Deletion of Hoxa Genes in Adult Mice Affects Hematopoietic Stem Cell and B Cell Progenitors

Abstract Abstract 2359 BACKGROUND AND OBJECTIVE: Functional compensation between homeodomain proteins has hindered the ability to unravel their role in hematopoiesis using single gene knock-outs. Although several Hox genes can expand hematopoietic stem cells (HSC) when overexpressed, it remains unclear whether these genes are required for proper adult hematopoiesis. Moreover, it has been shown that HoxB genes are dispensable for hematopoiesis, and that expression of most HoxA genes is ten-fold superior to genes from other Hox clusters in HSC enriched fetal liver populations (Bijl, 2006). Using a haploinsufficient mice for the entire HoxA cluster (HoxA+/−), we have shown that adult HSCs and progenitors are particularly sensitive to HoxA gene levels (Lebert-Ghali, 2010). Thus, we hypothesize that HoxA genes have a crucial function in definitive hematopoiesis. MATERIALS AND METHODS: To assess the role of HoxA genes in definitive hematopoiesis, we used a conditional mutant mouse model for the entire HoxA cluster in combination with an inducible Mx-Cre model. The functional effect of HoxA cluster deletion on hematopoietic cells was analysed by culture and repopulation assays. RESULTS: Highly efficient excision of HoxA cluster was achieved by 7 doses of poly(I):poly(C) treatment (91–100%). Mice (control, n=3 and Mx-CreHoxAflox/flox, n=3) were sacrificed and analysed three days after the last injection. Immunophenotyping showed a 3 to 4 fold increase of CD150+/CD48-/CD244-/Sca+/c-kit+/Lin- hematopoietic stem cells. Despite the enhancement of the HoxA−/− HSC population, single cell cultures showed that their proliferative potential in response to growth factors was significantly reduced (p=0.036) as growth was observed only for 16.6 ± 14.4% of HoxA−/− compared to 42.4 ±10.3% of control HSCs after 3-weeks of culturing. In contrast, the number of multipotent progenitor (MPP) cells (CD34+/CD135+/Sca+/c-kit+/Lin-) was reduced, indicating a partial block from the short-term HSC (CD34+/CD135-/Sca+/c-kit+/Lin-) to the MPP transition. Colony forming cell assays showed a dramatic decrease of B-cell progenitors in the bone marrow (BM) (10-fold, p=0.0079), while myeloid progenitors were not affected by the deletion. Transplantation assays demonstrated that grafts composed of > 91% HoxA−/− HSCs have slower repopulation kinetics compared to control HSCs and strongly reduced long-term engraftment (37 ± 28% and 92 ± 6% for HoxA−/− and control, respectively, 20 weeks post-transplantation). Genotyping of engrafted donor cells is currently analyzed to confirm repopulation by HoxA−/− cells. Consistent with the observations in primary mice, peripheral blood analysis revealed also a dramatic reduction of B220+ B-cell population in mice transplanted with HoxA−/− BM cells compared to control (7.1 ± 8.5% and 56.2 ±4.8% respectively p=0,000002) Altogether, in vitro assays and transplantation assays revealed that the functions of HoxA−/− HSC seem to be impaired. CONCLUSION: Together, these results show that HoxA cluster genes are required for both HSC function and B cell development, indicating that these genes are important regulators of adult hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Inability to Express HOXA Cluster and BCL11A Genes Compromises Self-Renewal and Multipotency of hESC-Derived Hematopoietic Cells

Blood ◽

10.1182/blood.v120.21.1190.1190 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1190-1190 ◽

Cited By ~ 1

Author(s):

Diana R Dou ◽

Arazin Minasian ◽

Maria I Sierra ◽

Pamela Saarikoski ◽

Jian Xu ◽

...

Keyword(s):

Stem Cells ◽

B Cells ◽

Proliferative Potential ◽

Pluripotent Cells ◽

Hoxa Cluster ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Hoxa Genes

Abstract Abstract 1190 The inability to derive functional hematopoietic stem cells (HSCs) in vitro from pluripotent cells prevents widespread utilization of HSCs in the clinic; however, the molecular defects compromising the in vitro generated hematopoietic stem/progenitor cells (HSPCs) are unknown. Using a two-step differentiation method in which human embryonic stem cells (hESCs) were first differentiated into embryo bodies (EBs) and then CD34+ cells from hEBs were co-cultured on OP9M2 bone marrow mesenchymal stem cell (MSC) stroma (hEB-OP9), we were able to derive HSPCs expressing the HSC immunophenotype (CD34+CD38−CD90+CD45+) (hereafter termed CD90+HSPCs). Colony forming and stroma co-culture assays demonstrated that the hEB-OP9 CD90+HSPCs were able to differentiate into myelo-erythroid lineages and T-cells. However, when comparing CD90+HSPCs from hEB-OP9 to those from fetal liver (FL)—an in vivo source of HSCs—the former remained severely functionally limited in their proliferative potential and ability to differentiate into B-cells. To identify the basis of the proliferative and differentiation defects, we performed microarray analysis to define gene expression differences between CD90+HSPCs derived from hEB-OP9, FL, early 3–5 week placenta (PL) and an earlier stage of hESC differentiation (hEB). This analysis revealed establishment of the general hematopoietic transcription factor network (e.g. SCL, RUNX1, CMYB, ETV6, HOXB4, MYB), demonstrating the successful differentiation and identification of hematopoietic cells using our two-step culturing techniques and immunophenotype criteria. Moreover, evaluation of Spearman coefficients confirmed CD90+HSPCs isolated from hEB-OP9 culture were brought into closer resemblance of the hFL CD90+HSPCs as compared to to the developmentally immature hEB and hPL CD90+HSPCs. Encouragingly, hEB-OP9 CD90+HSPCs displayed downregulation of expression of genes related to hemogenic endothelium development associated with hEB and hPL while genes critical in HSPC function, including DNA repair and chromatin modification, were upregulated to levels comparable to hFL-HSPCs. However, a subgroup of FL HSPC genes could not be induced in hEB-OP9 HSPCs, including the HOXA cluster genes and BCL11A—implicated in HSC self-renewal and B-cell formation, respectively. Interestingly, absence of HOXA genes and BCL11A and poor proliferative potential were also observed in HSPCs from early placenta, suggesting these defects are not in vitro artifacts but instead reflect an inability of hEB-OP9 HSPCs to complete developmental maturation. To validate the necessity of HOXA genes and BCL11A in proliferation potential and multipotency, we next utilized shRNAs to target MLL—the upstream regulator of the HOXA cluster—, individual HOXA genes, or BCL11A in FL-HSPCs to test whether knockdown was sufficient to recapitulate the defects observed in hESC-derived HSPCs. Knockdown of HOXA7 resulted in the loss of CD34+ cells while HOXA9 shRNA-treated cells displayed a loss of more differentiated CD38hi cells. MLL knockdown depleted both CD38+ and CD34+ populations. BCL11A silencing resulted in the loss of B-cells. These studies identify HOXA genes and BCL11A as developmentally regulated genes essential for generating self-renewing, multipotent HSCs from pluripotent cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

GPI-80 Defines Self-Renewal Ability In Hematopoietic Stem Cells During Human Development

Blood ◽

10.1182/blood.v122.21.4839.4839 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4839-4839

Author(s):

Sacha L. Prashad ◽

Vincenzo Calvanese ◽

Catherine Yao ◽

Joshua Kaiser ◽

Rajkumar Sasidharan ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Human Development ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Surface Protein ◽

Surface Proteins ◽

Proliferative Potential ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Advances in pluripotent stem cell and reprogramming technologies have provided hope of generating transplantable hematopoietic stem cells (HSC) in culture. However, better understanding of the identity and regulatory mechanisms that define the self-renewing HSC during human development is required. We discovered that the glycophosphatidylinositol-anchored surface protein GPI-80 (Vanin-2), previously implicated in neutrophil diapedesis, distinguishes a functionally distinct subpopulation of human fetal hematopoietic stem and progenitor cells (HSPC) that possess self-renewal ability. CD34+CD90+CD38-GPI80+ HSPCs were the only population that could maintain proliferative potential and undifferentiated state in co-culture on supportive stroma, and displayed engraftment potential in sublethally irradiated NSG mice. GPI-80 expression also enabled tracking of human HSC during development as they migrate across fetal hematopoietic niches, including early fetal liver and bone marrow. Microarray analysis comparing CD34+CD90+CD38-GPI80+ HSPC to their immediate progeny (CD34+CD90+CD38-GPI80-) identified novel candidate self-renewal regulators. Knockdown of GPI80, or the top enriched transcripts encoding surface proteins (ITGAM) or transcription factors (HIF3a) documented the necessity of all three molecules in sustaining human fetal HSC self-renewal. These findings provide new insights to the poorly understood regulation of human HSC development and suggest that human fetal HSCs utilize common mechanisms with leukocytes to enable cell-cell interactions critical for HSC self-renewal. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Development of Multi-Lineage Hematopoiesis in Two Novel Zebrafish runx1 mutants

Blood ◽

10.1182/blood.v126.23.2373.2373 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2373-2373

Author(s):

Erica Bresciani ◽

Blake Carrington ◽

Erika Mijin Kwon ◽

Marypat Jones ◽

Stephen Wincovitch ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Time Lapse ◽

Loss Of Function ◽

Transcription Activator ◽

Zebrafish Model ◽

Hematopoietic Stem ◽

Definitive Hematopoiesis

Abstract Long term hematopoietic stem cells are essential for the life-long maintenance of the hematopoietic system of an organism. The transcription factor RUNX1 is required for the emergence of definitive hematopoietic stem cells (HSCs) from the hemogenic endothelium during the embryo development. Runx1 knockout mouse embryos lack all definitive blood lineages and cannot survive past embryonic day 13. However, we previously showed that zebrafish homozygous for an ENU-induced nonsense mutation in runx1 (runx1W84X/W84X) were able to recover from a larval "bloodless" phase and develop to fertile adults with multi-lineage hematopoiesis, suggesting the formation of runx1-independent adult HSCs. However, our finding was based on a single zebrafish model, which requires verification in additional, independent models. In order to further investigate if a RUNX1-independent pathway exists for the formation of adult HSCs, we generated two new runx1 mutants, a deletion of 8 bp (runx1del8/del8) and a deletion of 25 bp (runx1del25/del25) within exon 4 of runx1, respectively, using the Transcription activator-like effector nucleases (TALENs) technology. These mutations cause frameshifts and premature terminations within the runt-homology domain,, resulting in loss of function of runx1 (runx1-/-). Both runx1del8/del8 and runx1del25/del25 mutant embryos had normal primitive hematopoiesis but failed to develop definitive hematopoiesis. Time-lapse recordings with confocal microscopy revealed that, indeed, there was no emergence of HSCs from the ventral wall of dorsal aorta in the runx1-/- embryos. The runx1-/- larvae gradually lost circulating primitive blood cells and became bloodless between 8 and 14 days post fertilization (dpf). However they gradually regained circulating blood cells between 15 and 20 dpf. Eventually, about 40% of runx1del8/del8 and runx1del25/del25 mutants developed to fertile adults with circulating blood cells of multi-lineages. Taken together, our data is consistent with the previously described runx1W84X/W84X phenotype and supports the possibility of a runx1-independent mechanism for HSC formation and definitive hematopoiesis. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Variant Pcgf-PRC1 Regulates Susceptibility of PRC2 Mediated H3K27me3 to Safeguard B Cell Fate of Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood-2019-129518 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3717-3717

Author(s):

Junichiro Takano ◽

Yaeko Nakajima-Takagi ◽

Shinsuke Ito ◽

Haruhiko Koseki ◽

Atsushi Iwama ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

B Cell ◽

Progenitor Cells ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Biological Significance ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

The Impact

Polycomb repressive complex (PRC) resides in two major complexes PRC1 and PRC2. They cooperate with each other to coordinate proper developmental process by silencing target genes; PRC1 posits H2AK119ub1 and PRC2 catalyzes trimethylation of H3K27 (H3K27me3). The PRC1 component BMI1/PCGF4 has long been recognized to be essential in the maintenance of normal and malignant hematopoietic stem cells (HSCs). Recently, diversity of PRC1 has been noticed and PRC1 is now classified into six alternative complexes depending on PCGF proteins. In embolic stem cells, PRC1 which contains PCGF1 (PCGF1-PRC1) has been demonstrated to serve upstream of the BMI1/PCGF4-PRC1. However, the impact of BCOR, which is a component of PCGF1-PRC1 on hematopoiesis is clearly different from BMI1/PCGF4; the mice deficient for BCOR exhibited normal HSC activities and BCOR rather prevented leukemic transformation of HSCs, suggesting the previously unappreciated gene control mechanisms of PCGF1-PRC1. To tackle this issue, we focused on the roles of PCGF1 in hematopoiesis. Loss of Pcgf1 in hematopoietic stem cells led to severe reduction of B lineage cells with an expansion of myeloid progenitors due to defects in lymphoid-primed multipotent progenitor (LMPP) cells. To explore the molecular mechanisms, we have established Id3-overexpressing hematopoietic progenitor cells (IdHPs) which correspond to LMPP-like cells (Ikawa et al. 2015) from bone marrow of ERT2-Cre Pcgf1 flox mice. The ChIP-seq analysis of normal IdHPs identified 1274 genes whose promoters were associated with PCGF1 peaks and 37% of them exhibited enrichment of H3K27me3 and binding of SUZ12 (PRC2). Deletion of Pcgf1 destabilized H3K27me3 levels, resulting in re-activation of genes associated with PCGF1 and SUZ12 peaks, whereas the chromatin occupancy of SUZ12 was not affected. Intriguingly, proteomic analysis demonstrated that PCGF1 interacts with key factors responsible for the organization of nucleosomes and PCGF1 loss triggered a decline of nucleosome-densities in promoters of genes occupied by PCGF1 and SUZ12 peaks. Since enzymatic activity of PRC2 is dependent on nucleosome-densities, PCGF1 is likely to regulate the susceptibility of H3K27me3 by PRC2 through determination of the nucleosome-densities. Furthermore, genes which were downregulated by PCGF1-nucleosome-H3K27me3 axis entailed many myeloid-related genes and knock down of one of those myeloid genes partially restored the B cell differentiation potential of Pcgf1-KO hematopoietic stem/progenitor cells (HSPCs), supporting the biological significance of the PCGF1-nucleosome-H3K27me3 axis. Collectively, these results indicate PCGF1 determines cellular fate of HSPCs through stabilization of nucleosomal organization. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Deletion of Bak and Bax in Tie2+ BM Hematopoietic Stem Cells Induces a B Cell Lymphoproliferative Disorder.

Blood ◽

10.1182/blood.v114.22.1247.1247 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1247-1247

Author(s):

Phuong L. Doan ◽

J. Lauren Russell ◽

Sarah K. Meadows ◽

Heather A. Himburg ◽

Pamela Daher ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

B Cell ◽

Lymphoproliferative Disorder ◽

Conflicts Of Interest ◽

Flow Cytometric Analysis ◽

Wild Type ◽

Hematopoietic Stem ◽

Normal Peripheral Blood ◽

Targeted Deletion

Abstract Abstract 1247 Poster Board I-269 Chronic lymphocytic leukemia (CLL) is characterized by a monoclonal, mature B-cell lymphocytosis, lymphadenopathy and splenomegaly. The pathogenesis of CLL is thought to be related to abnormal programmed cell death mechanisms but the precise contribution of several pro-apoptotic genes remains unknown. Elucidation of the pathogenesis of CLL is also limited by the lack of animal models which reproduce the CLL phenotype. We present a transgenic mouse model in which conditional, tissue-specific deletion of Bax is achieved in Tie2+ hematopoietic stem cells (HSCs) via Cre-LoxP recombination. In this model, Tie2Cre;Bak-/-;BaxFl/- mice carried constitutive deletion of Bak and targeted deletion of Bax in Tie2+ BM HSCs. Surprisingly, the combined deletion of Bak and Bax in Tie2+ cells caused a pronounced leukocytosis with total WBCs>100,000 in 8-week old mice. In contrast, Tie2Cre;Bak-/-;BaxFl/+ mice, which retained one Bax allele, had normal peripheral blood WBCs comparable to wild type C57Bl6 mice (range 4-10,000;p<0.01 and p<0.01). Tie2Cre;Bak-/-;BaxFl/- mice had massive splenomegaly with a 6-fold greater spleen weight compared to Tie2Cre;Bak-/-; BaxFl/+ controls. Review of PB smears demonstrated a preponderance of “smudge” cells (70% of total cells per HPF) in the Tie2Cre;Bak-/-;BaxFl/- mice, whereas smudge cells were not observed in Tie2Cre;Bak-/-;BaxFl/+ mice. Flow cytometric analysis of PB cell differential revealed a significant increase in B cells (B220+, p=0.0001), a decrease in T cells (Thy 1.2+, p=0.0002), decreased myeloid cells (Mac1/Gr1+ cells, p=0.02), and decreased erythroid cells (Ter119+, p=0.01) compared to Tie2Cre;Bak-/-;BaxFl/- mice. The B cell-to-T cell ratio was also aberrantly increased (p=0.0007) suggesting a predominant B-cell lymphocytosis. Interestingly, while Tie2Cre;Bak-/-;BaxFl/- mice demonstrated no increase in committed BM colony forming cells (CFCs) or colony forming unit-spleen day 12 (CFU-S12), these mice contained significantly increased numbers of BM long term culture-initiating cells (LTC-ICs, p=0.04), suggesting that the combined deletion of Bak and Bax in Tie2+ cells yielded an expansion of primitive BM HSCs. Lastly, in order to confirm whether the development of the lymphoproliferative disorder was autonomous to deletion of Bak and Bax in Tie2+ HSCs, we transplanted 4 × 106 BM cells from Tie2Cre;Bak-/-;BaxFl/- mice into lethally irradiated (950 cGy) wild type B6.SJL mice. At 12 weeks post-transplant, the recipient mice were chimeric with Bak and Bax deletion in BM hematopoietic cells and Bak and Bax retained in the BM microenvironment. Remarkably, the chimeric recipient mice demonstrated a significant reduction in PB WBCs (range 23-60,000, p=0.001) and a 2.4-fold decrease in spleen size compared to Tie2Cre;Bak-/-;BaxFl/- mice (p=0.02), suggesting that the development of the lymphoproliferative disorder was modulated by Tie2+ BM endothelial cells. Taken together, these data demonstrate that the targeted deletion of both Bak and Bax, but not Bak deletion alone, in Tie2+ HSCs produces a profound lymphoproliferative disorder in mice. While it has been previously shown that constitutive deletion of Bak and Bax caused a lymphoproliferative disorder in mice (Lindsten T et al. Mol Cell 2000;6:1389), the data presented here suggest that deletion of Bak and Bax in BM HSCs may be sufficient to induce a lymphoproliferative disorder and this phenotype may be significantly modulated by the BM microenvironment. We are currently testing whether mice bearing deletion of Bak and Bax in Tie2+ cells have a monoclonal B cell leukemia and are utilizing Cre-LoxP recombination methods to determine at which point in HSC differentiation that the Bak and Bax deletions are required in order for the lymphoproliferative disorder to occur. Given the profound phenotype of the Tie2Cre;Bak-/-;BaxFl/- mice, this may be a useful animal model for the study of the pathogenesis of lymphoproliferative disorders. Disclosures No relevant conflicts of interest to declare.

Download Full-text

G0/G1 Switch Gene 2 (G0S2) Is a Positive Regulator of Quiescence in Hematopoietic Stem Cells and Naïve T Cells.

Blood ◽

10.1182/blood.v114.22.2538.2538 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2538-2538

Author(s):

Takeshi Yamada ◽

Chun Shik Park ◽

Leon Bae ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Receptor Activation ◽

Brdu Incorporation ◽

Proliferative Potential ◽

Hematopoietic Stem

Abstract Abstract 2538 Poster Board II-515 The G0/G1 switch gene 2 (G0S2) was identified in blood mononuclear cells activated with mitogens. G0S2 is a small basic protein located in ER/Golgi in mouse fibroblast. Even though G0S2 is upregulated in inflammatory processes (i.e. rheumatoid arthritis, psoriasis, endometriosis) and by all-trans-retinoic acid treatment in acute promyelocytic leukemia cells, its role in the proliferation of hematopoietic stem cells (HSC) and T lymphocytes is largely unknown. Similarly to HSC, a delicate balance between quiescence and homeostatic proliferation maintains the T cell pool over time. An emerging paradigm suggests that cellular quiescence is an actively regulated state. G0S2 expression in bone marrow (BM) cells is downregulated following cytoablation, suggesting a role in the maintenance of stem cell quiescence. Gain-of function experiments, using retroviral gene transfer and BM transplantation, showed that G0S2 increased number of Lin- Sca-1+ c-kit+ cells in BM by inhibiting their proliferation at steady state. In addition, ectopic G0s2 expression inhibits homeostatic expansion of CD11b+ Gr-1+ and CD3+ cells. Competitive BM transplantation showed that cells overexpressing G0S2 are outcompeted by wild type BM cells due to lower proliferative potential, based on in vivo BrdU incorporation experiments. Inhibition of T cell proliferation by ectopic G0S2 expansion is consistent with suppression of G0S2 transcription in naïve CD8+ T cells upon T-cell receptor activation via MAPK and calcium-calmodulin pathways, suggesting that G0S2 also maintains quiescence of naïve T cells. G0S2 expression is silenced in myeloid and lymphoid leukemic cell lines by gene methylation, supporting a leukemogenic role. Collectively, our study uncovered an important role of G0S2 in normal and malignant proliferation of HSC and T cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Role of hSET1 Complex in Epigenetic Controls of HoxB4 Expression and Development of Hematopoietic Stem Cells

Blood ◽

10.1182/blood.v118.21.212.212 ◽

2011 ◽

Vol 118 (21) ◽

pp. 212-212 ◽

Cited By ~ 1

Author(s):

Changwang Deng ◽

Ying Li ◽

Shermi Liang ◽

Tal Salz ◽

Yi Qiu ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Hox Genes ◽

Conflicts Of Interest ◽

Epigenetic Mechanism ◽

Stem Cell Population ◽

Dominant Negative Mutant ◽

Hematopoietic Stem ◽

H3k4 Trimethylation ◽

Mes Cells

Abstract Abstract 212 Hox genes play an important role in embryonic development and are sequentially activated in a temporal and spatial fashion. Among them, HoxB4 regulates the self-renewal ability of adult and embryonic hematopoietic stem cells. It was shown that USF proteins positively regulate the transcription of HoxB4 gene. However, the epigenetic mechanism specifically controlling HoxB4 transcription during HSC formation remains unknown. In this report, we found that USF1 interacts with histone H3K4 methylatransferase SET1 complex, but not MLL proteins. The interaction is mediated by SET1/MLL core component, ASH2L. Both USF1 and hSET1 are colocalized at the HoxB4 promoter in hematopoietic progenitors, which is accompanied by H3K4 trimethylation and transcriptional activation. siRNA-mediated knockdown of both SET1 and HCF1 specifically impaired H3K4 trimetylation and the HoxB4 promoter-driven reporter gene activity. Overexpression of AUSF1, a dominant negative mutant of USF1 in which the basic DNA recognition domain is deleted and thereby, inhibits endogenous USF DNA binding activity, leads to dramatically decrease in HoxB4 transcription, disruption of embryonic body formation and inhibition of hematopoietic stem cell population in mouse ES (mES) cells and the AUSF1 transgenic mice. The suppression of USF activity drastically reduces SET1 recruitment, H3K4 trimethylation, and pol II binding at the HoxB4 promoter. Furthermore, knock-down of SET1, an enzymatic subunit of the SET1 complex, also resulted in reduction of HoxB4 transcription and impairment of hematopoietic differentiation capacity of mES cells that are accompanied by reducing the HoxB4 promoter H3K4 methylation and recruitment of RNA PolII machinery. Thus, our data reveal that recruitment of hSET1 complex and its H3K4 methyltransferase activity by USF1 is essential for HoxB4 transcription and expansion of hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Hematopoietic Stem Cells with High Proliferative Potential

Journal of Clinical Investigation ◽

10.1172/jci110611 ◽

1982 ◽

Vol 70 (2) ◽

pp. 242-253 ◽

Cited By ~ 82

Author(s):

Dane R. Boggs ◽

Sallie S. Boggs ◽

Debra F. Saxe ◽

Lora A. Gress ◽

Don R. Canfield

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Proliferative Potential ◽

Hematopoietic Stem

Download Full-text

Scl isoforms act downstream of etsrp to specify angioblasts and definitive hematopoietic stem cells

Blood ◽

10.1182/blood-2009-09-244640 ◽

2010 ◽

Vol 115 (26) ◽

pp. 5338-5346 ◽

Cited By ~ 42

Author(s):

Xi Ren ◽

Gustavo A. Gomez ◽

Bo Zhang ◽

Shuo Lin

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Critical Role ◽

Vascular Endothelial ◽

Hematopoietic Stem ◽

Vegf Signaling ◽

Endothelial Growth Factor ◽

Endothelial Development ◽

Definitive Hematopoiesis ◽

Runx1 Expression

Abstract Recent lineage studies suggest that hematopoietic stem cells (HSCs) may be derived from endothelial cells. However, the genetic hierarchy governing the emergence of HSCs remains elusive. We report here that zebrafish ets1-related protein (etsrp), which is essential for vascular endothelial development, also plays a critical role in the initiation of definitive hematopoiesis by controlling the expression of 2 stem cell leukemia (scl) isoforms (scl-α and scl-β) in angioblasts. In etsrp morphants, which are deficient in endothelial and HSC development, scl-α alone partially rescues angioblast specification, arterial-venous differentiation, and the expression of HSC markers, runx1 and c-myb, whereas scl-β requires angioblast rescue by fli1a to restore runx1 expression. Interestingly, when vascular endothelial growth factor (Vegf) signaling is inhibited, HSC marker expression can still be restored by scl-α in etsrp morphants, whereas the rescue of arterial ephrinb2a expression is blocked. Furthermore, both scl isoforms partially rescue runx1 but not ephrinb2a expression in embryos deficient in Vegf signaling. Our data suggest that downstream of etsrp, scl-α and fli1a specify the angioblasts, whereas scl-β further initiates HSC specification from this angioblast population, and that Vegf signaling acts upstream of scl-β during definitive hematopoiesis.

Download Full-text

CD4 is expressed on murine pluripotent hematopoietic stem cells

Blood ◽

10.1182/blood.v80.7.1717.1717 ◽

1992 ◽

Vol 80 (7) ◽

pp. 1717-1724 ◽

Cited By ~ 38

Author(s):

JP Wineman ◽

GL Gilmore ◽

C Gritzmacher ◽

BE Torbett ◽

CE Muller-Sieburg

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

B Cell ◽

B Cell Differentiation ◽

Cd4 Cells ◽

Hematopoietic Stem ◽

Significant Life ◽

High Concentrations ◽

First Time

Abstract We show here for the first time that pluripotent hematopoietic stem cells express the CD4 antigen. CD4+ cells isolated from mouse marrow repopulated all hematopoietic lineages in both the long-term repopulation assay and the competitive repopulation assay. This finding indicates that the CD4+ population contains primitive stem cells with extensive repopulation capacity. Interestingly, the CD4- population had significant life-sparing activity, even though this population was depleted of long-term repopulating stem cells when compared with CD4+ cells. The majority of the cells that respond to the stroma in Whitlock- Witte cultures with B-cell differentiation were recovered in the CD4- population. Thus, this bone marrow (BM)-derived B-cell precursor lacks CD4, which is in contrast to myeloid precursors and thymus-derived lymphoid precursors that reportedly express CD4. We show further that the CD4 molecule expressed on BM cells is similar in molecular weight and epitope makeup to the CD4 antigen found on thymocytes. Detection of CD4 on BM cells is dependent on using high concentrations of antibodies. Thus, it is not surprising that expression of CD4 on pluripotent stem cells has been missed previously. Taken together, our data suggest that the CD4 molecule may play an important role in lineage definition in early hematopoietic differentiation.

Download Full-text