scholarly journals Aid is a key regulator of myeloid/erythroid differentiation and DNA methylation in hematopoietic stem/progenitor cells

Blood ◽  
2017 ◽  
Vol 129 (13) ◽  
pp. 1779-1790 ◽  
Author(s):  
Hiroyoshi Kunimoto ◽  
Anna Sophia McKenney ◽  
Cem Meydan ◽  
Kaitlyn Shank ◽  
Abbas Nazir ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Genetic Loci ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points

Key Points Aid loss leads to altered differentiation, transcription, and methylation in specific genetic loci in hematopoietic stem/progenitor cells. Aid loss does not contribute to enhanced HSC self-renewal or cooperate with Flt3-ITD in myeloid leukemogenesis.

Dnmt3a is Required For Aberrant Self-Renewal Driven by PML-RARA

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 477-477
Author(s):  
Christopher B Cole ◽  
Angela M. Verdoni ◽  
David H Spencer ◽  
Timothy J. Ley
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Myeloid Cells ◽  
Cd11b Expression ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Marrow Cells

We previously identified recurrent mutations in the DNA methyltransferase DNMT3A in patients with acute myeloid leukemia (AML). DNMT3A and the highly homologous gene DNMT3B encode the two methyltransferases that are primarily responsible for mediating de novo methylation of specific CpG residues during differentiation. Loss of Dnmt3a in hematopoietic stem cells impairs their ability to differentiate into committed progenitors (Challen et al Nat Gen 44:23, 2011). Importantly, DNMT3A mutations are mutually exclusive of the favorable prognosis AML-initiating translocations, including the t(15;17) translocation (which creates the PML-RARA fusion gene), and translocations involving MLL. PML-RARA has been shown to interact with DNMT3A in vitro (Di Croce et al Science 295:1079,2002), and to require DNMT3A to induce methylation and transcriptional silencing of a subset of specific target genes. These findings, and the lack of DNMT3A mutations in APL patients, suggest that PML-RARA may require functional DNMT3A to initiate leukemia. To investigate this possibility, we utilized a well-characterized transgenic mouse model (in a pure B6 background) in which expression of PML-RARA is driven in hematopoietic stem/progenitor cells by the mouse Cathepsin G locus (Ctsg-PML-RARA+/- mice). These mice spontaneously develop acute promyelocytic leukemia (APL) with high penetrance and long latency, and also exhibit a preleukemic phenotype marked by the accumulation of myeloid cells in bone marrow and spleen. In addition, myeloid progenitor cells derived from these mice have the ability to serially replate in methylcellulose cultures, demonstrating aberrant self-renewal. We generated Ctsg-PML-RARA+/- mice lacking Dnmt3a (PML-RARA+/- x Dnmt3a-/-) as well as mice in which conditional ablation of Dnmt3b in hematopoietic cells is driven by Vav-Cre (PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+). Loss of Dnmt3a completely abrogated the ex vivo replating ability of PML-RARA bone marrow (Figure 1). Although colonies from both PML-RARA+/- and PML-RARA+/- x Dnmt3a-/- mice appeared similar in morphology and number on the first plating, PML-RARA+/- x Dnmt3a-/- marrow ceased to form colonies with subsequent replating (see Figure), and cultured cells lost the expression of the myeloid marker CD11b. The same phenotype was also observed using bone marrow from both genotypes that was secondarily transplanted into wild type recipients, indicating that it is intrinsic to transplantable hematopoietic progenitors. Reintroduction of DNMT3A into bone marrow cells derived from PML-RARA+/- x Dnmt3a-/- mice with retroviral transduction restored replating ability and CD11b expression. Competitive repopulation experiments with PML-RARA+/- x Dnmt3a-/- marrow revealed a decreased contribution to peripheral lymphoid and myeloid cells at 4 weeks, relative to PML-RARA+/- or WT control animals. Finally, 12 weeks after transplantation, recipients of PML-RARA+/- x Dnmt3a-/- bone marrow did not display an accumulation of myeloid cells in the bone marrow and spleen. Importantly, bone marrow from PML-RARA+/- x Dnmt3b fl/fl x Vav-Cre+/- mice displayed no replating deficit or loss of CD11b expression ex vivo, indicating different functions for Dnmt3a versus Dnmt3b in this model. Finally, we interrogated the effect of Dnmt3a loss on bone marrow DNA methylation patterns using a liquid phase DNA capture technique that sampled ∼1.9 million mouse CpGs at >10x coverage. Loss of Dnmt3a caused a widespread loss of DNA methylation in whole bone marrow cells, with 36,000 CpGs that were highly methylated (methylation value >0.7) in the PML-RARA+/- and WT mice, but hypomethylated (methylation value <0.4) in Dnmt3a-/- and PML-RARA+/- x Dnmt3a-/- mice. Characterization of the effect of Dnmt3a loss on leukemia latency, penetrance, and phenotype in PML-RARA+/- mice is currently being defined in a tumor watch. In summary, we have demonstrated that PML-RARA requires functional Dnmt3a (but not Dnmt3b) to drive aberrant self-renewal of myeloid progenitors ex vivo, and that loss of Dnmt3a leads to widespread DNA hypomethylation in bone marrow cells, and abrogates preleukemic changes in mice expressing PML-RARA. This data may explain why DNMT3A mutations are not found in patients with APL initiated by PML-RARA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals CXCR2 and CXCL4 regulate survival and self-renewal of hematopoietic stem/progenitor cells

Blood ◽  
2016 ◽  
Vol 128 (3) ◽  
pp. 371-383 ◽  
Author(s):  
Amy Sinclair ◽  
Laura Park ◽  
Mansi Shah ◽  
Mark Drotar ◽  
Simon Calaminus ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Murine Models ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Genetic Ablation ◽  
Key Points ◽  
Chemokine Ligands ◽  
Quiescent Hscs

Key Points Chemokine ligands CXCL1-4, 6, 10, 11, and 13 are upregulated in human quiescent HSCs with CXCR2 and CXCL4 regulating their survival. Genetic ablation of Cxcr2 or Cxcl4 in murine models induces initial expansion but eventual exhaustion of HSC in transplantation assays.

scholarly journals Oncogenic N-Ras and Tet2 haploinsufficiency collaborate to dysregulate hematopoietic stem and progenitor cells

2018 ◽  
Vol 2 (11) ◽  
pp. 1259-1271 ◽  
Author(s):  
Xi Jin ◽  
Tingting Qin ◽  
Meiling Zhao ◽  
Nathanael Bailey ◽  
Lu Liu ◽  
...  
Keyword(s):  
Overall Survival ◽  
Progenitor Cells ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Stem And Progenitor Cells

Key Points N-RasG12D and haploinsufficient Tet2 collaborate to induce lethal and highly penetrant CMML in mice with shortened overall survival. N-RasG12D and haploinsufficient Tet2 together promote balanced proliferation and enhanced competitiveness and self-renewal in HSPCs.

Tet2 Negatively Regulates Homeostasis and Differentiation of Hematopoietic Stem Cells in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2354-2354
Author(s):  
Myunggon Ko ◽  
Hozefa S. Bandukwala ◽  
Jungeun An ◽  
Edward D. Lamperti ◽  
Elizabeth C. Thompson ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Enzymatic Activity ◽  
Cancer Cells ◽  
Progenitor Cells ◽  
Loss Of Function ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Macrophage Lineage

Abstract Abstract 2354 Epigenetic alterations in cancer cells include aberrant DNA methylation and histone modifications. Specifically, cancer cells display global hypomethylation associated with genomic instability as well as promoter hypermethylation associated with inactivation of tumor suppressor, cell cycle or repair-related genes. In the hematopoietic system, whole-genome sequencing and other genetic analyses have led to the discovery of recurrent somatic alterations that contribute to the pathogenesis of a variety of myeloid malignancies by perturbing the epigenetic landscape of cancer cells. Ten-Eleven-Translocation (TET) family enzymes, TET1, TET2, and TET3 modify DNA methylation status by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in a 2-oxoglutarate and Fe2+-dependent manner. TET2 is located in chromosome 4q24, a region undergoing frequent microdeletions and uniparental disomy in patients with a wide spectrum of myeloid malignancies. Somatic mutations in TET2 are some of the most prevalent acquired mutations in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML) and secondary AML (sAML). We previously showed that missense mutations of TET2 in myeloid malignancies are loss-of-function mutations that compromise its dioxygenase activity. TET2 mutations correlate with decreased levels of 5hmC in patients. Tet2-depleted mouse hematopoietic precursor cells are preferentially committed to differentiation towards monocyte/ macrophage lineages in culture. The levels of DNA methylation in patients with high 5hmC versus healthy controls are similar, however, samples from patients with low 5hmC show hypomethylation relative to controls at the majority of differentially methylated CpG sites. Although it is postulated that impaired TET2 activity may potentiate myeloid transformation by influencing hematopoietic stem/progenitor cells (HSPCs), it has yet to be directly tested whether Tet2 mutations or deletions are implicated in abnormal hematopoiesis in vivo. To clarify the function of Tet2 in hematopoietic development, we generated mice with targeted disruption of the Tet2 catalytic domain and found that Tet2 is critical for self-renewal and differentiation of hematopoietic stem cells (HSCs). Ablation of Tet2 specifically repressed Tet2 expression with no effect on the other Tet family members, Tet1 and Tet3. Dot blot analysis showed that Tet2-deficient cells contain significantly diminished levels of genomic 5hmC in several organs examined. Tet2 deficiency augmented the frequency and absolute number of HSPC compartment in a cell-autonomous manner. In competitive transplantation assays, Tet2-deficient HSCs were capable of multi-lineage reconstitution and possessed a competitive advantage over wild type HSCs, resulting in enhanced hematopoiesis into both lymphoid and myeloid lineages. In vitro differentiation assays showed that Tet2 restrains HSCs from undergoing differentiation, as assessed by expression of lineage markers upon differentiation. Despite this antagonizing effect, however, the number of monocyte/ macrophage cells was greater in Tet2−/− cultures compared with controls, and immature Tet2−/− progenitor cells differentiated prematurely into the monocyte/macrophage lineage. These results indicate that Tet2 deficiency alters stem/progenitor cell properties to delay HSC differentiation and induce developmental skewing towards the monocyte/macrophage lineage. Taken together, these studies indicate that Tet2 has a critical role in regulating the expansion and self-renewal of HSCs. Our data suggest that cell fate decisions of HSPC are affected by TET2 mutations that decrease enzymatic activity, and that this phenomenon has a crucial role in the pathogenesis of diverse myeloid malignancies. We are testing whether Tet2 deficiency synergises with other recurrent mutations to promote myeloid malignancies. Because loss-of-function mutations in TET1 or TET3 have not been reported in most TET2-mutated cancer and TET2 loss-of-function seems to facilitate myeloid transformation because of impaired 5hmC production, it might be beneficial from the perspective of cancer therapies to develop strategies to activate the enzymatic activity of other TET proteins. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ex vivo expansion of human hematopoietic stem and progenitor cells

Blood ◽  
2011 ◽  
Vol 117 (23) ◽  
pp. 6083-6090 ◽  
Author(s):  
Ann Dahlberg ◽  
Colleen Delaney ◽  
Irwin D. Bernstein
Keyword(s):  
Stem Cell ◽  
Growth Factors ◽  
Notch Signaling ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Growth Factors ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

AbstractDespite progress in our understanding of the growth factors that support the progressive maturation of the various cell lineages of the hematopoietic system, less is known about factors that govern the self-renewal of hematopoietic stem and progenitor cells (HSPCs), and our ability to expand human HSPC numbers ex vivo remains limited. Interest in stem cell expansion has been heightened by the increasing importance of HSCs in the treatment of both malignant and nonmalignant diseases, as well as their use in gene therapy. To date, most attempts to ex vivo expand HSPCs have used hematopoietic growth factors but have not achieved clinically relevant effects. More recent approaches, including our studies in which activation of the Notch signaling pathway has enabled a clinically relevant ex vivo expansion of HSPCs, have led to renewed interest in this arena. Here we briefly review early attempts at ex vivo expansion by cytokine stimulation followed by an examination of our studies investigating the role of Notch signaling in HSPC self-renewal. We will also review other recently developed approaches for ex vivo expansion, primarily focused on the more extensively studied cord blood–derived stem cell. Finally, we discuss some of the challenges still facing this field.

scholarly journals Human leukemia mutations corrupt but do not abrogate GATA-2 function

2018 ◽  
Vol 115 (43) ◽  
pp. E10109-E10118 ◽  
Author(s):  
Koichi R. Katsumura ◽  
Charu Mehta ◽  
Kyle J. Hewitt ◽  
Alexandra A. Soukup ◽  
Isabela Fraga de Andrade ◽  
...  
Keyword(s):  
Dna Binding ◽  
Progenitor Cells ◽  
Zinc Finger ◽  
Erythroid Differentiation ◽  
Cell Types ◽  
Myeloid Differentiation ◽  
Human Leukemia ◽  
Hematopoietic Stem ◽  
Amino Terminal ◽  
Disease Mutations

By inducing the generation and function of hematopoietic stem and progenitor cells, the master regulator of hematopoiesis GATA-2 controls the production of all blood cell types. Heterozygous GATA2 mutations cause immunodeficiency, myelodysplastic syndrome, and acute myeloid leukemia. GATA2 disease mutations commonly disrupt amino acid residues that mediate DNA binding or cis-elements within a vital GATA2 intronic enhancer, suggesting a haploinsufficiency mechanism of pathogenesis. Mutations also occur in GATA2 coding regions distinct from the DNA-binding carboxyl-terminal zinc finger (C-finger), including the amino-terminal zinc finger (N-finger), and N-finger function is not established. Whether distinct mutations differentially impact GATA-2 mechanisms is unknown. Here, we demonstrate that N-finger mutations decreased GATA-2 chromatin occupancy and attenuated target gene regulation. We developed a genetic complementation assay to quantify GATA-2 function in myeloid progenitor cells from Gata2 −77 enhancer-mutant mice. GATA-2 complementation increased erythroid and myeloid differentiation. While GATA-2 disease mutants were not competent to induce erythroid differentiation of Lin−Kit+ myeloid progenitors, unexpectedly, they promoted myeloid differentiation and proliferation. As the myelopoiesis-promoting activity of GATA-2 mutants exceeded that of GATA-2, GATA2 disease mutations are not strictly inhibitory. Thus, we propose that the haploinsufficiency paradigm does not fully explain GATA-2–linked pathogenesis, and an amalgamation of qualitative and quantitative defects instigated by GATA2 mutations underlies the complex phenotypes of GATA-2–dependent pathologies.

scholarly journals Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2812-2821 ◽  
Author(s):  
Fabiana Perna ◽  
Nadia Gurvich ◽  
Ruben Hoya-Arias ◽  
Omar Abdel-Wahab ◽  
Ross L. Levine ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Myeloproliferative Neoplasms ◽  
Erythroid Differentiation ◽  
Polycomb Group ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cd34 Cells

Abstract L3MBTL1, the human homolog of the Drosophila L(3)MBT polycomb group tumor suppressor gene, is located on chromosome 20q12, within the common deleted region identified in patients with 20q deletion-associated polycythemia vera, myelodysplastic syndrome, and acute myeloid leukemia. L3MBTL1 is expressed within hematopoietic CD34+ cells; thus, it may contribute to the pathogenesis of these disorders. To define its role in hematopoiesis, we knocked down L3MBTL1 expression in primary hematopoietic stem/progenitor (ie, CD34+) cells isolated from human cord blood (using short hairpin RNAs) and observed an enhanced commitment to and acceleration of erythroid differentiation. Consistent with this effect, overexpression of L3MBTL1 in primary hematopoietic CD34+ cells as well as in 20q− cell lines restricted erythroid differentiation. Furthermore, L3MBTL1 levels decrease during hemin-induced erythroid differentiation or erythropoietin exposure, suggesting a specific role for L3MBTL1 down-regulation in enforcing cell fate decisions toward the erythroid lineage. Indeed, L3MBTL1 knockdown enhanced the sensitivity of hematopoietic stem/progenitor cells to erythropoietin (Epo), with increased Epo-induced phosphorylation of STAT5, AKT, and MAPK as well as detectable phosphorylation in the absence of Epo. Our data suggest that haploinsufficiency of L3MBTL1 contributes to some (20q−) myeloproliferative neoplasms, especially polycythemia vera, by promoting erythroid differentiation.

scholarly journals A GPI-Anchored Protein, CD109, Protects Hematopoietic Progenitor Cells from Erythroid Differentiation Induced By TGF-β

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3736-3736
Author(s):  
Tanabe Mikoto ◽  
Nguyen Hoang Maianh ◽  
Kohei Hosokawa ◽  
Noriharu Nakagawa ◽  
Luis Espinoza ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Leukemia Cell Line ◽  
Serum Free Medium ◽  
Free Medium ◽  
Hematopoietic Stem ◽  
Serum Free ◽  
Company Limited ◽  
Immune Mediated

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may have some roles in the negative regulation of the HSPC commitment induced by inflammatory cytokines given the fact that progenies of GPI(-) HSPC are often detected in patients with immune-mediated bone marrow (BM) failure. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs remains unknown. We previously demonstrated that TGF-β induced erythroid differentiation of TF-1 cells, a myeloid leukemia cell line that expresses CD109, in a dose-dependent manner and that knockout of the CD109 gene resulted in erythroid differentiation of TF-1 cells cultured in fetal bovine serum-containing medium, suggesting an inhibitory role of CD109 in the erythroid differentiation of HSPCs induced by low levels of TGF-β (Blood, 2018. 132 (Suppl.1) :3874). However, as most CD109 KO TF-1 cells changed into erythroid cells, they were unsuitable for investigating the role of CD109 in the erythroid differentiation induced by TGF-β. To overcome this issue, we prepared TF-1 cells and cord blood (CB) HSPCs in which the CD109 expression was transiently downregulated, and attempted to further clarify the role of CD109. [Methods] TF-1 cells and CD34+ cells isolated from CB mononuclear cells were treated with siRNA that was complementary to CD109 mRNA. CD109 knockdown cells were cultured for 4 days in serum-free medium supplemented with stem cell factor, thrombopoietin, and erythropoietin with or without TGF-β. In separate experiments, TF-1 cells were treated with phosphatidylinositol-specific phospholipase C (PIPL-C) treatment for 1 hour and were incubated in the presence or absence of TGF-β. CD109 KO TF-1 cells were incubated in serum-free medium (StemPro-34 SFM) for 14 days and their phenotype was determined using flow cytometry (FCM). The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) and iron staining. [Results] The down-regulation of CD109 in TF-1 cells by the siRNA treatment increased GPA expression in response to 12 ng/ml of TGF-β from 1.77% to 35.6%. The transient depletion of GPI-APs by PIPL-C also augmented the GPA expression induced by TGF-β from 1.27% to 6.77%. In both BM of healthy individuals and CB, CD109 was more abundantly expressed in Lin-CD34+CD38-CD90+CD45RA- hematopoietic stem cells (HSCs) than in Lin-CD34+CD38-CD90-CD45RA- multipotent progenitors (MPPs) and Lin-CD34+CD38+ HSPCs (Fig. 1). The treatment of CB cells with siRNA reduced the CD109 expression in Lin-CD34+CD38+ cells from 55.9% to 23.1%. TGF-β induced the expression of GPA in Lin-CD34+CD38+CD123-CD45RA- megakaryocyte-erythrocyte progenitor cells (MEPs) of CD109 knockdown cells to a greater degree than the control counterpart (Fig. 2). During 14-day serum-free culture, GPA-positive CD109 KO TF-1 cells died, and similarly to WT TF-1 cells, most surviving CD109 KO TF-1 cells were GPA-negative. TGF-β treatment induced erythroid differentiation in CD109 KO TF-1 cells to a greater degree than in WT TF-1 cells. [Conclusions] CD109 plays a key role in the inhibition of TF-1 erythroid differentiation in response to TGF-β. CD109 may suppress TGF-β signaling, and the lack of CD109 may make PIGA-mutated HSPCs more sensitive to TGF-β, thus leading to the preferential commitment of the mutant erythroid progenitor cells to mature red blood cells in immune-mediated BM failure. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy.

scholarly journals Functional Investigation of the Argonaute Proteins in Human Hematopoietic Stem and Progenitor Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 32-32
Author(s):  
Gordon G. L. Wong ◽  
Gabriela Krivdova ◽  
Olga I. Gan ◽  
Jessica L. McLeod ◽  
John E. Dick ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Erythroid Differentiation ◽  
Lineage Commitment ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Erythroid Precursor ◽  
Lineage Differentiation ◽  
Erythroid Precursors

Micro RNA (miRNA)-mediated gene silencing, largely mediated by the Argonaute (AGO) family proteins, is a post-transcriptional gene expression control mechanism that has been shown to regulate hematopoietic stem and progenitor cells (HSPCs) quiescence, self-renewal, proliferation, and differentiation. Interestingly, only the function of AGO2 in hematopoiesis has been investigated. O'Carroll et al. (2007) showed that AGO2 knockout in mice bone marrow cells interferes with B220low CD43- IgM-pre-B cells and peripheral B cell differentiation and impairs Ter119high, CD71high erythroid precursors maturation. However, the functional significance of other AGO proteins in the regulation of stemness and lineage commitment remains unclear. AGO submembers, AGO1-4 in humans, are traditionally believed to act redundantly in their function. However, our previous proteomic analysis from sorted populations of the human hematopoietic hierarchy shows each sub-member is differentially expressed during HSPCs development, suggesting each sub-member may have a specialized function in hematopoiesis. Here, we conducted CRISPR-Cas9 mediated knockout of AGO1-4 in human cord blood derived long-term (LT-) and short-term hematopoietic stem cells (ST-HSCs) and investigated the impact of the loss of function of individual AGOs in vitro and in vivo in xenograft assays. From the in vitro experiment, we cultured CRISPR-edited LT- or ST-HSCs in a single cell manner on 96-well plates pre-cultured with murine MS5 stroma cells in erythro-myeloid differentiation condition. The colony-forming capacity and lineage commitment of each individual HSC is assessed on day 17 of the culture. Initial data showed that AGO1, AGO2 and AGO3 knockout decreased the colony formation efficacy of both LT- and ST-HSCs, suggesting AGO1, AGO2 and AGO3 are involved in LT- and ST-HSCs proliferation or survival. As for lineage output, AGO1 knockout increases CD56+ natural killer cell commitment in LT-HSCs and erythroid differentiation in ST-HSCs; AGO2 knockout increases erythroid differentiation in both LT- and ST-HSCs and decreases myeloid differentiation in ST-HSCs; while AGO4 knockout seems to decrease erythroid output. For the in vivo experiment, we xenotransplanted AGO1 and AGO2 knockout LT-HSCs in irradiated immunodeficient NSG mice and assessed the change in LT-HSCs engraftment level and lineage differentiation profile at 12- and 24-week time points. We found that AGO2 knockout increased CD45+ engraftment at both 12- and 24-weeks. Aligning with our in vitro data, AGO2 knockout increases GlyA+ erythroid cells at 12- and 24-weeks. The increase in GlyA+ erythroid cells is a consequence of the 2-fold increase in GlyA+ CD71+ erythroid precursor cells, recapitulating previous findings that AGO2 knockout in mice impairs CD71high erythroid precursor maturation leading to the accumulation of undifferentiated CD71+ erythroid precursors (O'Carroll et al., 2007). Accumulation of early progenitors of the erythroid lineage, including the common myeloid progenitors (CMPs) and myelo-erythroid progenitor (MEPs) were observed, as well as their progeny including CD33+ myeloid and CD41+ megakaryocytes. For the myeloid lineage, AGO2 knockout shifts myeloid differentiation toward CD66b+ granulocytes from CD14+ monocytes. For lymphoid, AGO2 knockout decreases CD19+ CD10- CD20+ mature B-lymphoid cells, which again aligns with previous AGO2 knockout mice results. On the other hand, AGO1 knockout LT-HSCs share some similar phenotype with AGO2 knockout LT-HSCs, where AGO1 knockout increases CD71+ erythroid precursors. However, AGO1 knockout in LT-HSCs also results in unique phenotypes, with a decrease in neutrophil formation and an increase in CD4+ CD8+ T progenitor cells are observed. AGO3 and AGO4 knockout experiments are in progress. In summary, our AGO2 knockout experiments recapitulate the reported results from murine studies but also illustrate a more complete role of AGO2 in hematopoietic lineage differentiation. Moreover, AGO knockout experiments of individual submembers are revealing novel insights into their role in the regulation of stemness and lineage commitment of LT-HSCs and ST-HSCs. These data point to a unique role of different AGO isoforms in lineage commitment in human HSCs and argue against redundant functioning. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.

The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal

Blood ◽  
2021 ◽  
Author(s):  
Yuqing Yang ◽  
Andrew J Kueh ◽  
Zoe Grant ◽  
Waruni Abeysekera ◽  
Alexandra L Garnham ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Hematopoietic Stem Cell ◽  
Cell Function ◽  
Bone Marrow Cells ◽  
High Rate ◽  
Embryonic Patterning ◽  
Self Renewal ◽  
Hematopoietic Stem

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac) and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used two complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1 null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow two to six weeks after Hbo1 deletion. Hbo1 deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors (HPCs). The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1 and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

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