Targeted ShRNA Screening Identified Critical Role of Pleckstrin-2 in Erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3199-3199
Author(s):  
Ganesan Keerthivasan ◽  
Jing Yang ◽  
Piu Wong ◽  
John Doench ◽  
David E. Root ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Late Stage ◽  
Large Scale ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Critical Role ◽  
Erythroid Cells ◽  
Novel Genes ◽  
Erythroid Precursor ◽  
In Erythroid Cells

Abstract Abstract 3199 Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursor; induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. Recent genome wide transcriptional profile study demonstrated that over 500 genes are up-regulated during erythropoiesis. Many of these genes encode erythroid specific proteins that play well-known functions in red cells. However, the functions of the most other genes in the erythroid cells are still unknown. To identify novel genes in erythropoiesis, we infected mouse fetal liver erythroblasts with lentiviruses containing mammalian shRNA knockdown library that selectively includes the most highly upregulated 100 genes with unknown functions in erythroid cells. The infected cells were cultured in two different conditions for the characterization of early and late stage erythropoiesis using a high throughput flow cytometry based analysis. With these methods, we identified 33 novel genes that regulate cell differentiation or apoptosis in early stage erythropoisis; 20 genes play important roles in late stage erythropoiesis including enucleation. Significantly, there is an overlap of 16 genes that function in both early and late stage erythropoiesis. We focused on pleckstrin-2, which is specifically and abundantly expressed in erythroid cells, to further characterize its detailed functions in red cell development. We found that knockdown of pleckstrin-2 leads to dramatic apoptosis in early stage erythropoiesis. Knockdown of pleckstrin-2 in late stage erythropoiesis blocks enucleation with no apparent effects on cell differentiation, proliferation or apoptosis. We further discovered that pleckstrin-2 deficiency in early and late erythroblasts disrupts normal actin cytoskeleton as evidenced by super-resolution immunofluorescence microscope. To elucidate the detailed mechanisms of the functions of pleckstrin-2 in different stages of erythropoiesis, we performed proteomic studies and identified candidate proteins that interact with pleckstrin-2 that may contribute to the phenotypes of apoptosis and enucleation defects. In summary, our study identified pleckstrin-2 as a critical regulator of mammalian erythropoiesis and proved the significance of large-scale shRNA screening in the discovery of novel genes in development. Disclosures: No relevant conflicts of interest to declare.

A High Throughput Screening System for the Identification of Novel Genes in the Regulation of Mammalian Erythropoiesis

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2093-2093
Author(s):  
Peng Ji ◽  
Piu Wong ◽  
Harvey F. Lodish
Keyword(s):  
Cell Differentiation ◽  
High Throughput ◽  
High Throughput Screening ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Megaloblastic Anemia ◽  
Chromatin Condensation ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Novel Genes

Abstract Abstract 2093 Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursors. This induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. A recent genome wide transcriptional profile study demonstrated that over 600 genes are up-regulated during erythropoiesis. Surprisingly, the roles that most of the identified genes play in erythroid cells are still unknown. Understanding the functions of these unknown genes in the erythroid cells is necessary to elucidate the pathogenesis of red cell disorders such as congenital dyserythropoietic anemias, fanconi anemia, aplastic anemia, megaloblastic anemia, as well as leukemia and myelodysplastic syndromes with leukemic or dysplastic erythroid features. The goal of our study is to identify novel genes involved in different stages of erythropoiesis. To achieve this goal, we developed a high-throughput flow cytometry based assay that simultaneously detects erythroid cell differentiation and enucleation. In this assay, mouse fetal liver erythroblasts were purified and infected with lentiviruses containing a mammalian shRNA knockdown library obtained from the Broad Institute. The infected cells were cultured in a 96-well plate. Over the following two days the unaffected cells fully differentiate with approximately 60% of the cells enucleated. However, those cells in which shRNAs have knocked down genes critical for erythropoiesis are expected to show alterations in differentiation and/or enucleation. The system was validated using lentiviruses expressing shRNAs against Gata1 and mDia2, known proteins that are involved in the early and late stages of erythropoiesis, which showed inhibitions of differentiation and enucleation, respectively. We have pre-screened the most up-regulated 100 genes that play unknown functions in the erythroid cells. Future studies will be focused on the identified novel genes on their functions in erythroid lineage commitment, Epo mediated signaling pathways, hemoglobin enrichment, chromatin condensation, and enucleation. Disclosures: No relevant conflicts of interest to declare.

Distinct Roles of TET Proteins in the Regulation of Normal and Disordered Human Erythropoiesis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 159-159
Author(s):  
Hongxia Yan ◽  
Yaomei Wang ◽  
Jie Li ◽  
Xiaoli Qu ◽  
Yumin Huang ◽  
...  
Keyword(s):  
Epigenetic Regulation ◽  
Late Stage ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Erythroid Differentiation ◽  
Erythroid Cells ◽  
Phenotypic Changes ◽  
Tet Proteins ◽  
Cd34 Cells

Abstract TET family proteins (TET1, TET2 and TET3) have recently emerged as important epigenetic modifiers by catalyzing the conversion of 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmc). Although they have been documented to play important roles in a variety of biological processes, their function in erythroid differentiation has yet to be defined. In the present study, we show that of the three TET family members, TET2 and TET3 but not TET1 are expressed in erythroid cells and that TET3 is more abundantly expressed than TET2. Using shRNA-mediated knockdown approach we explored the role of TET proteins in erythroid differentiation of CD34+ human cells. We first showed that consistent with their role in the production of 5hmc, knockdown of either TET2 or TET3 led to a decrease in global 5hmc levels as assessed by mass spectrometric analysis. However, knockdown of TET2 or TET3 resulted in distinctly different phenotypic changes during erythropoiesis. Knockdown of TET3 in human CD34+ cells resulted in impaired cell growth which is accompanied by increased apoptosis of late stage erythroblasts. Knockdown of TET3 also led to generation of bi/multinucleated polychromatic/orthochromatic erythroblasts which is accompanied by impaired enucleation. To explore the molecular mechanisms for the observed phenotypic changes, we performed RNA-seq analysis on control and TET3 knockdown erythroblasts at same stages of development. Bioinformatics analysis revealed that the expression levels of several apoptosis-promoting genes such as FOXO1, TNFRSF10B, TGFB1 and BTG1 are increased and that of a mitosis/cytokinesis associated gene KLHDC8B is decreased in polychromatic and orthochromatic erythroblasts following TET3 knockdown. Measurement of 5hmc and 5mc at promoter region of KLHDC8B locus revealed decreased 5hmc level concurrent with increased 5mc level. Importantly, knockdown of KLHDC8B in CD34+ cells as with knockdown on TET3 led to generation of increased numbers of bi/multinucleated polychromatic/orthochromatic erythroblasts and impaired enucleation implying a role for this protein in cytokinesis of late stage but not early stage erythroblasts. These findings demonstrate that TET3 regulates erythropoiesis in a stage-specific manner by targeting different set of genes. Importantly, knockdown of TET2 led to phenotypic changes that were very different from that seen following knockdown of TET3 but the observed changes are similar to the erythroid development defects noted in myelodysplastic syndromes (MDS). These include hyper-proliferation of early stage erythroid cells; delayed terminal erythroid differentiation and increased apoptosis of late stage erythroblasts. Together with the fact that TET2 gene mutation is one of the most common mutations in MDS and dyserythropoiesis is a hallmark of this disorder, our findings suggest that TET2 gene mutations can directly account for dyserythropoiesis of MDS. Our findings demonstrate distinct and important roles for TET2 and TET3 in regulating erythropoiesis and provide significant new and novel insights into epigenetic regulation of erythropoiesis at distinct development stages. The findings are likely to be very useful for furthering our understanding of epigenetic regulation of normal and disordered human erythropoiesis. Disclosures No relevant conflicts of interest to declare.

Metagenomic and Metatranscriptomic Responses of Chemical Dispersant Application during a Marine Dilbit Spill

2022 ◽  
Author(s):  
Yiqi Cao ◽  
Baiyu Zhang ◽  
Charles W. Greer ◽  
Kenneth Lee ◽  
Qinhong Cai ◽  
...  
Keyword(s):  
Microbial Community ◽  
Late Stage ◽  
Large Scale ◽  
Community Dynamics ◽  
Early Stage ◽  
Small Scale ◽  
Rrna Gene ◽  
Shotgun Metagenomics ◽  
Microcosm Studies ◽  
Over Time

The global increase in marine transportation of dilbit (diluted bitumen) can increase the risk of spills, and the application of chemical dispersants remains a common response practice in spill events. To reliably evaluate dispersant effects on dilbit biodegradation over time, we set large-scale (1500 mL) microcosms without nutrients addition using low dilbit concentration (30 ppm). Shotgun metagenomics and metatranscriptomics were deployed to investigate microbial community responses to naturally and chemically dispersed dilbit. We found that the large-scale microcosms could produce more reproducible community trajectories than small-scale (250 mL) ones based on the 16S rRNA gene amplicon sequencing. In the early-stage large-scale microcosms, multiple genera were involved into the biodegradation of dilbit, while dispersant addition enriched primarily Alteromonas and competed for the utilization of dilbit, causing depressed degradation of aromatics. The metatranscriptomic based Metagenome Assembled Genomes (MAG) further elucidated early-stage microbial antioxidation mechanism, which showed dispersant addition triggered the increased expression of the antioxidation process genes of Alteromonas species. Differently, in the late stage, the microbial communities showed high diversity and richness and similar compositions and metabolic functions regardless of dispersant addition, indicating the biotransformation of remaining compounds can occur within the post-oil communities. These findings can guide future microcosm studies and the application of chemical dispersants for responding to a marine dilbit spill. Importance In this study, we employed microcosms to study the effects of marine dilbit spill and dispersant application on microbial community dynamics over time. We evaluated the impacts of microcosm scale and found that increasing the scale is beneficial for reducing community stochasticity, especially in the late stage of biodegradation. We observed that dispersant application suppressed aromatics biodegradation in the early stage (6 days) whereas exerting insignificant effects in the late stage (50 days), from both substances removal and metagenomic/metatranscriptomic perspectives. We further found that Alteromonas species are vital for the early-stage chemically dispersed oil biodegradation, and clarified their degradation and antioxidation mechanisms. The findings would help to better understand microcosm studies and microbial roles for biodegrading dilbit and chemically dispersed dilbit, and suggest that dispersant evaluation in large-scale systems and even through field trails would be more realistic after marine oil spill response.

Alterations In the Signaling Profile of Leukemic Progenitors Can Predict the Response of Myelodysplastic Syndrome (MDS) Patients to Azacytidine

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2921-2921
Author(s):  
Paraskevi Miltiades ◽  
Irene Bouchliou ◽  
Evangelia Nakou ◽  
Emmanuil Spanoudakis ◽  
Sotirios Papageorgiou ◽  
...  
Keyword(s):  
Treatment Response ◽  
Late Stage ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Response To Therapy ◽  
Biological Behavior ◽  
Transfusion Rate ◽  
Gm Csf ◽  
Transfusion Requirements ◽  
Kinetics Of

Abstract Abstract 2921 STAT3 and STAT5 regulate fundamental cellular processes and comprise the most studied signaling molecules of both normal and malignant hematopoiesis. Deregulation of STAT signaling contributes to leukemogenesis and may serve as a treatment target. In leukemic progenitors (LPs), the clustering of STAT3 and STAT5 phosphorylation patterns, both basal and after growth factor stimulation, can be achieved by flow cytometry, leading to the identification of distinct signaling profiles (SPs). In acute myeloid leukemia patients, SPs reflect the biological behavior of the LPs and can distinguish patient subgroups with worse prognosis and/or resistance to treatment. As epigenetic defects of genes involved in cell signaling are frequently observed in cancer cells we investigated the alterations in the SPs of MDS progenitors during azacytidine treatment and their correlation with response, cytogenetics and transfusion requirements. Bone marrow samples of 24 high risk MDS patients were obtained before and 15 days after the initiation of azacytidine in order to assess potential changes in SP before the disappearance of the LPs. According to the IWG response criteria patients were divided into group A (CR, PR and HI, n=10) and group B (stable disease and failure, n=14). Immunomagnetically purified LPs were either left untreated or stimulated with G-CSF and GM-CSF for 15` and then stained intracellularly with monoclonal antibodies against STAT3 and STAT5. The comparisons of basal and potentiated responses before and 15 days after azacytidine initiation were made with Mann-Whitney U-test. Clustering of SPs was performed with hierarchical cluster analysis and was correlated with treatment response, cytogenetics and transfusion dependence by using Chi square or Fisher Exact tests as appropriate. All analyses were performed using SPSS 14.0 software (SPSS Science, Chicago, IL). By clustering the SPs before and 15 days after the initiation of azacytidine we distinguished two subgroups of patients based on both the basal levels and potentiated response to growth factors. Patients with generally weak expression of STAT3 and STAT5 had significantly better response to azacytidine compared to those with strong expression of the same molecules (p=0.035), whereas there were no correlation of SPs with the karyotype (p=0.45) and transfusion rate (p=0.39). In line with the above, we further identified a STAT3+STAT5+ double positive population of MDS progenitors whose pretreatment levels after G-CSF and GM-CSF stimulation were inversely associated with treatment response (figure 1). Additionally, SP kinetics were following the disease course and response to therapy. In two late-stage MDS patients who achieved complete remission the SP was restored to early-stage MDS levels in day 15 after azacytidine initiation (figure 2A). In contrast, the SPs in the majority of non-responding patients remained unaltered (figure 2B), whereas the SP of a relapsing patient reverted to pretreatment levels after an initial restoration to early-stage MDS levels (figure 3). Figure 1. Significantly lower pretreatment levels of STAT3+STAT5+ MDS progenitors after G and GM-CSF stimulation in responding patients. (A) Representative plots of a patient who failed azacytidine (i, ii) and one who achieved CR (iii, iv). (B) Cumulative results in responding (A) and non-responding (B) patients. Figure 1. Significantly lower pretreatment levels of STAT3+STAT5+ MDS progenitors after G and GM-CSF stimulation in responding patients. . / (A) Representative plots of a patient who failed azacytidine (i, ii) and one who achieved CR (iii, iv). (B) Cumulative results in responding (A) and non-responding (B) patients. Figure 2. The kinetics of SPs follow the response to azacytidine. (A) The SP of a late-stage MDS patient who attained PR reverted to early-stage MDS levels at day 15 after the first cycle of azacytidine. (B) By contrast, a patient who failed treatment displayed no SP changes. Figure 2. The kinetics of SPs follow the response to azacytidine. . / (A) The SP of a late-stage MDS patient who attained PR reverted to early-stage MDS levels at day 15 after the first cycle of azacytidine. (B) By contrast, a patient who failed treatment displayed no SP changes. Figure 3. Kinetics of the SP in a relapsing patient Plots of a patient who achieved CR but relapsed 4 months after the discontinuation of azacytidine. Figure 3. Kinetics of the SP in a relapsing patient . / Plots of a patient who achieved CR but relapsed 4 months after the discontinuation of azacytidine. In conclusion, we demonstrate that SP alterations of MDS progenitors during azacytidine treatment can predict clinical response. Moreover, it appears that azacytidine can restore the leukemic signaling in MDS by modifying both the basal and potentiated expression of STAT3 and STAT5. Our findings advocate the differentiating activity of hypomethylating agents, potentially via epigenetic reprogramming of pivotal signaling networks of leukemic progenitors. Disclosures: No relevant conflicts of interest to declare.

Regulation Of Erythro-Megakaryocytic Lineage Bifurcation By The Gfi1b Gene Target Rgs18

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1191-1191
Author(s):  
Ananya Sengupta ◽  
Ghanshyam Upadhyay ◽  
Asif Chowdhury ◽  
Shireen Saleque
Keyword(s):  
Conflicts Of Interest ◽  
Integration Site ◽  
Fetal Liver ◽  
Cell Types ◽  
Mapk Signaling ◽  
Erythroid Cells ◽  
Erythroid Progenitor ◽  
Gene Target ◽  
Over Expression ◽  
In Erythroid Cells

Abstract The molecular basis for the divergence of the erythroid (red blood cell) and megakaryocyte (platelet) lineages from a common bipotent MEP (megakaryocyte-erythroid progenitor) remains undefined. We now demonstrate that Rgs18 (regulator of G protein signaling 18), a GAP (GTPase activating protein) factor and a transcriptional gene target of the Gfi1b transcriptional repressor complex, likely arbitrates this critical lineage decision downstream of Gfi1b. Rgs18 was identified in a chromatin immunoprecipitation (ChIP on chip) screen for Gfi1b/LSD1/Rcor1 targets in erythroid cells. Accordingly, Rgs18 expression was found to be up-regulated in LSD1 inhibited, and Gfi1b deficient erythroid cells confirming repression of this gene by Gfi1b and its co-factors in this lineage. In contrast, Rgs18 expression was comparable in megakaryocytic cells derived from wild type and gfi1b-/-hematopoietic progenitors indicating Gfi1b independent expression of Rgs18 in these cells. Manipulation of Rgs18 expression produced opposite effects in the erythroid and megakaryocytic lineages. Rgs18 inhibition retarded megakaryocytic differentiation while its ectopic over-expression promoted differentiation at the expense of proliferation. The reverse was observed in erythroid cells where Rgs18 inhibition produced an enhancement of differentiation while over-expression impaired erythropoiesis. Since Rgs signaling regulates the activity of downstream MAPK pathways we determined the status of these pathways in Rgs18 manipulated cells. Inhibition of Rgs18 stimulated ERK phosphorylation in megakaryocytes but diminished it in erythroid cells. In contrast, Rgs18 inhibition in erythroid cells elevated p38MAPK protein and phosphorylation levels. The opposite effects of Rgs18 manipulation on MAPK signaling in erythroid versus megakaryocytic cells while intriguing are consistent with the changes in differentiation and proliferation observed in each lineage, respectively. Although Rgs18 manipulation produced opposite effects in erythroid and megakaryocytic cells, the level and activity of this factor correlated similarly with those of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1) and KLF1/EKLF (Kruppel like factor1) in both cell types. In both lineages, Rgs18 protein levels correlated directly with Fli1, and inversely with KLF1, message levels. Since Fli1 promotes megakaryocytic, and KLF1 erythroid, development; these results demonstrate that Rgs18 promotes the emergence of megakaryocytic cells from bipotent MEPs by modulating MAPK signaling and altering Fli1/KLF1 stoichiometries. Although it is unclear why Gfi1b mediated repression of Rgs18 is erythroid specific even though the former is expressed in both lineages, these results demonstrate that repression of Rgs18 by Gfi1b in fetal liver MEPs limits megakaryopoiesis and augments erythropoiesis. However following megakaryocytic commitment, robust Gfi1b independent expression of Rgs18 drives differentiation of this lineage while continued repression of Rgs18 by Gfi1b in erythroid cells ensures their proper maturation. Disclosures: No relevant conflicts of interest to declare.

p19INK4d Modulates Human Terminal Erythroid Differentiation By Post-Transcriptionally Regulating GATA1 Expression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 697-697
Author(s):  
Xu Han ◽  
Jieying Zhang ◽  
Yuanliang Peng ◽  
Huiyong Chen ◽  
Xiao Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Late Stage ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ectopic Expression ◽  
Erythroid Differentiation ◽  
Hematopoietic Stem ◽  
Erk Activation ◽  
Protein Levels ◽  
Terminal Erythroid Differentiation

Abstract Erythropoiesis is a process during which hematopoietic stem cell (HSCs) are first committed to erythroid progenitors, which subsequently undergo terminal erythroid differentiation to produce mature red blood cells. During terminal erythroid differentiation, proerythroblasts undergo 4-5 mitoses to sequentially generate basophilic erythroblasts, polychromatic erythroblasts and orthochromatic erythroblasts that expel their nuclei to produce enucleated reticulocytes. Terminal erythropoiesis is a tightly regulated process. The most well studied regulatory mechanisms include EPO/EPOR mediated signal transduction and transcription factors among which GATA1 plays critical role. Terminal erythroid differentiation is also tightly coordinated with cell cycle exit, which is regulated by cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors (CDKI), yet their roles in erythropoiesis remain largely undefined. Our RNA-seq of human terminal erythroid differentiation shows that of seven CDKI members, only three of them, p18INK4c, p19INK4d and p27KIP1, are abundantly expressed in erythroid cells and their expressions are significantly upregulated in late stage erythroblasts, which were further confirmed by western blotting analysis. In contrast to demonstrated roles of p18INK4c and p27KIP1 in terminal erythroid differentiation, the function of p19INK4d this process has not been studied. To explore the role of p19INK4d during human erythropoiesis, we employed a shRNA-mediated knockdown approach in CD34+ cells and found that p19INK4d knockdown delayed erythroid differentiation, inhibited cell growth, led to increased apoptosis and generation of abnormally nucleated late stage erythroblasts. Unexpectedly, p19INK4d knockdown did not affect cell cycle. Rather it led to decreased GATA1 protein levels. Importantly, the differentiation and nucleus defects were rescued by ectopic expression of GATA1. As GATA1 protein is protected by nuclear HSP70, to explore the mechanism for the decreased GATA1 protein levels, we examined the effects of p19INK4d knockdown on HSP70 and found p19INK4d knockdown led to decreased nuclear localization of HSP70 due to reduced ERK activation. Further biochemical analysis revealed that p19INK4d directly binds to Ras kinase inhibitor PEBP1 and that p19INK4d knockdown increased the expression of PEBP1 which in turn led to reduced ERK activation. These results demonstrate that p19INK4d maintains GATA1 protein levels through PEBP1-pERK-HSP70-GATA1 pathway. Our findings identify previously unknown and unexpected roles for p19INK4d in human terminal erythroid differentiation via a novel pathway. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Condensin II Complex Subunit NCAPH2 Is Essential for Erythropoiesis and Regulates Erythroid Gene Expression

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 286-286
Author(s):  
Zachary C. Murphy ◽  
Maeve Wells ◽  
Kristin Murphy ◽  
Michael Getman ◽  
Laurie A. Steiner
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Conflicts Of Interest ◽  
Chromatin Condensation ◽  
Differentially Expressed ◽  
Erythroid Cells ◽  
Dramatic Decline ◽  
Upregulated Genes ◽  
Mutant Cells ◽  
In Erythroid Cells

Abstract Erythropoiesis requires dramatic changes in gene expression in a cell that is rapidly proliferating and undergoing progressive nuclear condensation in anticipation of enucleation. Disruption of this process is associated with myelodysplastic syndromes and congenital anemias. Our lab has demonstrated that Setd8, the sole histone methyltransferase that can generate H4K20me1, plays an essential role in this process (Malik 2019). H4K20me1 accumulates during terminal erythroid maturation (Murphy Blood 2021) and can regulate chromatin structure and gene expression through interaction with multiple partners, including the Condensin II Complex. The Condensin II complex is a ring-like structure composed of two conserved SMC components (SMC2 and SMC4), two HEAT subunits (NCAPG2 and NCAPD3), and a kleisin subunit NCAPH2. The Condensin II complex plays an important role in chromatin condensation during mitosis, and establishing higher-order chromatin interactions in interphase cells, with some studies suggesting it also regulates gene expression (Yuen Science Adv 2017; Iwasaki Nature Comm 2019). Similar to Setd8, many subunits of the Condensin II complex are highly expressed in erythroid cells compared to most other cell types (biogps.org). We hypothesized that the Condensin II complex, in conjunction with Setd8 and H4K20me1, is important for establishing appropriate patterns of chromatin architecture and gene expression in maturing erythroblasts. To address this hypothesis, we deleted the NCAPH2 subunit in erythroid cells by crossing mice with floxed alleles of NCAPH2 with mice expressing cre-recombinase under the direction of the Erythropoietin receptor promotor (EpoRCre). Homozygous disruption of NCAPH2 was embryonic lethal by E13.5. NCAPH2 mutant embryos were similar in appearance to littermate controls until E12.5 when they developed notable pallor and a dramatic decline in the number of benzidine positive cells. Cell cycle analyses demonstrated that an accumulation of cells in G2/M preceded the dramatic decline in erythroblast numbers at E12.5. In contrast to cells from littermate controls, the NCAPH2 mutant cells were very heterogenous in cell and nuclear size and morphology. Surpisingly, most NCAPH2 mutant cells appeared to be hemoglobinized, suggesting sufficient iron accumulation and heme synthesis. In vitro cultures derived from primitive erythroid progenitors replicated in vivo findings, including normal early erythropoeisis, with significant abnormalities during mid- to late- maturation. Western blot in cycloheximide treated primitive erythroid cultures revealed that NCAPH2 has a long half-life, which likely contributes to the relative normalicy of early primitive erythoproesis. NCAPH2 mutant embryos also had a dramatic failure of definitive erythropoiesis, as evidenced by loss of mature erythroblasts in the fetal liver at E13.5. To gain insights into the mechanisms underlying these findings, we performed RNA-seq of NCAPH2 mutant, NCAPH2 het, and NCAPH2 WT erythroblasts from E11.5 embryos. Comparing NCAPH2 mutant and NCAPH2 WT erythroblasts there were 1121 down regulated genes and 743 upregulated genes (adj p-value <0.05). As expected, the downregulated genes were enriched for pathways related to cell cycle, such as Mitotic Spindle Organization (adj pvalue 5e-42). The upregulated genes were enriched for a variety of pathways including p53 transcriptional network (adj pvalue 4e-10), neutrophil mediated immunity (2e-9), DNA-binding transcription factor (adj pvalue 7e-5), and regulation of erythrocyte differentiation (adj pvalue 5e-4). Intriguingly, 91/340 genes differentially expressed in Setd8 mutant erythroblats were also differentially expressed in NCAPH2 mutant cells, including genes typically repressed early in erythroid commitment, such as GATA2 and SPI1. Cut&Tag in CD34+ derived erythroblasts demonstrated occupancy of H4K20me1 at these loci. Mass spectrometry of proteins isolated via mono-mehtylated H4K20 peptides in erythroid extracts identified Condensin II components, supporting a model where the Condensin II complex directly interacts with H4K20me1. Together, these results demonstrate that the Condensin II complex is essential for erythropoiesis, and may work in conjunction with Setd8 and H4K20me1 to establish appropriate patterns of gene expression in maturing erythroblasts. Disclosures No relevant conflicts of interest to declare.

Tumors Are a Major Source of Tissue Factor-Positive Microparticles in a Mouse Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3190-3190
Author(s):  
Jianguo Wang ◽  
Nigel Mackman
Keyword(s):  
Mouse Model ◽  
Tissue Factor ◽  
Late Stage ◽  
Tumor Size ◽  
Conflicts Of Interest ◽  
Early Stage ◽  
Membrane Vesicles ◽  
Pancreatic Tumors ◽  
Pancreatic Metastasis ◽  
Tumor Bearing

Abstract Abstract 3190 Poster Board III-127 Tissue factor (TF) is present in blood in the form of cell-membrane vesicles called microparticles (MPs). Elevated levels of TF-positive MPs may increase the risk of venous thromboembolism (VTE) in patients with cancer. In mice containing human tumors, levels of human TF antigen significantly correlate with both levels of thrombin-antithrombin (TAT) and tumor size. The objectives of this study were to measure levels of MP TF activity in the plasma of tumor-bearing mice and analyze the relative contribution of host versus tumor to this pool of MPs. Human pancreatic cells (Hpaf-II, 2×106 cells per mouse) were injected subcutaneously into female SCID mice. Five weeks later, whole blood was collected and plasma was prepared. The median tumor weight was 1.03 ± 0.64 grams (n=9). MP TF activity was measured using our recent described method (Wang et al. JTH 2009:1092-1098). The species-specific antibodies 1H1 and HTF-1 were used to inhibit mouse TF activity in MPs derived from the host and human TF activity in MPs derived from the tumor, respectively. We also measured levels of human TF antigen and TAT in the plasma. In control mice injected with saline, levels of human TF antigen and human TF activity in MPs were undetectable, whereas both human TF antigen (318.6 ± 256.2 pg/mL, n=9) and human TF activity in MPs (1.08 ± 1.18 pg/mL, n=9) were dramatically increased in tumor-bearing mice. Consistent with previous reports, there was a significant correlation between levels of human TF antigen and tumor size (r=0.67, n=9, p=0.030). Importantly, we found that levels of human TF activity in MPs significantly correlated with levels of human TF antigen (r=0.883, n=9, p=0.002) and tumor size (r=0.667, n=9, p=0.029). Furthermore, tumor-bearing mice had significant higher levels of TAT (7.5 ± 1.8 ng/mL, n=9) than control mice (3.9 ± 0.7 ng/mL, n=3) (p=0.008). Four of the 9 mice had metastatic tumors in the pancreas. Therefore, based on metastasis and tumor size, the tumor-bearing mice were classified into two groups: early stage (no pancreatic metastasis or tumor size ≤ 1 gram, 0.70 ± 0.35 gram, n=4) and late stage (pancreatic metastasis or tumor size ≥1 gram, 1.30 ± 0.72 gram, n=5). As expected, mice in the late stage group have significant higher levels of human TF antigen (468.1 ± 228.0 vs 131.8 ± 148.3 pg/ml, p=0.039) and human TF activity in MPs (1.74 ± 1.24 pg/ml vs 0.25 ± 0.08 pg/mL, p=0.049) than those in the early stage group. Finally, mice in the late stage group have significantly higher levels of human TF activity in MPs derived from the tumor (1.95 ± 1.54 pg/mL) than mouse TF activity in MPs derived from the host (0.20 ± 0.26 pg/mL, p=0.0362, n=5). In conclusion, mice containing human pancreatic tumors have significant elevated levels of total MP TF activity and TAT. Levels of human TF activity in MPs correlate with levels of human TF antigen and activation of coagulation. Tumors are a major source of TF-positive MPs in a mouse model. Disclosures No relevant conflicts of interest to declare.

Transcriptional Regulation of Transferrin Receptor by Heme in Erythroid Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2049-2049
Author(s):  
Matthias Schranzhofer ◽  
Nam-Chun Lok ◽  
Manfred Schifrer ◽  
Ernst W Muellner ◽  
Prem Ponka
Keyword(s):  
Mrna Stability ◽  
Aminolevulinic Acid ◽  
Conflicts Of Interest ◽  
Erythroid Cells ◽  
Knock Out ◽  
Iron Regulatory Proteins ◽  
Iron Incorporation ◽  
Knock Out Mice ◽  
Feedback Regulator ◽  
In Erythroid Cells

Abstract Abstract 2049 Erythroid cells are the largest consumers of iron which is delivered to them by tansferrin (Tf) by its cognate receptor (TfR). In contrast to other cells, developing red blood cells (RBC) regulate TfR expression not only at the level of mRNA stability via the iron regulatory proteins (IRP) 1 and 2, but also by transcription (Lok & Ponka, J Biol Chem 275:24185-90, 2000). Here we provide evidence that TfR expression and cellular uptake of iron from Tf is stimulated by enhanced heme synthesis. Incubation of erythroid cells with 5-aminolevulinic acid (ALA) increased TfR expression accompanied by increased iron incorporation into heme. This effect of ALA can be completely prevented by the inhibitors of heme biosynthesis (succinylacetone [blocks ALA dehydratase] or N-methylprotoporphyrin [blocks ferrochelatase]), indicating that the effect of ALA requires its metabolism to heme. The induction of TfR mRNA expression by ALA is mainly a result of increased mRNA synthesis since the effect of ALA can be abolished by actinomycin D. Recently, IRP2 was proposed to play a role in maintaining TfR mRNA stability in developing RBC (Cooperman et al., Blood 106:1084-91; 2005; Galy et al., Blood 106:2580-9, 2005). Importantly, we have demonstrated that ALA added to cultures of erythroid cells derived from IRP2 knock out mice restores the expression of TfR to levels observed in cells obtained from wild type mice. In conclusion, our results indicate that in erythroid cells heme serves as a positive feedback regulator that maintains high TfR levels thus ensuring adequate iron availability for hemoglobin synthesis. Disclosures: No relevant conflicts of interest to declare.

HSP70, the Key to Account for Erythroid Tropism of Diamond-Blackfan Anemia?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 671-671
Author(s):  
Marc Gastou ◽  
Sarah Rio ◽  
Mickael Dussiot ◽  
Narjesse Karboul ◽  
Thierry Leblanc ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Critical Role ◽  
Erythroid Differentiation ◽  
Expression Level ◽  
Hsp70 Expression ◽  
Erythroid Cells ◽  
Diamond Blackfan Anemia ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
P53 Activation

Abstract Diamond-Blackfan anemia (DBA) was the first ribosomopathy identified and is characterized by a moderate to severe, usually macrocytic aregenerative anemia associated with congenital malformations in 50% of the DBA cases. This congenital rare erythroblastopenia is due to a blockade in erythroid differentiation between the BFU-e and CFU-e stages. The link between a haploinsufficiency in a ribosomal protein (RP) gene that now encompass 15 different RP genes and the erythroid defect is still to be fully defined. Recently, mutations in TSR2 and GATA1 genes have been identified in a few DBA families. The GATA1 gene encodes for the major transcription factor critical for erythropoiesis and mutation in this gene that lead to loss of expression of the long form of the protein, necessary for the erythroid differentiation accounts for erythroblastopenia of DBA phenotype. Our group and others (Dutt et al., Blood 2011) have shown previously that p53 plays an important role in the DBA erythroblastopenia, inducing cell cycle arrest in G0/G1 and depending on the nature of RP gene mutation, a delayed erythroid differentiation and an increased apoptosis. Indeed, we identified two distinct DBA phenotypes (H. Moniz, M. Gastou, Cell Death Dis, 2012): a haploinsufficiency in RPL5 or RPL11 reduced dramatically the erythroid proliferation, delayed the erythroid differentiation, and markedly increased apoptosis, while RPS19 haploinsufficiency while reduced the extent of erythroid proliferation without inducing significant apoptosis. While p53 pathway has been found to be activated in RP haploinsufficient erythroid cells in DBA patients or shRNA-RPS19, -RPL5, or -RPL11 infected CD34+ erythroid cells, the intensity of the p53 activation pathway (p21, BAX, NOXA) is different depending on the mutated RP gene. Since the differences between the two phenotypes involved the degree of apoptosis we hypothesized that HSP70, a chaperone protein of GATA1 may play a key role in the erythroid defect of DBA. Indeed, HSP70 protects GATA1 from the cleavage by the caspase 3, a protease activated during erythroid differentiation and as such reduced levels of HSP70 related to a RP haploinsufficiency could account for increased apoptosis and delayed erythroid differentiation of erythroid cells in DBA. Indeed, a defect in RPL5 or RPL11 decreased dramatically the expression level of HSP70 and GATA1 in primary human erythroid cells from DBA patients and following in vitro knockdown of the proteins in CD34+ cells by RPL5 or RPL11 shRNA. Importantly, RPS19 haploinsufficiency did not exhibit this effect in conjunction with normal levels of HSP70 expression. Furthermore, we found that the decreased expression level of HSP70 was independent on the p53 activation. Strikingly, HSP70 was noted to be degraded by the proteasome since the bortezomib, the MG132, or the lactacystin were able to restore both the HSP70 expression level and intracellular localization in the cell. The lentiviral infection of haploinsufficient RPL5 or RPL11 cord blood CD34+ cells with a wild type HSP70 cDNA restored both the erythroid proliferation and differentiation confirming a critical role for HSP70 in the erythroid proliferation and differentiation defect in the RPL5 or RPL11 DBA phenotypes. The loss of HSP70 may explain the loss of GATA1 in DBA and also the erythroid tropism of the DBA disease. Restoration of the HSP70 expression level may be a viable and novel therapeutic option for management of this debilitating and difficult to manage erythroid disorder. Disclosures No relevant conflicts of interest to declare.

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