bluuebunny/crossref_metadata_2025_split huggingface dataset:
Prevention of Human Interferon-alpha-Induced Thrombocytopenia by a Non-Peptidyl Human Thrombopoietin Receptor Activator, NIP-004.
Authors: Yamane Akiko , Nakamura Takanori , Ito Mamoru , Ohnishi Yasuyuki , Ikeda Yasuo , Miyakawa Yoshitaka 
Published: April 2020

Abstract
Human interferon-α (IFN) is useful for the treatment of chronic hepatitis C and neoplastic disorders; however, one major adverse effect is that thrombocytopenia often leads to a dose reduction or discontinuation of therapy. Since around 40% of patients with chronic hepatitis C will develop liver cancer, it is very important to prevent the transition from chronic hepatitis to liver cirrhosis and liver cancer with IFN treatment. However, some patients cannot receive IFN treatment because of thrombocytopenia resulting from liver cirrhosis and autoimmunity. This prompted us to evaluate the effects of a novel non-peptidyl human thrombopoietin (TPO) receptor (c-Mpl) activator, NIP-004, to prevent IFN-induced thrombocytopenia. We recently reported that NIP-004 at 30 μg/kg/day for 2 weeks increased human platelets 4-fold in immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) mice transplanted with 1 × 105 human cord blood-derived CD34+ cells (Blood, 2006). When PEG-hIFN-α2b (IFN-α2b) at 30 μg/kg was administered three times a week for 3 weeks to NOG mice transplanted with human CD34+ cells, the number of human platelets was significantly reduced by 40% compared to the control mice. Moreover, the number of human platelets was restored to the same level as the control mice with daily administration of 30 mg/kg NIP-004 for 2 weeks during the course of IFN treatment. These results indicated that NIP-004 is effective in preventing IFN-α2b-induced thrombocytopenia in vivo. Although IFN is believed to directly inhibit megakaryopoiesis, its mechanisms are not clearly understood. We analyzed the inhibitory effects of IFN-α2b on the colony formation of human megakaryocytes (CFU-MK) and the intracellular signaling induced by IFN and NIP-004. NIP-004 prevented the inhibition of CFU-MK formation by IFN-α2b in a dose dependent manner. IFN-α2b modestly inhibited the proliferation of human leukemia UT-7/EPO cells expressing human c-Mpl, and they were stimulated with TPO and NIP-004, respectively. We analyzed some form of cross-talk between TPO and IFN receptors by Western blot and quantitative RT-PCR analysis. NIP-004 induced the expression of SOCS-3 and CIS in UT-7/EPO-hMpl cells. We suggest that NIP-004 induces inhibitory signaling such as SOCS-3 and CIS and inhibits IFN-induced intracellular signaling. In conclusion, NIP-004 was shown to prevent IFN-α2b-induced thrombocytopenia in vivo and in vitro, suggesting the future clinical application of non-peptidyl c-Mpl activators for the treatment of patients with chronic hepatitis C by IFN.
Epo-Independent Proliferation and Differentiation of Genetically Modified Erythroid Progenitor Cells Using a Chemical Inducer of Dimerization.
Authors: Miller Chris P. , Zheng Songmao , Blau C. Anthony 
Published: April 2020

Abstract
Recombinant human erythropoietin (Epo) revolutionized the care of anemia in cancer. In light of emerging (albeit controversial) roles for Epo in supporting tumor growth, it may be useful to develop Epo-independent methods for regulating red blood cell production. Previous studies in this laboratory established that marrow transduced with conditional derivatives of fibroblast growth factor receptor-1 (F36VFGFR1) and the thrombopoietin receptor (F36VMpl) can support the chemical inducer of dimerization (CID)-dependent production of erythroid cells in transplanted mice. In the current study, we used human CD34+ cord blood (CB) cells to test whether CID-regulated red cell production might require collaboration between CID-initiated signals and those provided by Epo. CD34+ CB cells cultured in the absence of Epo did not proliferate and retained expression of the myeloid marker CD33. Epo (5U/mL) promoted proliferative expansion (66.2-fold in 12 days) and CD33 expression was lost as the cells differentiated to mature, glycophorin A+ erythroid cells (92.9 +/− 1.8%, n=3). Addition of a soluble human Epo receptor extracellular domain (shEpoR) at a concentration of 3ug/mL completely blocked Epo-dependent proliferation and similar to cells cultured without Epo, they retained expression of CD33. Addition of CID (100nM AP20187) to F36VMpl-transduced CD34+ CB cells in the absence of Epo promoted proliferative expansion (89.8-fold in 12 days) and differentiation as glycophorin A+ erythroid cells (77.2 +/− 4.8%, n=3). These CID responses were not significantly affected by the addition of shEpoR, as CID induced both mitogenic expansion (84.5 fold in 12 days) and differentiation as glycophorin A+ erythroid cells (74.2 +/− 6.7%, n=3) in the presence of concentrations of shEpoR that blocked Epo responses. These data suggest that F36VMpl does not require Epo signaling to support the proliferation and differentiation of human erythroid progenitor cells, and further define an Epo-independent method of erythroid cell production.
Leukemia Associated Mutants of SHP2 Protein Tyrosine Phosphatase Inhibit Nf1-Expression by Decreasing ICSBP and IRF2 Phosphorylation.
Authors: Huang Weiqi , Horvath Elizabeth , Eklund Elizabeth A. 
Published: April 2020

Abstract
Neurofibromin 1 (Nf1) is a ubiquitous protein with Ras-GAP activity. In myeloid cells, Nf1 down regulates Ras which has been activated by hematopoietic cytokines. Congenital absence of Nf1 is associated with the myeloproliferative/dysplastic disorder Juvenile Chronic Myelomonocytic Leukemia (JMMoL). JMMoL is characterized by hypersensitivity to cytokines such as GM-CSF, SCF and M-CSF. In previous studies, we found that cytokine stimulation of myeloid progenitor cells increases transcription of the NF1-gene, thereby increasing Nf1-expression. We identified a cis element in the NF1-promoter which is activated by the interferon consensus sequence binding protein (ICSBP). This is of interest because ICSBP-deficiency confers hypersensitivity to the same cytokines as does Nf1-deficiency. In the current studies, we find that interferon regulatory factor 2 (IRF2) cooperates with ICSBP to activate the NF1-cis element in differentiating myeloid cells. This interaction requires specific phosphorylation of conserved tyrosine residues in the IRF-domains of IRF2 and ICSBP. Phosphorylation of both proteins occurs in differentiating myeloid progenitor cells and myeloid cell lines. In myeloid progenitor cells, ICSBP and IRF2 are maintained in a non-phosphorylated state by the protein tyrosine phosphatases SHP1 and SHP2. During myeloid differentiation, activity of these PTPs decreases, permitting increased phosphorylation of ICSBP and IRF2. Leukemia associated, activating mutants of SHP2-PTP have been recently described in JMMoL, MDS and AML. Such mutations induce conformational changes in SHP2, resulting in constitutive activation. When introduced into myeloid progenitor cells, such SHP2-mutants induced a profile of cytokine hypersensitivity which is similar to those seen in Nf1-deficiency or ICSBP-deficiency. We find that expression of one of these leukemia associated SHP2 mutants blocks cytokine-induced phosphorylation of ICSBP and IRF2, impairing Nf1-expression in differentiating myeloid cells. This results in hypersensitivity and sustained proliferation in response to differentiating cytokines. Therefore, our studies mechanistically associate three molecular defects that have been associated with myeloproliferation and cytokine hypersensitivity; ICSBP-deficiency, Nf1-deficiency and expression of activated SHP2 mutants. Our results suggest that hematopoietic cytokine signaling induces tyrosine phosphorylation of IRF2 and ICSBP. These phospho-proteins activate transcription of the NF1-gene. Increased Nf1-expression down regulates cytokine-activated Ras. However, expression of constitutively active forms of SHP2-PTP blocks this process by impairing cytokine induced phosphorylation of IRF2 and ICSBP, decreasing Nf1-expression, and resulting in unopposed Ras activation. These results may be important to targeting this pathway in myeloid disorders characterized by cytokine hypersensitivity and myeloproliferation.
Essential Role of Kindlin 3 in Zebrafish Thrombopoiesis.
Authors: Mann James J. , Langer Nathaniel B. , Woo Andrew , Moran Tyler B. , Schindler Yocheved , Sullivan Matthew , Paw Barry H. , Cantor Alan B. 
Published: April 2020

Abstract
The zinc finger transcription factor GATA-1 is required for proliferative inhibition and terminal maturation of megakaryocytes, and is mutated in Down Syndrome Transient Myeloproliferative Disorder (TMD) and Acute Megakaryoblastic Leukemia (DS-AMKL). Yet the molecular mechanisms that regulate GATA-1 activity in megakaryopoiesis remain incompletely understood. Many transcription factors, in addition to binding DNA, make important protein-protein interactions that modulate their activity. In order to further understand GATA-1’s function, and possibly identify new factors involved in megakaryopoiesis, we purified GATA-1 containing multiprotein complexes from the murine L8057 megakaryocytic cell line. We generated stable L8057 cell lines expressing metabolically biotinylated and FLAG epitope tagged GATA-1, and then performed a tandem anti-FLAG immunoaffinity and streptavidin affinity purification. Using mass spectrometry (LC/MS/MS), we identified the known GATA-1 associated proteins Friend of GATA-1 (FOG-1), SCL, Ldb1, Runx-1/Cbf-β. SP1 and all components of the NuRD complex (which binds FOG-1) as co-purifying proteins. In addition, we reproducibly obtained several novel proteins. We previously reported the identification of the kruppel-type zinc finger transcription factor zfp148 (also called ZBP-89), and showed that it plays an essential role in megakaryopoiesis and definitive erythropoiesis. Here we report the identification of Kindlin 3 (also called URP2 for UNC-112 related protein 2), a member of a family of PH and FERM domain containing proteins that are thought to play a role in integrin-mediated processes. Expression of Kindlin 3 is restricted to hematopoietic cells, principally megakaryocytes and lymphocytes. It is first expressed at ~E9.5 during murine embryogenesis, and is abundant in fetal liver megakaryocytes by day E14.5. In order to begin to assess the role of Kindlin 3 in megakaryopoiesis in vivo, we performed morpholino-mediated knockdown of Kindlin 3 expression in CD41-GFP transgenic zebrafish embryos. In contrast to control embryos, embryos injected with Kindlin 3 specific morpholinos exhibited nearly complete loss of GFP+ thrombocytes (equivalent to mammalian megakaryocyte/platelets). Erythroid development (equivalent to mammalian primitive erythropoiesis at this stage of development) was not significantly affected, similar to embryos injected with zfp148-specific morpholinos. Given the role of integrin outside-to-inside signaling in megakaryopoiesis, we propose that Kindlin 3 may play a role linking extracellular signals to megakaryocyte maturation and growth control via GATA-1 transcription complexes. Further analysis in murine systems is underway to test this hypothesis.
Acetylation of GATA-1 Is Required for Chromatin Occupancy.
Authors: Lamonica Janine M. , Vakoc Christopher R. , Blobel Gerd A. 
Published: April 2020

Abstract
All three hematopoietic GATA transcription factors GATA-1, GATA-2, and GATA-3 are acetylated, although the in vivo role of this modification remains unclear. It has been proposed that acetylation of GATA-1 increases its affinity for DNA in vitro, although this finding has not been observed by others. To study the role of GATA-1 acetylation, we examined the functions of an acetylation-defective mutant of GATA-1 in maturing erythroid cells. We found that removal of the acetylation sites in GATA-1 largely abrogates its biological activity but does not impair its nuclear localization, steady state protein levels, or its ability to bind naked GATA elements in vitro. However, chromatin immunoprecipitation (ChIP) experiments revealed that mutant GATA-1 was dramatically impaired in binding to its cellular target sites in vivo, including genes that are normally activated (α- and β-globin, EKLF, FOG-1, Band3, and AHSP) and repressed (GATA-2 and c-kit) by GATA-1. Together, these results suggest that acetylation is required for GATA-1 chromatin occupancy. These findings point to a novel function for transcription factor acetylation, perhaps by facilitating protein interactions required for stable association with chromatin templates in vivo. To identify proteins that interact with acetylated GATA-1, we performed peptide affinity chromatography using acetylated GATA-1 peptides. Using this technique coupled with mass spectrometry, several proteins that bind to GATA-1 peptides in an acetylation-dependent manner were identified. The identified proteins contain known acetyl-lysine binding modules (bromodomains) consistent with their binding properties. The in vivo role of these proteins with regard to GATA-1 function is being examined and will be discussed.
Hypomorphic Alleles of PU.1 Result in Impaired Myelopoiesis and Target Gene Expression.
Authors: Houston Isaac B. , Kamath Meghana B. , Schweitzer Brock L. , Chlon Timothy M. , Ondr Jennifer K. , DeKoter Rodney P. 
Published: April 2020

Abstract
PU.1 is an Ets-family transcription factor that is critical for normal hematopoiesis. Knock-out studies of PU.1 demonstrate that it is required for the development of myeloid and lymphoid cells. PU.1 is expressed at graded levels in the immune system, with higher expression in myeloid lineage cells. Previous research has suggested that differences in PU.1 concentration are involved in hematopoietic cell fate decisions. However, the mechanism by which target genes in developing cells sense and respond to different PU.1 concentrations is unknown. To address this question, we inserted a beta-lactamase reporter gene and a neomycin resistance cassette into the first coding exon of PU.1. Mice homozygous for the targeted allele (PU.1BN), and an allele in which neomycin was removed (PU.1BLAC), had graded reductions in PU.1 expression and function. PU.1BN/BN mice display a substantial reduction in fetal and neonatal myelopoiesis and are macrophage deficient, resulting in a failure of vascular regression in the developing eye. A fraction of these mice survive until weaning and have an accumulation of c-Kit-expressing immature myeloid cells in the spleen and bone marrow. In addition, these mice acquire a myelomonocytic infiltration in the retina and hyaloid vessels of the eye. Mice homozygous for the second allele (PU.1BLAC/BLAC) survive less than one week and have a severe reduction in PU.1 activity and a block to myeloid development. Fetal livers from both types of mice lack any discernable mature myeloid cells and demonstrate a graded reduction in colony forming ability. To determine if these two alleles have an effect on gene expression, we established IL-7-dependent pro-B cell lines or IL-3-dependent cell lines from these mice as well as from wild type or PU.1 null controls. We found that PU.1-dependent target genes in these cell lines are expressed in a graded manner. In particular, expression of the gene encoding the low-affinity IgG Fc receptor (FcγRIIb) is directly responsive to PU.1 activity. In summary, these results demonstrate the critical importance of an appropriate threshold of PU.1 activity for normal hematopoiesis. The hypomorphic PU.1 alleles provide an excellent model for determining the mechanism of graded PU.1 levels in hematopoiesis.
STAT5 Is a Negative Regulator of Megakaryopoiesis.
Authors: Drayer Lyndsay , Olthof Sandra , Schuringa Jan Jacob , Vellenga Edo 
Published: April 2020

Abstract
The transcription factor STAT5 plays a critical role in self-renewal and lineage commitment of hematopoietic stem cels (HSCs). We have recently shown in CB CD34+ cells that persistent activation of STAT5 results in enhanced self-renewal and induces erythroid differentiation, while myelopoiesis was severely impaired. In this study we analyzed the function of STAT5 during megakaryocyte (MK) differentiation. Using RNA interference we downregulated STAT5 expression in peripheral blood CD34+ cells. Cells were transduced with a lentiviral construct encoding eGFP and a short-hairpin RNA for STAT5 or with a control vector expressing YFP. Transduction efficiencies were determined by flow cytometric detection of eGFP/YFP and ranged from 40%–80%. Decreased STAT5 expression was verified by Western blot and quantitative RT-PCR (65% lower expression in STAT5 RNAi cells versus YFP+ control cells, p=&lt;0.01). To assess the effects of decreased STAT5 expression on the progenitor pool, the transduced populations were sorted and enumerated in colony assays. Downregulation of STAT5 significantly increased the number of MK progenitors (1.9-fold, p=0.02) and resulted in a decrease of erythroid progenitors (0.6-fold, p&lt;0.01), but did not affect the number of granulocyte/monocyte progenitors. Prospective isolation of immature and commited progenitors are currently being performed to distinguish if certain progenitor subsets are re-directed to the MK lineage by low STAT5 expression. Next, we analyzed the role of STAT5 during MK differentiation in unilineage suspension cultures. Transduced cells were cultured in serum-free medium containing TPO and SCF and scored for cell counts and expression of MK markers weekly. The percentage of transduced cells and their number did not differ when STAT5 RNAi cells were compared to controls, indicating that downregulation of STAT5 provides no proliferative advantage or disadvantage. However, the expression of glycoproteins IIb/IIIa (CD41) and Ib (CD42) was upregulated, and an increase in large, polyploid cells was observed in STAT5 RNAi cultures (15.2% polyploid cells versus 4.9% in control cells at day 7, p&lt;0.01). RT-PCR analysis of transcription factors predominantly expressed in MK and erythroid lineages revealed that Runx1 and Erg were increased in STAT5 RNAi cells, and the level of Scl was decreased compared to control cells. These observations correlate with the increased MK differentiation observed in STAT5 downregulated cells. Together, these data demonstrate that the expression level of STAT5 has important effects regarding the type of hematopoietic cell development, with high levels favouring erythroid development and low levels enhancing MK differentiation and maturation.
Regulation of RUNX1 Transcriptional Function by GATA-1 Kinase Recruitment: A Novel Pathway for Megakaryocytic Gene Regulation.
Authors: Elagib Kamaleldin E. , Mihaylov Ivailo S. , Delehanty Lorrie L. , Gonias Sara L. , Caronia Jill F. , Goldfarb Adam N. 
Published: April 2020

Abstract
RUNX1 and GATA-1 both play essential roles in the transcriptional programming of normal mammalian megakaryocytic development, deficiencies of either factor having similar phenotypic consequences. We have previously characterized physical and functional interactions between these two factors, and others have confirmed analogous cooperations in Drosophila and Danio homologs. We now present data on molecular mechanisms for the cooperation of these two factors in the transcriptional activation of the megakaryocytic aIIb integrin promoter. In these studies, GATA-2 also physically interacted with RUNX1 but failed to cooperate in transcriptional activation. In fact, increasing amounts of GATA-2 repressed the functional interplay between GATA-1 and RUNX1. Through generation of GATA-2/GATA-1 chimeras, we identified a conserved subdomain within the GATA-1 amino terminus that was both necessary and sufficient for transcriptional cooperation with RUNX1. Coexpression of wild type GATA-1 or of cooperating GATA-2/GATA-1 chimeras, but not of GATA-2 or of non-cooperating chimeras, induced a mobility shift in wild type RUNX1. Using immunoprecipitation followed by immunoblot with a panel of phosphospecific antibodies, we found GATA-1 to induce RUNX1 phosphorylation at recognition sites for cyclin-dependent kinases (cdks). Treatment of cells with roscovitine, a specific cdk inhibitor, blocked the transcriptional cooperation of GATA-1 with RUNX1 and eliminated the RUNX1 mobility shift caused by GATA-1 coexpression. Mutagenesis of RUNX1 identified a cluster of serine/threonine-proline (S/TP) sites collectively required for the transcriptional augmentation and mobility shift induced by GATA-1. In addition, intact DNA binding by RUNX1 was required for cooperation with GATA-1. These results provide a new paradigm for cooperation of interacting transcription factors, in which one partner recruits a kinase leading to phosphorylation and activation of the other partner. Furthermore, these results provide a biochemical basis for the previously inexplicable functional differences between GATA-1, which promotes megakaryocytic maturation, and GATA-2 which promotes proliferation without maturation.
Orientation-Dependent Regulation of RUNX1 and GATA-1 Transcriptional Function by the p300/CBP Coactivators.
Authors: Mihaylov Ivailo S. , Elagib Kamaleldin E. , Delehanty Lorrie L. , Gonias Sara L. , Caronia Jill F. , Goldfarb Adam N. 
Published: April 2020

Abstract
The transcription factors RUNX1 and GATA-1, as well as the coactivators p300/CBP, have been implicated in the regulation of primary megakaryopoiesis through studies of knockout mice. In particular, p300/CBP has previously been shown to serve as a coactivator for both RUNX1 and GATA-1 in the transactivation of hematopoietic target genes. Coactivator orientation within transactivating complexes is generally not considered to influence the degree of transcriptional activity, reflecting an inherent flexibility in the spatial requirements for coactivator function. Experiments to further explore this issue showed coexpression of p300 to enhance cooperative transcriptional activation by wild type RUNX1 and GATA-1. Enforced recruitment of p300/CBP to GATA-1 through fusion of GATA-1 with the p300/CBP docking module of adenoviral E1A, E1A(1–89), enhanced GATA-1 activity alone, regardless of whether the fusion was to the amino or carboxy terminal of GATA-1. However, enforced recruitment of p300/CBP to the amino terminus of GATA-1 completely eliminated cooperation with RUNX1, and enforced recruitment of p300/CBP to the carboxy terminus of GATA-1 diminished cooperation with RUNX1. Both GATA-1 fusions retained physical interaction with RUNX1. Similarly, fusion of E1A(1–89) directly to the amino terminus of RUNX1 completely eliminated its transcriptional activity, while fusion to the carboxy terminus diminished RUNX1 transcriptional activity. For both the GATA-1 and the RUNX1 fusions, these repressive effects were attributable to the ectopic recruitment of p300/CBP because a mutation within E1A(1–89) known to specifically diminish p300/CBP recruitment, R2G, rescued the transcriptional activities. Addressing the mechanism of repression by ectopic p300/CBP, we found that E1A(1–89)-GATA-1 caused diminished serine phosphorylation within RUNX1, an effect opposite to that of wild type GATA-1 which enhanced RUNX1 phosphorylation. Similarly, E1A(1–89)-RUNX1 showed complete loss of phosphorylation on cdk target sites, as compared with wild type RUNX1. RUNX1-E1A(1–89) showed diminished phosphorylation on cdk target sites, as compared with wild type RUNX1. From these results, we conclude that p300/CBP may function as a coactivator for the RUNX1-GATA-1 complex when recruited to endogenous, wild type domains. By contrast, ectopic recruitment of p300/CBP to RUNX1, particularly to the amino terminus, targets RUNX1 for repression through inhibition or reversal of phosphorylation. Our results thus offer a novel paradigm in which the function of p300/CBP, coactivator versus repressor, may be determined by its mode of recruitment to certain transcriptional complexes. Notably, some transcription factors, such as GATA-1, have relaxed requirements for the topology of coactivator recruitment, whereas other transcription factors, such as RUNX1, have stringent requirements in this regard.