Runx1 Is Expressed in Adult Mouse Hematopoietic Stem Cells and Differentiating Myeloid and Lymphoid Cells, But Not in Maturing Erythroid Cel
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《干细胞学杂志》
a Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, USA;
b (Current address for Dr. de Bruijn) MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom
Key Words. Runx1/AML1/CBF2 ? Hematopoietic stem cells ? Gene expression ? Transcription factor
Marella F.T.R. de Bruijn, Ph.D., MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. Telephone: 44-1865-222397; Fax: 44-1865-222500; e-mail: marella.debruijn@imm.ox.ac.uk
ABSTRACT
Runx1 belongs to the small family of core-binding factors (CBFs). Runx1 binds DNA through a conserved sequence, the Runt domain, and forms together with the non-DNA binding CBF? subunit, the heterodimeric transcription factor Runx1-CBF?. Runx1-CBF? has been found to play a crucial role in definitive hematopoiesis during ontogeny . The high frequencies of RUNX1 and CBFB mutations in acute leukemias and myelodysplasia and the demonstration that haploinsufficiency of RUNX1 causes familial platelet disorder suggest a role for this factor in postnatal hematopoietic differentiation as well . Potential Runx1-CBF? target genes have been identified in the erythroid, myeloid, and lymphoid lineages . Runx1 can either activate or repress transcription, depending on which other proteins bind to Runx1 or adjacent sites in target promoter/enhancer regions . Although both Runx1 and CBF? are expressed in adult bone marrow (BM), thymus, and peripheral lymphoid organs , a detailed characterization and functional analysis of Runx1-expressing cells in adult hematopoiesis has been lacking. Comprehensive mapping of Runx1 expression in hematopoietic stem and progenitor cell populations would facilitate functional studies of the role of Runx1 in normal and aberrant adult hematopoiesis.
We previously generated a Runx1 allele in which two coding exons were replaced with lacZ coding sequences (Runx1lz) . The Runx1-?-galactosidase fusion protein produced from this modified Runx1lz allele contains the N-terminus of Runx1 through amino acid 242 (64 amino acids C-terminal to the DNA-binding Runt domain). Runx11z is a nonfunctional Runx1 allele . The fusion of ?-galactosidase to the DNA-binding domain of Runx1 could potentially interfere with endogenous core-binding factors by creating a protein that can bind DNA but not activate transcription. The fidelity of Runx1-?-galactosidase expression is supported by the observation that it correlates with the presence of Runx1 protein and mRNA in Runx1lz/+ embryos, as detected by immunohistochemistry and in situ hybridization . Here, mice heterozygous for this Runx1lz allele were used to isolate and characterize Runx1-expressing cells. We show that in the adult mouse, Runx1 was expressed in all BM hematopoietic stem cells (HSCs), in spleen colony-forming units (CFU-S), and in the majority of progenitor cells generating myeloid, erythroid and mixed colonies in culture. We also characterized Runx1 expression in more differentiated hematopoietic cells by flow cytometry and compared the BM and peripheral blood compositions of normal mice with those of mice heterozygous for two different mutated Runx1 alleles to determine which lineages are affected by reduced Runx1 dosage.
MATERIALS AND METHODS
Runx1-?-Galactosidase Fusion Protein Levels Are Similar to Wild-Type Runx1 Protein Levels
We utilized the ?-galactosidase enzymatic reaction in cells from Runx1lz/+ mice to analyze Runx1 expression in various hematopoietic cell populations. ?-galactosidase activity from the Runx1-?-galactosidase fusion protein should be a relatively sensitive indicator of Runx1 protein. However, it is possible that the stability of the Runx1-?-galactosidase fusion protein may differ from that of endogenous Runx1. To at least determine whether the steady-state levels of the Runx1-?-galactosidase fusion protein substantially differed from those of endogenous Runx1, we performed a Western blot analysis on thymic extracts prepared from Runx1lz/+ mice (Fig. 1) using a monoclonal antibody that recognizes the Runt domain. We found that the Runx1-?-galactosidase fusion protein did not accumulate to higher levels than the endogenous Runx1 protein.
Figure 1. Western blot analysis of thymic extracts. Thymocytes of Runx1lz/+ and Runx1+/+ mice were analyzed for endogenous and mutant Runx1 protein expression. Lanes 1, 2, and 6: thymic extracts of three Runx1lz/+ mice; lanes 3–5: thymic extracts of three Runx1+/+ mice. The position of Runx1 and Runx1-?-galactosidase proteins is indicated. Molecular weight markers in kilodaltons (kd) are listed on the left.
Adult BM-Derived Hematopoietic Stem and Progenitor Cells Express Runx1
Approximately 80% of cells in the adult Runx1lz/+ BM and 85% of the Lin-c-kit+ BM population, which is enriched for stem and progenitor cells, expressed Runx1 (Fig. 2A and 2B; Table 1). Runx1-expressing (Runx1+) and nonexpressing (Runx1-) cells, isolated from total BM and from the c-kit+ and Lin-c-kit+ BM fractions, were assayed for the presence of HSCs. Upon intravenous transfer of 105 sorted cells into irradiated adult recipients, we found long-term multilineage high-level hematopoietic reconstitution, indicative of HSC activity, only in the Runx1+ populations (Table 1). The extent of the hematopoietic reconstitution was similar to that observed after transplantation of 105 unsorted BM cells (7 of 10 recipients reconstituted, 45%-73% donor-derived BM cells; not shown). No detectable donor cell reconstitution was observed after transfer of Runx1- cells. Thus, virtually all long-term repopulating HSCs express Runx1, beginning with their generation in ontogeny and extending into adult life. We also determined that all progenitor cells capable of forming spleen colonies (CFU-S11) expressed Runx1 (Table 1). In addition, in vitro clonogenic progenitors such as CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM), CFU-granulocyte-macrophage (GM), and BFU-E, were found almost exclusively among Runx1+ cells. The small number of CFU-C in the Runx1- fraction may be due to contamination with Runx1+ cells, or may reflect the presence of some CFU-C (sort purity was 98%).
Figure 2. Flow cytometric analysis of Runx1 expression in BM cells. FDG was used as a fluorescent substrate for ?-galactosidase. In all experiments, Runx1+/+ cells were used as a negative control for FDG loading (as shown in A, panel 1). Gates or markers for the FDG-negative cell populations in A-F were set based on wild-type FDG levels within the different lineage populations (as shown in B panel 2). Appropriate isotype control antibodies were used to determine background fluorescence. Dead cells were always excluded from analysis on the basis of propidium iodide, Hoechst 33258, or TO-PRO3 uptake. A) BM cells from Runx1lz/+ and control +/+ mice were isolated and loaded with FDG. Sort gates are indicated, with representative percentages of cells within each gate. Reanalysis of sorted Runx1+ and Runx1- cells revealed cell purities of 95%-99%. Data are representative of 11 experiments. B) Analysis of Runx1 expression within the Lin- c-kit+ BM population. BM cells were loaded with FDG and labeled with lineage (Mac-1, Gr-1, TER-119, B220, CD3, CD4, CD8) and c-kit antibodies. Sort gates are shown, with percentages of cells within each gate. Data are representative of four experiments. C) Changes in Runx1 expression during myeloid differentiation. Based on differential CD31 and Ly6-C expression , a population of CD31+Ly-6C+ cells containing mainly (approximately 80%) myeloid progenitors and some erythroid progenitors, CD31-Ly-6Cmed neutrophilic granulocytes (from band stage onwards), and CD31-Ly-6Chi monocytes (from promonocyte stage onwards) can be distinguished. Histograms represent Runx1 (FDG) expression within each subset, and are representative of four experiments; average percentages are listed in Table 6. The lower proportion of Runx1-expressing cells in the CD31+Ly-6C+ population most likely resulted from the presence of erythroid cells that were largely Runx1- (Figure 1D). D) Changes in Runx1 expression during erythroid differentiation. BM cells were loaded with FDG and labeled with CD71 and TER-119 antibodies. In the spleen, this combination of antibodies has been shown to detect a maturation series of erythroid precursors with the order CD71+ TER-119- CD71hi TER-119+ CD71med TER-119+ CD71lo TER-119+, representing proerythroblasts, basophilic erythroblasts, chromatophilic erythroblasts, and normoblasts, respectively . In Runx1lz/+ BM, similar phenotypic populations were identified. Morphological inspection of these BM populations after sort and Wright/Giemsa staining showed that they represent the same maturation sequence (data not shown). Histograms show Runx1 (FDG) expression within the four erythroid cell subsets and are representative of six experiments; see Table 6 for average percentages. (E) Levels of Runx1 expression during T-cell maturation. Runx1lz/+ CD4/8 DN, CD4/8 DP, CD4 SP, and CD8 SP thymocyte populations were analyzed for Runx1 expression. Representative FDG histograms of 1 out of 16 experiments are shown. See Table 6 for average Runx1 expression. F) Levels of Runx1 expression during B-cell maturation. Early B220+CD43+, and later B220+CD43- stages of B-cell maturation were analyzed for Runx1 expression. Representative FDG plots are from 1 experiment out of 14; see Table 6 for average percentages of Runx1 expression. G) Schematic of percentages of Runx1+ cells during T-cell maturation. Pie charts represent individual maturation stages; grey shaded areas represent the average percentages of Runx1-expressing cells within each cell population. The percentages of Runx1+ cells (±SD) within the T-cell lineage are shown below the pies for thymic DN1 lymphoid progenitors (CD3-4-8-44+25-), DN2 pro-T (CD3-4-8-44+25+), DN3 early pre-T (CD3-4-8-44-25+), DN4 late pre-T (CD3-4-8-44-25-), DP (CD4+8+), CD4 SP (CD4+8-), CD8 SP (CD4-8+), spleen CD4 SP (CD4+8-), spleen CD8 SP (CD4-8+), thymic NK cell (CD3-4-8- NK1.1+), and T cells (CD4-8- TCR+). No obvious changes compared with wild type were observed in the relative sizes of thymic NK and T cells, or in the relative sizes of DN1-4 subsets (data not shown). Data are from a total of 14 experiments. H) Percentages of Runx1+cells (±SD) within B-cell differentiation are shown for pre-pro-B (B220+ CD43+ HSA- BP1-), pro-B (B220+ CD43+ HSA+ BP1-), large pre-B (B220+ CD43+ HSA+ BP1+), small pre-B (B220+ CD43- IgM-), immature B (B220+ CD43- IgM+ IgD-), mature B (B220+ CD43- IgM+ IgD+), and spleen mature B (B220+ CD43- IgM+ IgD+) cells. No difference in Runx1 expression was observed between the different spleen IgM+ IgD+ cells: IgMhigh IgDlow (62% ± 6% Runx1+), IgMhigh IgDhigh (65% ± 9%) and IgMlow IgDhigh (58% ± 6%) (not shown). Data are from a total of 10 experiments.
Table 1. BM hematopoietic stem and progenitor cells express Runx1
Decreased Cellularity in Hematopoietic Organs of Runx1lz/+ Mice
We consistently observed a 25% overall reduction in cellularity in BM and thymus from Runx1lz/+ mice compared with age- and sex-matched Runx1+/+ control mice (Table 2), while no significant decrease was observed in the spleen. In peripheral blood, no significant decrease was found either (Table 2), although we observed that four of seven mice analyzed had leukocyte counts between 1.1 and 1.8 x 106/ml, low counts that were not observed among the six wild-type mice analyzed.
Table 2. Cellularity* of hematopoietic organs and peripheral blood in Runx1lz/+ and Runx1rd/+ mice
The Runx1-?-galactosidase fusion protein produced from the Runx1lz allele has the potential to bind DNA and dominantly inhibit Runx1-CBF? function. To exclude the possibility that the consistent decrease in BM and thymus cellularity was specific to the Runx1lz allele, mice heterozygous for a nonfunctional Runx1 allele, in which an exon encoding a portion of the DNA-binding runt domain was deleted (Runx1rd/+ ), were also examined. A similar decrease in the cellularity of BM and thymus was seen in Runx1rd/+ and Runx1lz/+ mice (Table 2). In addition, the decrease in spleen cell count observed in the Runx1rd/+ mice versus wild-type mice was statistically significant. The difference in peripheral blood leukocyte counts between the Runx1rd/+ mice and wild-type mice was, like the difference between Runx1lz/+ and wild-type counts, not significant. However, we did find low leukocyte counts (1.1 and 1.6 x 106 nucleated cells/ml) in two of five Runx1rd/+ mice analyzed.
Small changes were seen in the cellular composition of BM and peripheral blood from Runx1lz/+ and Runx1rd/+ mice compared with BM and blood from wild-type mice (Tables 3 and 4). In BM there was a small but significant increase in the proportions of monocyte and granulocyte precursors (Mac-1 and Gr-1 cells), while for most other populations there were decreases in frequency (Table 3). In addition, we tested the effect of Runx1 gene dosage on early progenitor cells in functional assays. Similar frequencies of CFU-S, CFU-GEMM, CFU-GM, and BFU-E were obtained from both wild-type and Runx1lz/+ BM (Table 5). The adult stem cell compartment was not examined.
Table 3. Changes in bone marrow composition detected in Runx1lz/+ and Runx1rd/+ mice
Table 4. Changes in peripheral blood composition detected in Runx1lz/+ and Runx1rd/+ mice
Table 5. Comparison of CFU-S* and CFU-C# frequencies in Runx1+/+ and Runx1lz/+ BM
Granulocytes and Monocytes Differ in Runx1 Expression Levels
Many of the oncogenic fusion genes involving Runx1 are associated with myeloid leukemias . The defect is in part the failure of progenitor cells to differentiate properly, suggesting a role for Runx1 in myeloid differentiation, at least in the context of secondary mutations . In BM myeloid differentiation, the large majority of myeloid blast cells, granulocytes from the band stage onwards, and monocytes from the promonocytic stage onwards expressed Runx1 (Fig. 2C). This is consistent with the reported broad expression of Runx1 mRNA and CBF family proteins in BM myeloid cells and the high frequency of Runx1+ cells detected among Mac-1+ and Gr-1+ cells in BM and peripheral blood (Tables 3 and 4). Notably, the level of Runx1 expression in BM granulocytes was approximately 4.5-fold lower than in monocytes (Fig. 2C). Runx1+ cells were also found among mature Mac3+ macrophages and CD11c+ dendritic cells in BM, albeit at lower frequencies (on average 43%-58%; Table 3). In the megakaryocytic lineage, on average, 50% of BM CD62P+ megakaryocytes (Table 3) and 59% ± 12% of peripheral blood CD41+ platelets (n = 4; not shown) expressed Runx1. CBF? expression has also been reported in BM myeloid cells from CFU-GM through fully differentiated cells , supporting a role for Runx1-CBF? throughout myeloid differentiation.
Runx1 Expression Decreases Upon Erythroid Maturation
Almost all BFU-E expressed Runx1 (Table 1), yet erythroid cells in BM are reported to express little or no RUNX1 protein and mRNA or other proteins of the CBF family . Indeed, only 26% ± 3% of TER-119+ cells were found to express Runx1 (Table 3). Examination of Runx1 expression during erythroid maturation showed that while, on average, 64% of proerythroblasts still expressed Runx1, this percentage decreased upon maturation to only 16% ± 4% of normoblasts (Fig. 2D, Table 6); the levels of Runx1 expression in individual cells also decreased (Fig. 2D). A similar decrease in CBF? expression was reported . Thus, Runx1-CBF? could be involved in the initial activation of erythroid-specific genes at the CFU-GEMM or BFU-E stages , but is probably not required for terminal erythroid maturation .
Table 6. Runx1 expression and cellular composition of myeloid, erythroid, and lymphoid lineages from Runx1lz/+ and Runx1+/+ mice
Fewer Mature Than Immature Lymphoid Cells Express Runx1
Runx1 has been shown to play a role at distinct stages in T-cell development , and Runx1 expression has been detected in the thymic cortex, the major CD4/CD8-defined thymocyte populations, and spleen T cells . In addition, a translocation involving RUNX1 has recently been reported in acute T-lymphoblastic leukemia, and proviral insertion studies indicated Runx1 as a dominant oncogene in T-cell lymphoma . CBF? protein expression was reported in all stages of T-cell differentiation .
T-cell development in the thymus is characterized by the maturation of CD4 and CD8 single-positive (SP) cells from a lymphoid precursor population. Hayashi et al. showed an effect of decreased Runx1 dosage in thymocyte maturation characterized by reduced numbers of CD4 and CD8 SP T cells and a relative increase in the percentage of CD4/CD8 double-positive (DP) T cells. In accordance with this, we observed similar changes in CD4/CD8 populations in thymocytes and reduced numbers of CD4 and CD8 SP cells in BM and peripheral blood from Runx1lz/+ mice (Tables 3, 4, and 6). We examined Runx1 expression within the distinct CD4/CD8 thymocyte populations. On average, 44% ± 11% of double negative (DN) cells expressed Runx1 (Fig. 2E; Table 6). The majority of cells in the first three DN stages expressed Runx1 (Fig. 2G), with no observed differences in levels of Runx1 expression between the three subsets (data not shown). In the fourth DN stage, the percentage of Runx1-expressing cells decreased dramatically (Figure 2G). The percentage of Runx1+ cells increases at later stages of T-cell differentiation (Fig. 2E and 2G; Tables 3, 4, and 6).
A role for Runx1 in B lymphopoiesis is suggested by the association of the t(12;21) TEL-Runx1 fusion protein with pediatric acute B-lymphocytic leukemia . We found Runx1 expressed at all stages of differentiation (Fig. 2F and 2H; Table 6) and in peripheral blood B cells (Table 4). During B-cell differentiation in the BM, on average, 48%-68% of the cells are Runx1+ (Fig. 2F and 2H; Table 6), with no obvious differences in the levels of Runx1 expression between immature and more mature B-cell populations (Fig. 2F, and data not shown).
DISCUSSION
We thank Dr. Alice Givan, Gary Ward, Ann Atzberger, and Christiane Kuhl for their help with flow cytometry, and Drs. Rudi Hendriks, Tariq Enver, Catherine Porcher, and Paresh Vyas for helpful discussions. T.E.N. and C.J.M. were supported by T32GM08704 from the NIH/GM and N.A.S. by Public Health Service grant R01CA58343. M.F.T.R.d.B. was supported by a fellowship from the Dutch Cancer Society. Flow cytometry at Dartmouth Medical School was done in The Herbert C. Englert Cell Analysis Laboratory, established by a grant from the Fannie E. Rippel Foundation and supported in part by the Core Grant of the Norris Cotton Cancer Center (CA 23108).
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b (Current address for Dr. de Bruijn) MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom
Key Words. Runx1/AML1/CBF2 ? Hematopoietic stem cells ? Gene expression ? Transcription factor
Marella F.T.R. de Bruijn, Ph.D., MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. Telephone: 44-1865-222397; Fax: 44-1865-222500; e-mail: marella.debruijn@imm.ox.ac.uk
ABSTRACT
Runx1 belongs to the small family of core-binding factors (CBFs). Runx1 binds DNA through a conserved sequence, the Runt domain, and forms together with the non-DNA binding CBF? subunit, the heterodimeric transcription factor Runx1-CBF?. Runx1-CBF? has been found to play a crucial role in definitive hematopoiesis during ontogeny . The high frequencies of RUNX1 and CBFB mutations in acute leukemias and myelodysplasia and the demonstration that haploinsufficiency of RUNX1 causes familial platelet disorder suggest a role for this factor in postnatal hematopoietic differentiation as well . Potential Runx1-CBF? target genes have been identified in the erythroid, myeloid, and lymphoid lineages . Runx1 can either activate or repress transcription, depending on which other proteins bind to Runx1 or adjacent sites in target promoter/enhancer regions . Although both Runx1 and CBF? are expressed in adult bone marrow (BM), thymus, and peripheral lymphoid organs , a detailed characterization and functional analysis of Runx1-expressing cells in adult hematopoiesis has been lacking. Comprehensive mapping of Runx1 expression in hematopoietic stem and progenitor cell populations would facilitate functional studies of the role of Runx1 in normal and aberrant adult hematopoiesis.
We previously generated a Runx1 allele in which two coding exons were replaced with lacZ coding sequences (Runx1lz) . The Runx1-?-galactosidase fusion protein produced from this modified Runx1lz allele contains the N-terminus of Runx1 through amino acid 242 (64 amino acids C-terminal to the DNA-binding Runt domain). Runx11z is a nonfunctional Runx1 allele . The fusion of ?-galactosidase to the DNA-binding domain of Runx1 could potentially interfere with endogenous core-binding factors by creating a protein that can bind DNA but not activate transcription. The fidelity of Runx1-?-galactosidase expression is supported by the observation that it correlates with the presence of Runx1 protein and mRNA in Runx1lz/+ embryos, as detected by immunohistochemistry and in situ hybridization . Here, mice heterozygous for this Runx1lz allele were used to isolate and characterize Runx1-expressing cells. We show that in the adult mouse, Runx1 was expressed in all BM hematopoietic stem cells (HSCs), in spleen colony-forming units (CFU-S), and in the majority of progenitor cells generating myeloid, erythroid and mixed colonies in culture. We also characterized Runx1 expression in more differentiated hematopoietic cells by flow cytometry and compared the BM and peripheral blood compositions of normal mice with those of mice heterozygous for two different mutated Runx1 alleles to determine which lineages are affected by reduced Runx1 dosage.
MATERIALS AND METHODS
Runx1-?-Galactosidase Fusion Protein Levels Are Similar to Wild-Type Runx1 Protein Levels
We utilized the ?-galactosidase enzymatic reaction in cells from Runx1lz/+ mice to analyze Runx1 expression in various hematopoietic cell populations. ?-galactosidase activity from the Runx1-?-galactosidase fusion protein should be a relatively sensitive indicator of Runx1 protein. However, it is possible that the stability of the Runx1-?-galactosidase fusion protein may differ from that of endogenous Runx1. To at least determine whether the steady-state levels of the Runx1-?-galactosidase fusion protein substantially differed from those of endogenous Runx1, we performed a Western blot analysis on thymic extracts prepared from Runx1lz/+ mice (Fig. 1) using a monoclonal antibody that recognizes the Runt domain. We found that the Runx1-?-galactosidase fusion protein did not accumulate to higher levels than the endogenous Runx1 protein.
Figure 1. Western blot analysis of thymic extracts. Thymocytes of Runx1lz/+ and Runx1+/+ mice were analyzed for endogenous and mutant Runx1 protein expression. Lanes 1, 2, and 6: thymic extracts of three Runx1lz/+ mice; lanes 3–5: thymic extracts of three Runx1+/+ mice. The position of Runx1 and Runx1-?-galactosidase proteins is indicated. Molecular weight markers in kilodaltons (kd) are listed on the left.
Adult BM-Derived Hematopoietic Stem and Progenitor Cells Express Runx1
Approximately 80% of cells in the adult Runx1lz/+ BM and 85% of the Lin-c-kit+ BM population, which is enriched for stem and progenitor cells, expressed Runx1 (Fig. 2A and 2B; Table 1). Runx1-expressing (Runx1+) and nonexpressing (Runx1-) cells, isolated from total BM and from the c-kit+ and Lin-c-kit+ BM fractions, were assayed for the presence of HSCs. Upon intravenous transfer of 105 sorted cells into irradiated adult recipients, we found long-term multilineage high-level hematopoietic reconstitution, indicative of HSC activity, only in the Runx1+ populations (Table 1). The extent of the hematopoietic reconstitution was similar to that observed after transplantation of 105 unsorted BM cells (7 of 10 recipients reconstituted, 45%-73% donor-derived BM cells; not shown). No detectable donor cell reconstitution was observed after transfer of Runx1- cells. Thus, virtually all long-term repopulating HSCs express Runx1, beginning with their generation in ontogeny and extending into adult life. We also determined that all progenitor cells capable of forming spleen colonies (CFU-S11) expressed Runx1 (Table 1). In addition, in vitro clonogenic progenitors such as CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM), CFU-granulocyte-macrophage (GM), and BFU-E, were found almost exclusively among Runx1+ cells. The small number of CFU-C in the Runx1- fraction may be due to contamination with Runx1+ cells, or may reflect the presence of some CFU-C (sort purity was 98%).
Figure 2. Flow cytometric analysis of Runx1 expression in BM cells. FDG was used as a fluorescent substrate for ?-galactosidase. In all experiments, Runx1+/+ cells were used as a negative control for FDG loading (as shown in A, panel 1). Gates or markers for the FDG-negative cell populations in A-F were set based on wild-type FDG levels within the different lineage populations (as shown in B panel 2). Appropriate isotype control antibodies were used to determine background fluorescence. Dead cells were always excluded from analysis on the basis of propidium iodide, Hoechst 33258, or TO-PRO3 uptake. A) BM cells from Runx1lz/+ and control +/+ mice were isolated and loaded with FDG. Sort gates are indicated, with representative percentages of cells within each gate. Reanalysis of sorted Runx1+ and Runx1- cells revealed cell purities of 95%-99%. Data are representative of 11 experiments. B) Analysis of Runx1 expression within the Lin- c-kit+ BM population. BM cells were loaded with FDG and labeled with lineage (Mac-1, Gr-1, TER-119, B220, CD3, CD4, CD8) and c-kit antibodies. Sort gates are shown, with percentages of cells within each gate. Data are representative of four experiments. C) Changes in Runx1 expression during myeloid differentiation. Based on differential CD31 and Ly6-C expression , a population of CD31+Ly-6C+ cells containing mainly (approximately 80%) myeloid progenitors and some erythroid progenitors, CD31-Ly-6Cmed neutrophilic granulocytes (from band stage onwards), and CD31-Ly-6Chi monocytes (from promonocyte stage onwards) can be distinguished. Histograms represent Runx1 (FDG) expression within each subset, and are representative of four experiments; average percentages are listed in Table 6. The lower proportion of Runx1-expressing cells in the CD31+Ly-6C+ population most likely resulted from the presence of erythroid cells that were largely Runx1- (Figure 1D). D) Changes in Runx1 expression during erythroid differentiation. BM cells were loaded with FDG and labeled with CD71 and TER-119 antibodies. In the spleen, this combination of antibodies has been shown to detect a maturation series of erythroid precursors with the order CD71+ TER-119- CD71hi TER-119+ CD71med TER-119+ CD71lo TER-119+, representing proerythroblasts, basophilic erythroblasts, chromatophilic erythroblasts, and normoblasts, respectively . In Runx1lz/+ BM, similar phenotypic populations were identified. Morphological inspection of these BM populations after sort and Wright/Giemsa staining showed that they represent the same maturation sequence (data not shown). Histograms show Runx1 (FDG) expression within the four erythroid cell subsets and are representative of six experiments; see Table 6 for average percentages. (E) Levels of Runx1 expression during T-cell maturation. Runx1lz/+ CD4/8 DN, CD4/8 DP, CD4 SP, and CD8 SP thymocyte populations were analyzed for Runx1 expression. Representative FDG histograms of 1 out of 16 experiments are shown. See Table 6 for average Runx1 expression. F) Levels of Runx1 expression during B-cell maturation. Early B220+CD43+, and later B220+CD43- stages of B-cell maturation were analyzed for Runx1 expression. Representative FDG plots are from 1 experiment out of 14; see Table 6 for average percentages of Runx1 expression. G) Schematic of percentages of Runx1+ cells during T-cell maturation. Pie charts represent individual maturation stages; grey shaded areas represent the average percentages of Runx1-expressing cells within each cell population. The percentages of Runx1+ cells (±SD) within the T-cell lineage are shown below the pies for thymic DN1 lymphoid progenitors (CD3-4-8-44+25-), DN2 pro-T (CD3-4-8-44+25+), DN3 early pre-T (CD3-4-8-44-25+), DN4 late pre-T (CD3-4-8-44-25-), DP (CD4+8+), CD4 SP (CD4+8-), CD8 SP (CD4-8+), spleen CD4 SP (CD4+8-), spleen CD8 SP (CD4-8+), thymic NK cell (CD3-4-8- NK1.1+), and T cells (CD4-8- TCR+). No obvious changes compared with wild type were observed in the relative sizes of thymic NK and T cells, or in the relative sizes of DN1-4 subsets (data not shown). Data are from a total of 14 experiments. H) Percentages of Runx1+cells (±SD) within B-cell differentiation are shown for pre-pro-B (B220+ CD43+ HSA- BP1-), pro-B (B220+ CD43+ HSA+ BP1-), large pre-B (B220+ CD43+ HSA+ BP1+), small pre-B (B220+ CD43- IgM-), immature B (B220+ CD43- IgM+ IgD-), mature B (B220+ CD43- IgM+ IgD+), and spleen mature B (B220+ CD43- IgM+ IgD+) cells. No difference in Runx1 expression was observed between the different spleen IgM+ IgD+ cells: IgMhigh IgDlow (62% ± 6% Runx1+), IgMhigh IgDhigh (65% ± 9%) and IgMlow IgDhigh (58% ± 6%) (not shown). Data are from a total of 10 experiments.
Table 1. BM hematopoietic stem and progenitor cells express Runx1
Decreased Cellularity in Hematopoietic Organs of Runx1lz/+ Mice
We consistently observed a 25% overall reduction in cellularity in BM and thymus from Runx1lz/+ mice compared with age- and sex-matched Runx1+/+ control mice (Table 2), while no significant decrease was observed in the spleen. In peripheral blood, no significant decrease was found either (Table 2), although we observed that four of seven mice analyzed had leukocyte counts between 1.1 and 1.8 x 106/ml, low counts that were not observed among the six wild-type mice analyzed.
Table 2. Cellularity* of hematopoietic organs and peripheral blood in Runx1lz/+ and Runx1rd/+ mice
The Runx1-?-galactosidase fusion protein produced from the Runx1lz allele has the potential to bind DNA and dominantly inhibit Runx1-CBF? function. To exclude the possibility that the consistent decrease in BM and thymus cellularity was specific to the Runx1lz allele, mice heterozygous for a nonfunctional Runx1 allele, in which an exon encoding a portion of the DNA-binding runt domain was deleted (Runx1rd/+ ), were also examined. A similar decrease in the cellularity of BM and thymus was seen in Runx1rd/+ and Runx1lz/+ mice (Table 2). In addition, the decrease in spleen cell count observed in the Runx1rd/+ mice versus wild-type mice was statistically significant. The difference in peripheral blood leukocyte counts between the Runx1rd/+ mice and wild-type mice was, like the difference between Runx1lz/+ and wild-type counts, not significant. However, we did find low leukocyte counts (1.1 and 1.6 x 106 nucleated cells/ml) in two of five Runx1rd/+ mice analyzed.
Small changes were seen in the cellular composition of BM and peripheral blood from Runx1lz/+ and Runx1rd/+ mice compared with BM and blood from wild-type mice (Tables 3 and 4). In BM there was a small but significant increase in the proportions of monocyte and granulocyte precursors (Mac-1 and Gr-1 cells), while for most other populations there were decreases in frequency (Table 3). In addition, we tested the effect of Runx1 gene dosage on early progenitor cells in functional assays. Similar frequencies of CFU-S, CFU-GEMM, CFU-GM, and BFU-E were obtained from both wild-type and Runx1lz/+ BM (Table 5). The adult stem cell compartment was not examined.
Table 3. Changes in bone marrow composition detected in Runx1lz/+ and Runx1rd/+ mice
Table 4. Changes in peripheral blood composition detected in Runx1lz/+ and Runx1rd/+ mice
Table 5. Comparison of CFU-S* and CFU-C# frequencies in Runx1+/+ and Runx1lz/+ BM
Granulocytes and Monocytes Differ in Runx1 Expression Levels
Many of the oncogenic fusion genes involving Runx1 are associated with myeloid leukemias . The defect is in part the failure of progenitor cells to differentiate properly, suggesting a role for Runx1 in myeloid differentiation, at least in the context of secondary mutations . In BM myeloid differentiation, the large majority of myeloid blast cells, granulocytes from the band stage onwards, and monocytes from the promonocytic stage onwards expressed Runx1 (Fig. 2C). This is consistent with the reported broad expression of Runx1 mRNA and CBF family proteins in BM myeloid cells and the high frequency of Runx1+ cells detected among Mac-1+ and Gr-1+ cells in BM and peripheral blood (Tables 3 and 4). Notably, the level of Runx1 expression in BM granulocytes was approximately 4.5-fold lower than in monocytes (Fig. 2C). Runx1+ cells were also found among mature Mac3+ macrophages and CD11c+ dendritic cells in BM, albeit at lower frequencies (on average 43%-58%; Table 3). In the megakaryocytic lineage, on average, 50% of BM CD62P+ megakaryocytes (Table 3) and 59% ± 12% of peripheral blood CD41+ platelets (n = 4; not shown) expressed Runx1. CBF? expression has also been reported in BM myeloid cells from CFU-GM through fully differentiated cells , supporting a role for Runx1-CBF? throughout myeloid differentiation.
Runx1 Expression Decreases Upon Erythroid Maturation
Almost all BFU-E expressed Runx1 (Table 1), yet erythroid cells in BM are reported to express little or no RUNX1 protein and mRNA or other proteins of the CBF family . Indeed, only 26% ± 3% of TER-119+ cells were found to express Runx1 (Table 3). Examination of Runx1 expression during erythroid maturation showed that while, on average, 64% of proerythroblasts still expressed Runx1, this percentage decreased upon maturation to only 16% ± 4% of normoblasts (Fig. 2D, Table 6); the levels of Runx1 expression in individual cells also decreased (Fig. 2D). A similar decrease in CBF? expression was reported . Thus, Runx1-CBF? could be involved in the initial activation of erythroid-specific genes at the CFU-GEMM or BFU-E stages , but is probably not required for terminal erythroid maturation .
Table 6. Runx1 expression and cellular composition of myeloid, erythroid, and lymphoid lineages from Runx1lz/+ and Runx1+/+ mice
Fewer Mature Than Immature Lymphoid Cells Express Runx1
Runx1 has been shown to play a role at distinct stages in T-cell development , and Runx1 expression has been detected in the thymic cortex, the major CD4/CD8-defined thymocyte populations, and spleen T cells . In addition, a translocation involving RUNX1 has recently been reported in acute T-lymphoblastic leukemia, and proviral insertion studies indicated Runx1 as a dominant oncogene in T-cell lymphoma . CBF? protein expression was reported in all stages of T-cell differentiation .
T-cell development in the thymus is characterized by the maturation of CD4 and CD8 single-positive (SP) cells from a lymphoid precursor population. Hayashi et al. showed an effect of decreased Runx1 dosage in thymocyte maturation characterized by reduced numbers of CD4 and CD8 SP T cells and a relative increase in the percentage of CD4/CD8 double-positive (DP) T cells. In accordance with this, we observed similar changes in CD4/CD8 populations in thymocytes and reduced numbers of CD4 and CD8 SP cells in BM and peripheral blood from Runx1lz/+ mice (Tables 3, 4, and 6). We examined Runx1 expression within the distinct CD4/CD8 thymocyte populations. On average, 44% ± 11% of double negative (DN) cells expressed Runx1 (Fig. 2E; Table 6). The majority of cells in the first three DN stages expressed Runx1 (Fig. 2G), with no observed differences in levels of Runx1 expression between the three subsets (data not shown). In the fourth DN stage, the percentage of Runx1-expressing cells decreased dramatically (Figure 2G). The percentage of Runx1+ cells increases at later stages of T-cell differentiation (Fig. 2E and 2G; Tables 3, 4, and 6).
A role for Runx1 in B lymphopoiesis is suggested by the association of the t(12;21) TEL-Runx1 fusion protein with pediatric acute B-lymphocytic leukemia . We found Runx1 expressed at all stages of differentiation (Fig. 2F and 2H; Table 6) and in peripheral blood B cells (Table 4). During B-cell differentiation in the BM, on average, 48%-68% of the cells are Runx1+ (Fig. 2F and 2H; Table 6), with no obvious differences in the levels of Runx1 expression between immature and more mature B-cell populations (Fig. 2F, and data not shown).
DISCUSSION
We thank Dr. Alice Givan, Gary Ward, Ann Atzberger, and Christiane Kuhl for their help with flow cytometry, and Drs. Rudi Hendriks, Tariq Enver, Catherine Porcher, and Paresh Vyas for helpful discussions. T.E.N. and C.J.M. were supported by T32GM08704 from the NIH/GM and N.A.S. by Public Health Service grant R01CA58343. M.F.T.R.d.B. was supported by a fellowship from the Dutch Cancer Society. Flow cytometry at Dartmouth Medical School was done in The Herbert C. Englert Cell Analysis Laboratory, established by a grant from the Fannie E. Rippel Foundation and supported in part by the Core Grant of the Norris Cotton Cancer Center (CA 23108).
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