GATA Factor Switching during Erythroid Differentiation Is Facilitated By FBW7 Mediated Clearance of GATA2

Abstract Erythroid differentiation is controlled by the dynamic exchange of GATA family transcription factors. During early erythroid maturation, high GATA2 levels activate progenitor genes such as c-kit-, c-myc, and GATA2 itself. In contrast, GATA1 levels are low in early progenitor cells, but rise during terminal maturation. During this process GATA1 turns off GATA2 controlled early progenitor genes and activates terminal maturation genes, such as globin genes, heme biosynthesis enzymes, and iron transporters. This involves the exchange of GATA1 for GATA2 at key chromatin sites, the so-called "GATA factor switch". GATA factor switching is facilitated by the much shorter half-life of GATA2 (~30-60 min) compared to GATA1 (>4-6 hrs). We and others recently demonstrated that the E3 ubiquitin ligase adaptor protein FBW7 contributes to GATA2's relative instability. This prompted us to dissect the role of FBW7 during GATA switching and erythroid differentiation. We deleted the Fbw7 gene using CRISPR/Cas9 gene editing in the inducible G1-ER murine erythroid cell line. This resulted in the delayed clearance of GATA2 during differentiation. RNA-seq analysis at an early time points (7 hr) demonstrated impaired repression of GATA2 regulated genes and reduced activation of GATA1 target genes. Globally, altered gene expression was enriched for GATA factor switch genes. This ultimately resulted in delayed erythroid maturation. We also found that Fbw7 mRNA transcript levels increase during erythroid maturation in wild type cells. We identified a site ~40kb upstream of the Fbw7 gene transcriptional start site, which is itself a GATA factor switch site. We propose that FBW7 facilitates GATA factor switching by promoting the clearance of GATA2 from GATA factor switch sites. Moreover, we suggest that GATA factor switching at the Fbw7 locus itself reinforces the commitment of erythroid cells to terminal maturation, by enhancing the clearance of GATA2 and other Fbw7 progenitor target gene proteins such as c-Myc and c-Myb. As Fbw7 recognition of GATA2 requires phosphorylation of GATA2's degron motif, this suggests that signaling pathways, acting through Fbw7, may modulate erythroid maturation kinetics. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Regulation of Erythropoiesis by Histone Methyltransferase EZH2 Inhibitor 3-Deazaneplanocin A (DZNep)

Blood ◽

10.1182/blood.v120.21.982.982 ◽

2012 ◽

Vol 120 (21) ◽

pp. 982-982

Author(s):

Tohru Fujiwara ◽

Haruka Saitoh ◽

Yoko Okitsu ◽

Noriko Fukuhara ◽

Yasushi Onishi ◽

...

Keyword(s):

Molecular Mechanisms ◽

Target Genes ◽

Conflicts Of Interest ◽

K562 Cells ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Mrna Levels ◽

Wide Range ◽

The Impact ◽

Chip Analysis

Abstract Abstract 982 Background. EZH2, a core component of Polycomb repressive complex 2 (PRC2), plays a role in transcriptional repression through mediating trimethylation of histone H3 at lysine 27 (H3K27), and is involved in various biological processes, including hematopoiesis. Overexpression of EZH2 has been identified in a wide range of solid tumors as well as hematological malignancies. Recent studies indicated that 3-deazaneplanocin A (DZNep), an inhibitor of EZH2, preferentially induces apoptosis in cancer cells, including acute myeloid leukemia and myelodysplastic syndromes, implying that EZH2 may be a potential new target for epigenetic treatment. On the other hand, whereas PRC2 complex has been reported to participate in epigenetic silencing of a subset of GATA-1 target genes during erythroid differentiation (Yu et al. Mol Cell 2009; Ross et al. MCB 2012), the impact of DZNep on erythropoiesis has not been evaluated. Method. The K562 erythroid cell line was used for the analysis. The cells were treated with DZNep at doses of 0.2 and 1 microM for 72 h. Quantitative ChIP analysis was performed using antibodies to acetylated H3K9 and GATA-1 (Abcam). siRNA-mediated knockdown of EZH2 was conducted using Amaxa nucleofection technology™ (Amaxa Inc.). For transcription profiling, SurePrint G3 Human GE 8 × 60K (Agilent) and Human Oligo chip 25K (Toray) were used for DZNep-treated and EZH2 knockdown K562 cells, respectively. Gene Ontology was analyzed using the DAVID Bioinformatics Program (http://david.abcc.ncifcrf.gov/). Results. We first confirmed that DZNep treatment decreased EZH2 protein expression without significantly affecting EZH2 mRNA levels, suggesting that EZH2 was inhibited at the posttranscriptional level. We also confirmed that DZNep treatment significantly inhibited cell growth. Interestingly, the treatment significantly induced erythroid differentiation of K562 cells, as determined by benzidine staining. Transcriptional profiling with untreated and DZNep-treated K562 cells (1 microM) revealed that 789 and 698 genes were upregulated and downregulated (> 2-fold), respectively. The DZNep-induced gene ensemble included prototypical GATA-1 targets, such as SLC4A1, EPB42, ALAS2, HBA, HBG, and HBB. Concomitantly, DZNep treatment at both 0.2 and 1 microM upregulated GATA-1 protein level as determined by Western blotting, whereas the effect on its mRNA levels was weak (1.02- and 1.43-fold induction with 0.2 and 1 microM DZNep treatment, P = 0.73 and 0.026, respectively). Furthermore, analysis using cycloheximide treatment, which blocks protein synthesis, indicated that DZNep treatment could prolong the half-life of GATA-1 protein, suggesting that DZNep may stabilize GATA-1 protein, possibly by affecting proteolytic pathways. Quantitative ChIP analysis confirmed significantly increased GATA-1 occupancy as well as increased acetylated H3K9 levels at the regulatory regions of these target genes. Next, to examine whether the observed results of DZNep treatment were due to the direct inhibition of EZH2 or hitherto unrecognized effects of the compound, we conducted siRNA-mediated transient knockdown of EZH2 in K562 cells. Quantitative RT-PCR analysis demonstrated that siRNA-mediated EZH2 knockdown had no significant effect on the expression of GATA-1 as well as erythroid-lineage related genes. Furthermore, transcription profiles of the genes in the quantitative range of the array were quite similar between control and EZH2 siRNA-treated K562 cells, with a correlation efficient of 0.977. Based on our profiling results, we are currently exploring the molecular mechanisms by which DZNep promotes erythroid differentiation of K562 cells. Conclusion. DZNep promotes erythroid differentiation of K562 cells, presumably through a mechanism not directly related to EZH2 inhibition. Our microarray analysis of DZNep-treated K562 cells may provide a better understanding of the mechanism of action of DZNep. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Fetal Inactivation of the Nuclear Interacting SET Domain Protein 1 Impairs Terminal Erythroid Maturation and Results in Acute Erythroleukemia in Mice

Blood ◽

10.1182/blood.v130.suppl_1.713.713 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 713-713

Author(s):

Katharina Leonards ◽

Marwa Almosailleakh ◽

Samantha Tauchmann ◽

Helene Mereau ◽

Sabine Juge ◽

...

Keyword(s):

Target Genes ◽

Conflicts Of Interest ◽

Erythroid Differentiation ◽

Regulatory Elements ◽

Genetic Alterations ◽

Wild Type ◽

Set Domain ◽

Erythroid Maturation ◽

Domain Protein ◽

Differentiation Block

Abstract The nuclear receptor binding SET domain protein 1 (NSD1) histone (H3K36) methyltransferase is target of recurrent genetic alterations in human hematological malignancies and certain solid cancers. To study its role in normal hematopoiesis, we inactivated the Nsd1 gene in the blood forming system of the mouse. Vav1-iCre mediated cleavage of floxed exon 5 resulted in almost complete loss of Nsd1 expression at the mRNA and protein level. After a latency of 7-17 weeks, all Vav1-iCre;Nsd1fl/fl mice developed signs of disease, whereas heterozygous littermates expressed normal Nsd1 levels and remained healthy. Symptomatic mice presented anemia, reticulocytosis and thrombocytopenia with presence of erythroblasts on peripheral blood smears. Diseased mice had significant splenomegaly and infiltration of erythroblasts in spleen, liver and lung. Bone marrow (BM) transplantation from symptomatic mice was able to fully propagate the disease in wild type recipients, alone or in competition with normal cells. The majority of the BM and spleen cells of diseased mice expressed modest levels of the transferrin receptor (CD71dim) and variable amounts of c-Kit and FcγR-II/III, but no TER119, CD34, B220 or Sca-1. The cells formed abnormal BFU-like dense reddish and partially hemoglobinized colonies in erythropoietin (EPO) containing methylcellulose that could be replated up to 4 rounds. In Vav1-iCre;Nsd1fl/fl fetal livers, we also observed accumulation of abnormal erythroid progenitors, deficient for Nsd1, suggesting a prenatal origin of the phenotype. RNA sequencing of lineage marker-negative, Sca1+, c-Kit+ (LSK) cells revealed aberrant regulation of genes associated with/functioning in erythroid differentiation. Indeed, in vitro differentiation of Vav1-iCre;Nsd1fl/fl erythroblasts was significantly impaired. Retroviral expression of Nsd1 was able to partially rescue the erythroid differentiation block. Interestingly, Vav1-iCre;Nsd1fl/fl erythroblasts constitutively expressed significantly increased protein levels of the erythroid master transcriptional regulator GATA1 independent whether they were expanded as immature cells or induced for terminal maturation. Impaired terminal maturation of Vav1-iCre;Nsd1fl/fl erythroblasts was associated with reduced transactivation of GATA1 positively-regulated targets (HbbA, HbbB, Gpa, Bcl2l1, p21), while expression of GATA1-repressed target genes (Gata2, c-kit, Spi1) was not affected. Strikingly, exogenous expression of Gata1 was able to overcome the differentiation block of Vav1-iCre;Nsd1fl/fl erythroblasts depending on the integrity of the N- and C-zinc fingers, as seen in colony forming assays and liquid cultures. Chromatin immunoprecipitation (ChIP) revealed that binding of GATA1 to HbbA1 locus and to a lesser extent to regulatory elements in the Gata1 promoter region (G1-HE site) was impaired in Vav1-iCre;Nsd1fl/fl erythroblasts, but restored upon overexpression of exogenous Gata1 . However, we found no significant changes of global H3K36me1 and H3K36me2 marks in wild type and Vav1-iCre;Nsd1fl/fl erythroblasts. In addition, published ChIP-seq data revealed no enrichment for H3K36me1/2 marks near critical GATA1 binding sites in HbbA1 or Gata1 suggesting that NSD1 most likely regulates GATA1 activity by direct modification or by interfering with GATA1 interacting partners necessary for efficient transactivation of critical mediators of terminal erythroid maturation. Taken together, our work unraveled NSD1 as a critical regulator of terminal erythroid differentiation. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Epigenetic Determinants of Erythropoiesis: Role of the Histone Methyltransferase SetD8 in Promoting Erythroid Cell Maturation and Survival

Molecular and Cellular Biology ◽

10.1128/mcb.01422-14 ◽

2015 ◽

Vol 35 (12) ◽

pp. 2073-2087 ◽

Cited By ~ 25

Author(s):

Andrew W. DeVilbiss ◽

Rajendran Sanalkumar ◽

Bryan D. R. Hall ◽

Koichi R. Katsumura ◽

Isabela Fraga de Andrade ◽

...

Keyword(s):

Cell Biology ◽

Target Genes ◽

Histone Methyltransferase ◽

Cell Maturation ◽

Erythroid Cell ◽

Histone H4 ◽

Loss Of Function ◽

Gata Factor ◽

Function Strategy ◽

Erythroid Maturation

Erythropoiesis, in which committed progenitor cells generate millions of erythrocytes daily, involves dramatic changes in the chromatin structure and transcriptome of erythroblasts, prior to their enucleation. While the involvement of the master-regulatory transcription factors GATA binding protein 1 (GATA-1) and GATA-2 in this process is established, the mechanistic contributions of many chromatin-modifying/remodeling enzymes in red cell biology remain enigmatic. We demonstrated that SetD8, a histone methyltransferase that catalyzes monomethylation of histone H4 at lysine 20 (H4K20me1), is a context-dependent GATA-1 corepressor in erythroid cells. To determine whether SetD8 controls erythroid maturation and/or function, we used a small hairpin RNA (shRNA)-based loss-of-function strategy in a primary murine erythroblast culture system. In this system, SetD8 promoted erythroblast maturation and survival, and this did not involve upregulation of the established regulator of erythroblast survival Bcl-xL. SetD8 catalyzed H4K20me1 at a criticalGata2 ciselement and restricted occupancy by an enhancer ofGata2transcription, Scl/TAL1, thereby repressingGata2transcription. Elevating GATA-2 levels in erythroid precursors yielded a maturation block comparable to that induced by SetD8 downregulation. As lowering GATA-2 expression in the context of SetD8 knockdown did not rescue erythroid maturation, we propose that SetD8 regulation of erythroid maturation involves multiple target genes. These results establish SetD8 as a determinant of erythroid cell maturation and provide a framework for understanding how a broadly expressed histone-modifying enzyme mediates cell-type-specific GATA factor function.

Download Full-text

Dynamic Change in the Stochiometry of ETO2 and SCL Governs the Transition to Terminal Differentiation in Erythroid Progenitors.

Blood ◽

10.1182/blood.v108.11.1169.1169 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1169-1169

Author(s):

Julie A. Lambert ◽

Nicolas Goardon ◽

Patrick Rodriguez ◽

Sabine Herblot ◽

Pierre Thibault ◽

...

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Chromatin Immunoprecipitation ◽

Target Genes ◽

Terminal Differentiation ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Erythroid Progenitors ◽

The Core ◽

Undifferentiated Cells

Abstract As highly proliferative erythroid progenitors commit to terminal differentiation, they also progressively undergo growth arrest. To determine the mechanisms underlying the appropriate timing of erythroid gene expression and those associated with growth cessation, we analyzed the dynamical composition of the multiprotein complex nucleated by the bHLH transcription factor SCL, a crucial regulator of erythropoiesis that absolutely requires interaction with other factors to activate transcription. In progenitor cells, the SCL complex marks a subset of erythroid specific genes (alpha-globin, P4.2, glycophorin A) that are transcribed later in differentiating cells, conducting cells toward terminal maturation. To unravel the regulation of transcription by SCL, we used tagging/proteomics approaches in two differentiation-inducible erythroid cell lines, coupled with binding assays to immobilized DNA templates and chromatin immunoprecipitation. Our analyses reveal that the core complex comprised of known proteins (SCL, GATA-1, LMO2, Ldb1 and E2A) and two additional E protein family members, HEB and E2-2, did not change with differentiation. Strikingly, this complex recruits HDAC1-2 in undifferentiated cells which were exchanged with TRRAP, a chromatin remodelling factor, upon differentiation, suggesting an epigenetic regulation of erythroid differentiation mediated by the core SCL complex. Finally, we identified the corepressor ETO2 targeted via this complex through direct interaction with E2A/HEB. In vivo, ETO2 represses the transcription of SCL target genes both in transient assays and in chromatin. During erythroid differentiation, ETO2 remains associated with the SCL complex bound to erythroid promoters. However, the stoichiometry of ETO2 and SCL/HEB changes as SCL and HEB levels increase with erythroid differentiation, both in nuclear extracts and on DNA. To determine the functional consequence of this imbalance in activator to co-repressor ratio, we delivered ETO2 siRNA in primary hematopoietic cells and found an accelerated onset of SCL target genes on induction of erythroid differentiation, and conversely, these genes were decreased following ectopic ETO2 expression. Strikingly, inhibition of ETO2 expression in erythroid progenitors arrests cell proliferation, indicating that ETO2 is required for their expansion. We therefore analyzed gene expression in purified erythroid progenitors and differentiating erythroid cells (E1-E5) and found an inverse correlation between the mRNA levels of ETO2 and cyclin-dependent kinase inhibitors. Moreover, ETO2 siRNA treatment of primary erythroid progenitors results in increased p21 CDKI and Gfi1b expression, as assessed by real-time PCR. Finally, we show by chromatin immunoprecipitation that Gfi-1b, p21 and p27, are direct targets of the SCL- ETO2 complex. We therefore conclude that ETO2 regulates the erythroid lineage fate by repressing SCL marked erythroid genes in undifferentiated cells, and by controlling the expansion of erythroid progenitors. Our study elucidates the dual function of ETO2 in the erythroid lineage and sheds light on epigenetic mechanisms coordinating red blood cell proliferation and differentiation.

Download Full-text

Klf1 Regulatory Networks in Primary Erythroid Cells.

Blood ◽

10.1182/blood.v114.22.1462.1462 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1462-1462

Author(s):

Michael Tallack ◽

Thomas Whitington ◽

Brooke Gardiner ◽

Eleanor Wainwright ◽

Janelle Keys ◽

...

Keyword(s):

Regulatory Networks ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Es Cells ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Gene Promoters ◽

Erythroid Cells ◽

Red Cell Membrane ◽

Sequencing Platform

Abstract Abstract 1462 Poster Board I-485 Klf1/Eklf regulates a diverse suite of genes to direct erythroid cell differentiation from bi-potent progenitors. To determine the local cis-regulatory contexts and transcription factor networks in which Klf1 works, we performed Klf1 ChIP-seq using the SOLiD deep sequencing platform. We mapped more than 10 million unique 35mer tags and found ∼1500 sites in the genome of primary fetal liver erythroid cells are occupied by endogenous Klf1. Many reside within well characterised erythroid gene promoters (e.g. b-globin) or enhancers (e.g. E2f2 intron 1), but some are >100kb from any known gene. We tested a number of Klf1 bound promoter and intragenic sites for activity in erythroid cell lines and zebrafish. Our data suggests Klf1 directly regulates most aspects of terminal erythroid differentiation including synthesis of the hemoglobin tetramer, construction of a deformable red cell membrane and cytoskeleton, bimodal regulation of proliferation, and co-ordination of anti-apoptosis and enucleation pathways. Additionally, we suggest new mechanisms for Klf1 co-operation with other transcription factors such as those of the gata, ets and myb families based on over-representation and spatial constraints of their binding motifs in the vicinity of Klf1-bound promoters and enhancers. Finally, we have identified a group of ∼100 Klf1-occupied sites in fetal liver which overlap with Klf4-occupied sites in ES cells defined by Klf4 ChIP-seq. These sites are associated with genes controlling the cell cycle and proliferation and are Klf4-dependent in skin, gut and ES cells, suggesting a global paradigm for Klfs as regulators of differentiation in many, if not all, cell types. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Studies on Heme Oxygenase 1 During Erythroid Differentiation

Blood ◽

10.1182/blood.v116.21.4254.4254 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4254-4254

Author(s):

Daniel Garcia Santos ◽

Jesse Eisenberg ◽

Matthias Schranzhofer ◽

Prem Ponka

Keyword(s):

Aminolevulinic Acid ◽

Conflicts Of Interest ◽

Tissue Injury ◽

Erythroid Differentiation ◽

The Body ◽

Cellular Damage ◽

Erythroid Cell ◽

Heme Oxygenase 1 ◽

Erythroid Cells ◽

Murine Erythroleukemia

Abstract Abstract 4254 Heme is indispensable for the function of all aerobic cells as a prosthetic group of innumerable proteins. However, “free heme” (uncommitted) can initiate the formation of free radicals and cause lipid peroxidation, which can lead to cellular damage and tissue injury. Therefore, the rate of heme biosynthesis and catabolism must be well balanced by tight control mechanisms. The highest amounts of organismal heme (75-80%) are present in circulating red blood cells (RBC), whose precursors synthesize heme with rates that are at least one order of magnitude higher (on the per cell basis) than those in the liver – the second most active heme producer in the body. The degradation of heme is exclusively carried out by heme oxygenases 1 and 2 (HO1 and HO2), which catalyze the rate-limiting step in the oxidative degradation of heme. Although the heme-inducible HO isoform, HO1, has been extensively studied in hepatocytes and many other non-erythroid cells, virtually nothing is known about the expression of HO1 in developing RBC. Similarly, it is unknown whether HO1 plays any role in erythroid cell development under physiological or pathophysiological conditions. Using both a murine erythroleukemia cell line (MEL) and primary erythroid cells isolated from mouse fetal livers, we have demonstrated that during erythroid differentiation HO1 is up-regulated at both mRNA and protein levels. This increase in HO1 can be prevented by succinylacetone (SA), an inhibitor of heme synthesis that blocks 5-aminolevulinic acid dehydratase. These data suggest that in developing RBC, in addition to the continuous assembly of heme with globin chains, there is an increase in levels of uncommitted heme, which upregulates HO1 expression. Additionally, we have shown that down-regulation of HO1 via siRNA increased hemoglobinization in differentiating MEL cells. In contrast, induction of HO1 expression by NaAsO2 reduced the hemoglobinization of MEL cells. This effect could be reversed to control levels by the addition of HO1 inhibitor tin-protophorphyrin (SnPP). These results show that in differentiating erythroid cells the balance between levels of heme and HO1 have to be tightly regulated to maintain hemoglobinization at appropriate levels. Our results lead us to propose that disturbances in HO1 expression could play a role in some pathophysiological conditions such as thalassemias. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Complete Block of Early B Cell Differentiation in Mice Lacking the Endosomal Adaptor Protein p14

Blood ◽

10.1182/blood.v126.23.1026.1026 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1026-1026

Author(s):

Marcin Lyszkiewicz ◽

Daniel Kotlarz ◽

Natalia Zietara ◽

Gudrun Brandes ◽

Jana Diestelhorst ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Target Genes ◽

Conflicts Of Interest ◽

Adaptor Protein ◽

Cell Development ◽

B Cell Development ◽

B Cell Differentiation ◽

Knock Out ◽

And Function

Abstract Human primary immunodeficiency caused by a point mutation in the 3' untranslated region of the endosomal adaptor protein p14 (also known as Lamtor2) resulted in severely impaired function of neutrophils, B cells, T cells and melanocytes. However, complexity of the phenotype and scarcity of human material preclude in-depth studies. Therefore, to gain insight into the role of p14 in B cell development and function, we generated loxP conditional knock-out mice. Using mb-1-Cre mice we demonstrated that loss of p14 at the preB1 stage lead to a complete block of B cell development, resulting in the absence of IgM-positive B cells. Further, to test the significance of p14 deficiency in peripheral organs, we took advantage of CD19-Cre mice, which have limited efficiency in deleting target genes in the bone marrow, but reach up to 95% efficiency in spleen. Thus, we could demonstrate that later in B cell development, p14 was essential for the generation and activation of mature B lymphocytes. While B1 cell development was maintained, splenic follicular B cells were massively reduced in the absence of p14. Furthermore, activation of B cell receptor (BCR) resulted in impaired intracellular signalling and proliferation of p14 deficient B cells. In particular, lack of p14 lead to delayed internalization of BCR and endosomal processing associated with impaired mobilization of Ca++ from intracellular stores as well as aberrant phosphorylation of BCR-associated kinases. In conclusion, our data revealed that p14 is a critical regulator of B cell development and function, which acts by modulating BCR signalling. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Mitochondria Clearance Mechanisms Via Interaction with Macrophages in Erythropoiesis

Blood ◽

10.1182/blood-2018-99-110049 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2320-2320

Author(s):

Chong Yang ◽

Toshio Suda ◽

Xiaoxuan Lin ◽

Mitsuhiro Endo

Keyword(s):

Large Scale ◽

Conflicts Of Interest ◽

Terminal Differentiation ◽

Erythroid Differentiation ◽

Transcriptional Pattern ◽

Mitochondria Transfer ◽

Erythroid Maturation ◽

Terminal Erythroid Differentiation ◽

Late Stages

Abstract Adult erythropoiesis involves a series of well-coordinated events resulting in the production of mature red blood cells. One of such events is the mitochondria clearance, which is known to occur cell-autonomously via autophagy-dependent mechanisms. Interestingly, we identified a sequential changes in the transcriptional pattern during terminal erythroid differentiation based on the expression of several macroautophagy (e.g. Atg3, Atg5, Atg7 and Atg10) and non-canonical mitophagy (e.g. Pink1, Park2, Bnip3l/Nix, P62 and Ulk1) genes. Hence we hypothesize that the progressive reduction in mitochondria during terminal erythroid differentiation is directed by distinct transcriptionally-regulated programs. Notably, we revealed a gradual reduction of the expression of lysosome related genes (e.g. Lamp1, CD63, and Atp6v) and lysosomal activities from early to late stages of terminal differentiation. On the other hand, P62-Pink1-Parkin mediated ubiquitin proteasome degradation of mitochondria proteins seems to be more prominent during late stage erythropoiesis. Hence our data suggest that mitochondria clearance is predominantly mediated by canonical autophagy during early stages of terminal differentiation, whereas non-canonical mitophagy pathway seem to play a more predominant role to regulate late stages erythroid maturation. Next, we discovered mitochondria transfer activities from erythroblasts to their niche. In the context of erythropoiesis, macrophages are known to interact closely with erythroblasts to provide a specialized niche for erythroid precursors to proliferate, differentiate and enucleate. We showed defective erythropoiesis after macrophage depletion in the bone marrow. Subsequently, we identified a tendency for early erythroblasts to associate with macrophages and isolated those erythroblasts from mito-dendra2 mice with trackable mitochondria to establish a murine primary cell co-culture system. Then we report mitochondria transfer activities in the erythroid niche via different modes including direct uptake, micro-vesicle transfer and tunnelling nanotubes (TNT). Interestingly, interchangeable structures between micro-vesicles and TNTs have been observed, suggesting an interplay between cytoskeleton and membrane lipid molecules in the mitochondria transfer mechanisms. Furthermore, mitochondria transfer activities have also been observed in the co-culture of mito-dendra2 erythroid cells with a macrophage cell line, RAW cells, and are significantly enhanced by the activation of the RAW cells via Tfe3 activation. In summary, our findings may provide insight into the mitochondria clearance machineries that mediates erythroid maturation to fulfil the clinical demand for large scale transfusable blood cell production in vitro. Disclosures No relevant conflicts of interest to declare.

Download Full-text

A Screen In Primary Erythroblasts Reveals a MicroRNA-Centered Homeostatic Mechanism for Regulating Cyclin E Activity.

Blood ◽

10.1182/blood.v116.21.1543.1543 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1543-1543

Author(s):

Yanfei Xu ◽

Tanushri Sengupta ◽

Alexander C. Minella

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Conflicts Of Interest ◽

Cyclin E ◽

Erythroid Differentiation ◽

Erythroid Cell ◽

Mrna Levels ◽

Homeostatic Mechanism ◽

Regulated Expression

Abstract Abstract 1543 A growing body of evidence highlights the importance of microRNAs in regulating the expression of mediators of cell cycle progression. A theme emerging from these studies is that microRNAs participate in feedback or feed-forward circuits to provide bistability for key transition points in the cell cycle. We previously have shown that proper regulation of cyclin E activity is required for normal erythroid cell maturation in vivo, using cyclin ET74AüT393A knock-in mice, which have markedly dysregulated cyclin E due to its failure to interact with the Fbw7 ubiquitin ligase complex. We hypothesized that we could identify novel, microRNA-based molecular circuitry for maintaining appropriate levels of cyclin E activity by screening cyclin E knock-in erythroblasts for alterations in microRNA expression. We analyzed data we obtained from multiplex real-time PCR arrays comparing the expression of over 500 microRNAs in cyclin ET74A T393A knock-in versus wild-type erythroblasts (Ter119+/CD71+) and found down-regulated expression of a number of microRNAs targeting CDK inhibitors. We also identified down-regulated expression of potential microRNA regulators of Fbw7 expression. We found that overexpression of miR-223, in particular, significantly reduces Fbw7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability. We next confirmed that miR-223 targets the Fbw7 3’ untranslated region. We then found that reduced miR-223 expression leads to increased Fbw7 expression and decreased cyclin E activity. Finally, we found that miR-223 expression in K562 cells is responsive to acute alterations in cyclin E regulation by the Fbw7 pathway and that dysregulated Fbw7 expression alters the erythroid differentiation capacity of these cells. Mir-223 plays an important role in myeloid and erythroid differentiation by regulating multiple substrates involved in these maturation programs. Here, we identify Fbw7 as a novel target of miR-223. Our data also indicate that miR-223 modulates Fbw7 expression as part of a homeostatic mechanism to regulate cyclin E activity and provide the first evidence that activity of the SCFFbw7 ubiquitin ligase can be controlled by the microRNA pathway. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The Hbo1-Brd1/Brpf2 HAT Complex Is Required for Erythropoiesis In Fetal Liver

Blood ◽

10.1182/blood.v116.21.3872.3872 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3872-3872

Author(s):

Yuta Mishima ◽

Satoru Miyagi ◽

Atsunori Saraya ◽

Masamitsu Negishi ◽

Mitsuhiro Endoh ◽

...

Keyword(s):

Transcription Factor ◽

Target Genes ◽

Conflicts Of Interest ◽

Fetal Liver ◽

Flow Cytometric Analysis ◽

K562 Cells ◽

Erythroid Differentiation ◽

Pwwp Domain ◽

Transcription Factor Genes ◽

Chip Analysis

Abstract Abstract 3872 Bromodomain-containing protein 1 (Brd1, initially designated as BR140-LIKE; BRL) contains a bromodomain, two plant homology domain (PHD) zinc fingers, and a proline-tryptophan-tryptophan-proline (PWWP) domain, three types of modules characteristic of chromatin regulators. Recently, BRD1 appeared to belong to the BRPF family which includes BRPF1, BRD1/BRPF2, and BRPF3. Among them, BRPF1 is known to be a subunit of the MOZ H3 histone acetyltransferase (HAT) complex. BRD1 has been proposed to be additional subunit of the MOZ H3 HAT complex on the analogy of BRPF1. However, its molecular function remains elusive. To elucidate the biological functions of BRD1, we generated Brd1-null mice and found that they die in utero. Brd1-/- embryos were alive and recovered at nearly the expected Mendelian ratio at 12.5 days postcoitum (dpc) but died by 15.5 dpc. Brd1-/- embryos at 12.5 dpc were pale and the cell number of fetal livers, in which fetal hematopoiesis occurs, was decreased to about 20% of the control. Cytological analysis revealed that Brd1-/- fetal livers had profoundly fewer erythroblasts at maturation stages beyond proerythroblasts compared to wild-type fetal livers. Flow cytometric analysis of Brd1-/- fetal livers revealed a significant accumulation of CD71+Ter119- proerythroblasts and a reduction in CD71+Ter119+ and CD71-Ter119+ maturating erythroblasts. A drastic increase in AnnexinV+ apoptotic cells was detected in the CD71+Ter119+ and CD71-Ter119- cell fractions in Brd1-/- fetal livers. These findings suggested that severe anemia caused by compromised differentiation and/or survival of erythroblasts accounts for embryonic lethality of Brd1-/- embryos. To understand the mechanism underlying defective erythropoiesis in Brd1-null embryos, we performed biochemical analyses and found that Brd1 bridges the HAT, HBO1 but not MOZ, and its activator protein, ING4, to form an enzymatically active HAT complex. Forced expression of Brd1 promoted erythroid differentiation of K562 cells, while Brpf1, which preferentially binds to MOZ, had no significant effect. Correspondingly, depletion of Hbo1 by Hbo1 knockdown perturbed erythroid differentiation of mouse fetal liver progenitors. Of note, the level of global acetylation of histone H3 at lysine 14 (H3K14) was specifically decreased in Brd1-deficient erythroblasts. These results collectively implied that acetylation of H3K14 catalyzed by the Hbo1-Brd1 complex has a crucial role in fetal liver erythropoiesis. To identify the downstream targets for the HBO1-BRD1 complex, we performed the ChIP-on-chip analysis in K562 cells and found that BRD1 and HBO1 largely co-localize on the genome, especially on the promoters of erythroid transcription factor genes. ChIP analysis revealed that acetylation of H3K14 at the promoters of erythroid transcription factor genes, including Gata1, Gata2, Tal1, Stat5a, and ETO2, were profoundly diminished in the Brd1-deficient erythroblasts. Among these target genes, we focused on Gata1, which plays a central role in erythropoiesis, and carried out complementation experiments with Gata1 using a Gata1 retrovirus. Exogenous Gata1, but not Bcl-xL, efficiently improved proliferative capacity and survival of Brd1-deficient erythroid progenitors and also restored, at least partially, their impaired differentiation. These results clearly showed that the Hbo1-Brd1 complex is required for the acetylation of H3K14 at the promoters of erythroid transcription factor genes, thereby is crucial for erythropoiesis in fetal liver. Disclosures: No relevant conflicts of interest to declare.

Download Full-text