PKA Mediated Phosphorylation of TAL1 Regulates Its Interaction with LSD1 During Hematopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2599-2599
Author(s):  
Ying Li ◽  
Changwang Deng ◽  
Xin Hu ◽  
Xueqi Fu ◽  
Yi Qiu ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Transcriptional Activation ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Erythroid Progenitor ◽  
Functional Link ◽  
Dynamic Regulation ◽  
Helix Loop Helix ◽  
Interacting Domain

Abstract Abstract 2599 TAL1 is a member of the basic helix-loop-helix (bHLH) family of transcription factors and is required for the development of all hematopoietic cell lineages. TAL1 is a phosphorylated protein and its activities are mediated by the corepressors and coactivators that associate with TAL1. However, the functional link between phosphorylation and the recruitment of co-regulators by TAL1 is currently unknown. We showed that TAL1 dynamically interacts with LSD1 complex containing both histone H3K4 demethylase and deacetylase activities during hematopoiesis (Proc. Natl. Acad. Sci. USA 106: 10141–10146). To further understand the molecular mechanism that regulates the TAL1 and LSD1 interaction during hematopoiesis, we determined whether TAL1 directly interacts with LSD1 and characterized the domains required for this interaction. TAL1 directly interacts with LSD1, and the interacting domain encompasses amino acids 142–185 proximal to the bHLH domain, which contains a serine 172 residue that becomes phosphorylated by Protein kinase A (PKA) during the transcriptional activation of TAL1. The PKA inhibitor, H89, stimulated TAL1 interaction with LSD1 in hematopoietic cells, while Forskolin, an activator of PKA, completely abolished TAL-LSD1 interaction. We further mutated serine 172 of TAL1 to Alanine (Ala) or to Aspartic acid (Asp) to mimic unphosphorylated or phosphorylated TAL1, respectively. While the TAL1Ser172Ala mutant remains interacted with LSD1, TAL1Ser172Asp specifically loses its interaction with LSD1 indicating that serine 172 phosphorylation of TAL1 by PKA destabilizes the TAL1 and LSD1 interaction. Our previous results indicate that LSD1 inhibits TAL1 mediated erythroid differentiation. To further test whether the activity of TAL1 is mediated through an interaction with LSD1, we expressed the TAL1 mutant that deleted the LSD1 interacting domain in erythroid progenitor cells and showed that the deletion of the LSD1 interacting domain of TAL1 leads to a promotion of erythroid differentiation and H3K4 hypermethylation of the P4.2 promoter. In contrast, the expression of the TAL1Ser172Ala mutant and TAL1-LSD1 chimerical fusion enhanced cellular proliferation and colony formation ability of the hematopoietic progenitor cells while these constructs inhibited erythroid differentiation. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and erythroid differentiation. The results suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs. Disclosures: No relevant conflicts of interest to declare.

Serine Phosphorylation On TAL1 Regulates Its Interaction with Histone Demethylase LSD1.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1460-1460
Author(s):  
Ying Li ◽  
Xin Hu ◽  
River Ybarra ◽  
Xueqi Fu ◽  
Yi Qiu ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Erythroid Differentiation ◽  
Histone Demethylase ◽  
Serine Phosphorylation ◽  
Rapid Decline ◽  
Erythroid Progenitor ◽  
Functional Link ◽  
Inhibition Of Proliferation ◽  
Interacting Domain

Abstract Abstract 1460 Poster Board I-483 TAL1, originally identified by virtue of its involvement in a T-cell acute lymphoblastic leukemia (T-ALL)-specific chromosomal translocation, is a member of the basic helix-loop-helix (bHLH) family of transcription factors and is required for the development of all hematopoietic cell lineages. TAL1 is a phosphorylated protein and its activities are mediated by the corepressors and coactivators that associate with TAL1. However, the functional link between phosphorylation and the recruitment of co-regulators by TAL1 is currently unknown. We undertook the biochemical purification of the TAL1 containing complexes and showed that TAL1 is associated with histone demethylase complexes containing LSD1, CoREST, HDAC1 and HDAC2. This complex mediates TAL1 directed transcriptional repression during hematopoiesis. The interaction between TAL1 and LSD1 are dynamically regulated and is required for TAL1's function in erythroid differentiation (Proc. Natl. Acad. Sci. USA 106: 10141-10146). To further understand the molecular mechanism that regulates the TAL1 and LSD1 interaction during hematopoiesis, we determined whether TAL1 directly interacts with LSD1 and characterized the domains required for this interaction. TAL1 and its various deletion mutants were tested for their ability to interact with LSD1 in vitro. TAL1 directly interacts with LSD1, and the interacting domain encompasses amino acids 142-185 proximal to the bHLH domain, which contains a serine 172 residue that becomes phosphorylated during transcriptional activation of TAL1. We further mutated serine 172 of TAL1 to Alanine (Ala) or to Aspartic acid (Asp) to mimic unphosphorylated or phosphorylated TAL1, respectively. While the TAL1Ser172Ala mutant remains the interaction with LSD1, TAL1Ser172Asp specifically loses its interaction with LSD1 indicating that serine 172 phosphorylation of TAL1 destabilizes the TAL1 and LSD1 interaction. Given that our previous results indicated that LSD1 inhibits TAL1 mediated erythroid differentiation, to further test whether the activity of TAL1 is mediated through interaction with LSD1, we expressed the TAL1 mutant that deleted the LSD1 interacting domain in erythroid progenitor cells and showed that the deletion of the LSD1 interacting domain of TAL1 lead to a promotion of erythroid differentiation and inhibition of proliferation. Furthermore, consistent with the rapid decline of TAL1-associated LSD1 complex during differentiation, the ChIP and ChIP-seq data showed that H3K4 di- and tri-methylation are enriched at the promoters of TAL1 target genes upon erythroid differentiation. Thus, our data revealed that histone lysine demethylase LSD1 may negatively regulate TAL1-mediated transcription and erythroid differentiation. The results suggest that the dynamic regulation of TAL1-associated LSD1/HDAC1 complex may determine the onset of erythroid differentiation programs. * These authors contribute equally to this work. Disclosures No relevant conflicts of interest to declare.

An RNA Interference Model of RPS19 Deficiency in Diamond Blackfan Anemia Recapitulates Defective Erythropoiesis and Rescue by Dexamethasone: Identification of Dexamethasone Responsive Genes by Microarray.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 721-721 ◽  
Author(s):  
Benjamin L. Ebert ◽  
Michele Lee ◽  
Jennifer Pretz ◽  
Todd R. Golub ◽  
Colin Sieff
Keyword(s):  
Progenitor Cells ◽  
Molecular Basis ◽  
Cellular Proliferation ◽  
Erythroid Differentiation ◽  
Regulatory Genes ◽  
Cell Surface Expression ◽  
Erythroid Progenitor ◽  
Diamond Blackfan Anemia ◽  
Erythroid Progenitor Cells ◽  
Cd34 Cells

Abstract Diamond Blackfan Anemia (DBA), a congenital erythroblastopenia, is a model disease for the study of erythroid differentiation, but is poorly understood. RPS19 is the only gene yet to have been associated with DBA, but the relevance of this ubiquitously expressed ribosomal gene to erythroid differentiation is unclear. Glucocorticoids are the primary pharmacological therapy for patients with DBA, but the molecular basis for the activity of glucocorticoids in erythroid differentiation has not been identified. Through retroviral expression of short hairpin RNAs (shRNAs), we demonstrate that targeted degradation of the RPS19 gene in cultured human CD34+ cells blocks the proliferation and differentiation of erythroid progenitor cells. Decreased RPS19 expression limited production of erythroid colony formation on methylcellulose, decreased cell surface expression of glycophorin-A, and decreased cellular proliferation. Treatment of RPS19 deficient cells with dexamethasone restored erythroid differentiation to normal levels. We investigated the molecular basis of pharmacologic therapies for DBA using oligonucleotide microarrays to survey gene expression in CD34+ cells treated with combinations of erythropoietin, dexamethasone, IL-3, and SCF. None of these agents had a direct effect on the expression of RPS19 or on the coordinate expression of other ribosomal genes. Instead, dexamethasone activated a genetic program that includes a set of key hematopoietic regulatory genes, including Flt-3 and PLZF, that increase proliferation of hematopoietic progenitor cells. Genes specific to erythroid progenitor cells were up-regulated by dexamethasone, while genes specific to non-erythroid lineages were powerfully down-regulated. Deficiency of RPS19 therefore blocks proliferation of immature erythroid progenitor cells, and dexamethasone activates proliferation of the same cell population through mechanisms independent of RPS19. Identification of the key regulatory genes affected by dexamethasone may aid in the development of novel therapeutics that de-couple the beneficial hematopoietic effects of dexamethasone from detrimental non-hematopoietic side effects.

scholarly journals Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1576-1582 ◽  
Author(s):  
M Silva ◽  
D Grillot ◽  
A Benito ◽  
C Richard ◽  
G Nunez ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Apoptotic Cell ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Erythroid Progenitor ◽  
Survival Factor ◽  
Erythroid Progenitor Cells ◽  
Survival Signal ◽  
Proliferation And Differentiation

Abstract Erythropoietin (Epo), the hormone that is the principal regulator of red blood cell production, interacts with high-affinity receptors on the surface of erythroid progenitor cells and maintains their survival. Epo has been shown to promote cell viability by repressing apoptosis; however, the molecular mechanism involved is unclear. In the present studies we have examined whether Epo acts as a survival factor through the regulation of the bcl-2 family of apoptosis-regulatory genes. We addressed this issue in HCD-57, a murine erythroid progenitor cell line that requires Epo for proliferation and survival. When HCD-57 cells were cultured in the absence of Epo, Bcl-2 and Bcl-XL but not Bax were downregulated, and the cells underwent apoptotic cell death. HCD-57 cells infected with a retroviral vector encoding human Bcl-XL or Bcl-2 rapidly stopped proliferating but remained viable in the absence of Epo. Furthermore, endogenous levels of bcl-2 and bcl-XL were downregulated after Epo withdrawal in HCD-57 cells that remained viable through ectopic expression of human Bcl-XL, further indicating that Epo specifically maintains the expression of bcl-2 and bcl-XL. We also show that HCD-57 rescued from apoptosis by ectopic expression of Bcl-XL can undergo erythroid differentiation in the absence of Epo, demonstrating that a survival signal but not Epo itself is necessary for erythroid differentiation of HCD-57 progenitor cells. Thus, we propose a model whereby Epo functions as a survival factor by repressing apoptosis through Bcl-XL and Bcl-2 during proliferation and differentiation of erythroid progenitors.

scholarly journals Stem cell factor retards differentiation of normal human erythroid progenitor cells while stimulating proliferation

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 572-580 ◽  
Author(s):  
K Muta ◽  
SB Krantz ◽  
MC Bondurant ◽  
CH Dai
Keyword(s):  
Stem Cell ◽  
Cell Viability ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Single Cells ◽  
Erythroid Differentiation ◽  
Tyrosine Kinase Receptor ◽  
Total Production ◽  
Erythroid Progenitor ◽  
Erythroid Progenitor Cells

Stem cell factor (SCF), the ligand for the c-kit tyrosine kinase receptor, markedly stimulates the accumulation of erythroid progenitor cells in vitro. We now report that SCF delays erythroid differentiation among the progeny of individual erythroid progenitors while greatly increasing the proliferation of these progeny. These effects appear to be independent of an effect on maintenance of cell viability. Highly purified day-6 erythroid colony-forming cells (ECFC), consisting mainly of colony-forming units-erythroid (CFU-E), were generated from human peripheral blood burst-forming units-erythroid (BFU-E). Addition of SCF to the ECFC in serum-free liquid culture, together with erythropoietin (EP) and insulin-like growth factor 1 (IGF-1), resulted in a marked increase in DNA synthesis, associated with a delayed peak in cellular benzidine positivity and a delayed incorporation of 59Fe into hemoglobin compared with cultures without SCF. In the presence of SCF, the number of ECFC was greatly expanded during this culture period, and total production of benzidine-positive cells plus hemoglobin synthesis were ultimately increased. To determine the effect of SCF on individual ECFC, single-cell cultures were performed in both semisolid and liquid media. These cultures demonstrated that SCF, in the presence of EP and IGF-1, acted on single cells and their descendants to delay erythroid differentiation while substantially stimulating cellular proliferation, without an enhancement of viability of the initial cells. This was also evident when the effect of SCF was determined using clones of ECFC derived from single BFU-E. Our experiments demonstrate that SCF acts on individual day-6 ECFC to retard erythroid differentiation while simultaneously providing enhanced proliferation by a process apparently independent of an effect on cell viability or programmed cell death.

Constitutive Activation of STAT5 and Bcl-XL Overexpression Can Induce Endogenous Erythroid Colony Formation in Human Primary Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3135-3135
Author(s):  
Loïc Garçon ◽  
Chloe James ◽  
Catherine Lacout ◽  
Valérie Camara-Clayette ◽  
Valérie Ugo ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Progenitor Cells ◽  
Colony Formation ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Primary Cells ◽  
Erythroid Progenitor ◽  
Constitutive Activation ◽  
Human Primary Cells

Abstract In contrast with secondary erythrocytosis, progenitor cells from polycythemia vera (PV) patients can undergo in vitro erythroid differentiation despite absence of erythropoietin (EPO) and presence of such endogenous erythroid colonies (EEC) is routinely used as a diagnostic assay. Recent focus on the JAK2 mutation V617F in PV patients argue for a direct implication of JAK2 dependent signaling pathways in EEC formation. Because STAT5 is the principal target of JAK2 in erythroid cells, we investigated whether EEC formation was only dependent on STAT5 activation or required other signaling pathways that would be activated by JAK2. For this purpose, we transduced a retroviral vector coding for a constitutively active form of STAT5 (MIGR-STAT5CA) in UT7 cells, a leukemic cell line with erythroid properties. We observed in cells transduced with the MIGR-STAT5CA vector a spontaneous induction of erythroid differentiation in comparison with cells infected with the empty vector MIGR, as assessed by GPA staining. We next investigated effects of STAT5CA on erythroid differentiation of human primary progenitors. Purified CD34+ cells obtained from peripheral blood (PB) of patients treated with G-CSF were transduced with the STA5CA vector, the CD36+/GPA− erythroid progenitor cells were sorted and cultured in presence of SCF alone. When expressing STAT5CA, they both proliferate and undergo erythroid terminal differentiation despite the absence of EPO. We concluded that a phosphorylated form of STAT5 was sufficient to support in vitro erythroid differentiation of human primary cells. Because STAT5 has been shown to play a crucial role in erythropoiesis via induction of the antiapoptotic protein Bcl-xL, we next investigated whether effects of STAT5CA on erythroid maturation was dependent on Bcl-xL induction. Tansduction of human CD36+/GPA− cells with a retrovirus containing the coding sequence of human Bcl-xL progenitors allowed survival, proliferation and GPA acquisition despite the absence of EPO. We next investigated whether STAT5CA or Bcl-xL overexpression in normal primary cells could reproduce the malignant phenotype observed in PV patients, i.e. induction of EEC formation. CD36+/GPA− transduced with either the STAT5 CA or the Bcl-XL vectors were plated in methylcellulose in the absence of EPO. Bcl-xL as well as STAT5CA vectors could both induce endogenous erythroid colony formation. Regardless to these results, we hypothesized that the EEC formation observed in myeloproliferative disorders could be at least partially due to the JAK2 dependent activation of the STAT5/Bcl-XL pathway. Thus, both constitutive activation of STAT5 and Bcl-xL overexpression could substitute to EPO to induce terminal differentiation of human primary erythroid progenitors.

Chromatin Modifying Agents Promote the Ex Vivo Production of Functional Human Erythroid Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 340-340
Author(s):  
Pratima Chaurasia ◽  
Dmitriy Berenzon ◽  
Ronald Hoffman
Keyword(s):  
Progenitor Cells ◽  
Histone Deacetylase Inhibitors ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Significant Proportion ◽  
Erythroid Progenitor ◽  
Erythroid Precursor ◽  
Cell Product ◽  
Erythroid Progenitor Cells ◽  
Cd34 Cells

Abstract Abstract 340 Presently, blood transfusion products (TP) are composed of terminally differentiated cells with a finite life span. We attempted to develop an alternative TP which would be capable of generating additional red blood cells (RBC). Several histone deacetylase inhibitors (HDACIs) were used in vitro to reprogram cord blood (CB) CD34+ cells to differentiate to erythroid progenitor cells (EPC). We demonstrated that CB CD34+ cells in the presence of HDACIs (SAHA, VPA and TSA), and a combination of cytokines SCF, IL-3, TPO and FLT3, promoted expansion of CD34+ cells and CD34+CD90+ cells as compared to cultures containing cytokines alone. Addition of VPA resulted in the greatest expansion of CD34+ cells, CD34+CD90+cells+ (59.4 fold, p=0.01; 66.7 fold, p=0.02, respectively) as compared to SAHA and TSA. VPA also led to the generation of the greatest absolute number of EPC cells (14.9×106, p=0.002), approximately a 5500 fold in the numbers of assayable EPC, as compared to primary CB. The single cell analyses of CB CD34+ cells (Day0) and single CD34+ reisolated from ex-vivo cultures pretreated with cytokines alone or cytokines+VPA demonstrated an skewed differentiation program of CD34+ cells to EPC (>94%, p=0.003) compared to CB CD34+(50%) and cytokines alone (29%). We investigated the expression of lineage specific phenotypic markers expressed by CD34+ cells exposed to cytokines alone or cytokines plus VPA. The FACS analyses showed a significantly greater proportion of CD34+CD36+ (52.4% vs 21.0%) CD36+CD71+(44.5% vs7.6%), CD36+GPA+(12.8% Vs 4.0%) and CD71+GPA+(22.2% vs 6.3%) cells with lower numbers of CD19+(2.8% vs 13.6%) cells, CD14+(2.0% vs 8.9%), CD15+(1.8 vs 6.9%) in VPA treated CD34+ cells as compared to cytokines alone. We monitored the relative expression of a group of genes characteristic of both primitive HPC and erythroid commitment (Bmi1, Dnmt1, Ezh2, Smad5, Eklf, GATA1, GATA2, EpoR and Pu.1). Q-PCR was performed on CD34+cells reisolated from cultures treated with cytokines alone or cytokines plus VPA and compared to primary CB CD34+ cells. The expression of genes associated with retention of the biological properties of the primitive HPC (Bmi1-2.6 fold, Dnmt1-10.3 fold and Ezh2-4.8 fold) and erythroid lineage specific genes (Smad5-6.2 fold, GATA2-3.7 fold) were upregulated and Pu.1 (0.6-fold), GATA1(1.9 fold) were downregulated as compared to cytokines alone. However, expression of EpoR and Eklf were similar in the two cell populations Histone acetylation study showed that the CB CD34+ cells and VPA treated CD34+ cells had a significant proportion of acetylated H3K9 cells, 52.2% and 56.1% respectively, while this population was virtually absent in CD34+ cells exposed to cytokines alone (1.3%, p=0.001). ChIP assay demonstrated a varying degree of H3K9/14 and H3K27 acetylation within the promoters of VPA treated CD34+ cells for GATA2 (7.4 fold, 7.2 fold), Eklf (7.4 fold, 9.7 fold), Pu.1(4.5fold, 4.8 fold), EpoR (2.3 fold, 4.7 fold) and GATA1(4.7 fold, 2.9 fold). The acetylation of cytokines treated CD34+ cells were much lower than VPA treated CD34+ cells. The VPA treated cell product after 9 days (supplemented with SCF, Epo and IL-3 for 2 additional days) compared to 7 days contained a greater percentage of EPC and erythroid precursor cells CD34+CD36+(24.9% vs 23.0%), CD36+GPA+(33.9% vs 18.8%), CD36+. CD71+(55.8% vs 37.8%), CD71+GPA+(33.9% vs 20.5%) and CD34+CXCR4+(28.8% vs 21.0 %). The TP contained very limited number of CD19+(1.4%), CD14+(11.11%) or CD15+(6.8%) of cells. Approximately 50 % of the cells present in the TP expressed the chemokine receptor CXCR4. We next evaluated the behavior of ex vivo expanded cell product following transfusion into sublethally irradiated NOD/SCID mice. FACS analyses of mice peripheral blood (PB) on serial days showed evidence of circulating nucleated erythroid and enucleated red cells. The greatest number of circulating human RBC (12.4%±6.8%) was observed on day5. RT-PCR analyses on the PB of mice on day 15 revealed the presence of erythroid cells containing both human adult and fetal hemoglobin. On day 15 the mice were sacrificed and the degree of human cells engraftment in the marrow were predominately hu -CD45+ (7.4%), CD34-CD36+(1.8%), CD36 (4.5%) and GPA+(1.7%) with no evidence of CD33+, CD14+, CD19+ and CD41+ cells. The ex vivo generated EPC-TP likely represents a paradigm shift in transfusion medicine due to its continued ability to generate additional RBC. Disclosures: No relevant conflicts of interest to declare.

ATG5 and ATG7 Fulfill Essential and Non-Redundant Roles in Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4316-4316
Author(s):  
Hendrik Folkerts ◽  
Maria Catalina Gomez Puerto ◽  
Albertus T.J. Wierenga ◽  
Koen Schepers ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Target Genes ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Erythroid Progenitor ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Human Hscs

Abstract Macroautophagy is a catabolic process by which intracellular contents are delivered to lysosomes for degradation. ATG5 and ATG7 play an essential role in this process. Recent studies have shown that mouse hematopoietic stem cells (HSCs) lacking ATG7 were unable to survive in vivo, however, the role of macroautophagy in proliferation and survival of human HSCs has not yet been defined. Here, we demonstrate that autophagy is functional in human hematopoietic stem/progenitor cells. Robust accumulation of the autophagy markers LC3 and p62 were observed in cord blood (CB)-derived CD34+ cells treated with bafilomycin-A1 (BAF) or hydroxychloroquine (HCQ), as defined by Western blotting. When these cells were subsequently differentiated towards the myeloid or erythroid lineage, a decreased accumulation of LC3 was observed. In addition, CB CD34+CD38- cells showed enhanced accumulation of cyto-ID (a marker for autophagic vesicles) compared to CD34+CD38+ progenitor cells upon BAF or HCQ treatment. In line with these results, also more mature CB CD33+ and CD14+ myeloid cells or CD71+CD235+ erythroid cells showed reduced levels of cyto-ID accumulation upon BAF or HCQ treatment. These findings indicate that human hematopoietic stem and progenitor cells (HSPCs) have a higher basal autophagy flux compared to more differentiated cells. To study the functional consequences of autophagy in human HSCs and their progeny, ATG5 and ATG7 were downregulated in CB-derived CD34+ cells, using a lentiviral shRNA approach which resulted in 80% and 70% reduced expression, respectively. Downmodulation of ATG5 or ATG7 in CB CD34+ cells resulted in a significant reduction of erythroid progenitor frequencies, as assessed by colony forming cell (CFC) assays (shATG5 2.2 fold, p<0.05 or shATG7 1.4 fold p<0.05). Additionally, a strong reduction in expansion was observed when transduced cells were cultured under myeloid (shATG5 17.9 fold, p<0.05 or shATG7 12.3 fold, p<0.05) or erythroid permissive conditions (shATG5 6.7 fold, p<0.05 or shATG7 1.7 fold, p<0.05), whereby differentiation was not affected. The phenotype upon knockdown of ATG5 or ATG7 could not be reversed by culturing the cells on a MS5 stromal layer. In addition to progenitor cells, HSCs were also affected since long term culture-initiating cell (LTC-IC) assays in limiting dilution revealed a 3-fold reduction in stem cell frequency after ATG5 and ATG7 knockdown. The inhibitory effects of shATG5 and shATG7 in cultured CD34+ cells were at least in part due to a decline in the percentage of cells in S phase and (shATG5 1.4 fold, p<0.01 and shATG7 1.3 fold, p<0.01) and an increase of Annexin V positive cells. The changes in cell cycle and apoptosis coincided with a marked increase in expression of the cell cycle-dependent kinase inhibitor p21, an increase in p53 levels, and an increase in proapoptotic downstream target genes BAX, PUMA and PHLDA3. Additionally, ROS levels were increased after ATG5 and ATG7 knockdown. The increased apoptosis in shATG5 and shATG7 transduced cells might be triggered by elevated ROS levels. Taken together, our data demonstrate that autophagy is an important survival mechanism for human HSCs and their progeny. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mutant N-RAS Induces Erythroid Lineage Dysplasia in Human CD34+ Cells

1997 ◽  
Vol 185 (7) ◽  
pp. 1337-1348 ◽  
Author(s):  
Richard L. Darley ◽  
Terence G. Hoy ◽  
Paul Baines ◽  
Rose Ann Padua ◽  
Alan K. Burnett
Keyword(s):  
Myelodysplastic Syndrome ◽  
Progenitor Cells ◽  
Marker Gene ◽  
Erythroid Differentiation ◽  
Multiparameter Flow Cytometry ◽  
Lacz Gene ◽  
Erythroid Progenitor ◽  
Stage Of Development ◽  
Cd34 Cells ◽  
Mutational Activation

RAS mutations arise at high frequency (20–40%) in both acute myeloid leukemia and myelodysplastic syndrome (which is considered to be a manifestation of preleukemic disease). In each case, mutations arise predominantly at the N-RAS locus. These observations suggest a fundamental role for this oncogene in leukemogenesis. However, despite its obvious significance, little is known of how this key oncogene may subvert the process of hematopoiesis in human cells. Using CD34+ progenitor cells, we have modeled the preleukemic state by infecting these cells with amphotropic retrovirus expressing mutant N-RAS together with the selectable marker gene lacZ. Expression of the lacZ gene product, β-galactosidase, allows direct identification and study of N-RAS–expressing cells by incubating infected cultures with a fluorogenic substrate for β-galactosidase, which gives rise to a fluorescent signal within the infected cells. By using multiparameter flow cytometry, we have studied the ability of CD34+ cells expressing mutant N-RAS to undergo erythroid differentiation induced by erythropoietin. By this means, we have found that erythroid progenitor cells expressing mutant N-RAS exhibit a proliferative defect resulting in an increased cell doubling time and a decrease in the proportion of cells in S + G2M phase of the cell cycle. This is linked to a slowing in the rate of differentiation as determined by comparative cell-surface marker analysis and ultimate failure of the differentiation program at the late-erythroblast stage of development. The dyserythropoiesis was also linked to an increased tendency of the RAS-expressing cells to undergo programmed cell death during their differentiation program. This erythroid lineage dysplasia recapitulates one of the most common features of myelodysplastic syndrome, and for the first time provides a causative link between mutational activation of N-RAS and the pathogenesis of preleukemia.

scholarly journals The Histone Methyltransferase Setd8 Alters the Chromatin Landscape and Regulates the Expression of Key Transcription Factors during Erythroid Differentiation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2568-2568
Author(s):  
Jacquelyn Lillis ◽  
Jeffrey Malik ◽  
Tyler A Couch ◽  
Michael Getman ◽  
Laurie A. Steiner
Keyword(s):  
Transcription Factors ◽  
Progenitor Cells ◽  
Transcriptional Repression ◽  
Conflicts Of Interest ◽  
Large Fraction ◽  
Erythroid Differentiation ◽  
Cell Number ◽  
P Value ◽  
Histone H4 ◽  
And Control

Abstract Setd8 is the sole methyltransferase capable of mono-methylating histone H4, lysine 20. Setd8 mRNA is expressed ~10-fold higher in erythroid cells than any other cell type (biogps.org) and Setd8 protein levels increase in concert with GATA1 levels during erythroid differentiation of CD34+ HSPCs, suggesting Setd8 may have a role regulating the erythroid transcriptome. Consistent with this hypothesis, erythroid deletion of Setd8 is embryonic lethal by embryonic day 11.5 (E11.5) due to profound anemia and global transcriptomic analyses of sorted populations of E10.5 Sed8 null and control erythroblasts demonstrated a profound defect in transcriptional repression, with 340/345 differentially expressed genes (DEG) expressed at higher levels in the Setd8 null cells than controls (Malik Cell Reports 2017). Primitive erythroblasts mature and enucleate in a semi-synchronous manner in circulation. To better understand the function of Setd8 in regulating the erythroid transcriptome, we extended our transcriptomic analyses by performing RNA-seq in sorted E9.5 Sed8 null (EpoRCre+; Setd8 Δ/Δ) and control (EpoRCre+; Setd8 Δ/+) erythroblasts. The Setd8 null cells failed to repress 20/137 (15%) of the genes that are down regulated in control cells from E9.5 to E10.5. Although relatively few genes were impacted, those genes were enriched for the pathway "Oxidative Stress" (adjusted p-value 0.009) suggesting that Setd8 may regulate specific functions during terminal erythroid maturation. We next compared the DEG in Setd8 null erythroblasts to transcriptomic changes that occur as a cell transcends the hematopoietic hierarchy, gaining lineage specificity while suppressing the multi-lineage transcriptome (GSE14833). A large fraction, 105/345 (~30%), of genes up-regulated in Setd8 null erythroblasts, are also up-regulated in multipotent progenitors compared to proerythroblasts. In contrast, only 16/345 (5%) were also up-regulated in granulocyte-monocyte progenitors suggesting that Setd8 does not repress other lineage restricted signatures. Together, these results suggest that Setd8 regulates repression of the multi-lineage transcriptome during erythroid differentiation from multipotent progenitor cells. To gain insights into how Setd8 regulates the erythroid transcriptome, we performed ATAC-seq (Buenrostro Nature Methods 2013) on sorted populations of erythroblasts from E10.5 Sed8 null and control embryos. Cell number for the Setd8 null samples was limited due to anemia, with ~1000 cells used for each replicate. Setd8 and control replicates were aggregated and accessible regions were identified using MACS2. Regions more accessible in Setd8 null cells were identified by computing a log2 ratio between Setd8 null and control samples using deepTools bamCompare. In addition, we utilized ChIPmentation (Schmidl Nature Methods 2015) to assay H3K27me3 occupancy across the genome of WT E10.5 erythroblasts to identify regions of heterochromatin in maturing erythroblasts. Two replicates were performed using 2.5-5x105 cells per assay, and peak called was done using MACS2. A total of 157 genes were identified that had more accessible chromatin in Setd8 null cells and contained an enrichment for H3K27me3 in WT cells suggesting that these genes should be repressed during normal erythropoiesis. Among these were several DEG that were up-regulated in the Setd8 null cells including Hhex, Cd63, and Gata2. Genomic data integration also identified several additional transcriptional regulators that are active in earlier hematopoietic progenitors but typically silenced during erythroid differentiation including Notch1 and Cebpa. Pathway analysis of the 157 genes identified several stemness-related pathways including "Transcriptional regulation of pluripotent stem cells" and "OCT4, SOX2, NANOG repress genes related to differentiation" (adjusted p-values 0.005 and 0.008, respectively). The chromatin regions that were more accessible in the Setd8 null cells were enriched for the DNA binding motifs of the transcription factor ERG (p-value 1-257), SCL (p-value 1e-193), and NRF1 (p-value 1e-101). Taken together, these data suggest that Setd8 works in concert with erythroid transcription factors to repress the transcriptional network in stem and progenitor cells and establish appropriate patterns of gene expression during erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

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

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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