《Nature》：抑制造血干细胞功能的机制
这个帖子发布于 20 年零 241 天前，其中的信息可能已发生改变或有所发展。
造血干细胞（ＨＳＣ）在我们的一生中维持血细胞的生产。只需一个细胞就足以恢复一个经过致命辐射的肌体的血液供应，所以，当只需要少量新细胞时，控制ＨＳＣ的这种增殖能力就显得非常重要。怎样才能做到这一点呢？研究人员在本期Ｎａｔｕｒｅ上报告说，他们发现，小鼠的Ｇｆｉ－１（独立生长因子－１）基因限制ＨＳＣ的增殖。这一发现为控制ＨＳＣ增殖提供了线索。研究人员曾发现，白血病与Ｇｆｉ－１受损有关，但该基因的这种抗增殖功能却是未曾料到的。Ｇｆｉ－１基因发生突变的小鼠产生很多ＨＳＣ，尽管它们在重新增加血细胞方面效果不像来自非突变动物的细胞那么好。
Nature \ 431, 1002 - 1007 (21 October 2004); doi:10.1038/nature02994
Nature AOP, published online 29 September 2004
Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells
HANNO HOCK1,2,3, MELANIE J. HAMBLEN1,4, HEATHER M. ROOKE1,4, JEFFREY W. SCHINDLER1, SHIREEN SALEQUE1, YUKO FUJIWARA1,4 & STUART H. ORKIN1,3,4
1 Division of Hematology/Oncology, Children's Hospital,
2 Department of Medical Oncology and
3 Department of Pediatric Oncology, Dana Farber Cancer Institute, Harvard Medical School and the
4 Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA
Correspondence and requests for materials should be addressed to S.H.O. (stuart_orkin@dfci.harvard.edu).
Haematopoietic stem cells (HSCs) sustain blood production throughout life. HSCs are capable of extensive proliferative expansion, as a single HSC may reconstitute lethally irradiated hosts1. In steady-state, HSCs remain largely quiescent and self-renew at a constant low rate, forestalling their exhaustion during adult life2, 3. Whereas nuclear regulatory factors promoting proliferative programmes of HSCs in vivo and ex vivo have been identified4-6, transcription factors restricting their cycling have remained elusive. Here we report that the zinc-finger repressor Gfi-1 (growth factor independent 1), a cooperating oncogene in lymphoid cells7, 8, unexpectedly restricts proliferation of HSCs. After loss of Gfi-1, HSCs display elevated proliferation rates as assessed by 5-bromodeoxyuridine incorporation and cell-cycle analysis. Gfi-1-/- HSCs are functionally compromised in competitive repopulation and serial transplantation assays, and are rapidly out-competed in the bone marrow of mouse chimaeras generated with Gfi-1-/- embryonic stem cells. Thus, Gfi-1 is essential to restrict HSC proliferation and to preserve HSC functional integrity.
The Gfi-1 locus (originally known as pal-1) is among the most frequent sites of retroviral integration contributing to the development of lymphoid tumours in mice8-10. Its product, a SNAG-domain-containing zinc-finger transcriptional repressor, promotes the proliferation of T cells (hence, growth factor independent 1)8-10. In the molecular pathogenesis of lymphoma Gfi-1 is a member of the same complementation group as the polycomb gene Bmi-1, suggesting involvement in a similar, or parallel, pathway of oncogenesis11. Recently, Bmi-1 has been shown to be required for proliferation and maintenance of HSCs4, 5. Gfi-1 is also expressed in HSCs (ref. 12 and data not shown) as well as lymphoid and myeloid cells13. Thus, in analogy to the similar involvement of Bmi-1 and Gfi-1 in lymphomagenesis, Gfi-1 might be anticipated to serve as a positive regulator of HSC proliferation.
To address the role of Gfi-1 in HSCs we first determined the immunophenotype and HSC number in Gfi-1-/- mice13 compared with wild-type littermates. The marker profile of Gfi-1-/- HSCs was preserved, albeit with a slight but consistent decrease in the intensity of c-kit expression (Fig. 1a). We confirmed that the Lin-, Sca1+, c-kit+ population of both Gfi-1-/- and control mice was similarly enriched (> 250-fold) in high proliferative potential colony progenitors (HPPCs), which correlate with stem cell activity14, and that Gfi-1-/- Lin-, Sca1+, c-kit+, Flt3- bone marrow cells are capable of lymphomyeloid reconstitution after injection into lethally irradiated recipients (Supplementary Fig. 1). Notably, the proportion of Flt3+ cells within HSCs (Lin-, Sca1+, c-kit+) was markedly reduced (Fig. 1a). In this population Flt3 expression is associated with increased lymphoid repopulating potential but loss of long-term repopulating potential15, 16. Reduced Flt3 expression in this compartment may signify an early lesion in lymphoid development as Gfi-1-/- mice have reduced lymphopoiesis13, 17. Surprisingly, however, the total number of phenotypic, long-term repopulating HSCs (Lin-, Sca1+, c-kit+, Flt3-) was increased in Gfi-1-/- bone marrow (Fig. 1b). This appeared to be in contrast to findings in Bmi-1-/- mice, where phenotypic HSCs are markedly reduced in frequency (10–45-fold)4, and suggested that despite assignment of Gfi-1 to the same complementation group as Bmi-1 based on cooperation in lymphomagenesis, its requirement in HSCs might be distinct.
Figure 1 Immunophenotypic analysis of HSCs in Gfi-1-/- mice. Full legend
High resolution image and legend (69k)
To assess the functional properties of Gfi-1-/- HSCs we transplanted bone marrow from Gfi-1-/- mice or littermate controls into irradiated host animals. In the absence of competitor marrow, a large dose (1.8 106) of Gfi-1-/- bone marrow cells rescued recipients from lethal irradiation and sustained haematopoiesis for >3 months. The reported haematological abnormalities of Gfi-1-/- mice—notably absence of mature granulocytes and the presence of immature myeloid cells—were evident in these reconstituted mice (Fig. 2a). Eventually, such rescued recipients became pancytopenic, succumbed to infections, or residual host haematopoiesis entirely out-competed Gfi-1-/- cells (data not shown). Notably, Gfi-1-/- bone marrow cells displayed a marked lack of activity in serial transplantation: transfer of 5 106 bone marrow cells collected from transplant recipients 6 weeks after primary transplantation of 5 106 bone marrow cells resulted in complete rescue and establishment of long-term haematopoiesis in lethally irradiated secondary recipients when wild-type donor cells were used. However, secondary recipients of the same dose of Gfi-1-/- bone marrow cells succumbed to severe anaemia after 6–8 weeks and failed to display evidence of lymphomyeloid engraftment (data not shown).
Figure 2 Function of Gfi-1-/- HSCs is compromised in bone marrow transplantation assays. Full legend
High resolution image and legend (53k)
To assess HSC functions in a quantitative setting, competitive repopulation experiments were performed with limiting dilutions of bone marrow from Gfi-1-/- mice or wild-type littermates (both CD45.2) transplanted into lethally irradiated recipients along with a constant, small dose (1.8 105) of competitor marrow (CD45.1). Three months after transplantation lymphomyeloid reconstitution was assessed in peripheral blood B cells and granulocytes (Fig. 2b). In groups of mice that had received 7,400 wild-type cells, CD45.2+ (donor) B cells and granulocytes were readily detected. When 6 105 wild-type donor cells were administered, engraftment of B cells and granulocytes from competitor marrow was undetectable. In marked contrast, even 1.8 106 Gfi-1-/- donor cells failed to contribute to myelopoiesis and provided only minimal contribution to B cells. These data indicate that the reconstitution of lymphomyeloid haematopoiesis by Gfi-1-/- HSCs is reduced at least 200-fold compared with controls. Analysis of representative mice confirmed these findings in bone marrow granulocytes, thymic T cells and bone marrow B cells (Fig. 2c). Similarly, haemoglobin isoform analysis confirmed that Gfi-1-/- donor cells provided donor-derived haemoglobin in the absence, but not the presence, of competitor marrow (data not shown). As erythroid cells do not express Gfi-1 (ref. 13), these findings further support the presumption that competition occurred at an early level of haematopoietic development. To corroborate that competition occurred at the stem cell level and did not reflect combined effects in the differentiated lineages, we performed CD45-isotype analysis on cells recovered from HPPCs, which are believed to arise from HSCs or their early progeny14 (Fig. 2c). Again, even in mice that had received 1.8 106 Gfi-1-/- cells, HPPCs were exclusively of competitor origin (Fig. 2c). Thus, competitive repopulation bone marrow transplant assays demonstrate that normal HSC activity is critically dependent on Gfi-1.
As young Gfi-1-/- mice are not anaemic and have sustained myelopoiesis, we infer that Gfi-1 is dispensable for the emergence and embryonic expansion of HSCs13. This conclusion is consistent with phenotypic enumeration of Gfi-1-/- HSCs. Later in life, marrow function deteriorates in Gfi-1-/- animals, at which time HSC function is difficult to assess because of concomitant myeloid abnormalities (ref. 13 and data not shown). To assess HSCs without prior transplantation and in the context of otherwise normal haematopoiesis, we generated chimaeric mice by injection of Gfi-1-/- or Gfi-1+/- (control) embryonic stem (ES) cells into wild-type blastocysts (Fig. 3a, b). In young chimaeras (< 3 weeks of age) progeny of Gfi-1+/- and Gfi-1-/- ES cells were detected in the bone marrow and thymus by Southern blot analysis (Fig. 3c), and ES-cell-derived haemoglobin was observed in peripheral blood (Fig. 3d). In contrast, in chimaeras >2 months, DNA from Gfi-1-/- cells was no longer detected by Southern blot analysis in bone marrow or thymus, and was sharply reduced in spleen, whereas contribution of Gfi-1-/- cell DNA was preserved in non-haematopoietic tissues (Fig. 3e). Gfi-1+/- ES cells consistently contributed to all tissues (Fig. 3e). Haemoglobin isoform analysis confirmed that progeny of Gfi-1+/- but not Gfi-1-/- ES cells contributed to haemoglobin production in adult chimaeras supporting an early haematopoietic defect (Fig. 3f). A defect at the level of HSCs was corroborated by the finding that only Gfi-1+/- but not Gfi-1-/- cells contributed to HPPCs derived from the bone marrow of chimaeric mice (Fig. 3g). Systematic analysis of peripheral blood from chimaeras of different ages revealed that Gfi-1-/- ES-cell-derived haemoglobin was detected during the first month of life, but was subsequently lost in all animals (Fig. 3h). Thus, Gfi-1-/- HSCs initiate, but do not sustain, bone marrow haematopoiesis in chimaeric mice. Taken together, the transplantation and chimaera experiments provide persuasive evidence that the defective function of Gfi-1-/- HSCs is intrinsic to the HSCs themselves rather than a secondary consequence of an impaired haematopoietic microenvironment.
Figure 3 Gfi-1-/- HSCs initiate, but do not sustain, bone marrow haematopoiesis in chimaeric mice. Full legend
High resolution image and legend (35k)
In principle, failure to maintain HSC function, as observed after Gfi-1 loss in both chimaera and transplant settings, might reflect decreased proliferative capacity and depletion of HSCs, as reported for loss of Bmi-1 (ref. 4). However, the number of phenotypic HSCs in young Gfi-1-/- mice was not severely reduced but instead was slightly elevated (Fig. 1a), a finding most consistent with impaired function rather than mere absence. Of note, loss of HSC function has been ascribed to excessive proliferation. Specifically, forced stem cell expansion driven by serial transplantation18, 19 or by genetic disruption of a negative regulatory component of the cell-cycle machinery20 results in functional compromise without depletion of immunophenotyped HSCs19, 20. To address a role for Gfi-1 in HSC proliferation directly we measured DNA synthesis by determining the fraction of cells that had incorporated 5-bromodeoxyuridine (BrdU) during 3-day in vivo exposure (Fig. 4a). Bone marrow was stained with antibodies to identify HSCs (Lin-, Sca1+, c-kit+) and detect nuclear BrdU simultaneously. In agreement with previous data3 28% of HSCs from wild-type mice incorporated BrdU and therefore had proliferated during the exposure. Surprisingly, more than double the percentage (67%) of HSCs from Gfi-1-/- mice proliferated during the same interval. Furthermore, we demonstrated that HSC hyperproliferation is intrinsic to the haematopoietic system via bone marrow transplantation of Gfi-1-/- or control marrow into wild-type mice. As previously reported21, we observed that bone marrow transplant of wild-type HSCs resulted in a sustained increase in HSC proliferation, but in the absence of Gfi-1 this was markedly more pronounced (Supplementary Fig. 2). Interestingly, HSC frequencies were markedly decreased in recipients of Gfi-1-/- marrow, potentially due to exhaustion after the added proliferative stress of transplantation (Supplementary Fig. 2b). As expression of Gfi-1 promotes proliferation of lymphoid cells7-9, we considered whether our results might reflect differences in experimental approach (overexpression versus loss of function) or rather signify authentic context-specific, divergent involvement of Gfi-1 in controlling cellular proliferation. Therefore, we isolated representative haematopoietic cells from Gfi-1-/- and control mice, and assessed cell-cycle stages by measuring DNA content after propidium iodide staining (Fig. 4b). In the thymus of Gfi-1-/- mice, in agreement with markedly reduced overall cellularity13, the proportion of cells in non-proliferative stages of the cell cycle (G0 and G1) was slightly increased whereas cells in active proliferation (S, G2, M) were reduced in number. These findings are consistent with the previous conclusion that Gfi-1 promotes proliferation of lymphoid cells. We also analysed Gr1+ Mac1+ myeloid cells from bone marrow. Granulocytic precursors of Gfi-1-/- mice, although arrested in differentiation and not overtly malignant, progressively expand in bone marrow as mice age13. Remarkably, these cells in Gfi-1-/- mice are highly proliferative as compared with Gr1+ Mac1+ of wild-type mice. Consistent with the BrdU incorporation data, the proportion of cycling long-term-repopulating HSCs (Lin-, Sca1+, c-kit+, Flt3-)—which were sorted by fluorescence-activated cell sorting (FACS) twice to exclude contamination by other cell types—was appreciably increased in Gfi-1-/- mice as compared with controls. Thus, Gfi-1 differentially affects proliferation and cell-cycle progression of haematopoietic cells, dependent on cell lineage and state of differentiation.
Figure 4 Gfi-1 restricts proliferation of HSCs in a cell-context-specific manner. Full legend
High resolution image and legend (130k)
To elucidate potential mechanisms through which Gfi-1 might control HSC growth we used quantitative, real-time polymerase chain reaction with reverse transcription (RT–PCR) to assess transcript levels for candidate molecules previously implicated in HSC or progenitor proliferation. Bmi-1, c-Myc and p27 were expressed at similar levels in wild-type and mutant HSCs. Gata-2, which is thought to promote proliferation, was slightly upregulated. Of note, expression of cyclin-dependent kinase inhibitor and G1 checkpoint regulator p21Cip1/Waf1 was markedly downregulated (at least tenfold) in Gfi-1-/- HSCs (Fig. 4c). Interestingly, p21Cip1/Waf1, a generic regulator of the cell cycle, is required to maintain HSCs (lineage-, Hoechst 33342low) in G0 (distinguished from G1 by low RNA content)20. In the absence of p21Cip1/Waf1, HSCs exhibit impaired serial transplantation capacity. Although the effects of p21Cip1/Waf1 loss on HSC function are less pronounced than those described here, our data argue that p21Cip1/Waf1 is one mediator of Gfi-1 in stem cells. In view of the more extreme phenotype on its loss, we anticipate that Gfi-1 functions through additional pathways.
In keeping with our findings, retroviral insertions into the Gfi-1 locus (which typically trans-activate and cause overexpression) are almost exclusively found in lymphoid tumours in mice10. Thus far, there is no evidence that loss of Gfi-1 function may also contribute to oncogenesis in other settings by releasing proliferative control at the level of HSCs or after commitment to the myeloid lineage. Our data, however, warrant consideration of this possibility. The versatile role of Gfi-1 in proliferation highlights the critical importance of context in transcriptional regulation in vivo, where its function may be modified by cell-type-specific events such as post-translational modifications, interaction with lineage-specific partner molecules, or differential access to regulatory elements of critical target genes. The paralogue of Gfi-1, Gfi-1b, is co-expressed with Gfi-1 in HSCs12, 22 and is slightly ( twofold) upregulated in Gfi-1-/- HSCs (assessed by quantitative PCR; data not shown). Yet, we demonstrated by competitive repopulation and serial transplantation assays using donor cells from Gfi-1b-/- fetal liver that in marked contrast to Gfi-1, Gfi-1b is dispensable for HSC function (Supplementary Fig. 3).
The severe disturbance of HSC function that we have documented in the absence of Gfi-1 is probably the result of excessive cycling. Yet, we cannot exclude the possibility that it reflects additional roles of Gfi-1 in HSCs. Nevertheless, our data provide molecular insight into how the transcriptional machinery protects HSCs by curbing excessive or unnecessary proliferation. Elucidation of the role of Gfi-1 in HSCs provides a novel entry point into the molecular mechanics of the HSC 'brake' for proliferation, and may ultimately generate opportunities for its manipulation.
Methods
Flow cytometry MoFlo, FACS-Calibur or FACS-Scan flow cytometers were used for analysis and sorting. Antibody conjugates and matched isotype controls were obtained from PharMingen or eBioscience. HSC stain: lineage markers CD4 (RM4-5), CD8 (53-6), CD3 (145-2C11), TCR (H54-597), B220 (RA3-6B2), CD19 (6D5), Gr1 (RB6-8C5), Mac-1 (M1/70), Ter119 (all Cy-Chrome or PE-Cy5); CD117 (c-kit, 2B8, APC), CD135 (Flt3, A2F10, PE), Sca-1 (E13-161, fluorescein isothiocyanate), propidium iodide (1 µg ml-1; Molecular Probes). CD45 isotype analysis: B220 (RA3-6B2, APC), Gr1 (RB6-8C5, APC), Mac1 (M1/70, PE), CD45.1 (104, fluorescein isothiocyanate), CD45.2 (A20, PE, fluorescein isothiocyanate).
Bone marrow transplantation Bone marrow transplantations were performed by intravenous injection of donor cells from 4-week-old Gfi-1-/- mice or littermates (C57bl6/129 mixed) and/or competitor marrow (B6.SJL; 002014, Jackson) into recipients that had received 1,200 cGy of radiation as a split dose. Donor cells from Gfi-1b-/- fetal livers were obtained on day 12.5 of embryonic development after timed mating of heterozygotes22. Recipients were B6/129SF1/J (101043; Jackson) in experiments shown in Fig. 2. Similar results were obtained using B6.SJL mice as recipients, which were also used in experiments shown in Supplementary Figs 1, 2 and 3. Southern blot, automated blood counts and bone marrow morphology was as described13.
Colony-forming assays HPPCs were generated by seeding bone marrow cells into methylcellulose-containing media (MethoCult 3234; Stem Cell Technologies) in the presence of SCF, interleukin (IL)-3, EPO, IL-6, IL-1 granulocyte–macrophage colony-stimulating factor and macrophage colony-stimulating factor (all R&D Systems). Colonies with a tight centre and a diameter 1 mm at day 12 were isolated using a capillary under an inverted microscope, pooled and processed for FACS staining or DNA extraction.
Generation of chimaeras Targeting of Gfi-1 in ES cells has been described13. ES cells homozygous for Gfi-1 deletion were obtained by selecting clones that grew despite increased neomycin concentration (1.7 mg ml–1) owing to crossing over of the targeted allele. Subsequent Southern blot analysis confirmed duplication of the targeted mutation and absence of wild-type Gfi-1 (probe B13). Chimaeras were obtained by injecting Gfi-1+/- or Gfi-1-/- ES clones into C57/bl6 blastocysts. Chimaerism was assessed by agouti coat-colour contribution. Only animals with contribution >70% were selected for analysis. Southern blot analysis of selected tissues after DNA extraction (internal probe13) and haemoglobin isoform analysis was performed as described23.
BrdU incorporation Analysis of BrdU incorporation was performed using the FITC BrdU Flow Kit (PharMingen) after a single intraperitoneal injection of BrdU (PharMingen, 1 mg per 6 g of mouse weight) and admixture of 1 mg ml-1 of BrdU (Sigma, 1 mg ml-1) to drinking water for 3 days (or 2 days after transplant). Mice were 4–6 weeks of age. Surface stain consisted of lineage markers as above, CD117 (c-kit, 2B8, APC) and Sca-1 (E13-161, PE).
Cell-cycle analysis Cell-cycle analysis of thymocytes, or sorted myeloid and HSC populations was performed after ethanol fixation, RNase digestion and propidium iodide staining (Roche, 1399861) using BD ModFit LT software.
Gene expression analysis RNA was isolated from HSCs (8,100 cells; derived from pooled bone marrow of 6–7 mice) using the RNeasy Micro Kit (Qiagen) and recommended protocol. First-strand complementary DNA synthesis was primed with OligodT (Invitrogen) from sample RNA (2 µl) or wild-type bone marrow RNA (100 ng) using the Sensiscript RT Kit (Qiagen). For real-time PCR analysis, cDNA (1.5 µl) was mixed with iQ SYBR green supermix reagent (BioRad), primers (Supplementary Table 1) and amplification plots were generated using the iCycler Thermal cycler and iQ 4-colour real-time detection system (BioRad). To generate standard curves, cDNA from wild-type bone marrow RNA (100 ng) was used as template in a threefold dilution series (0–81-fold). Sample cDNA was used undiluted. Relative expression was calculated using the standard curve method (ABI Prism 7700 Sequence Detection System, user Bulletin 2, PE Applied Biosystems). Primers as in Supplementary Table 1.
Supplementary information accompanies this paper.
Received 14 May 2004;accepted 10 August 2004
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Nature \ 431, 1002 - 1007 (21 October 2004); doi:10.1038/nature02994
Nature AOP, published online 29 September 2004
Gfi-1 restricts proliferation and preserves functional integrity of haematopoietic stem cells
HANNO HOCK1,2,3, MELANIE J. HAMBLEN1,4, HEATHER M. ROOKE1,4, JEFFREY W. SCHINDLER1, SHIREEN SALEQUE1, YUKO FUJIWARA1,4 & STUART H. ORKIN1,3,4
1 Division of Hematology/Oncology, Children's Hospital,
2 Department of Medical Oncology and
3 Department of Pediatric Oncology, Dana Farber Cancer Institute, Harvard Medical School and the
4 Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA
Correspondence and requests for materials should be addressed to S.H.O. (stuart_orkin@dfci.harvard.edu).
Haematopoietic stem cells (HSCs) sustain blood production throughout life. HSCs are capable of extensive proliferative expansion, as a single HSC may reconstitute lethally irradiated hosts1. In steady-state, HSCs remain largely quiescent and self-renew at a constant low rate, forestalling their exhaustion during adult life2, 3. Whereas nuclear regulatory factors promoting proliferative programmes of HSCs in vivo and ex vivo have been identified4-6, transcription factors restricting their cycling have remained elusive. Here we report that the zinc-finger repressor Gfi-1 (growth factor independent 1), a cooperating oncogene in lymphoid cells7, 8, unexpectedly restricts proliferation of HSCs. After loss of Gfi-1, HSCs display elevated proliferation rates as assessed by 5-bromodeoxyuridine incorporation and cell-cycle analysis. Gfi-1-/- HSCs are functionally compromised in competitive repopulation and serial transplantation assays, and are rapidly out-competed in the bone marrow of mouse chimaeras generated with Gfi-1-/- embryonic stem cells. Thus, Gfi-1 is essential to restrict HSC proliferation and to preserve HSC functional integrity.
The Gfi-1 locus (originally known as pal-1) is among the most frequent sites of retroviral integration contributing to the development of lymphoid tumours in mice8-10. Its product, a SNAG-domain-containing zinc-finger transcriptional repressor, promotes the proliferation of T cells (hence, growth factor independent 1)8-10. In the molecular pathogenesis of lymphoma Gfi-1 is a member of the same complementation group as the polycomb gene Bmi-1, suggesting involvement in a similar, or parallel, pathway of oncogenesis11. Recently, Bmi-1 has been shown to be required for proliferation and maintenance of HSCs4, 5. Gfi-1 is also expressed in HSCs (ref. 12 and data not shown) as well as lymphoid and myeloid cells13. Thus, in analogy to the similar involvement of Bmi-1 and Gfi-1 in lymphomagenesis, Gfi-1 might be anticipated to serve as a positive regulator of HSC proliferation.
To address the role of Gfi-1 in HSCs we first determined the immunophenotype and HSC number in Gfi-1-/- mice13 compared with wild-type littermates. The marker profile of Gfi-1-/- HSCs was preserved, albeit with a slight but consistent decrease in the intensity of c-kit expression (Fig. 1a). We confirmed that the Lin-, Sca1+, c-kit+ population of both Gfi-1-/- and control mice was similarly enriched (> 250-fold) in high proliferative potential colony progenitors (HPPCs), which correlate with stem cell activity14, and that Gfi-1-/- Lin-, Sca1+, c-kit+, Flt3- bone marrow cells are capable of lymphomyeloid reconstitution after injection into lethally irradiated recipients (Supplementary Fig. 1). Notably, the proportion of Flt3+ cells within HSCs (Lin-, Sca1+, c-kit+) was markedly reduced (Fig. 1a). In this population Flt3 expression is associated with increased lymphoid repopulating potential but loss of long-term repopulating potential15, 16. Reduced Flt3 expression in this compartment may signify an early lesion in lymphoid development as Gfi-1-/- mice have reduced lymphopoiesis13, 17. Surprisingly, however, the total number of phenotypic, long-term repopulating HSCs (Lin-, Sca1+, c-kit+, Flt3-) was increased in Gfi-1-/- bone marrow (Fig. 1b). This appeared to be in contrast to findings in Bmi-1-/- mice, where phenotypic HSCs are markedly reduced in frequency (10–45-fold)4, and suggested that despite assignment of Gfi-1 to the same complementation group as Bmi-1 based on cooperation in lymphomagenesis, its requirement in HSCs might be distinct.
Figure 1 Immunophenotypic analysis of HSCs in Gfi-1-/- mice. Full legend
High resolution image and legend (69k)
To assess the functional properties of Gfi-1-/- HSCs we transplanted bone marrow from Gfi-1-/- mice or littermate controls into irradiated host animals. In the absence of competitor marrow, a large dose (1.8 106) of Gfi-1-/- bone marrow cells rescued recipients from lethal irradiation and sustained haematopoiesis for >3 months. The reported haematological abnormalities of Gfi-1-/- mice—notably absence of mature granulocytes and the presence of immature myeloid cells—were evident in these reconstituted mice (Fig. 2a). Eventually, such rescued recipients became pancytopenic, succumbed to infections, or residual host haematopoiesis entirely out-competed Gfi-1-/- cells (data not shown). Notably, Gfi-1-/- bone marrow cells displayed a marked lack of activity in serial transplantation: transfer of 5 106 bone marrow cells collected from transplant recipients 6 weeks after primary transplantation of 5 106 bone marrow cells resulted in complete rescue and establishment of long-term haematopoiesis in lethally irradiated secondary recipients when wild-type donor cells were used. However, secondary recipients of the same dose of Gfi-1-/- bone marrow cells succumbed to severe anaemia after 6–8 weeks and failed to display evidence of lymphomyeloid engraftment (data not shown).
Figure 2 Function of Gfi-1-/- HSCs is compromised in bone marrow transplantation assays. Full legend
High resolution image and legend (53k)
To assess HSC functions in a quantitative setting, competitive repopulation experiments were performed with limiting dilutions of bone marrow from Gfi-1-/- mice or wild-type littermates (both CD45.2) transplanted into lethally irradiated recipients along with a constant, small dose (1.8 105) of competitor marrow (CD45.1). Three months after transplantation lymphomyeloid reconstitution was assessed in peripheral blood B cells and granulocytes (Fig. 2b). In groups of mice that had received 7,400 wild-type cells, CD45.2+ (donor) B cells and granulocytes were readily detected. When 6 105 wild-type donor cells were administered, engraftment of B cells and granulocytes from competitor marrow was undetectable. In marked contrast, even 1.8 106 Gfi-1-/- donor cells failed to contribute to myelopoiesis and provided only minimal contribution to B cells. These data indicate that the reconstitution of lymphomyeloid haematopoiesis by Gfi-1-/- HSCs is reduced at least 200-fold compared with controls. Analysis of representative mice confirmed these findings in bone marrow granulocytes, thymic T cells and bone marrow B cells (Fig. 2c). Similarly, haemoglobin isoform analysis confirmed that Gfi-1-/- donor cells provided donor-derived haemoglobin in the absence, but not the presence, of competitor marrow (data not shown). As erythroid cells do not express Gfi-1 (ref. 13), these findings further support the presumption that competition occurred at an early level of haematopoietic development. To corroborate that competition occurred at the stem cell level and did not reflect combined effects in the differentiated lineages, we performed CD45-isotype analysis on cells recovered from HPPCs, which are believed to arise from HSCs or their early progeny14 (Fig. 2c). Again, even in mice that had received 1.8 106 Gfi-1-/- cells, HPPCs were exclusively of competitor origin (Fig. 2c). Thus, competitive repopulation bone marrow transplant assays demonstrate that normal HSC activity is critically dependent on Gfi-1.
As young Gfi-1-/- mice are not anaemic and have sustained myelopoiesis, we infer that Gfi-1 is dispensable for the emergence and embryonic expansion of HSCs13. This conclusion is consistent with phenotypic enumeration of Gfi-1-/- HSCs. Later in life, marrow function deteriorates in Gfi-1-/- animals, at which time HSC function is difficult to assess because of concomitant myeloid abnormalities (ref. 13 and data not shown). To assess HSCs without prior transplantation and in the context of otherwise normal haematopoiesis, we generated chimaeric mice by injection of Gfi-1-/- or Gfi-1+/- (control) embryonic stem (ES) cells into wild-type blastocysts (Fig. 3a, b). In young chimaeras (< 3 weeks of age) progeny of Gfi-1+/- and Gfi-1-/- ES cells were detected in the bone marrow and thymus by Southern blot analysis (Fig. 3c), and ES-cell-derived haemoglobin was observed in peripheral blood (Fig. 3d). In contrast, in chimaeras >2 months, DNA from Gfi-1-/- cells was no longer detected by Southern blot analysis in bone marrow or thymus, and was sharply reduced in spleen, whereas contribution of Gfi-1-/- cell DNA was preserved in non-haematopoietic tissues (Fig. 3e). Gfi-1+/- ES cells consistently contributed to all tissues (Fig. 3e). Haemoglobin isoform analysis confirmed that progeny of Gfi-1+/- but not Gfi-1-/- ES cells contributed to haemoglobin production in adult chimaeras supporting an early haematopoietic defect (Fig. 3f). A defect at the level of HSCs was corroborated by the finding that only Gfi-1+/- but not Gfi-1-/- cells contributed to HPPCs derived from the bone marrow of chimaeric mice (Fig. 3g). Systematic analysis of peripheral blood from chimaeras of different ages revealed that Gfi-1-/- ES-cell-derived haemoglobin was detected during the first month of life, but was subsequently lost in all animals (Fig. 3h). Thus, Gfi-1-/- HSCs initiate, but do not sustain, bone marrow haematopoiesis in chimaeric mice. Taken together, the transplantation and chimaera experiments provide persuasive evidence that the defective function of Gfi-1-/- HSCs is intrinsic to the HSCs themselves rather than a secondary consequence of an impaired haematopoietic microenvironment.
Figure 3 Gfi-1-/- HSCs initiate, but do not sustain, bone marrow haematopoiesis in chimaeric mice. Full legend
High resolution image and legend (35k)
In principle, failure to maintain HSC function, as observed after Gfi-1 loss in both chimaera and transplant settings, might reflect decreased proliferative capacity and depletion of HSCs, as reported for loss of Bmi-1 (ref. 4). However, the number of phenotypic HSCs in young Gfi-1-/- mice was not severely reduced but instead was slightly elevated (Fig. 1a), a finding most consistent with impaired function rather than mere absence. Of note, loss of HSC function has been ascribed to excessive proliferation. Specifically, forced stem cell expansion driven by serial transplantation18, 19 or by genetic disruption of a negative regulatory component of the cell-cycle machinery20 results in functional compromise without depletion of immunophenotyped HSCs19, 20. To address a role for Gfi-1 in HSC proliferation directly we measured DNA synthesis by determining the fraction of cells that had incorporated 5-bromodeoxyuridine (BrdU) during 3-day in vivo exposure (Fig. 4a). Bone marrow was stained with antibodies to identify HSCs (Lin-, Sca1+, c-kit+) and detect nuclear BrdU simultaneously. In agreement with previous data3 28% of HSCs from wild-type mice incorporated BrdU and therefore had proliferated during the exposure. Surprisingly, more than double the percentage (67%) of HSCs from Gfi-1-/- mice proliferated during the same interval. Furthermore, we demonstrated that HSC hyperproliferation is intrinsic to the haematopoietic system via bone marrow transplantation of Gfi-1-/- or control marrow into wild-type mice. As previously reported21, we observed that bone marrow transplant of wild-type HSCs resulted in a sustained increase in HSC proliferation, but in the absence of Gfi-1 this was markedly more pronounced (Supplementary Fig. 2). Interestingly, HSC frequencies were markedly decreased in recipients of Gfi-1-/- marrow, potentially due to exhaustion after the added proliferative stress of transplantation (Supplementary Fig. 2b). As expression of Gfi-1 promotes proliferation of lymphoid cells7-9, we considered whether our results might reflect differences in experimental approach (overexpression versus loss of function) or rather signify authentic context-specific, divergent involvement of Gfi-1 in controlling cellular proliferation. Therefore, we isolated representative haematopoietic cells from Gfi-1-/- and control mice, and assessed cell-cycle stages by measuring DNA content after propidium iodide staining (Fig. 4b). In the thymus of Gfi-1-/- mice, in agreement with markedly reduced overall cellularity13, the proportion of cells in non-proliferative stages of the cell cycle (G0 and G1) was slightly increased whereas cells in active proliferation (S, G2, M) were reduced in number. These findings are consistent with the previous conclusion that Gfi-1 promotes proliferation of lymphoid cells. We also analysed Gr1+ Mac1+ myeloid cells from bone marrow. Granulocytic precursors of Gfi-1-/- mice, although arrested in differentiation and not overtly malignant, progressively expand in bone marrow as mice age13. Remarkably, these cells in Gfi-1-/- mice are highly proliferative as compared with Gr1+ Mac1+ of wild-type mice. Consistent with the BrdU incorporation data, the proportion of cycling long-term-repopulating HSCs (Lin-, Sca1+, c-kit+, Flt3-)—which were sorted by fluorescence-activated cell sorting (FACS) twice to exclude contamination by other cell types—was appreciably increased in Gfi-1-/- mice as compared with controls. Thus, Gfi-1 differentially affects proliferation and cell-cycle progression of haematopoietic cells, dependent on cell lineage and state of differentiation.
Figure 4 Gfi-1 restricts proliferation of HSCs in a cell-context-specific manner. Full legend
High resolution image and legend (130k)
To elucidate potential mechanisms through which Gfi-1 might control HSC growth we used quantitative, real-time polymerase chain reaction with reverse transcription (RT–PCR) to assess transcript levels for candidate molecules previously implicated in HSC or progenitor proliferation. Bmi-1, c-Myc and p27 were expressed at similar levels in wild-type and mutant HSCs. Gata-2, which is thought to promote proliferation, was slightly upregulated. Of note, expression of cyclin-dependent kinase inhibitor and G1 checkpoint regulator p21Cip1/Waf1 was markedly downregulated (at least tenfold) in Gfi-1-/- HSCs (Fig. 4c). Interestingly, p21Cip1/Waf1, a generic regulator of the cell cycle, is required to maintain HSCs (lineage-, Hoechst 33342low) in G0 (distinguished from G1 by low RNA content)20. In the absence of p21Cip1/Waf1, HSCs exhibit impaired serial transplantation capacity. Although the effects of p21Cip1/Waf1 loss on HSC function are less pronounced than those described here, our data argue that p21Cip1/Waf1 is one mediator of Gfi-1 in stem cells. In view of the more extreme phenotype on its loss, we anticipate that Gfi-1 functions through additional pathways.
In keeping with our findings, retroviral insertions into the Gfi-1 locus (which typically trans-activate and cause overexpression) are almost exclusively found in lymphoid tumours in mice10. Thus far, there is no evidence that loss of Gfi-1 function may also contribute to oncogenesis in other settings by releasing proliferative control at the level of HSCs or after commitment to the myeloid lineage. Our data, however, warrant consideration of this possibility. The versatile role of Gfi-1 in proliferation highlights the critical importance of context in transcriptional regulation in vivo, where its function may be modified by cell-type-specific events such as post-translational modifications, interaction with lineage-specific partner molecules, or differential access to regulatory elements of critical target genes. The paralogue of Gfi-1, Gfi-1b, is co-expressed with Gfi-1 in HSCs12, 22 and is slightly ( twofold) upregulated in Gfi-1-/- HSCs (assessed by quantitative PCR; data not shown). Yet, we demonstrated by competitive repopulation and serial transplantation assays using donor cells from Gfi-1b-/- fetal liver that in marked contrast to Gfi-1, Gfi-1b is dispensable for HSC function (Supplementary Fig. 3).
The severe disturbance of HSC function that we have documented in the absence of Gfi-1 is probably the result of excessive cycling. Yet, we cannot exclude the possibility that it reflects additional roles of Gfi-1 in HSCs. Nevertheless, our data provide molecular insight into how the transcriptional machinery protects HSCs by curbing excessive or unnecessary proliferation. Elucidation of the role of Gfi-1 in HSCs provides a novel entry point into the molecular mechanics of the HSC 'brake' for proliferation, and may ultimately generate opportunities for its manipulation.
Methods
Flow cytometry MoFlo, FACS-Calibur or FACS-Scan flow cytometers were used for analysis and sorting. Antibody conjugates and matched isotype controls were obtained from PharMingen or eBioscience. HSC stain: lineage markers CD4 (RM4-5), CD8 (53-6), CD3 (145-2C11), TCR (H54-597), B220 (RA3-6B2), CD19 (6D5), Gr1 (RB6-8C5), Mac-1 (M1/70), Ter119 (all Cy-Chrome or PE-Cy5); CD117 (c-kit, 2B8, APC), CD135 (Flt3, A2F10, PE), Sca-1 (E13-161, fluorescein isothiocyanate), propidium iodide (1 µg ml-1; Molecular Probes). CD45 isotype analysis: B220 (RA3-6B2, APC), Gr1 (RB6-8C5, APC), Mac1 (M1/70, PE), CD45.1 (104, fluorescein isothiocyanate), CD45.2 (A20, PE, fluorescein isothiocyanate).
Bone marrow transplantation Bone marrow transplantations were performed by intravenous injection of donor cells from 4-week-old Gfi-1-/- mice or littermates (C57bl6/129 mixed) and/or competitor marrow (B6.SJL; 002014, Jackson) into recipients that had received 1,200 cGy of radiation as a split dose. Donor cells from Gfi-1b-/- fetal livers were obtained on day 12.5 of embryonic development after timed mating of heterozygotes22. Recipients were B6/129SF1/J (101043; Jackson) in experiments shown in Fig. 2. Similar results were obtained using B6.SJL mice as recipients, which were also used in experiments shown in Supplementary Figs 1, 2 and 3. Southern blot, automated blood counts and bone marrow morphology was as described13.
Colony-forming assays HPPCs were generated by seeding bone marrow cells into methylcellulose-containing media (MethoCult 3234; Stem Cell Technologies) in the presence of SCF, interleukin (IL)-3, EPO, IL-6, IL-1 granulocyte–macrophage colony-stimulating factor and macrophage colony-stimulating factor (all R&D Systems). Colonies with a tight centre and a diameter 1 mm at day 12 were isolated using a capillary under an inverted microscope, pooled and processed for FACS staining or DNA extraction.
Generation of chimaeras Targeting of Gfi-1 in ES cells has been described13. ES cells homozygous for Gfi-1 deletion were obtained by selecting clones that grew despite increased neomycin concentration (1.7 mg ml–1) owing to crossing over of the targeted allele. Subsequent Southern blot analysis confirmed duplication of the targeted mutation and absence of wild-type Gfi-1 (probe B13). Chimaeras were obtained by injecting Gfi-1+/- or Gfi-1-/- ES clones into C57/bl6 blastocysts. Chimaerism was assessed by agouti coat-colour contribution. Only animals with contribution >70% were selected for analysis. Southern blot analysis of selected tissues after DNA extraction (internal probe13) and haemoglobin isoform analysis was performed as described23.
BrdU incorporation Analysis of BrdU incorporation was performed using the FITC BrdU Flow Kit (PharMingen) after a single intraperitoneal injection of BrdU (PharMingen, 1 mg per 6 g of mouse weight) and admixture of 1 mg ml-1 of BrdU (Sigma, 1 mg ml-1) to drinking water for 3 days (or 2 days after transplant). Mice were 4–6 weeks of age. Surface stain consisted of lineage markers as above, CD117 (c-kit, 2B8, APC) and Sca-1 (E13-161, PE).
Cell-cycle analysis Cell-cycle analysis of thymocytes, or sorted myeloid and HSC populations was performed after ethanol fixation, RNase digestion and propidium iodide staining (Roche, 1399861) using BD ModFit LT software.
Gene expression analysis RNA was isolated from HSCs (8,100 cells; derived from pooled bone marrow of 6–7 mice) using the RNeasy Micro Kit (Qiagen) and recommended protocol. First-strand complementary DNA synthesis was primed with OligodT (Invitrogen) from sample RNA (2 µl) or wild-type bone marrow RNA (100 ng) using the Sensiscript RT Kit (Qiagen). For real-time PCR analysis, cDNA (1.5 µl) was mixed with iQ SYBR green supermix reagent (BioRad), primers (Supplementary Table 1) and amplification plots were generated using the iCycler Thermal cycler and iQ 4-colour real-time detection system (BioRad). To generate standard curves, cDNA from wild-type bone marrow RNA (100 ng) was used as template in a threefold dilution series (0–81-fold). Sample cDNA was used undiluted. Relative expression was calculated using the standard curve method (ABI Prism 7700 Sequence Detection System, user Bulletin 2, PE Applied Biosystems). Primers as in Supplementary Table 1.
Supplementary information accompanies this paper.
Received 14 May 2004;accepted 10 August 2004
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