The Expression of Polycomb Group Genes Products, Bmi1 and Mel-18, Regulate the Function of Murine Primitive Hematopoietic Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1261-1261
Author(s):  
Teruyuki Kajiume ◽  
Takashi Sato ◽  
Masao Kobayashi
Keyword(s):  
Gene Expression ◽  
Hematopoietic Cells ◽  
Inverse Function ◽  
Complex Form ◽  
Negative Control ◽  
Polycomb Group ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Polycomb Group Genes ◽  
Marrow Cells

Abstract The Polycomb group (PcG) genes (bmi1 and mel-18) known as negative control factors of the Hox gene is thought to regulate the differentiation and self-renewal of hematopoietic stem cells (HSCs). The loss of mel-18 results in the promotion of HSC self-renewal, and the increase of mel-18 expression inversely leads to the differentiation of HSCs. On the other hand, the loss of bmi1 does not lead to self-renewal activity of HSCs. In this study we examined the effect of expression of bmi1 and mel-18 on the role of function in murine HSCs. Lineage-negative, Sca1-positive, and cKit-positive primitive hematopoietic cells were purified and the expression of PcG protein was evaluated from the intra-nuclear distribution of PcG proteins. The Bmi1-positive hematopoietic cells barely contained Mel-18, and the Mel-18-positive cells barely contained Bmi1. the frequency of positive cells for both Bmi1 and Mel-18 was less than 0.5% of purified primitive hematopoietic cells. The expression levels of the PcG genes, bmi1 and mel-18, in HSCs were knocked down by siRNA and then gene expression was assessed by quantitative real-time PCR. The introduction of siRNA against bmi1 or mell-18 resulted in approximate 50 to 60% decrease of each gene expression without affecting another gene expression. Primary colony-forming activity of knocked down cells in response to stem cell factor, thrombopoietin and the ligand for flt3 was not affected by the induction of siRNA. However, secondary colony-forming activity from primary colony-forming cells in bmi1-knockdown cells was significantly decreased when compared with that of control cells. Conversely, the mel-18-knockdown cells significantly increased, suggesting that mel-18-knockdown cells are capable of proliferating activity. Finally, bone marrow reconstitutive activity was examined by using Ly5.1 and Ly5.2 system. While the bmi1-knockdown marrow cells decreased the reconstitutive activity, the mel-18-knockdown marrow cells showed the increase of engraftment activity after 6 months of transplantation. From these results, we consider that mel-18 and bmi1 have reciprocal functions in HSCs. Mammalian PcG protein complexes can be classified into two distinct types, Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). The Mel-18 protein is a constituent of mammalian PRC1 together with M33, Bmi1 or rae28, and Scmh1. The Mel-18 protein is composed of 342 amino acids and the N-terminal region of the 102 amino acid, which includes the RING finger motif, shares 93% homology with Bmi1 protein. In addition, its secondary structure shows high homology with the Mel-18 and Bmi1 proteins. We hypothesized that the opposite function is expressed in HSCs because Mel-18 and Bmi1 share the same structure and compete when in the complex form. These results suggest that mel-18 and bmi1 have inverse function in HSCs and that the balance of Bmi1 and Mel-18 may regulate the fate of self-renewal and differentiation in HSCs.

scholarly journals Polycomb Group Gene rae28 Is Required for Sustaining Activity of Hematopoietic Stem Cells

2002 ◽  
Vol 195 (6) ◽  
pp. 759-770 ◽  
Author(s):  
Hideaki Ohta ◽  
Akihisa Sawada ◽  
Ji Yoo Kim ◽  
Sadao Tokimasa ◽  
Seiji Nishiguchi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Fetal Liver ◽  
Polycomb Group ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Polycomb Group Genes ◽  
Hematopoietic Activity

The rae28 gene (rae28), also designated as mph1, is a mammalian ortholog of the Drosophila polyhomeotic gene, a member of Polycomb group genes (PcG). rae28 constitutes PcG complex 1 for maintaining transcriptional states which have been once initiated, presumably through modulation of the chromatin structure. Hematopoietic activity was impaired in the fetal liver of rae28-deficient animals (rae28−/−), as demonstrated by progressive reduction of hematopoietic progenitors of multilineages and poor expansion of colony forming units in spleen (CFU-S12) during embryonic development. An in vitro long-term culture-initiating cell assay suggested a reduction in hematopoietic stem cells (HSCs), which was confirmed in vivo by reconstitution experiments in lethally irradiated congenic recipient mice. The competitive repopulating units (CRUs) reflect HSCs supporting multilineage blood-cell production. CRUs were generated, whereas the number of CRUs was reduced by a factor of 20 in the rae28−/− fetal liver. We also performed serial transplantation experiments to semiquantitatively measure self-renewal activity of CRUs in vivo. Self-renewal activity of CRUs was 15-fold decreased in rae28−/−. Thus the compromised HSCs were presumed to reduce hematopoietic activity in the rae28−/− fetal liver. This is the first report to suggest that rae28 has a crucial role in sustaining the activity of HSCs to maintain hematopoiesis.

scholarly journals Lethal myelofibrosis induced by Bmi1-deficient hematopoietic cells unveils a tumor suppressor function of the polycomb group genes

2012 ◽  
Vol 209 (3) ◽  
pp. 445-454 ◽  
Author(s):  
Hideyuki Oguro ◽  
Jin Yuan ◽  
Satomi Tanaka ◽  
Satoru Miyagi ◽  
Makiko Mochizuki-Kashio ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Hematopoietic Cells ◽  
Primary Myelofibrosis ◽  
Extramedullary Hematopoiesis ◽  
Polycomb Group ◽  
Tumor Suppressor Function ◽  
Hematopoietic Stem ◽  
Polycomb Group Genes ◽  
Suppressor Function ◽  
And Function

Polycomb-group (PcG) proteins form the multiprotein polycomb repressive complexes (PRC) 1 and 2, and function as transcriptional repressors through histone modifications. They maintain the proliferative capacity of hematopoietic stem and progenitor cells by repressing the transcription of tumor suppressor genes, namely Ink4a and Arf, and thus have been characterized as oncogenes. However, the identification of inactivating mutations in the PcG gene, EZH2, unveiled a tumor suppressor function in myeloid malignancies, including primary myelofibrosis (PMF). Here, we show that loss of another PcG gene, Bmi1, causes pathological hematopoiesis similar to PMF. In a mouse model, loss of Bmi1 in Ink4a-Arf−/− hematopoietic cells induced abnormal megakaryocytopoiesis accompanied by marked extramedullary hematopoiesis, which eventually resulted in lethal myelofibrosis. Absence of Bmi1 caused derepression of a cohort of genes, including Hmga2, which is an oncogene overexpressed in PMF. Chromatin immunoprecipitation assays revealed that Bmi1 directly represses the transcription of Hmga2. Overexpression of Hmga2 in hematopoietic stem cells induced a myeloproliferative state with enhanced megakaryocytopoiesis in mice, implicating Hmga2 in the development of pathological hematopoiesis in the absence of Bmi1. Our findings provide the first genetic evidence of a tumor suppressor function of Bmi1 and uncover the role of PcG proteins in restricting growth by silencing oncogenes.

Epigenetic Modification Can Regulate the Stem Cell State of Hematopoietic Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1356-1356
Author(s):  
Il-Hoan Oh ◽  
Hye-Jung Kim ◽  
Yun Shin Chung
Keyword(s):  
Dna Methylation ◽  
Stem Cell ◽  
De Novo ◽  
Epigenetic Modification ◽  
Hematopoietic Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Marrow Cells

Abstract The maintenance of undifferentiated state and ability for self-renewal constitutes key properties of hematopoietic stem cell (HSC). To explore the possibility that epigenetic modification contributes to regulation of HSC functions, we have studied whether distinctive epigenetic modification can be correlated to different functions of hematopoietic cells. As a first approach, the global DNA methylations in non-coding repetitive elements were examined using mIAP, mEtn, and mc.satellite regions as marker loci. Sequencing of sodium bisulfite-modified CpG islands in these loci showed that most primitive Lin-c-kit+CD34− cells displayed highest level of DNA methylation as compared to the progenitor-enriched Lin-c-kit+CD34+ cells or differentiated counterpart (Lin+ cells) in mc satellite regions (85.7%, 60 %, 65%, respectively) and mEtn regions (73%, 58%, 74%, respectively), but not in mIAP region. Interestingly, whereas most primitive Lin-c-kit+CD34− cells expressed highest level of methyl cytosine binding protein (MeCp2) or DNA methyl transferase 3 (DNMT3a, 3b), the Lin+ cells expressed bare to minimal level of these gene products despite their high maintenance of DNA methylation, suggesting a differential de-novo methylations between these cells. Similarly, the most primitive Lin-c-kit+CD34− cells exhibited highest level of total acetylated histone (Ac-H4) but these cells expressed also high levels of histone deacetylase (HDAC) as well as histone acetyl transferase (HAT). Subsequent pulse-labeling with C14-acetate demonstrated that immature bone marrow cells (lin-), but not mature Lin+ cells, exhibited active acetylation of H4 with higher turn-overs, thus showing active remodeling of chromatin structures in immature hematopoietic cells. Next, to explore whether alterations in epigenetic modification could influence HSC function, the effect of epigenetic blockers (5-Azacytidine or TSA) were examined for their influence on in-vivo self-renewing activity of transplanted HSCs. Thus, recipients that had been lethally irradiated and transplanted with congeneic HSCs were treated with blockers during first two weeks of recovery, and transplanted into secondary recipients 18 weeks later to determine CRU numbers regenerated. The result of this CRU assay revealed that HSCs had underwent 32-folds higher self-renewal with inhibition of HDAC (22 vs. 738 CRUs), and 11-folds higher self-renewal with inhibition of DNA methylation (22 vs. 248 CRUs) showing that self-renewing potential of “stimulated” HSC is regulated by epigenetic modification. Next, to see if stem cell fate can be also influenced by epigenetic reprogramming, the effects of epigenetic blockers during “stationary” phase of hematopoiesis were examined by treating donor mice with epigenetic blockers for 3 weeks before sacrifice. Limiting dilution transplantation of these marrows revealed that the CRU frequencies in the treated (5-Azacytidine + TSA) donor marrows were 7-folds higher compared to the un-treated donor marrow cells (1/22,9000 vs. 1/3000) in the absence of increase in total marrow cell numbers, suggesting de-novo generation of CRUs with epigenetic manipulations. Taken together, these results show that the epigenetic modification should be an important regulatory mechanism for self-renewal and fate decisions for normal HSCs in-vivo.

Epigenetic Regulation of Hematopoietic Stem Cell Self-Renewal by Polycomb Group Genes

2005 ◽  
Vol 81 (4) ◽  
pp. 294-300 ◽  
Author(s):  
Atsushi Iwama ◽  
Hideyuki Oguro ◽  
Masamitsu Negishi ◽  
Yuko Kato ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Epigenetic Regulation ◽  
Polycomb Group ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Polycomb Group Genes

scholarly journals Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Simple Procedure ◽  
Hematopoietic Cells ◽  
Stem Cell Population ◽  
Proliferative Potential ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

scholarly journals Hyperactive Nras and Dose Reduction of Tet2 Collaborate to Dys-Regulate Hematopoietic Stem Cells and Promote Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1204-1204
Author(s):  
Xi Jin ◽  
Tingting Qin ◽  
Nathanael G Bailey ◽  
Meiling Zhao ◽  
Kevin B Yang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Double Mutant ◽  
Mutant Mice ◽  
Cytokine Signaling ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Single Mutant ◽  
Tet2 Mutation

Abstract Activating mutations in RAS and somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) are frequently detected in hematologic malignancies. Global genomic sequencing revealed the co-occurrence of RAS and TET2 mutations in chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias (AMLs), suggesting that the two mutations collaborate to induce malignant transformation. However, how the two mutations interact with each other, and the effects of co-existing RAS and TET2 mutations on hematopoietic stem cell (HSC) function and leukemogenesis, remains unknown. In this study, we generated conditional Mx1-Cre+;NrasLSL-G12D/+;Tet2fl/+mice (double mutant) and activated the expression of mutant Nras and Tet2 in hematopoietic tissues with poly(I:C) injections. Double mutant mice had significantly reduced survival compared to mice expressing only NrasG12D/+ or Tet2+/-(single mutants). Hematopathology and flow-cytometry analyses showed that these mice developed accelerated CMML-like phenotypes with higher myeloid cell infiltrations in the bone marrow and spleen as compared to single mutants. However, no cases of AML occurred. Given that CMML is driven by dys-regulated HSC function, we examined stem cell competitiveness, self-renewal and proliferation in double mutant mice at the pre-leukemic stage. The absolute numbers of HSCs in 10-week old double mutant mice were comparable to that observed in wild type (WT) and single mutant mice. However, double mutant HSCsdisplayed significantly enhanced self-renewal potential in colony forming (CFU) replating assays. In vivo competitive serial transplantation assays using either whole bone marrow cells or 15 purified SLAM (CD150+CD48-Lin-Sca1+cKit+) HSCs showed that while single mutant HSCs have increased competitiveness and self-renewal compared to WT HSCs, double mutants have further enhanced HSC competitiveness and self-renewal in primary and secondary transplant recipients. Furthermore, in vivo BrdU incorporation demonstrated that while Nras mutant HSCs had increased proliferation rate, Tet2 mutation significantly reduced the level of HSC proliferation in double mutants. Consistent with this, in vivo H2B-GFP label-retention assays (Liet. al. Nature 2013) in the Col1A1-H2B-GFP;Rosa26-M2-rtTA transgenic mice revealed significantly higher levels of H2B-GFP in Tet2 mutant HSCs, suggesting that Tet2 haploinsufficiency reduced overall HSC cycling. Overall, these findings suggest that hyperactive Nras signaling and Tet2 haploinsufficiency collaborate to enhance HSC competitiveness through distinct functions: N-RasG12D increases HSC self-renewal, proliferation and differentiation, while Tet2 haploinsufficiency reduces HSC proliferation to maintain HSCs in a more quiescent state. Consistent with this, gene expression profiling with RNA sequencing on purified SLAM HSCs indicated thatN-RasG12D and Tet2haploinsufficiencyinduce different yet complementary cellular programs to collaborate in HSC dys-regulation. To fully understand how N-RasG12D and Tet2dose reduction synergistically modulate HSC properties, we examined HSC response to cytokines important for HSC functions. We found that when HSCs were cultured in the presence of low dose stem cell factor (SCF) and thrombopoietin (TPO), only Nras single mutant and Nras/Tet2 double mutant HSCs expanded, but not WT or Tet2 single mutant HSCs. In the presence of TPO and absence of SCF, HSC expansion was only detected in the double mutants. These results suggest that HSCs harboring single mutation of Nras are hypersensitive to cytokine signaling, yet the addition of Tet2 mutation allows for further cytokine independency. Thus, N-RasG12D and Tet2 dose reduction collaborate to promote cytokine signaling. Together, our data demonstrate that hyperactive Nras and Tet2 haploinsufficiency collaborate to alter global HSC gene expression and sensitivity to stem cell cytokines. These events lead to enhanced HSC competitiveness and self-renewal, thus promoting transition toward advanced myeloid malignancy. This model provides a novel platform to delineate how mutations of signaling molecules and epigenetic modifiers collaborate in leukemogenesis, and may identify opportunities for new therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

scholarly journals Slug deficiency enhances self-renewal of hematopoietic stem cells during hematopoietic regeneration

Blood ◽  
2010 ◽  
Vol 115 (9) ◽  
pp. 1709-1717 ◽  
Author(s):  
Yan Sun ◽  
Lijian Shao ◽  
Hao Bai ◽  
Zack Z. Wang ◽  
Wen-Shu Wu
Keyword(s):  
Hematopoietic Cells ◽  
Stress Conditions ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steady State Condition ◽  
Therapeutic Benefits ◽  
Evolutionarily Conserved ◽  
Novel Strategy

Abstract Both extrinsic and intrinsic mechanisms tightly govern hematopoietic stem cell (HSC) decisions of self-renewal and differentiation. However, transcription factors that can selectively regulate HSC self-renewal division after stress remain to be identified. Slug is an evolutionarily conserved zinc-finger transcription factor that is highly expressed in primitive hematopoietic cells and is critical for the radioprotection of these key cells. We studied the effect of Slug in the regulation of HSCs in Slug-deficient mice under normal and stress conditions using serial functional assays. Here, we show that Slug deficiency does not disturb hematopoiesis or alter HSC homeostasis and differentiation in bone marrow but increases the numbers of primitive hematopoietic cells in the extramedullary spleen site. Deletion of Slug enhances HSC repopulating potential but not its homing and differentiation ability. Furthermore, Slug deficiency increases HSC proliferation and repopulating potential in vivo after myelosuppression and accelerates HSC expansion during in vitro culture. Therefore, we propose that Slug is essential for controlling the transition of HSCs from relative quiescence under steady-state condition to rapid proliferation under stress conditions. Our data suggest that inhibition of Slug in HSCs may present a novel strategy for accelerating hematopoietic recovery, thus providing therapeutic benefits for patients after clinical myelosuppressive treatment.

scholarly journals Gene expression profile of a selection of Polycomb Group genes during zebrafish embryonic and germ line development

PLoS ONE ◽  
2018 ◽  
Vol 13 (7) ◽  
pp. e0200316 ◽  
Author(s):  
Naomi D. Chrispijn ◽  
Karolina M. Andralojc ◽  
Charlotte Castenmiller ◽  
Leonie M. Kamminga
Keyword(s):  
Gene Expression ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Germ Line ◽  
Polycomb Group ◽  
Polycomb Group Genes ◽  
Selection Of
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