bluuebunny/crossref_metadata_2025_split huggingface dataset:
C/EBPα Binds and Activates the Distal PU.1 Enhancer.
Authors: Yeamans Christine , Wang Dehua , Paz-Priel Ido , Tenen Daniel G. , Friedman Alan D. 
Published: April 2020

Abstract
C/EBPα and PU.1 are both key regulators of myeloid development. C/EBPα (−/−) mice have markedly reduced granulocyte-monocyte progenitors, and PU.1 (−/−) mice lack B cells and monocytes and have greatly reduced neutrophil numbers. Genetic evidence indicates that higher levels of PU.1 are required for monocyte as compared to granulocyte development. Exogenous PU.1 or C/EBPα converts B-lineage to monocytic cells and increases monocyte as compared to granulocyte production from transduced myeloid progenitors cultured in IL3/IL6/SCF or in GM-CSF. We previously demonstrated that activation of C/EBPα-ER with estradiol induces PU.1 mRNA within 4 hr even in the presence of cycloheximide and that C/EBPα binds and activates the PU.1 promoter via a site conserved in the murine and human genes. However, binding to the promoter site in a gel shift assay was weak compared to the strong binding site in the neutrophil elastase promoter. To obtain further evidence for regulation of the PU.1 gene by C/EBPα, we have now investigated regulation of the 240 bp proximal segment of the PU.1 distal enhancer located at −14 kb. We focused on the proximal segment of the enhancer due to is strong transcriptional activating activity when linked to the PU.1 promoter, compared to the repressive effect of the distal enhancer segment. Five sites within the proximal enhancer (C1–C5) fit the C/EBP consensus site T(T/G)NNGNAA(T/G) or differed from this consensus by 1 bp. Each was evaluated for binding to C/EBPα expressed transiently in 293T cells. Site C1 bound C/EBPα weakly, and C2 and C5 each bound C/EBPα strongly. Binding of C/EBPα to the endogenous proximal enhancer segment was also demonstrated in the HF1 myeloid cell line using the chromosomal immunoprecipitation (ChIP) assay. HF1 cells were employed for this assay due to their high-level expression of C/EBPα. Sites C1, C2, and C5 were mutated individually within the enhancer, which was then linked to the PU.1 promoter and the luciferase cDNA. Mutation of C1 minimally reduced the activity of this reporter, mutation of C2 reduced activity 2-fold, and mutation of C5 reduced activity 5-fold, on average, upon transient transfection into the more readily tranfectable 32DPKCδ cell line. These data provide further support for the conclusion that C/EBPα directly activates the PU.1 gene and that induction of PU.1 by C/EBPα, potentially in cooperation with additional factors such as c-Jun, helps direct bipotent myeloid progenitors to commit to the monocytic lineage.
MicroRNAs 155, -221 and -222 Control Megakaryopoiesis at Progenitor and Precursor Level through Ets-1 Multitargeting.
Authors: Marziali Giovanna , Lulli Valentina , Coppola Simona , Romania Paolo , Fontana Laura , Liuzzi Francesca , Biffoni Mauro , Felli Nadia , Croce Carlo Maria , Pelosi Elvira , Peschle Cesare 
Published: April 2020

Abstract
It is generally accepted that microRNAs (miRs) control basic biological functions, such as cell proliferation and differentiation. However, only few targets of the ~ 300 known mammalian miRs have been validated so far, thus hampering delineation of miR-based control circuitries. Particularly, little is known on miR function in mammalian hematopoiesis. We have investigated by microarray and Northern blot evalutation miR expression profiles in human megakarypoiesis, as evaluated in cord blood hematopoietic progenitor cell (HPC) unilineage culture through the megakaryopoietic (MK) differentiation-maturation pathway. These studies indicated that miR-155, -221 and -222 are abundant in HPCs, but sharply decline during megakaryopoiesis. The decline may favour megakaryopoiesis by unblocking translation of key functional target protein(s). Bioinformatic analysis predicted that miR-155, -221 and -222 target Ets-1, a transcription factor up-regulated in Mk differentiation, which transactivates relevant Mk genes (e.g., TPO receptor, PF4, CD42 and von Willebrand factor). Consistently, in megakaryocytic cells the increase of Ets-1 protein expression coincides with the miR-155, -221 and -222 decrease. To find out whether Ets-1 mRNA is a possible target of miR-155, -221 and -222, we cloned segments of the 3′UTR of the Ets-1 gene downstream of a firefly luciferase ORF. Luciferase assay confirmed a direct interaction between each of these miRs and the 3′UTR of Ets-1. Functional studies showed that enhanced expression of these three miRs impairs proliferation, differentiation and maturation of MK cells, at least in part via enhanced degradation of Ets1 mRNA and down-modulation of Ets-1 protein. Similar results were obtained by RNA interference against Ets-1. Finally, HPCs transfected with miR-155, -221, and -222 showed a significant reduction of their Mk clonogenic capacity, suggesting that down-modulation of these miRs favours MK progenitor recruitment and commitment. Altogether, these studies indicate that a novel regulatory circuitry, based on miR-155, -221, -222 multitargeting Ets-1 mRNA leading in turn to transactivation of Mk-specific genes, plays a key role in the control of megakaryopoiesis at both progenitor and precursor level.
Identification of Common Molecular Pathways in Granulopoiesis Associated with C/EBP Epsilon and Gfi-1-Deficient Neutrophil Specific Granule Deficiency (SGD).
Authors: Khanna-Gupta Arati , Halene Stephanie , Sun Hong , Dahl Richard , Boxer Laurence A. , Berliner Nancy 
Published: April 2020

Abstract
Neutrophil specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections of the skin and respiratory system. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and exhibit defects in chemotaxis and bactericidal activity. A mouse model deficient for the transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) manifests a similar phenotype to SGD patients, and functional mutations in the C/EBPε gene have been identified in two patients with SGD. However, other patients with a similar disease phenotype do not have functional C/EBPε mutations, suggesting that other genetic abnormalities in myelopoiesis can lead to SGD. Studies in our laboratory on one such patient lacking a functional C/EBPε mutation demonstrated elevated protein levels of C/EBPε and significantly decreased levels of the transcription factor Gfi-1 (Growth factor independent-1) in peripheral blood neutrophils from this SGD patient. However, no mutation has been found thus far in the coding region of the Gfi-1 gene from the SGD patient. SGD can therefore be classified as C/EBPε negative or C/EBPε positive. We hypothesize that during granulopoiesis defects in a common molecular pathway occur in both C/EBPε- positive and -negative SGD resulting in the SGD phenotype. In order to gain insight into the molecular features of the two SGD phenotypes in the face of severe limitations on primary material from SGD patients, we have generated and characterized two cell line models. Using bone marrow from both C/EBPε−/− and Gfi-1+/− mice and their corresponding wildtype (WT) littermates, we generated EML (erythroid, myeloid, lymphoid) cell lines by transducing bone marrow with a retroviral vector expressing the dominant negative RARα and selecting for immortalized cells. Upon induction of the knock out and WT EML cell lines with all-trans retinoic acid (ATRA) to the neutrophil stage, we confirmed that both C/EBPε−/− and Gfi-1+/− cells share morphologic and functional abnormalities corresponding to their respective knockout mice, and hence to SGD. To further characterize the molecular defects in SGD, we have initiated microarray and proteomics analyses using RNA and protein prepared from both knockout cell lines. Using this approach, we may delineate defective pathways in both knock out cell types and correlate these observations to the specific SGD type. Our study should shed light on the relationship between Gfi-1 and C/EBPε during neutrophil development and should address the issue as to whether common or divergent molecular pathways are responsible for C/EBPε-negative and -positive SGD.
MicroRNA Function in Human Hematopoiesis: Identification of Lineage- and Stage-Specific Expression Profiles, Pivotal Targets and Regulatory Circuitries.
Authors: Chicarella Simona , Felli Nadia , Ferracin Manuela , Pelosi Elvira , Morsilli Ornella , Calin George Adrian , Liu Chang-Gong , Negrini Massimo , Croce Carlo Maria , Peschle Cesare 
Published: April 2020

Abstract
MicroRNAs (miRs) play a key role in the regulation of gene expression in normal and abnormal cell growth/differentiation, via suppression of translation or degradation of target mRNAs mainly by base pairing to their 3′ UTR sequences. However, little is known on miR expression/function in normal hematopoiesis. We have assayed the level of miRs in serum-free unilineage culture of cord blood (CB) CD34+ hematopoietic progenitor cells (HPC) through the erythroid (E), megakaryocytic (Mk), granulocytic (G) and monocytic (Mo) series at discrete stages of differentiation up to terminal maturation. The assay included control CB T cells. The analysis was performed by microarray and confirmed by Northern blot. Altogether, 49 miRs are expressed at significant levels in hematopoiesis. Cluster analysis indicates lineage-specific expression patterns. Furthermore, miR levels are mostly downmodulated through HPC differentiation/maturation. A group of 12 single or clustered miRs is sharply modulated (&gt; 10-fold) in hematopoiesis according to specific patterns, thus suggesting their key functional role. This group includes:miR-223, downmodulated in the E series, but upmodulated in the other lineages;miR-221/222, miR-146 and miR-155 declining more sharply in E than other lineages;miR-17/20/106 and miR-25, miR-32, declining prevailingly in the G and Mo series;a miR group showing little downmodulation in the Mk or Mo lineages (miR-123, -130a or miR-181a, b, respectively);miR-99b, -100 downmodulated in all lineages;miR-210 upmodulated in all lineages.
Bioinformatic analysis suggests that these miRs have important targets, which have been in part verified by the luciferase target assay (see below). Relevant examples include miR-221/222 and -130a targeting kit receptor, miR-146 targeting SDFR/CXCR4, miR 181a, b, 99b and 100, 210 targeting diverse HOX genes (respectively, HOXB5, A1 and A3). Novel miR-based regulatory circuitries have been identified:(a) miR-221/222 modulate erythropoietic and erythroleukemia cell growth via kit receptor targeting (N. Felli et al, PNAS, 2005);(b) miR-17-5p/20a/106 function as a master gene complex controlling monocytopoiesis through AML1 targeting, leading to M-CSF receptor transactivation (L. Fontana et al, this Meeting);miR-155 and -221/222 control megakaryopoiesis at progenitor and precursor level through Ets-1 multitargeting (G. Marziali et al, this Meeting).
Elucidating the Oncogenic Potential of NPMc+ In Vitro and In Vivo.
Authors: Cheng Ke , Grisendi Silvia , Clohessy John G. , Majid Samia , Sportoletti Paolo , Pandolfi Pier Paolo 
Published: April 2020

Abstract
Nucleophosmin (NPM) is a nucleolar phosphoprotein that plays a key role in ribosome biogenesis, control of genomic stability and stabilization of tumor suppressors, such as ARF (Grisendi et al. Nature Review Cancer, 2006). We have recently shown that NPM can act as a tumor suppressor through the generation and characterization of an NPM hypomorphic series in vivo (Grisendi et al. Nature, 2005). NPM is one of the most frequent targets of genetic alterations in hematopoietic tumors, as about 60% of all adult Acute Myelogenous Leukemia with a normal karyotype harbor C-terminal frameshift mutations (NPMc+). As a consequence, NPMc+ aberrantly localizes to the cytoplasm of leukemic blasts.
It has been shown that NPMc+ can act as a dominant negative NPM mutant sequestering endogenous NPM to the cytosol. However, to date this mutant has never been shown to behave as an oncogene, which casts doubt on its role in leukemogenesis. To assess the oncogenic potential of NPMc+, we carried out soft agar and low-density seeding assays using primary mouse embryonic fibroblasts (MEFs) expressing NPMc+ in combination with a battery of oncogenes. Interestingly, we found that NPMc+, but not wild type NPM, cooperate with adenovirus E1A to transform primary MEFs in soft agar. NPMc+/E1A also consistently showed high efficiency at forming foci in low-density seeding assays comparable to RasV12/E1A. However, NPMc+ does not transform alone or in combination with RasV12 or c-Myc and does not transform p53−/−, p21−/− or Arf−/− primary MEFs. This suggests that two complementary growth signals from NPMc+ and E1A can cooperate to evade cellular senescence and apoptosis. These data are consistent with a model by which NPMc+, through the inhibition of NPM function, can block the ARF response elicited by E1A. In support of these observations, we found that in primary cells from our NPM hypomorhic series Arf level and localization are dramatically affected by the progressive reduction in Npm dose. To elucidate in vivo the crosstalk between NPM and ARF, genetic crosses using Npm+/− and Arf+/− mice are currently ongoing and data from this analysis will also be presented.
Constitutive Activation of Hemostasis in Children with Sickle Cell Disease.
Authors: Steele MacGregor , Williams Suzan , Pluthero Fred , Bang K.W. Annie , Goldman Ran , Friedman Jeremy , Freedman John , Kirby Melanie , Rand Margaret L. , Blanchette Victor S. , Kahr Walter H. 
Published: April 2020

Abstract
Recent studies of sickle cell disease (SCD) suggest that hemostatic activation is a key aspect of pathophysiology, leading to clinical consequences such as vaso-occlusive crises and stroke. In many of these studies a SCD-associated hypercoagulable state has been inferred from markers suggestive of increased hemostatic activity, such as elevated levels of plasma thrombin-antithrombin complexes and tissue factor-containing microparticles. As part of a study using a broad-spectrum approach to explore the relationships among various aspects of normal and abnormal hemostasis in SCD we used a whole-blood coagulation assay (thromboelastography, TEG) to directly assess global hemostatic activation in children with SCD defined by genotype (SS and SC), together with a group of unaffected children. We also assessed platelet activation and procoagulant surface expression in platelets and red blood cells (RBCs) using flow cytometry. Eligible SCD subjects included:patients with painful crisis assessed at two time points: hospital admission (crisis group) and clinic follow-up appointment (follow-up group);patients not in crisis attending a regular clinic appointment (steady-state group).
Patients were ineligible if they had received a recent blood transfusion, hydroxyurea, anticoagulants or aspirin. The results of TEG assays with citrated whole blood showed that compared to SC patients (n = 16) and normal children (n = 16), SS patients (n = 45) had significantly (p&lt;0.001) earlier clotting onset (mean R times were 4.5, 6.5 and 7.6 minutes for SS, SC and normals respectively) and significantly (p&lt;0.001) higher rates of clotting (mean maximum clotting rates were 16.8, 12.6 and 10.9 mm/min for SS, SC and normals respectively). TEG clotting onset and maximum clotting rates were not significantly different among steady-state, crisis and follow-up groups of children with SCD (both SS and SC genotypes), nor between sexes within each study group. Whole blood flow cytometry revealed that platelet GPIIb/IIIa activation (PAC-1 binding) was significantly elevated (p&lt;0.05) in SCD patients relative to normal children. In addition, markers of RBC procoagulant surface expression (RBC annexin A5 binding) and RBC-platelet aggregates were elevated in SCD patients compared to normal children. These results indicate that children with the SS genotype have an activated hemostatic system relative to normal and SC genotype children, and that this hypercoagulable state is maintained during sickle cell crisis as well as during steady state. It remains to be determined whether pharmacological interventions and/or RBC transfusions which improve clinical outcomes in SCD patients modify their constitutively hypercoagulable state.
Gene Expression Profiles during the γ- to β-Globin Switch in Erythroid Progenitors.
Authors: Li Wei , Pace Betty S. 
Published: April 2020

Abstract
The design and evaluation of therapies for sickle cell disease rely on our understanding of the sequential expression of the β-like globin genes during development. Drug inducers that reactivate γ-globin transcription after birth ameliorate symptoms in sickle cell patients. Therefore, the objective of our study is to identify trans-factors involved in γ-gene activation during the γ- to β-globin switch recapitulated in erythropoiesis. We previously established the one-phase liquid culture system using buffy coat mononuclear cells grown in erythropoietin (Epo; 4U/mL), stem cell factor (50ng/ml) and Interleukin-3 (IL-3; 10ng/ml). We observed high γ-globin transcription on day 7, equal γ- and β-globin mRNA levels by day 20 followed by high β-globin expression on day 28. Cell surface markers were measured by quantitative PCR (qPCR) and correlated with globin gene expression. During lineage commitment and erythroid maturation CD34+ cells decreased while the erythroid markers CD71, glycophorin A and Epo receptor increased 12 to 144-fold, followed by a decrease on day 28. We concluded that the γ- to β-globin switch was recapitulated in our system. Subsequent studies were performed with microarray and gene profiling by principle component analysis (PCA) to characterize proteins involved in switching. The Discover Chip from ArrayIt (Sunnyvale, CA) containing 380 human genes was used. cDNA from day 0/7, 7/21 and 21/28 were labeled with Cy3 and Cy5 respectively and used for standard chip hybridization. Fluorescence data was acquired using the Axon scanner and GenePixPro 6.0 software. By day 7, 88 genes were silenced and 68 activated; at day 28 an additional 38 genes were silenced and 47 activated. PCA defined the four major gene profiles consistent with developmental globin gene regulation. Profile 1 genes including IL-3, IL-9, heat shock protein 1a and neurofibromin were activated greater than 2-fold on day 7 suggesting positive regulators of γ-globin; trans-activators were not identified. Profile 2 genes were highly expressed around the switch on day 20. GATA-1, guanine nucleotide binding protein, ZF protein 132 and ERK1 expression peaked during this time. This data is consistent with a γ-globin silencer role previously demonstrated for ERK. Profile 3 genes also peaked on day 20 but continued to be highly expressed during β-globin transcription; EKLF, IL-8, Ras, Fos, and histone H3 were identified in this group. Experimental data support EKLF binding in the locus control region to compete with γ-globin expression. Finally Profile 4 genes increased steadily and peaked on day 28 consistent with positive β-globin regulators including NF-E2, ATF-2, IL-12, and thymosin β10. The microarray data were confirmed by qPCR in 90% of the genes tested. Our findings support the one-phase erythroid liquid culture system as a model for globin switching. We will expand gene profiling efforts using the Agilent 44K chip and PCA to confirm this model.
A Monoclonal Antibody to Lactadherin Inhibits Sickle Red Blood Cell Adhesion to Vascular Endothelial Cells in a Plasma Milieu.
Authors: Dasgupta Swapan K. , Guchhait Prasenjit , Le Anhquyen , Yellapragada Sarvari , Lopez Jose , Thiagarajan Perumal 
Published: April 2020

Abstract
Adherence of red blood cells to the endothelium initiates vaso-occlusion in sickle cell anemia. The increased adhesiveness of sickle erythrocytes is accompanied by several changes in the lipids of the erythrocyte membrane, including increased expression of phosphatidylserine (PS). One important PS-binding protein is lactadherin (also known as milk fat globule-EGF factor 8), a 45-kDa glycoprotein containing an Arg-Gly-Asp (RGD) sequence. It is secreted by macrophages and is present in normal plasma. Lactadherin promotes phagocytosis of PS-expressing apoptotic lymphocytes and sickle red blood cells by anchoring them to integrins on macrophages. Here, we investigated the role of endogenous lactadherin in adhesion of sickle erythrocytes to the endothelium. We developed a murine monoclonal antibody to human lactadherin, called L688, and investigated its effect on the adhesion of sickle red blood cells to histamine-stimulated human umbilical vein endothelial cells under hydrodynamic flow. In three experiments using washed erythrocytes resuspended in autologous plasma from three different patients with sickle cell anemia, L688 (20 μg/ml) inhibited adhesion by 24–30% (p&lt;0.01). Further evidence for an important role for lactadherin in sickle erythrocyte adhesion to endothelial cells was provided by the observation that exogenous lactadherin enhanced adhesion in a concentration-dependent manner. Lactadherin-mediated adhesion was also inhibited by monoclonal antibody abciximab, (c7E3, 10 μg/ml) which targets the β3 integrin subunit common to both αIIbβ3 and αVβ3. Control antibodies had no effect. Finally, the lactadherin-dependent adhesion of sickle erythrocytes to activated endothelium was inhibited by PS vesicles but not by phosphatidylcholine vesicles, confirming an important role for PS in sickle cell adhesion. Consistent with this, normal erythrocytes can be induced to adhere to stimulated HUVEC in a lactadherin-dependent manner by treatment with N-ethylmaleimide (10 mM) and calcium ionophore A23187 (4 μM) — treatment that exposes PS on the outer leaflet of the red cell membrane. Together, these results indicate that lactadherin mediates sickle cell adhesion to the endothelium by bridging PS on the erythrocytes with αVβ3 integrin on the endothelium. We propose that anemia in sickle cell disease is at least partially due to phagocytosis of lactadherin-coated sickle erythrocytes in the spleen, liver, and lymph nodes. Those erythrocytes that are not ingested immediately by macrophages will become more adhesive for endothelium. Thus, lactadherin appears to be involved both in sickle cell clearance from the circulation and in adhesion to endothelium.