Transformation Mechanisms of the Nfia-ETO2 Fusion Gene Associated with Pediatric Pure Acute Erythroleukemia

Pure erythroleukemia (PEL) is a very aggressive, but poorly understood form of acute myeloid leukemia characterized by malignant accumulation of erythroid progenitor cells. A novel t(1;16)(p31;q24) chromosomal translocation leading to expression of a fusion between the nuclear factor I A (NFIA) and the ETO2 transcriptional co-regulator (a.k.a. CBFA2T3 or MTG16) has been identified in pediatric patients with PEL. Based on the function of the fusion partners, we hypothesized that NFIA-ETO2 (N-E) might initiate PEL by interfering with erythroid differentiation. To investigate its function, we cloned a full-length ORF and retrovirally expressed N-E in primary mouse bone marrow (BM)- and fetal liver (FL)-derived erythroblasts (EB). N-E expression significantly increased proliferation and blocked differentiation of EB. N-E expressing BM-derived hematopoietic stem and progenitor cells (HSPC) could be plated in erythropoietin (EPO)-containing methylcellulose (MC) for up to 3 rounds. Expression of N-E deletion mutants lacking the NFIA DNA-binding, the ETO2 NHR2 or NHR4 (ΔNHR4) transcriptional repression domains were unable to block erythroid differentiation. Notably, interfering with the ETO2-NHR2 oligomerization domain by overexpressing a competing peptide overcame the N-E-mediated differentiation block. Transplantation of N-E-expressing BM-derived HSPC into irradiated syngenic mice did not induce any disease suggesting the need of genetic cooperation. As TP53 gain-of-function (GOF) mutations are molecular hallmarks of PEL, we explored functional cooperation by using a conditional TP53R248Q allele. Interestingly, the TP53 status did not affect EB in vitro proliferation or differentiation. However, N-E expression increased proliferation of TP53R248Q+ EB and resulted in the formation of abnormal round and dense colonies in MC that could be serially propagated. In addition, transplantation of N-E-expressing TP53R248Q+ EB into irradiated recipients induced a transplantable PEL-like disease after a median latency of 4 months. Symptomatic mice presented with anemia, thrombocytopenia, multi-organ tumor cell infiltration and increased white blood cell counts. To better understand the molecular mechanism, we compared the gene expression signatures before and 24 hours after induced differentiation of FL-derived EB expressing WT or the inactive ΔNHR4 N-E mutant, in presence or absence of TP53R248Q. Principal component analysis (PCA) revealed a clear separation between the transcriptomes of WT EB expressing either the active or the inactive ΔNHR4 N-E (PC1:54.7%) and by their erythroid differentiation stage (PC2:9.07%). Overall, we observed 3753 (FDR<0.05, logFC>1.5) differentially expressed genes. Many of the significantly higher expressed genes (2313/3753) were related to hematopoietic stemness (GSEAs, p<0.001). Almost 10% of the significantly lower expressed genes (92/1440) were linked to the erythroid lineage development and to erythropoietic targets of NFIA or the erythroid master regulator GATA1. Interestingly, we also found reduced expression of genes encoding for ETO2-interacting transcription factors including TAL1 and KLF1. Despite a critical role on disease progression, PCA showed only minimal changes in the N-E expression signature in presence or absence of TP53R248Q with only 12 genes differently expressed (FDR<0.05, logFC>1). These genes were previously shown to be oncogenic mediators of TP53-GOF mutations, related to metabolism and transcriptional regulation. Interestingly, the signature of differentially expressed genes in N-E transformed FL-derived EB were significantly differentially expressed in tumor cells from pediatric but not adult PEL patients (p=0.00045), indicating the pediatric origin of the fusion. Collectively, we found that the PEL-associated N-E fusion blocks erythroid differentiation, and cooperates with a TP53-GOF mutation to induce a PEL-like disease in mice that phenocopies the human disease. Mechanistically, its activity seems to correlate with repression of erythroid regulatory genes controlled by the fusion partners NFIA, ETO2, and the erythroid master regulator GATA1. Disclosures No relevant conflicts of interest to declare.

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Bone Marrow Oxidative Stress and Acquired Lineage-Specific Genotoxicity in Hematopoietic Stem/Progenitor Cells Exposed to 1,4-Benzoquinone

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17165865 ◽

2020 ◽

Vol 17 (16) ◽

pp. 5865

Author(s):

Ramya Dewi Mathialagan ◽

Zariyantey Abd Hamid ◽

Qing Min Ng ◽

Nor Fadilah Rajab ◽

Salwati Shuib ◽

...

Keyword(s):

Oxidative Stress ◽

Bone Marrow ◽

Dna Damage ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Protein Carbonyls ◽

Mouse Bone Marrow ◽

Tail Moment ◽

Erythroid Progenitor ◽

Hematopoietic Stem

Hematopoietic stem/progenitor cells (HSPCs) are susceptible to benzene-induced genotoxicity. However, little is known about the mechanism of DNA damage response affecting lineage-committed progenitors for myeloid, erythroid, and lymphoid. Here, we investigated the genotoxicity of a benzene metabolite, 1,4-benzoquinone (1,4-BQ), in HSPCs using oxidative stress and lineage-directed approaches. Mouse bone marrow cells (BMCs) were exposed to 1,4-BQ (1.25–12 μM) for 24 h, followed by oxidative stress and genotoxicity assessments. Then, the genotoxicity of 1,4-BQ in lineage-committed progenitors was evaluated using colony forming cell assay following 7–14 days of culture. 1,4-BQ exposure causes significant decreases (p < 0.05) in glutathione level and superoxide dismutase activity, along with significant increases (p < 0.05) in levels of malondialdehyde and protein carbonyls. 1,4-BQ exposure induces DNA damage in BMCs by significantly (p < 0.05) increased percentages of DNA in tail at 7 and 12 μM and tail moment at 12 μM. We found crucial differences in genotoxic susceptibility based on percentages of DNA in tail between lineage-committed progenitors. Myeloid and pre-B lymphoid progenitors appeared to acquire significant DNA damage as compared with the control starting from a low concentration of 1,4-BQ exposure (2.5 µM). In contrast, the erythroid progenitor showed significant damage as compared with the control starting at 5 µM 1,4-BQ. Meanwhile, a significant (p < 0.05) increase in tail moment was only notable at 7 µM and 12 µM 1,4-BQ exposure for all progenitors. Benzene could mediate hematological disorders by promoting bone marrow oxidative stress and lineage-specific genotoxicity targeting HSPCs.

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Impaired Erythropoiesis Of Gfi-1 Null Hematopoietic Progenitor Cells Is Rescued By Reducing Id2 Levels

Blood ◽

10.1182/blood.v122.21.737.737 ◽

2013 ◽

Vol 122 (21) ◽

pp. 737-737

Author(s):

Wonil Kim ◽

Kimberly D Klarmann ◽

Jonathan R Keller

Keyword(s):

Transcription Factors ◽

Progenitor Cells ◽

Molecular Mechanisms ◽

Early Stage ◽

Erythroid Cell ◽

Mouse Bone Marrow ◽

Short Term ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Erythroid Development

Abstract The survival, self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPC) are tightly regulated by extrinsic signals, and intrinsically by transcription factors and their regulatory networks. The molecular and cellular mechanisms, which regulate the complex process of hematopoiesis, depend upon the correct expression of transcription factors and their regulators. One such family of regulators is the inhibitor of DNA binding/differentiation (Id), which is helix-loop-helix proteins that function by acting as dominant negative regulators of transcription factors such as E proteins, ETS, Pax, and retinoblastoma proteins. Expression of Id2, one of the Id family proteins, is regulated by growth factor independence-1 (Gfi-1) encoding a transcriptional repressor. Gfi-1 is required for the development of multiple cell lineages including HSPC and ultimately differentiated blood cells. Although genes have been identified to mediate hematopoietic defects observed in Gfi-1 knockout (Gfi-1 KO) mice including the maturational and developmental defects in granulocyte (CSF-1, RasGRP1, and PU.1) and B cell (PU.1 or Id2), and myeloid hyperplasia (Id2 or HoxA9), Gfi-1-target genes that mediate the defects in radioprotection, maintenance of HSC, and erythroid hyperplasia in Gfi-1 KO mice are unknown. Since Id2 expression is elevated in HSPC of Gfi-1 KO mice and Id2 promotes cell proliferation, we hypothesized that lowering Id2 expression could rescue the HSPC defects in the Gfi-1 KO mice. By transplanting Gfi-1 KO mouse bone marrow cells (BMC) into lethally-irradiated recipient mice, we observed that short-term reconstituting cell (STRC) activity in Gfi-1 KO BMC is rescued by transplanting Gfi-1 KO; Id2 Het (heterozygosity at the Id2 locus) BMC, while the long-term reconstitution defect of HSC was not. Interestingly, lineage- Sca-1- c-Kithi HPC, which enriched for megakaryocyte-erythroid progenitor (MEP) as one of the STRC, were fully restored in mice transplanted with Gfi-1 KO; Id2 Het BMC, in contrast to lack of the HPC in Gfi-1 KO BM-transplanted mice. The restoration of donor c-Kithi HPC was directly correlated with increased red blood cell (RBC) levels in recipient mice, which was produced after donor BM engraftment. Furthermore, we identified that reduced Id2 levels restore erythroid cell development by rescuing short-term hematopoietic stem cell, common myeloid progenitor and MEP in the Gfi-1 KO mice. In addition, burst forming unit-erythroid (BFU-E) colony assay showed that hemoglobinized BFU-E development was restored in Gfi-1 KO BM and spleen by lowering Id2 levels. Unlike Id2 reduction, reducing other Id family (Id1 or Id3) levels in Gfi-1 KO mice does not rescue the impaired development of erythroid and other hematopoietic lineages including myeloid, T and B cells. Abnormal expansion of CD71+ Ter119-/low erythroid progenitor cells was rescued in Gfi-1 KO; Id2 Het BMC compared to those in Gfi-1 KO mice. Thus, we hypothesized that erythroid development was blocked at the early stage of erythropoiesis due to the ectopic expression of Id2 in Gfi-1 KO mice. Using Id2 promoter-driven YFP reporter mice, we found that Id2 is highly expressed in the CD71+ Ter119-/low erythroid progenitors, and decreases as the cells mature to pro-erythroblasts and erythroblasts, suggesting that repression of Id2 expression is required for proper erythroid differentiation in the later stages. The dramatic changes of Id2 expression during erythroid development support our findings that the overexpression of Id2 in the absence of Gfi-1-mediated transcriptional repression causes impaired erythropoiesis at the early stage. To identify the molecular mechanisms that could account for how reduced Id2 levels rescue erythropoiesis in Gfi-1 KO mice, we compared the expression of genes and proteins in Gfi-1 KO; Id2 Het and Gfi-1 KO BMC. Using microarray, qRT-PCR and western blot, we discovered that reduction of Id2 expression in Gfi-1 KO BMC results in increased expression of Gata1, EKlf, and EpoR genes, which are required for erythropoiesis. However, the expression levels of cell cycle regulators were not altered by lowering Id2 expression in Gfi-1 KO mice. These data suggest a novel molecular mechanism in which Gfi-1 modulates erythropoiesis by repressing the expression of Id2 that reduce the levels of Id2 protein, binding to E2A and inhibiting the formation of E2A/Scl transcription enhancer complex. Disclosures: No relevant conflicts of interest to declare.

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Regulatory association of long noncoding RNAs and chromatin accessibility facilitates erythroid differentiation

Blood Advances ◽

10.1182/bloodadvances.2021005167 ◽

2021 ◽

Author(s):

Yunxiao Ren ◽

Junwei Zhu ◽

Yuanyuan Han ◽

Pin Li ◽

Jing Wu ◽

...

Keyword(s):

Progenitor Cells ◽

Erythroid Differentiation ◽

Chromatin Accessibility ◽

Human Cord Blood ◽

Erythroid Progenitor ◽

Integrated Network ◽

Hematopoietic Stem ◽

Multipotent Progenitor Cells ◽

Tf Gene ◽

Terminal Erythroid Differentiation

Erythroid differentiation is a dynamic process regulated by multiple factors, while the interaction between long non-coding RNAs and chromatin accessibility and its influence on erythroid differentiation remains unclear. To elucidate this interaction, we employed hematopoietic stem cells, multipotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, and erythroblasts from human cord blood as an erythroid differentiation model to explore the coordinated regulatory functions of lncRNAs and chromatin accessibility by integrating RNA-Seq and ATAC-Seq data. We revealed that the integrated network of chromatin accessibility and lncRNAs exhibits stage-specific changes throughout the erythroid differentiation process, and that the changes at the EB stage of maturation are dramatic. We identified a subset of stage-specific lncRNAs and transcription factors (TFs) that associate with chromatin accessibility during erythroid differentiation, in which lncRNAs are key regulators of terminal erythroid differentiation via a lncRNA-TF-gene network. LncRNA PCED1B-AS1 was revealed to regulate terminal erythroid differentiation by coordinating GATA1 dynamically binding to the chromatin and interacting with cytoskeleton network during erythroid differentiation. DANCR, another lncRNA that is highly expressed at the MEP stage, was verified to promote erythroid differentiation by compromising megakaryocyte differentiation and coordinating with chromatin accessibility and TFs, such as RUNX1. Overall, our results identified the associated network of lncRNAs and chromatin accessibility in erythropoiesis and provide novel insights into erythroid differentiation and abundant resources for further study.

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RNA‐Seq analysis of differentially expressed genes relevant to mismatch repair in aging hematopoietic stem‐progenitor cells

Journal of Cellular Biochemistry ◽

10.1002/jcb.28417 ◽

2019 ◽

Vol 120 (7) ◽

pp. 11401-11408 ◽

Cited By ~ 3

Author(s):

Xiaolan Lian ◽

Yongpin Dong ◽

Mingyi Zhao ◽

Yajie Liang ◽

Weiwei Jiang ◽

...

Keyword(s):

Progenitor Cells ◽

Mismatch Repair ◽

Differentially Expressed Genes ◽

Differentially Expressed ◽

Rna Seq ◽

Hematopoietic Stem

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Clastogenicity and Aneugenicity of 1,4-Benzoquinone in Different Lineages of Mouse Hematopoietic Stem/Progenitor Cells

Toxics ◽

10.3390/toxics9050107 ◽

2021 ◽

Vol 9 (5) ◽

pp. 107

Author(s):

Paik Wah Chow ◽

Zariyantey Abd Hamid ◽

Ramya Dewi Mathialagan ◽

Nor Fadilah Rajab ◽

Salwati Shuib ◽

...

Keyword(s):

Progenitor Cells ◽

Bone Marrow Cells ◽

Mouse Bone Marrow ◽

Hematopoietic Progenitor ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Myeloid Progenitor ◽

Lineage Specificity ◽

B Lymphoid

Previous reports on hematotoxicity and leukemogenicity related to benzene exposure highlighted its adverse effects on hematopoiesis. Despite the reported findings, studies concerning the mechanism of benzene affecting chromosomal integrity in lineage-committed hematopoietic stem/progenitor cells (HSPCs) remain unclear. Here, we studied the clastogenicity and aneugenicity of benzene in lineage-committed HSPCs via karyotyping. Isolated mouse bone marrow cells (MBMCs) were exposed to the benzene metabolite 1,4-benzoquinone (1,4-BQ) at 1.25, 2.5, 5, 7, and 12 μM for 24 h, followed by karyotyping. Then, the chromosomal aberration (CA) in 1,4-BQ-exposed hematopoietic progenitor cells (HPCs) comprising myeloid, Pre-B lymphoid, and erythroid lineages were evaluated following colony-forming cell (CFC) assay. Percentage of CA, predominantly via Robertsonian translocation (Rb), was increased significantly (p < 0.05) in MBMCs and all progenitors at all concentrations. As a comparison, Pre-B lymphoid progenitor demonstrated a significantly higher percentage of CA (p < 0.05) than erythroid progenitor at 1.25, 2.5, and 7 μM as well as a significantly higher percentage (p < 0.05) than myeloid progenitor at 7 μM of 1,4-BQ. In conclusion, 1,4-BQ induced CA, particularly via Rb in both MBMCs and HPCs, notably via a lineage-dependent response. The role of lineage specificity in governing the clastogenicity and aneugenicity of 1,4-BQ deserves further investigation.

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KLF4 as a Mediator of Quiescence in Hematopoietic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v104.11.4146.4146 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4146-4146

Author(s):

Jonathan K. Alder ◽

Robert W. Georgantas ◽

Xiaobing Yu ◽

Curt I. Civin

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Progenitor Cells ◽

Lentiviral Vector ◽

Mouse Bone Marrow ◽

Quiescent State ◽

Cmv Promoter ◽

Hematopoietic Stem ◽

Cycle Arrest ◽

Primary Mouse

Abstract At any given time, only a very small fraction of the hematopoietic stem cells (HSCs) in an organism are actively dividing; the vast majority of these cells remain in a quiescent state. HSCs can escape this quiescent state to self-renew and give rise to progenitor cells (HPCs), which expand rapidly to form all of the mature progeny cells of the lympho-hematopoietic system. Despite the centrality of quiescence in hematopoiesis, the mechanisms by which quiescence is regulated are largely unknown. Several studies have shown that p21CIP/WAF1 is required for cell cycle arrest of HSCs, and deletion of p21 results in an increase in HSC pool size. However, little is known about the pathways upstream of p21. Recently, we identified 81 genes that were highly over-expressed by normal HSC-enriched CD34+/CD38− cells compared to HPC-enriched CD34+/CD38+ cells. One of the HSC-over-expressed genes was Krupple-Like Factor 4 (KLF4), a member of the Kruppel-like transcription factor family. KLF4 has been associated with a quiescent phenotype in T and B lymphocytes, and exogenous expression of KLF4 induces growth arrest in a colon cancer cell line. It has been shown that KLF4 is an important mediator of p21 in the G1/S cell cycle arrest following DNA damage. Thus, we hypothesized that KLF4 is an important upstream regulator of HSPC quiescence via p21. To begin to test this, we designed a dual promoter self-inactivating (SIN) lentiviral vector that expressed KLF4 under the control of the EF1α promoter and GFP under the CMV promoter. We transduced primary mouse bone marrow (BM) Lin− or human cord blood (CB) CD34+ HSPCs, and purified the resulting successfully transduced GFP+ cells by fluorescence-activated cell sorting. KLF4-transduced human or mouse HPCs displayed a major reduction in colony forming capacity, as compared to either GFP− HPCs transduced with the vector containing KLF4 and GFP or HPCs transduced with the parental control vector expressing only GFP (Fig 1 shows representative experiments). In suspension cultures, KLF4-transduced cell populations generated fewer total cells, accompanied by an increase in apoptic/necrotic cells. In the KLF4-transduced cells, a higher percent were CD34+. These results are consistent with KLF4 inhibiting HPC differentiation and inducing a p21-like pathway with cell cycle arrest and increased apoptosis. Further investigation of KLF4 cellular and molecular actions may not only lead to enhanced understanding of the molecular basis of HSPC quiescence, but also to novel methods for expansion of normal HSPCs. Figure Figure

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Cooperating Effect of Rps14, Csnk1a1 and miRNA145/miRNA146a Haploinsufficiency in the Activation of the Innate Immune System in Del(5q) MDS

Blood ◽

10.1182/blood.v126.23.356.356 ◽

2015 ◽

Vol 126 (23) ◽

pp. 356-356

Author(s):

Schneider K. Rebekka ◽

Monica Schenone ◽

Monica Ferreira Ventura ◽

Fabian Beier ◽

Tim H. Bruemmendorf ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Research Funding ◽

Erythroid Differentiation ◽

Innate Immune ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Innate Immune Signaling ◽

Stem And Progenitor Cells ◽

Cell Type Specific

Abstract Heterozygous deletion of RPS14 occurs in del(5q) MDS and has been linked to impaired erythropoiesis, characteristic of this disease subtype. We previously generated a mouse model with conditional inactivation of Rps14 and demonstrated a p53-dependent erythroid differentiation defect with apoptosis at the transition from polychromatic to orthochromatic erythroblasts resulting in age-dependent progressive anemia, megakaryocyte dysplasia, and loss of hematopoietic stem cell (HSC) quiescence. We now sought to determine the mechanistic basis for the anemia using unbiased, quantitative mass spectrometry in erythroid progenitor cells. We found powerful induction of proteins involved in innate immune signaling, particularly the danger associated molecular pattern (DAMP) heterodimeric S100A8/S100A9 proteins. We found significantly increased S100a8 in the erythroid progenitor populations affected by the differentiation block (RIII-RIV population) and in monocytes and macrophages of Rps14 haploinsufficient bone marrows, all representing cells of the erythroblastic niche. Recombinant S100A8 was sufficient to impair erythropoiesis in wild-type cells. We rescued the erythroid differentiation defect in Rps14 haploinsufficient HSCs by genetic inactivation of S100a8 expression using CRISPR/Cas-mediated gene inactivation in primary mouse Rps14 haploinsufficient HSPC. We validated the association between induction of S100A8 and a severe erythroid phenotype in bone marrow samples of patients with del(5q) MDS. To examine whether ribosomal haploinsufficiency also leads to activation of S100A8 in patients with del(5q) MDS, we measured S100A8 expression using immunofluorescence in bone marrow biopsies from MDS patients with and without del(5q). In del(5q) MDS, the frequency of S100A8-positive cells was associated with disease severity, as reflected by transfusion burden. RPS14, CSNK1A1 and miR-145 are universally co-deleted in the 5q- syndrome and each represent different clinical features of del(5q) MDS in murine models. Haploinsufficiency of miR-145 or -146a also induces inappropriate activation of innate immune signaling. To analyze the combinatorial effect of haploinsufficiency Rps14, Csnk1a1 and miRNA-145, we transduced hematopoietic stem and progenitor cells (HSPC) from compound haploinsufficient Rps14 and Csnk1a1 mice and stably knocked down both miR-145/miR-146a by retrovirus-mediated overexpression of respective target sequences. Compound haploinsufficiency of Rps14, Csnk1a1 and miR-145/146a led to a progressive anemia comparable to Rps14 haploinsufficiency with splenomegaly and an erythroid differentiation defect at the RIII/RIV population, indicating that the anemia is mainly driven by Rps14 haploinsufficiency. Bone marrow histology demonstrated the typical 5q-phenoytpe of megakaryocytes, in line with significant thrombocytosis. At 10 months of age, hematopoietic stem and progenitor cells were significantly increased (lineagelow ckit+ Sca1+; LSK), in particular multipotent progenitor cells (MPPs; lineagelow ckit+ Sca1+ CD48- CD150+) to significantly higher extents than in solely Rps14 or Csnk1a1 haploinsufficient cells. We next asked if compound haploinsufficiency of the three 5q-genes has combinatorial or synergistic effects on S100a8 expression. Compound haploinsufficiency of Csnk1a1, Rps14 and miR-145/146a induced the highest expression of S100a8 in monocytes, while haploinsufficiency of Rps14 alone induced the highest expression of S100a8 in the RIII erythroid population, suggesting that cell-type specific induction mediates the phenotype. Our data indicate an unexpected link between haploinsufficiency for a ribosomal gene, Rps14, activation of S100A8, and inhibition of erythropoiesis. We demonstrate that compound haploinsufficiency for Csnk1a1 and miR145/146a with Rps14 haploinsuffciency increases the expression of S100a8, mainly in monocytes, and recapitulates the phenotype of del(5q) MDS by cooperating, cell-type specific effects. Disclosures Platzbecker: Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Boehringer: Research Funding.

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The dynamic interactive network of long non-coding RNAs and chromatin accessibility facilitates erythroid differentiation

10.1101/2021.03.02.433076 ◽

2021 ◽

Author(s):

Yunxiao Ren ◽

Junwei Zhu ◽

Yuanyuan Han ◽

Pin Li ◽

Hongzhu Qu ◽

...

Keyword(s):

Progenitor Cells ◽

Specific Interaction ◽

Erythroid Differentiation ◽

Chromatin Accessibility ◽

List Type ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Interactive Network ◽

Non Coding Rnas ◽

Terminal Erythroid Differentiation

AbstractErythroid differentiation is a dynamic process regulated by multiple factors, while the interaction between long non-coding RNAs and chromatin accessibility and its influence on erythroid differentiation remains unclear. To elucidate this interaction, we employed hematopoietic stem cells, multipotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, and erythroblasts from human cord blood as an erythroid differentiation model to explore the coordinated regulatory functions of lncRNAs and chromatin accessibility in erythropoiesis by integrating RNA-Seq and ATAC-Seq data. We revealed that the integrated network of chromatin accessibility and LncRNAs exhibits stage-specific changes throughout the erythroid differentiation process, and that the changes at the EB stage of maturation are dramatic. We identified a subset of stage-specific lncRNAs and transcription factors (TFs) that coordinate with chromatin accessibility during erythroid differentiation, in which lncRNAs are key regulators of terminal erythroid differentiation via a lncRNA-TF-gene network. LncRNA PCED1B-AS1 was revealed to regulate terminal erythroid differentiation by coordinating GATA1 dynamically binding to the chromatin during erythroid differentiation. DANCR, another lncRNA that is highly expressed at the MEP stage, was verified to promote erythroid differentiation by compromising megakaryocyte differentiation and coordinating with chromatin accessibility and TFs, such as RUNX1. Overall, our results identified the interactive network of lncRNAs and chromatin accessibility in erythropoiesis and provide novel insights into erythroid differentiation and abundant resources for further study.Key PointsLncRNAs regulate erythroid differentiation through coordinating with chromatin accessibility.The integrative multi-omics analysis reveals stage-specific interaction network of LncRNAs and chromatin accessibility in erythropoiesis.

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Reveal the Neutrophil: Elucidating the Role of a Neutrophil-Specific JAK2-V617F Mutation

Blood ◽

10.1182/blood-2019-122660 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2965-2965

Author(s):

Tobias Ronny Haage ◽

Andreas Johann Müller ◽

Priyadharshini Arunachalam ◽

Thomas Fischer

Keyword(s):

Blood Cell ◽

Progenitor Cells ◽

Spleen Weight ◽

Β1 Integrin ◽

Normal Range ◽

Erythroid Progenitor ◽

Hematopoietic Stem ◽

Platelet Counts ◽

Cell Counts

Introduction: Myeloproliferative neoplasms (MPN) are characterized by monoclonal proliferation of myeloid cells. The clinical presentation of classic BCR-ABL negative MPN includes blood count changes, splenomegaly, chronic inflammation and, prognostically relevant, thrombosis. Within polycythemia vera, essential thrombocythemia and primary myelofibrosis, the most frequent genetic alteration is the point mutation V617F (VF) in the Jak2 gene. Due to an activation of leucocyte β1 and β2 integrins, in particular on neutrophils, JAK2-VF is driving thrombus formation (Edelmann et al., JCI, 2018). Thus, understanding the role of neutrophils within JAK2-VF-positive MPN is of special interest. We developed a murine JAK2-VF Catchup model of MPN that is characterized by the neutrophil-specific expression of JAK2-VF in combination with the fluorescent protein tdTomato allowing in-vivo two-photon microscopy. Neutrophil-specificity was achieved through a knock-in allele of Cre recombinase and tdTomato replacing exon 1 of Ly6G (Hasenberg et al., Nat Methods, 2015). Methods: The murine JAK2-VF Catchup model was developed by crossing JAK2+/loxP-VF-loxP mice and Ly6G+/Cre-tdTomato CatchupIVM-red mice. Experiments were performed in 12 weeks old JAK2+/loxP-VF-loxP Ly6G+/Cre-tdTomato CatchupIVM-red (JAK2+/VF Catchup) mice (n=5) and JAK2+/+ Ly6G+/Cre-tdTomato CatchupIVM-red (JAK2+/+ Catchup) mice (n=5) as wildtype (WT) controls. Whole blood cell counts and spleen weight were determined. Bone marrow (BM) cells were harvested from hind limbs followed by isolation of granulocytes using negative selection. In order to determine hematopoietic stem and progenitor cells (HSPCs), BM and spleen cell suspensions were analyzed by flow cytometry (FC). Using ICAM-1, VCAM-1, E-selectin and P-selectin, each with and without Fc-tag, adhesion properties of the isolated granulocytes were examined by either FC or microplate reader analysis. The expression of β1 integrin was further analyzed by FC. Results and Discussion: JAK2+/VF Catchup mice showed a slight but significant increase in platelet counts [1544±58.02 Gpt/l; WT: 1190±90.50 Gpt/l; p=0.011], with the mean value still remaining within the normal range. Hematocrit, red blood cell, white blood cell and neutrophil count as well as spleen weight were also within the normal range showing no differences from their WT controls. Interestingly, neutrophil and platelet counts of JAK2+/VF Catchup mice significantly correlated with one another [p=0.0251; R2=0.8529]. MPN-typical changes in number and composition of c-Kit+ HSPCs in either BM or spleen could not be found in JAK2+/VF Catchup mice. Directly compared to reliably MPN-developing Vav-1-Cre JAK2+/VF mice, JAK2+/VF Catchup mice showed significantly less megakaryocyte-erythroid progenitor (MEP), Lin- Sca1+ c-Kit+ (LSK) and multipotent progenitor cells (MPP) in the BM. In spleen of JAK2+/VF Catchup mice, the fractions of Lin- c-Kit+ (LK) and granulocyte-macrophage progenitor cells (GMP) were significantly lower compared to Vav-1-Cre JAK2+/VF mice. Thus, the neutrophil-specific JAK2-VF mutation does not lead to apparent numerical or compositional changes of HSPCs. These results show that a neutrophil-specific JAK2-VF mutation is not sufficient to induce a characteristic phenotype of JAK2-VF-positive MPN. The expression of β1 integrin on granulocytes was significantly increased in JAK2+/VF Catchup mice [fold change vs. control; 1.126±0.041; WT: 1.0±0.011; p=0.0194]. Unexpectedly, despite increased β1 integrin expression, the binding of granulocytes from JAK2+/VF Catchup mice to either ICAM-1, VCAM-1, E-selectin or P-selectin corresponded to that of their WT controls. Static adhesion and soluble ligand binding assays showed similar results. Since adhesion of granulocytes appeared unchanged in-vitro, we hypothesize that expression of JAK2-VF in neutrophils is not sufficient to overactivate integrins but secondary extrinsic signals (e.g. cytokines) may play a dominant role. Further experimentation is under way to elucidate this molecular mechanism. Conclusions: In order to dissect the properties of JAK2-VF-positive neutrophils, the JAK2-VF Catchup model represents a promising murine model of MPN. Herewith, the initiation of thrombosis and the development of other granulocyte-driven disease manifestations of JAK2-VF induced MPN can be thoroughly examined. Disclosures No relevant conflicts of interest to declare.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1813015115 ◽

2018 ◽

Vol 115 (43) ◽

pp. E10109-E10118 ◽

Cited By ~ 14

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.

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