, Olofsson T. Isolation and characterization of human myeloid progenitor populations–TpoR as discriminator between common myeloid and megakary-ocyte/erythroid progenitors. Exp Hematol. 2006; 34(5):599-609. PubMedhttps://doi.org/10.1016/j.exphem.2006.01.017Google Scholar
- Manz MG, Miyamoto T, Akashi K, Weissman IL. Prospective isolation of human clonogenic common myeloid progenitors. Proc Natl Acad Sci USA. 2002; 99(18):11872-7. PubMedhttps://doi.org/10.1073/pnas.172384399Google Scholar
- Argiropoulos B, Yung E, Xiang P, Lo CY, Kuchenbauer F, Palmqvist L. Linkage of the potent leukemogenic activity of Meis1 to cell-cycle entry and transcriptional regulation of cyclin D3. Blood. 2010; 115(20):4071-82. PubMedhttps://doi.org/10.1182/blood-2009-06-225573Google Scholar
- Jaatinen T, Hemmoranta H, Hautaniemi S, Niemi J, Nicorici D, Laine J. Global gene expression profile of human cord blood-derived CD133+ cells. Stem Cells. 2006; 24(3):631-41. PubMedhttps://doi.org/10.1634/stemcells.2005-0185Google Scholar
- Kumar AR, Li Q, Hudson WA, Chen W, Sam T, Yao Q. A role for MEIS1 in MLL-fusion gene leukemia. Blood. 2009; 113(8):1756-8. PubMedhttps://doi.org/10.1182/blood-2008-06-163287Google Scholar
- Cai M, Langer EM, Gill JG, Satpathy AT, Albring JC, Kc W. Dual actions of Meis1 inhibit erythroid progenitor development and sustain general hematopoietic cell proliferation. Blood. 2012; 120(2):335-46. PubMedhttps://doi.org/10.1182/blood-2012-01-403139Google Scholar
- Unnisa Z, Clark JP, Roychoudhury J, Thomas E, Tessarollo L, Copeland NG. Meis1 preserves hematopoietic stem cells in mice by limiting oxidative stress. Blood. 2012; 120(25):4973-81. PubMedhttps://doi.org/10.1182/blood-2012-06-435800Google Scholar
- Huang H, Cantor AB. Common features of megakaryocytes and hematopoietic stem cells: what’s the connection?. J Cell Biochem. 2009; 107(5):857-64. PubMedhttps://doi.org/10.1002/jcb.22184Google Scholar
- Calvo KR, Knoepfler PS, Sykes DB, Pasillas MP, Kamps MP. Meis1a suppresses differentiation by G-CSF and promotes proliferation by SCF: potential mechanisms of cooperativity with Hoxa9 in myeloid leukemia. Proc Natl Acad Sci USA. 2001; 98(23):13120-5. PubMedhttps://doi.org/10.1073/pnas.231115398Google Scholar
- Shen WF, Rozenfeld S, Kwong A, Kom ves LG, Lawrence HJ, Largman C. HOXA9 forms triple complexes with PBX2 and MEIS1 in myeloid cells. Mol Cell Biol. 1999; 19(4):3051-61. PubMedGoogle Scholar
- Berthelsen J, Kilstrup-Nielsen C, Blasi F, Mavilio F, Zappavigna V. The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH. Genes Dev. 1999; 13(8):946-53. PubMedhttps://doi.org/10.1101/gad.13.8.946Google Scholar
- Passegue E, Wagers AJ, Giuriato S, Anderson WC, Weissman IL. Global analysis of proliferation and cell cycle gene expression in the regulation of hematopoietic stem and progenitor cell fates. J Exp Med. 2005; 202(11):1599-611. PubMedhttps://doi.org/10.1084/jem.20050967Google Scholar
- Kozar K, Ciemerych MA, Rebel VI, Shigematsu H, Zagozdzon A, Sicinska E. Mouse development and cell proliferation in the absence of D-cyclins. Cell. 2004; 118(4):477-91. PubMedhttps://doi.org/10.1016/j.cell.2004.07.025Google Scholar
- Wang Z, Zhang Y, Kamen D, Lees E, Ravid K. Cyclin D3 is essential for megakaryocytopoiesis. Blood. 1995; 86(10):3783-8. PubMedGoogle Scholar